UvA-DARE (Digital Academic Repository) The …Histological polyp types Traditionally, colorectal...

UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl)

UvA-DARE (Digital Academic Repository)

The serrated neoplasia pathway: investigating the role of serrated polyps in colorectal cancerdevelopment

Boparai, K.S.

Link to publication

Citation for published version (APA):Boparai, K. S. (2011). The serrated neoplasia pathway: investigating the role of serrated polyps in colorectalcancer development.

General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s),other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).

Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, statingyour reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Askthe Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam,The Netherlands. You will be contacted as soon as possible.

Download date: 18 May 2020

https://dare.uva.nl/personal/pure/en/publications/the-serrated-neoplasia-pathway-investigating-the-role-of-serrated-polyps-in-colorectal-cancer-development(3653139c-93d7-4629-a35d-c27404022f1f).html

1

The serrated neoplasia pathway

Investigating the role of serrated

polyps in colorectal cancer development

Karam Singh Boparai

l

2

The Serrated Neoplasia Pathway Thesis, University of Amsterdam, the Netherlands ISBN: Cover: Forest in Corbett National Park, India Printed by: Ipskamp Drukkers BV, Amsterdam Copyright © K.S. Boparai, Amsterdam, the Netherlands The copyrights of the articles that have been accepted for publication published has been transferred to the respective journals The research described in this thesis was performed at the Departments of Gastroenterology & Hepatology and Pathology, AMC, Amsterdam The printing of this thesis was financially supported by AMC, Eurotec, Tramedico, Ferring, Olympus, Merck Sharp & Dohme B.V., US Endoscopy, Nederlandse Vereniging voor

Gastroenterologie,

l

3

The serrated neoplasia pathway

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam op gezag van de Rector Magnificus

prof. dr. D.C.van den Boom ten overstaan van een door het college voor promoties ingestelde

commissie in het openbaar te verdedigen in de Agnietenkapel op vrijdag 9 december 2011, te 12:00

door

Karam Singh Boparai

geboren te Purmerend

l

4

Promotiecommissie Promotores: Prof. dr. P. Fockens

Prof. dr. C.J.M. van Noesel Co-promotores: Dr. E. Dekker

Prof. dr. E.M.H. Mathus-Vliegen Overige leden: Prof. dr. G.A. Meijer Dr. F.M. Nagengast Prof dr. W.A. Bemelman Prof dr. G.J.A. Offerhaus Prof dr. F. Baas

Dr. J.E. East Faculteit der Geneeskunde

l

6

TABLE OF CONTENTS Chapter 1: General introduction and outline of 9

the thesis (submitted)

Part I: Clinical studies – Colorectal cancer in patients

and relatives Chapter 2: Multicentre cohort analysis: 41

colorectal cancer risk (Gut 2009)

Chapter 3: Increased CRC risk 1st degree 65

relatives in HPS (Gut 2010)

Part II: Molecular analyses – Etiology and colorectal

cancer pathways Chapter 4: Serrated polyps in MYH-associated 83

polyposis (Gastroenterology 2008)

Chapter 5: Serrated polyps in Lynch syndrome 101

(submitted) Chapter 6: Pathways leading to CRC in HPS 123

(American Journal of Pathology 2011) Part III: Endoscopic imaging – Detection and

differentiation of polyps Chapter 7: Increased polyp detection using NBI 153

(Endoscopy 2011) Chapter 8: Differentiation of polyps using ETMI in HPS 177

(Gastrointestinal Endoscopy 2009)

l

7

Summary and future perspectives 201 Samenvatting en toekomstperspectief 209 Acknowledgements 219 Curriculum Vitae 227

8

l

l

9

General introduction and outline of the thesis Submitted

Ch

ap

ter

General introduction and outline of the thesis

11

GENERAL INTRODUCTION Colorectal cancer causes

Colorectal cancer (CRC) ranks as the second most common

cause of cancer-related death in the western world.1 In general, CRC

can be subdivided into sporadic and familial cases. The overall

majority of individuals with CRC (~80%) are sporadic cases, lacking

any indication of an underlying inherited disorder. Approximately 20%

of cases are attributable to a familial (hereditary) disorder. However, in

only 5% of these patients the causative genetic defect is identified

such as in familial adenomatous polyposis syndrome (FAP), Lynch

syndrome or other rare CRC syndromes such as MUTYH-associated

polyposis (MAP), Peutz-Jeghers and juvenile polyposis (figure 1).

Recently a relatively new condition, called hyperplastic polyposis

syndrome (HPS), has been identified which is characterised by the

presence of multiple colorectal serrated polyps. This presumed

hereditary condition, has been shown to harbour a significantly

increased CRC risk.2-6

Although rare, the above mentioned hereditary disorders have

proven to be important models to study colorectal carcinogenesis in

general and based on these models major advances have occurred

regarding our understanding of the molecular events leading to CRC

and which potential pathways involving different polyp types are

implicated.

Chapte

r 1

Chapter I

12

Figure 1. Causes of colorectal cancer

Histological polyp types

Traditionally, colorectal polyps were divided in to two separate groups,

the adenoma which can be divided into tubular, tubulovillous and

villous subtypes7, and the hyperplastic polyp (HP), which displays

serration in the upper one-half to one-third of the crypt.8 Adenomas

have long been known to represent neoplastic precursor lesions of

most CRCs, especially those adenomas harbouring a villous

component. HPs on the other hand, which constitute the most common

occurring serrated polyps, were regarded until recently as innocuous

lesions lacking any premalignant potential. Untill very recently, clinical

guidelines did not recommend surveillance colonoscopies for

individuals with HPs because the associated CRC risk was not

believed to be higher than the risk in individuals without polyps.9


13

Recent morphological reappraisals of polyps revealed however that

serrated polyps can be further subdivided into different histological

entities with possibly different premalignant potential.

Hyperplastic polyps (HPs)

Colorectal HPs are typically diminutive polyps (<5mm in diameter)

which have a predilection for the sigmoid colon and rectum although

larger sized HPs with a proximal localization have also been

described.10 Besides being generally small these lesions are often flat

or sessile in shape, unremarkable in colour and often covered with

mucus.11, 12 Serration, which is caused by infolding of the crypt

epithelium leading to a saw-toothed appearance in longitudinal section

and a star-shaped appearance on cross-section, is limited to the upper

half to one-third of the crypt. Crypts are generally narrow and straight,

with a normal distribution of the proliferative zone in the base of the

crypts.8 HPs can be further subdivided into microvesicular, goblet cell-

rich and mucin-poor subtypes on the basis of mucin content of the

epithelial cells although the reproducibility hereof among pathologists

is doubtful and the clinical relevance questionable.

Sessile serrated lesion (SSL)

Torlakovic and Snover identified a new hyperplastic polyp variant,

formerly known as the sessile serrated adenoma.8, 13 These polyps are

currently referred to as sessile serrated lesions.109 These authors

showed that a subset of lesions that were originally diagnosed as HPs

in patients with Hyperplastic polyposis syndrome demonstrated a

different morphology with abnormal proliferation and concluded that

these SSLs were distinct lesions which should be differentiated from

HPs. SSLs are defined by predominantly architectural distortion with

Chapte

r 1

Chapter I

14

irregular dilated crypts, including dilatation of the base of the crypts

that often have a boot, L or inverted T shape. Serration can also be

identified at the base of the crypts as well as abnormal proliferation

and maturation with mature goblet or foveolar cells.14 Cytological

dysplasia is not usually demonstrated. However, if an area of dysplasia

is present, this should be specified. In contrast, HPs have narrow crypt

bases and serration is restricted to the upper half of the crypt.

Previous studies have shown that SSLs and HPs are

histologically hard to distinguish, leading to confusion with regard to

categorization of these serrated polyps. Indeed, it has recently been

shown that at re-evaluation the interobserver agreement for the

differentiation of serrated polyps remains only moderate.13, 15-17

Molecular analysis for the differentiation of these serrated polyps has

thus far shown that right-sided SSLs have increased MLH-1 and p16

methylation compared to left-sided SSLs and HPs.18 Some studies

have also shown that SSLs are more often BRAF mutated than HPs.11,

19 Nevertheless, unambiguous molecular differentiation between these

two lesions has as yet not been achieved.

Traditional serrated adenoma (TSA)

Longacre & Fenoglio Preiser, in a large polyp survey, re-evaluated a

group of polyps displaying mixed histological features of both

hyperplastic and adenomatous polyps. Rather than representing mixed

tumors, these lesions were considered to be adenomas with a serrated

configuration similar to HPs and were thus coined serrated adenomas

(currently named traditional serrated adenomas).20 Criteria for TSAs

include a pedunculated polyp shape with elongated villiform

projections uniformly lined with atypical cells having elongated nuclei

and eosinophilic cytoplasm.8, 21, 22 In the mentioned survey, TSAs


15

represented less than 1% of all colorectal polyps. Before re-evaluation,

approximately one third of these TSAs were originally diagnosed as

HP, one third as conventional adenoma and one third as intermediate

lesions. This variability underlines the difficulty in diagnosing these rare

lesions. TSAs are distinguishable from SSLs by their uniform

population of abnormal epithelial cells as well as their pedunculated

shape rather than sessile.

Colorectal carcinogenesis

CRC pathways

Notwithstanding this re-classification of serrated polyps,

conventional adenomas are considered to be the main lesions with an

undisputable premalignant potential. Clear histological evidence that

CRCs develop from adenomas was first established in 1975 by Muto

et al. which was further genetically described by Vogelstein et al. who

demonstrated in FAP patients that the adenoma-carcinoma sequence

is caused by aberrant activation of the Wnt signalling pathway.23, 24

This multi-step process of carcinogenesis is characterised by an initial,

bi-allelic inactivation of the adenomatous polyposis coli gene (APC)

followed by accumulation of genetic mutations in key oncogenes and

tumor-suppressor genes including KRAS, DCC and p53. These

events, in conjunction with associated chromosomal instability (CIN),

result in adenoma initiation and progression to CRC.

Subsequently, an alternative molecular pathway to CRC, the

microsatellite instability pathway, was discovered in patients with

Lynch syndrome.25 This pathway is initiated by deletion or inactivation

of one of the mismatch repair genes (MLH-1, MSH-2, MSH-6 or PMS-

2). This causes defective repair of replication errors in repetitive DNA

sequences which are most clearly demonstrated in so called

Chapte

r 1

Chapter I

16

microsatellite regions throughout the genome.26 Microsatellite regions

are abundant in non-encoding regions but are also present in encoding

regions of growth regulatory genes like tumor suppressor genes such

as TGFßRII, IGF2R and BAX. Alterations in such regions may then

cause inactivation of these genes and subsequent CRC formation with

microsatellite instability (MSI).27-29 MSI-CRCs can be the result of an

inherited germline defect of a mismatch-repair gene, as a part of Lynch

syndrome in 3% of all CRC cases, but can also be caused by a

somatic deletion or inactivation of a mismatch repair gene in up to 15%

of the sporadic CRCs (figure 1).30, 31

Alternative pathway to MSI CRC?

Both CRCs with CIN and CRCs with MSI from patients with Lynch

syndrome have been shown to harbour predominantly APC, KRAS

and P53 mutations.25, 32-36 In contrast, sporadic MSI CRCs infrequently

harbour these mutations which are typically found in conventional

adenomas.37-39 Instead, sporadic MSI CRCs are shown to have high

levels of mutations in the BRAF oncogene and extensive CPG- island

methylation of multiple genes (CIMP) including MLH-1, 40-44 which are

rare findings in sporadic adenomas34, 41, 43, 45, 46 but are commonly seen

in serrated polyps.11, 41, 45, 47 The association between serrated polyps

and specifically sporadic MSI-H CRCs is further emphasised by the

infrequent presence of BRAF mutations and CIMP in Lynch syndrome

MSI-H CRCs.42, 48 These findings suggested that serrated polyps,

instead of conventional adenomas, could represent precursor lesions

of sporadic MSI CRCs.

In parallel, clinicohistological evidence arose that serrated

polyps may also play a role in colorectal carcinogenesis. These reports

involve both sporadic HPs and HPs in the context of hyperplastic


17

polyposis syndrome and comprise CRCs in close vicinity of large

hyperplastic polyps49, 50; CRCs identified in mixed hyperplastic and

adenomatous polyps51; increased incidence of HPs and serrated

adenomas in patients with sporadic microsatellite-unstable CRCs52-54;

and a high prevalence of CRC in HPS patients13, 50, 55-57. More recent

cross-sectional association studies also showed a strong and

independent association between large (>1cm) sporadic serrated

polyps, i.e. HPs, SSLs and/or TSAs and synchronous advanced

CRC.58, 59 In another study, 5.8% of CRCs were found to have an

adjacent “serrated adenoma” which were more commonly MSI (38%)

than in CRCs without an adjacent serrated adenoma (11%).53

Currently however, proof of the existence of a serrated CRC pathway

data on the natural history of serrated polyps are lacking. Therefore,

the appropriate management of serrated polyps is unclear.

Serrated neoplasia pathway: proposed sequence of molecular events

While a single activating point mutation in BRAF (V600E) is an

oncogenetic hit in the mitogen-activated protein kinase (MAPK)

pathway resulting in augmented cell proliferation, survival and

inhibition of apoptosis, epigenetic alterations like CPG-island

methylation, in which the DNA sequence remains unaltered, can cause

inactivation of genes. CPG-islands are seen in approximately 50% of

human genes and consist of dense clusters of cytosine-guanosine

dinucleotides (CpG) that are susceptible to methylation of the cytosine

base resulting in gene silencing. Within the context of CRC, CPG-

island methylation can cause transcriptional repression of tumor

suppressor genes, like MLH1 in sporadic MSI CRCs.60-63 Despite their

different mechanisms of tumor development, BRAF mutations and

CIMP have been shown to be strongly associated in CRC, with an

Chapte

r 1

Chapter I

18

odds ratio of 203.64 This strong association between BRAF and CIMP

might be explained by the fact that mutations in the BRAF oncogene

alone induce an initial burst of proliferation followed by cell

senescence.64 Cell senescence is regulated by genes such as p16 and

p53 which can restrain the initially induced cell proliferation.65, 66

Further development to CRC may then be dependent on silencing of

these regulatory genes so that a stable senescent state is overcome. It

has thus been proposed that BRAF-mutant HPs may only progress to

CRC, possibly via SSLs, after a second molecular change such as

methylation-induced silencing of p16.67, 68 Interestingly, in a previous

study, analyzing the molecular differences between HPs and SSLs, it

was shown that p16 was significantly more methylated in SSLs than in

HPs, supporting the supposition that SSLs represent more advanced

lesions.18 Similarly, IGFBP7, a mediator of P53-induced senescence

has been shown to be methylated and silenced in BRAF mutated

CRCs.69, 70 Thus, it seems that the serrated pathway involves a

necessary multi-step process of genetic and epigenetic changes to

CRC.

Polyposis syndromes such as FAP and MAP are valuable

models for analyzing the molecular mechanisms of the adenoma-

carcinoma sequence due to the multitude of conventional adenomas

and coincident CRC in these patients. Similarly, HPS patients who

harbour multiple serrated polyps and have been shown to have

concurrent CRC in up to 50% of cases at clinical presentation3, 5, 6, 26, 51,

71-74, may provide a “scientific workbench” for further analysis of the

genetic sequence of events in the serrated neoplasia pathway.


19

Hyperplastic polyposis syndrome (HPS)

HPS is characterized by the presence of multiple serrated polyps

spread throughout the colorectum and has been defined by the World

Health Organization as the presence of at least five histologically

diagnosed HPs proximal to the sigmoid colon, of which 2 larger than

10mm in diameter, or more than 30 HPs distributed throughout the

colon.2 However, besides multiple HPs, SSLs and traditional

adenomas are common findings in this condition as well. HPs and

SSLs, both displaying a serrated phenotype, have been shown to be

histologically very similar and difficult to differentiate microscopically

with only moderate concordance.16-18, 75 Consequently, originally

diagnosed HPs may represent SSLs and vice versa. It was therefore

recently decided to rename the condition ‘serrated polyposis

syndrome’ or SPS, with inclusion of all serrated polyp subtypes.76

From a practical perspective, considering that most articles in this

thesis were published before this new definition was implemented, the

term hyperplastic polyposis syndrome (HPS) will be used throughout

this thesis.

From a clinical viewpoint, a further reclassification of HPS may

be beneficial to identify subtypes that have a higher risk of CRC

development. For example, it is assumable that HPS patients with a

higher number of polyps or larger polyps also have a higher CRC risk.

Also from an etiological viewpoint reclassification of HPS seems

prudent. The heterogeneous phenotype of HPS such as (i) the

presence of different polyp histologies and (ii) difference in polyp

localization and (iii) number of polyps, suggests that HPS represents

separate genetic conditions. Also molecular differences are found

between HPS phenotypes, such as more CPG-island methylation in

Chapte

r 1

Chapter I

20

right-sided serrated polyps as compared to left-sided serrated polyps,

however these differences are not distinctive.

Concerning the prevalence of HPS in the general population, a

previous screening sigmoidoscopy study in asymptomatic people aged

55 to 64 years estimated the prevalence to be 1:3000.77 However, in

this screening study only the rectosigmoid was investigated without

visualization of the remaining proximal colon. In addition, considering

that HPs are often diminutive in size, flat in shape and unremarkable in

colour, it is also possible that polyps were left undetected. This

suggests that the actual prevalence of HPS is probably higher than

currently estimated and in any case more frequent in occurrence than

other polyposis syndromes such as FAP (1:13.000).78 Concordantly, a

recent large prospective colonoscopy screening study involving 50.148

asymptomatic volunteers in Poland revealed 28 HPS patients leading

to an estimated incidence of 1:1800.79

HPS and CRC risk

HPS is associated with an increased CRC-risk. Previously published

case series report CRC at clinical presentation in up to 50% of HPS

patients.3, 5, 6, 26, 51, 71-74 However, because these findings could also be

interpreted as two-co-existent unrelated abnormalities, a causal

relationship between HPS and the development of CRC can not by

default be made but does seem presumable. Ideally, a large cohort of

asymptomatic HPS patients, derived from a population based

colonoscopy screening study, that is subsequently surveyed for

multiple years would be useful to adequately determine the risk of

CRC in these patients. Until such a study is performed, clinical

evidence for an increased CRC risk after HPS diagnosis is thus far

based on cohort studies with associated ascertainment bias. One such


21

study describes a case series of 13 HPS patients who were

prospectively followed and who received regular endoscopies. Three

of 13 (23%) patients developed CRC during follow-up, suggesting that

patients with HPS have an increased risk of malignant progression of

serrated polyps.5 Molecular supporting evidence include studies

showing a higher number of BRAF mutations and CIMP in serrated

polyps of HPS patients compared to sporadic serrated polyps.41, 80, 81

Moreover, HPS patients have far more serrated polyps than people in

the general population.

Nevertheless, considering that conventional adenomas, which

are traditionally considered premalignant polyps, are also common in

HPS, these polyps and the associated Wnt-pathway can not be

disregarded. Thus, based on the mixture of different precursor lesion

types, HPS patients represent a unique model to evaluate which CRC

pathways play a role in HPS and consequently which polyps are

clinically relevant and to obtain new evidence supporting the existence

of a serrated CRC pathway in humans.

Endoscopic diagnostics of HPS

Considering the presumed increased risk of malignant progression of

polyps in HPS, detection and removal of polyps seems necessary to

prevent CRC development in these patients. In HPS patients however,

serrated polyps, which are the overall majority, are generally small, flat

in shape and unremarkable in colour.82-84 These features are

associated with polyp miss-rates of up to 26% using standard

colonoscopy.85-87 Improved detection of these polyps seems therefore

desirable which can be achieved by improving bowel preparation;

clearing of fluid and debris; implementing a minimally accepted

withdrawal time of 6 minutes; visualizing the proximal sides of folds,

Chapte

r 1

Chapter I

22

flexures, and valves; and adequate colon distension.88-92 In addition,

chromoendoscopy has been shown to improve the detection of small

and flat lesions, specifically HPs, in patients undergoing surveillance

colonoscopy.86, 87, 93-95 Novel advanced endoscopic techniques, such

as narrow-band imaging (NBI) may also be an attractive alternative to

improve the detection of polyps in HPS. In contrast with

chromoendoscopy, which is a labour-intensive and time-consuming

technique, NBI (‘electronic chromoendoscopy’) is an easier push-of-a-

button technique that enhances mucosal and vascular detail without

the use of dyes. It has proven to be superior to high-resolution

endoscopy (HRE) for the detection of sporadic HPs but has not been

evaluated in HPS.96, 97

In addition, although removal of all larger polyps seems

necessary in any case, accurate differentiation of diminutive polyps

may aid the endoscopist in only removing advanced lesions (e.g.

conventional adenomas, SSLs and TSAs) and leaving ‘innocuous’

HPs, which display relatively lower levels of BRAF mutations and

CIMP, in situ.11, 16, 18, 19 Since HPS patients have many diminutive

polyps, this may save time and considerably prevent complications

from unnecessary polypectomies of HPs.3 Whereas chromoendoscopy

and NBI may also aid in the differentiation of HPs and adenomas, it is

unknown whether differentiation between HPs and SSLs is possible as

well.3, 98-101 Finally, autofluorescence imaging (AFI), which facilitates

differentiation of adenomas from non-adenomatous polyps based on

different fluorescence emission spectra102-104, may also be of value for

the differentiation of polyps in HPS.


23

Treatment of HPS

Due to this risk of malignant transformation of polyps, HPS patients are

either advised to undergo endoscopic surveillance with removal of

polyps or a surgical colonic resection. Recent expert opinions

recommend surveillance intervals ranging from one to three years.

Concerning the removal of polyps, advice varies from removal of only

proximally located polyps to complete removal of all polyps >5mm.3, 105

As a consequence, lack of clarity exists regarding the recommended

surveillance interval and which polyps should be removed. Therefore it

seems possible that a proportion of HPS patients may be insufficiently

treated and consequently be at risk of developing CRC under

surveillance (interval CRC). Conversely, over-treatment with

unnecessary removal of possibly harmless lesions which may result in

complications should also be avoided.

Familial risk

As opposed to FAP which has an autosomal dominant inheritance

pattern, in HPS the mode of inheritance is yet unknown. However,

although no genetic substrate has yet been identified, families have

previously been reported from which both an autosomal recessive and

autosomal dominant inheritance could be considered.3, 73, 106, 107 For

this reason, screening colonoscopies of first-degree relatives is

currently advised by some authorities.3, 108

The possible increased risk of developing CRC for first-degree

relatives is however unclear. In first-degree relatives CRC has been

reported, ranging in frequency from 0 to 50%.3, 4 In one retrospective

case series the incidence of polyps and CRC in first degree relatives

was studied.107 Of 17 first degree relatives who underwent screening

colonoscopy, 10/17 (59%) had polyps. In three patients HPs were

Chapte

r 1

Chapter I

24

detected, predominantly in the rectum and one patient (6%) was

diagnosed with HPS. This patient had multiple concomitant adenomas

as well. In the other 6 patients adenomas were discovered proximal to

the sigmoid colon. Of these patients, seven were under the age of 45

and the youngest was 32 years of age. No carcinoma was found.

However, these data were not prospectively acquired and instead of

performing a screening colonoscopy on all first-degree relatives of

patients with HPS, screening was primarily performed because of CRC

occurrence in the index-patient or because of an established family

history of CRC. To make an adequate estimate of the incidence of

polyps and HPS in first-degree relatives and the CRC-risk in this group

due to HPS, prospective screening of larger series of first-degree

relatives should be performed.


25

OUTLINE OF THE THESIS The foundation of this research project was laid at the endoscopy unit and out-patient clinic of the Academic Medical Center where an increasing number of patients with hyperplastic polyposis syndrome (HPS) have been diagnosed. HPS is characterized by the presence of multiple colorectal hyperplastic polyps which are traditionally considered to be benign lesions. The surprisingly common occurrence of colorectal cancer (CRC) in these patients prompted further research in to the clinical and molecular characteristics of HPS patients and the associated endoscopic management. Part I: Clinical analyses – Colorectal cancer risk The first part of the thesis focuses on the clinicopathological features of HPS patients and their first-degree relatives. Previously published case series report CRC at clinical presentation in up to 50% of HPS patients. Chapter 2 evaluates the risk of developing CRC after HPS diagnosis and which variables are associated with CRC in a large multi-centre cohort of HPS patients undergoing endoscopic surveillance. In chapter 3 the prevalence of polyps and CRC in first-degree relatives is described and compared with the background prevalence in the general population. Part II: Molecular analyses – Etiology and colorectal cancer pathways Although an underlying genetic disorder seems likely, thus far no genetic causes of HPS have been identified. However, previous case series of patients with a known genetic disorder have reported individuals with multiple serrated polyps. In chapter 4 and 5 we aimed to molecularly analyze whether serrated polyps identified in the setting of MYH-associated polyposis and Lynch syndrome are causally related to these respective disorders. Besides hyperplastic polyps (HPs), sessile serrated lesions (SSLs) and conventional adenomas are also common findings in HPS. Due to this heterogeneity, it is unknown which polyps eventually lead to CRC in HPS and thus are clinically relevant. In chapter 6 we aimed to analyze which polyps lead to CRC in HPS patients by performing combined immunohistological and molecular analyses.

Chapte

r 1

Chapter I

26

Part III: Endoscopic imaging – Detection and differentiation of polyps Considering the presumed increased risk of malignant progression of polyps in HPS, detection and removal of polyps seems necessary to prevent CRC development in these patients. Besides following general quality guidelines of colonoscopy, novel advanced endoscopic techniques, such as narrow-band imaging (NBI) may improve the detection of polyps in HPS. Chapter 7 involves a randomized cross-over study comparing NBI and high-resolution endoscopy with regard to their miss-rates of polyps. In addition to improved detection of polyps in HPS, accurate differentiation of HPs and SSLs, which appear endoscopically very similar, may aid the endoscopist in only removing SSLs and leaving HPs, which display comparatively lower levels of genetic mutations, in situ. In chapter 8 we evaluated high resolution white-light endoscopy, NBI and autofluorescence imaging for the endoscopic differentiation of polyps in HPS.


27

REFERENCES

1. Jemal A, Thun MJ, Ries LA, Howe HL, Weir HK, Center MM, Ward E,

Wu XC, Eheman C, Anderson R, Ajani UA, Kohler B, Edwards BK. Annual Report to the Nation on the Status of Cancer, 1975-2005, Featuring Trends in Lung Cancer, Tobacco Use, and Tobacco Control. J Natl Cancer Inst 2008.

2. Burt RW, Jass J. Hyperplastic polyposis. In: Hamilton SR and Aaltonen LA, eds. World Health Organisation Classification of Tumours Pathology and Genetics. Berlin: Springer-Verlag, 2000:135-136.

3. Chow E, Lipton L, Lynch E, D'Souza R, Aragona C, Hodgkin L, Brown G, Winship I, Barker M, Buchanan D, Cowie S, Nasioulas S, du SD, Young J, Leggett B, Jass J, Macrae F. Hyperplastic polyposis syndrome: phenotypic presentations and the role of MBD4 and MYH. Gastroenterology 2006;131:30-39.

4. Ferrandez A, Samowitz W, DiSario JA, Burt RW. Phenotypic characteristics and risk of cancer development in hyperplastic polyposis: case series and literature review. Am J Gastroenterol 2004;99:2012-2018.

5. Hyman NH, Anderson P, Blasyk H. Hyperplastic polyposis and the risk of colorectal cancer. Dis Colon Rectum 2004;47:2101-2104.

6. Rubio CA, Stemme S, Jaramillo E, Lindblom A. Hyperplastic polyposis coli syndrome and colorectal carcinoma. Endoscopy 2006;38:266-270.

7. Hamilton SR, Vogelstein B, Kudo S, et al. World Health Organization Classification of Tumours, Pathology and Genetics of Tumours of the Digestive System. Lyon, France: IARC Press, 2000:104-119.

8. Torlakovic E, Skovlund E, Snover DC, Torlakovic G, Nesland JM. Morphologic reappraisal of serrated colorectal polyps. Am J Surg Pathol 2003;27:65-81.

9. Winawer SJ, Zauber AG, Fletcher RH, Stillman JS, O'Brien MJ, Levin B, Smith RA, Lieberman DA, Burt RW, Levin TR, Bond JH, Brooks D, Byers T, Hyman N, Kirk L, Thorson A, Simmang C, Johnson D, Rex DK. Guidelines for colonoscopy surveillance after polypectomy: a consensus update by the US Multi-Society Task Force on Colorectal Cancer and the American Cancer Society. CA Cancer J Clin 2006;56:143-159.

Chapte

r 1

Chapter I

28

10. DiSario JA, Foutch PG, Mai HD, Pardy K, Manne RK. Prevalence and malignant potential of colorectal polyps in asymptomatic, average-risk men. Am J Gastroenterol 1991;86:941-945.

11. Spring KJ, Zhao ZZ, Karamatic R, Walsh MD, Whitehall VL, Pike T, Simms LA, Young J, James M, Montgomery GW, Appleyard M, Hewett D, Togashi K, Jass JR, Leggett BA. High prevalence of sessile serrated adenomas with BRAF mutations: a prospective study of patients undergoing colonoscopy. Gastroenterology 2006;131:1400-1407.

12. Yano T, Sano Y, Iwasaki J, Fu KI, Yoshino T, Kato S, Mera K, Ochiai A, Fujii T, Yoshida S. Distribution and prevalence of colorectal hyperplastic polyps using magnifying pan-mucosal chromoendoscopy and its relationship with synchronous colorectal cancer: prospective study. J Gastroenterol Hepatol 2005;20:1572-1577.

13. Torlakovic E, Snover DC. Serrated adenomatous polyposis in humans. Gastroenterology 1996;110:748-755.

14. Torlakovic EE, Gomez JD, Driman DK, Parfitt JR, Wang C, Benerjee T, Snover DC. Sessile Serrated Adenoma (SSA) vs. Traditional Serrated Adenoma (TSA). Am J Surg Pathol 2008;32:21-29.

15. Farris AB, Misdraji J, Srivastava A, Muzikansky A, Deshpande V, Lauwers GY, Mino-Kenudson M. Sessile serrated adenoma: challenging discrimination from other serrated colonic polyps. Am J Surg Pathol 2008;32:30-35.

16. Sandmeier D, Seelentag W, Bouzourene H. Serrated polyps of the colorectum: is sessile serrated adenoma distinguishable from hyperplastic polyp in a daily practice? Virchows Arch 2007;450:613-618.

17. Wong NA, Hunt LP, Novelli MR, Shepherd NA, Warren BF. Observer agreement in the diagnosis of serrated polyps of the large bowel. Histopathology 2009;55:63-66.

18. Sandmeier D, Benhattar J, Martin P, Bouzourene H. Serrated polyps of the large intestine: a molecular study comparing sessile serrated adenomas and hyperplastic polyps. Histopathology 2009;55:206-213.


29

19. Carr NJ, Mahajan H, Tan KL, Hawkins NJ, Ward RL. Serrated and non-serrated polyps of the colorectum: their prevalence in an unselected case series and correlation of BRAF mutation analysis with the diagnosis of sessile serrated adenoma. J Clin Pathol 2009;62:516-518.

20. Longacre TA, Fenoglio-Preiser CM. Mixed hyperplastic adenomatous polyps/serrated adenomas. A distinct form of colorectal neoplasia. Am J Surg Pathol 1990;14:524-537.

21. Snover DC, Jass JR, Fenoglio-Preiser C, Batts KP. Serrated polyps of the large intestine: a morphologic and molecular review of an evolving concept. Am J Clin Pathol 2005;124:380-391.

22. Snover DC. Serrated polyps of the large intestine. Semin Diagn Pathol 2005;22:301-308.

23. Muto T, Bussey HJ, Morson BC. The evolution of cancer of the colon and rectum. Cancer 1975;36:2251-2270.

24. Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M, Nakamura Y, White R, Smits AM, Bos JL. Genetic alterations during colorectal-tumor development. N Engl J Med 1988;319:525-532.

25. Aaltonen LA, Peltomaki P, Leach FS, Sistonen P, Pylkkanen L, Mecklin JP, Jarvinen H, Powell SM, Jen J, Hamilton SR, . Clues to the pathogenesis of familial colorectal cancer. Science 1993;260:812-816.

26. Iino H, Jass JR, Simms LA, Young J, Leggett B, Ajioka Y, Watanabe H. DNA microsatellite instability in hyperplastic polyps, serrated adenomas, and mixed polyps: a mild mutator pathway for colorectal cancer? J Clin Pathol 1999;52:5-9.

27. Souza RF, Appel R, Yin J, Wang S, Smolinski KN, Abraham JM, Zou TT, Shi YQ, Lei J, Cottrell J, Cymes K, Biden K, Simms L, Leggett B, Lynch PM, Frazier M, Powell SM, Harpaz N, Sugimura H, Young J, Meltzer SJ. Microsatellite instability in the insulin-like growth factor II receptor gene in gastrointestinal tumours. Nat Genet 1996;14:255-257.

28. Rampino N, Yamamoto H, Ionov Y, Li Y, Sawai H, Reed JC, Perucho M. Somatic frameshift mutations in the BAX gene in colon cancers of the microsatellite mutator phenotype. Science 1997;275:967-969.

Chapte

r 1

Chapter I

30

29. Markowitz S, Wang J, Myeroff L, Parsons R, Sun L, Lutterbaugh J, Fan RS, Zborowska E, Kinzler KW, Vogelstein B, . Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability. Science 1995;268:1336-1338.

30. Ionov Y, Peinado MA, Malkhosyan S, Shibata D, Perucho M. Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 1993;363:558-561.

31. Thibodeau SN, Bren G, Schaid D. Microsatellite instability in cancer of the proximal colon. Science 1993;260:816-819.

32. Fujiwara T, Stolker JM, Watanabe T, Rashid A, Longo P, Eshleman JR, Booker S, Lynch HT, Jass JR, Green JS, Kim H, Jen J, Vogelstein B, Hamilton SR. Accumulated clonal genetic alterations in familial and sporadic colorectal carcinomas with widespread instability in microsatellite sequences. Am J Pathol 1998;153:1063-1078.

33. Huang J, Papadopoulos N, McKinley AJ, Farrington SM, Curtis LJ, Wyllie AH, Zheng S, Willson JK, Markowitz SD, Morin P, Kinzler KW, Vogelstein B, Dunlop MG. APC mutations in colorectal tumors with mismatch repair deficiency. Proc Natl Acad Sci U S A 1996;93:9049-9054.

34. Konishi M, Kikuchi-Yanoshita R, Tanaka K, Muraoka M, Onda A, Okumura Y, Kishi N, Iwama T, Mori T, Koike M, Ushio K, Chiba M, Nomizu S, Konishi F, Utsunomiya J, Miyaki M. Molecular nature of colon tumors in hereditary nonpolyposis colon cancer, familial polyposis, and sporadic colon cancer. Gastroenterology 1996;111:307-317.

35. Losi L, Ponz de LM, Jiricny J, di GC, Benatti P, Percesepe A, Fante R, Roncucci L, Pedroni M, Benhattar J. K-ras and p53 mutations in hereditary non-polyposis colorectal cancers. Int J Cancer 1997;74:94-96.

36. Tomlinson I, Ilyas M, Johnson V, Davies A, Clark G, Talbot I, Bodmer W. A comparison of the genetic pathways involved in the pathogenesis of three types of colorectal cancer. J Pathol 1998;184:148-152.


31

37. Jass JR, Biden KG, Cummings MC, Simms LA, Walsh M, Schoch E, Meltzer SJ, Wright C, Searle J, Young J, Leggett BA. Characterisation of a subtype of colorectal cancer combining features of the suppressor and mild mutator pathways. J Clin Pathol 1999;52:455-460.

38. Salahshor S, Kressner U, Pahlman L, Glimelius B, Lindmark G, Lindblom A. Colorectal cancer with and without microsatellite instability involves different genes. Genes Chromosomes Cancer 1999;26:247-252.

39. Jass JR, Barker M, Fraser L, Walsh MD, Whitehall VL, Gabrielli B, Young J, Leggett BA. APC mutation and tumour budding in colorectal cancer. J Clin Pathol 2003;56:69-73.

40. Jass JR. Serrated adenoma of the colorectum and the DNA-methylator phenotype. Nat Clin Pract Oncol 2005;2:398-405.

41. Kambara T, Simms LA, Whitehall VL, Spring KJ, Wynter CV, Walsh MD, Barker MA, Arnold S, McGivern A, Matsubara N, Tanaka N, Higuchi T, Young J, Jass JR, Leggett BA. BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum. Gut 2004;53:1137-1144.

42. McGivern A, Wynter CV, Whitehall VL, Kambara T, Spring KJ, Walsh MD, Barker MA, Arnold S, Simms LA, Leggett BA, Young J, Jass JR. Promoter hypermethylation frequency and BRAF mutations distinguish hereditary non-polyposis colon cancer from sporadic MSI-H colon cancer. Fam Cancer 2004;3:101-107.

43. Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B, Velculescu VE. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature 2002;418:934.

44. Young J, Simms LA, Biden KG, Wynter C, Whitehall V, Karamatic R, George J, Goldblatt J, Walpole I, Robin SA, Borten MM, Stitz R, Searle J, McKeone D, Fraser L, Purdie DR, Podger K, Price R, Buttenshaw R, Walsh MD, Barker M, Leggett BA, Jass JR. Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings: parallel pathways of tumorigenesis. Am J Pathol 2001;159:2107-2116.

45. Chan TL, Zhao W, Leung SY, Yuen ST. BRAF and KRAS mutations in colorectal hyperplastic polyps and serrated adenomas. Cancer Res 2003;63:4878-4881.

Chapte

r 1

Chapter I

32

46. Ikehara N, Semba S, Sakashita M, Aoyama N, Kasuga M, Yokozaki H. BRAF mutation associated with dysregulation of apoptosis in human colorectal neoplasms. Int J Cancer 2005;115:943-950.

47. Yang S, Farraye FA, Mack C, Posnik O, O'Brien MJ. BRAF and KRAS Mutations in hyperplastic polyps and serrated adenomas of the colorectum: relationship to histology and CpG island methylation status. Am J Surg Pathol 2004;28:1452-1459.

48. Wang L, Cunningham JM, Winters JL, Guenther JC, French AJ, Boardman LA, Burgart LJ, McDonnell SK, Schaid DJ, Thibodeau SN. BRAF mutations in colon cancer are not likely attributable to defective DNA mismatch repair. Cancer Res 2003;63:5209-5212.

49. Azimuddin K, Stasik JJ, Khubchandani IT, Rosen L, Riether RD, Scarlatto M. Hyperplastic polyps: "more than meets the eye"? Report of sixteen cases. Dis Colon Rectum 2000;43:1309-1313.

50. Warner AS, Glick ME, Fogt F. Multiple large hyperplastic polyps of the colon coincident with adenocarcinoma. Am J Gastroenterol 1994;89:123-125.

51. Urbanski SJ, Kossakowska AE, Marcon N, Bruce WR. Mixed hyperplastic adenomatous polyps--an underdiagnosed entity. Report of a case of adenocarcinoma arising within a mixed hyperplastic adenomatous polyp. Am J Surg Pathol 1984;8:551-556.

52. Hawkins NJ, Ward RL. Sporadic colorectal cancers with microsatellite instability and their possible origin in hyperplastic polyps and serrated adenomas. J Natl Cancer Inst 2001;93:1307-1313.

53. Makinen MJ, George SM, Jernvall P, Makela J, Vihko P, Karttunen TJ. Colorectal carcinoma associated with serrated adenoma--prevalence, histological features, and prognosis. J Pathol 2001;193:286-294.

54. Goldstein NS, Bhanot P, Odish E, Hunter S. Hyperplastic-like colon polyps that preceded microsatellite-unstable adenocarcinomas. Am J Clin Pathol 2003;119:778-796.

55. Jeevaratnam P, Cottier DS, Browett PJ, Van de Water NS, Pokos V, Jass JR. Familial giant hyperplastic polyposis predisposing to colorectal cancer: a new hereditary bowel cancer syndrome. J Pathol 1996;179:20-25.


33

56. Kusunoki M, Fujita S, Sakanoue Y, Shoji Y, Yanagi H, Yamamura T, Utsunomiya J. Disappearance of hyperplastic polyposis after resection of rectal cancer. Report of two cases. Dis Colon Rectum 1991;34:829-832.

57. Shepherd NA. Inverted hyperplastic polyposis of the colon. J Clin Pathol 1993;46:56-60.

58. Li D, Jin C, McCulloch C, Kakar S, Berger BM, Imperiale TF, Terdiman JP. Association of large serrated polyps with synchronous advanced colorectal neoplasia. Am J Gastroenterol 2009;104:695-702.

59. Lazarus R, Junttila OE, Karttunen TJ, Makinen MJ. The risk of metachronous neoplasia in patients with serrated adenoma. Am J Clin Pathol 2005;123:349-359.

60. Ahuja N, Mohan AL, Li Q, Stolker JM, Herman JG, Hamilton SR, Baylin SB, Issa JP. Association between CpG island methylation and microsatellite instability in colorectal cancer. Cancer Res 1997;57:3370-3374.

61. Cunningham JM, Christensen ER, Tester DJ, Kim CY, Roche PC, Burgart LJ, Thibodeau SN. Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability. Cancer Res 1998;58:3455-3460.

62. Esteller M, Hamilton SR, Burger PC, Baylin SB, Herman JG. Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res 1999;59:793-797.

63. Kane MF, Loda M, Gaida GM, Lipman J, Mishra R, Goldman H, Jessup JM, Kolodner R. Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res 1997;57:808-811.

64. Weisenberger DJ, Siegmund KD, Campan M, Young J, Long TI, Faasse MA, Kang GH, Widschwendter M, Weener D, Buchanan D, Koh H, Simms L, Barker M, Leggett B, Levine J, Kim M, French AJ, Thibodeau SN, Jass J, Haile R, Laird PW. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet 2006;38:787-793.

Chapte

r 1

Chapter I

34

65. Collado M, Serrano M. The power and the promise of oncogene-induced senescence markers. Nat Rev Cancer 2006;6:472-476.

66. Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 1997;88:593-602.

67. Minoo P, Jass JR. Senescence and serration: a new twist to an old tale. J Pathol 2006;210:137-140.

68. Leggett B, Whitehall V. Role of the serrated pathway in colorectal cancer pathogenesis. Gastroenterology 2010;138:2088-2100.

69. Wajapeyee N, Serra RW, Zhu X, Mahalingam M, Green MR. Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7. Cell 2008;132:363-374.

70. Suzuki H, Igarashi S, Nojima M, Maruyama R, Yamamoto E, Kai M, Akashi H, Watanabe Y, Yamamoto H, Sasaki Y, Itoh F, Imai K, Sugai T, Shen L, Issa JP, Shinomura Y, Tokino T, Toyota M. IGFBP7 is a p53-responsive gene specifically silenced in colorectal cancer with CpG island methylator phenotype. Carcinogenesis 2010;31:342-349.

71. Carvajal-Carmona L, Howarth K, Lockett M, Polanco-Echeverry G, Volikos E, Gorman M, Barclay E, Martin L, Jones A, Saunders B, Guenther T, Donaldson A, Paterson J, Frayling I, Novelli M, Phillips R, Thomas H, Silver A, Atkin W, Tomlinson I. Molecular classification and genetic pathways in hyperplastic polyposis syndrome. J Pathol 2007;212:378-385.

72. Jass JR, Iino H, Ruszkiewicz A, Painter D, Solomon MJ, Koorey DJ, Cohn D, Furlong KL, Walsh MD, Palazzo J, Edmonston TB, Fishel R, Young J, Leggett BA. Neoplastic progression occurs through mutator pathways in hyperplastic polyposis of the colorectum. Gut 2000;47:43-49.

73. Rashid A, Houlihan PS, Booker S, Petersen GM, Giardiello FM, Hamilton SR. Phenotypic and molecular characteristics of hyperplastic polyposis. Gastroenterology 2000;119:323-332.

74. Oono Y, Fu K, Nakamura H, Iriguchi Y, Yamamura A, Tomino Y, Oda J, Mizutani M, Takayanagi S, Kishi D, Shinohara T, Yamada K, Matumoto J, Imamura K. Progression of a Sessile Serrated Adenoma to an Early Invasive Cancer Within 8 Months. Dig Dis Sci 2008.


35

75. Khalid O, Radaideh S, Cummings OW, O'Brien MJ, Goldblum JR, Rex DK. Reinterpretation of histology of proximal colon polyps called hyperplastic in 2001. World J Gastroenterol 2009;15:3767-3770.

76. Snover DC, Ahnen D, Burt R, Odze R.D. Serrated Polyps of the Colon and Rectum and Serrated Polyposis. WHO Classification of Tumours of the Digestive System. 4th edition ed. Lyon, France: IARC, 2010.

77. Lockett MJ, Atkin W.S. Hyperplastic polyposis: prevalence and cancer risk. 48 ed. 2001:A4.

78. Bisgaard ML, Fenger K, Bulow S, Niebuhr E, Mohr J. Familial adenomatous polyposis (FAP): frequency, penetrance, and mutation rate. Hum Mutat 1994;3:121-125.

79. Orlowska J, Kiedrowski M, Kaminski F.M., Regula J, Butruk E. Hyperplastic polyposis syndrome in asymptomatic patients: the results from the colorectal-cancer screening program. Virchows Arch 2009;Abstract: OP 13.8.

80. Beach R, Chan AO, Wu TT, White JA, Morris JS, Lunagomez S, Broaddus RR, Issa JP, Hamilton SR, Rashid A. BRAF mutations in aberrant crypt foci and hyperplastic polyposis. Am J Pathol 2005;166:1069-1075.

81. Chan AO, Issa JP, Morris JS, Hamilton SR, Rashid A. Concordant CpG island methylation in hyperplastic polyposis. Am J Pathol 2002;160:529-536.

82. Jaramillo E, Watanabe M, Rubio C, Slezak P. Small colorectal serrated adenomas: endoscopic findings. Endoscopy 1997;29:1-3.

83. Matsumoto T, Mizuno M, Shimizu M, Manabe T, Iida M, Fujishima M. Serrated adenoma of the colorectum: colonoscopic and histologic features. Gastrointest Endosc 1999;49:736-742.

84. Rubio CA, Jaramillo E. Flat serrated adenomas of the colorectal mucosa. Jpn J Cancer Res 1996;87:305-309.

85. van Rijn JC, Reitsma JB, Stoker J, Bossuyt PM, van Deventer SJ, Dekker E. Polyp miss rate determined by tandem colonoscopy: a systematic review. Am J Gastroenterol 2006;101:343-350.

Chapte

r 1

Chapter I

36

86. Brooker JC, Saunders BP, Shah SG, Thapar CJ, Thomas HJ, Atkin WS, Cardwell CR, Williams CB. Total colonic dye-spray increases the detection of diminutive adenomas during routine colonoscopy: a randomized controlled trial. Gastrointest Endosc 2002;56:333-338.

87. Hurlstone DP, Cross SS, Slater R, Sanders DS, Brown S. Detecting diminutive colorectal lesions at colonoscopy: a randomised controlled trial of pan-colonic versus targeted chromoscopy. Gut 2004;53:376-380.

88. Barclay RL, Vicari JJ, Doughty AS, Johanson JF, Greenlaw RL. Colonoscopic withdrawal times and adenoma detection during screening colonoscopy. N Engl J Med 2006;355:2533-2541.

89. Froehlich F, Wietlisbach V, Gonvers JJ, Burnand B, Vader JP. Impact of colonic cleansing on quality and diagnostic yield of colonoscopy: the European Panel of Appropriateness of Gastrointestinal Endoscopy European multicenter study. Gastrointest Endosc 2005;61:378-384.

90. Harewood GC, Sharma VK, de GP. Impact of colonoscopy preparation quality on detection of suspected colonic neoplasia. Gastrointest Endosc 2003;58:76-79.

91. Rex DK. Colonoscopic withdrawal technique is associated with adenoma miss rates. Gastrointest Endosc 2000;51:33-36.

92. Rex DK. Maximizing detection of adenomas and cancers during colonoscopy. Am J Gastroenterol 2006;101:2866-2877.

93. Lapalus MG, Helbert T, Napoleon B, Rey JF, Houcke P, Ponchon T. Does chromoendoscopy with structure enhancement improve the colonoscopic adenoma detection rate? Endoscopy 2006;38:444-448.

94. Le RM, Coron E, Parlier D, Nguyen JM, Canard JM, Alamdari A, Sautereau D, Chaussade S, Galmiche JP. High resolution colonoscopy with chromoscopy versus standard colonoscopy for the detection of colonic neoplasia: a randomized study. Clin Gastroenterol Hepatol 2006;4:349-354.

95. Park SY, Lee SK, Kim BC, Han J, Kim JH, Cheon JH, Kim TI, Kim WH. Efficacy of chromoendoscopy with indigocarmine for the detection of ascending colon and cecum lesions. Scand J Gastroenterol 2008;43:878-885.


37

96. Adler A, Pohl H, Papanikolaou IS, bou-Rebyeh H, Schachschal G, Veltzke-Schlieker W, Khalifa AC, Setka E, Koch M, Wiedenmann B, Rosch T. A prospective randomised study on narrow-band imaging versus conventional colonoscopy for adenoma detection: does narrow-band imaging induce a learning effect? Gut 2008;57:59-64.

97. Aschenbeck J, Adler A, Yenerim T, Mayr M, Aminalai A, Drossel R, Schroder A, Scheel M, Wiedenmann B, Rosch T. Narrow-Band Versus White-Light HDTV Endoscopic Imaging for Screening Colonoscopy: A Prospective Randomized Trial. Gastroenterology 2008.

98. Su MY, Hsu CM, Ho YP, Chen PC, Lin CJ, Chiu CT. Comparative study of conventional colonoscopy, chromoendoscopy, and narrow-band imaging systems in differential diagnosis of neoplastic and nonneoplastic colonic polyps. Am J Gastroenterol 2006;101:2711-2716.

99. Chiu HM, Chang CY, Chen CC, Lee YC, Wu MS, Lin JT, Shun CT, Wang HP. A prospective comparative study of narrow-band imaging, chromoendoscopy, and conventional colonoscopy in the diagnosis of colorectal neoplasia. Gut 2007;56:373-379.

100. East JE, Suzuki N, Bassett P, Stavrinidis M, Thomas HJ, Guenther T, Tekkis PP, Saunders BP. Narrow band imaging with magnification for the characterization of small and diminutive colonic polyps: pit pattern and vascular pattern intensity. Endoscopy 2008;40:811-817.

101. van den Broek FJ, Reitsma JB, Curvers WL, Fockens P, Dekker E. Systematic review of narrow-band imaging for the detection and differentiation of neoplastic and nonneoplastic lesions in the colon (with videos). Gastrointest Endosc 2009;69:124-135.

102. DaCosta RS, Andersson H, Cirocco M, Marcon NE, Wilson BC. Autofluorescence characterisation of isolated whole crypts and primary cultured human epithelial cells from normal, hyperplastic, and adenomatous colonic mucosa. J Clin Pathol 2005;58:766-774.

103. Haringsma J, Tytgat GN, Yano H, Iishi H, Tatsuta M, Ogihara T, Watanabe H, Sato N, Marcon N, Wilson BC, Cline RW. Autofluorescence endoscopy: feasibility of detection of GI neoplasms unapparent to white light endoscopy with an evolving technology. Gastrointest Endosc 2001;53:642-650.

Chapte

r 1

Chapter I

38

104. Wang TD, Van DJ, Crawford JM, Preisinger EA, Wang Y, Feld MS. Fluorescence endoscopic imaging of human colonic adenomas. Gastroenterology 1996;111:1182-1191.

105. East JE, Saunders BP, Jass JR. Sporadic and syndromic hyperplastic polyps and serrated adenomas of the colon: classification, molecular genetics, natural history, and clinical management. Gastroenterol Clin North Am 2008;37:25-46, v.


107. Lage P, Cravo M, Sousa R, Chaves P, Salazar M, Fonseca R, Claro I, Suspiro A, Rodrigues P, Raposo H, Fidalgo P, Nobre-Leitao C. Management of Portuguese patients with hyperplastic polyposis and screening of at-risk first-degree relatives: a contribution for future guidelines based on a clinical study. Am J Gastroenterol 2004;99:1779-1784.

108. Young J, Jenkins M, Parry S, Young B, Nancarrow D, English D, Giles G, Jass J. Serrated pathway colorectal cancer in the population: genetic consideration. Gut 2007;56:1453-1459

109 Vieth M, Quirke P, Lambert R, von KL, Risio M. Annex to Quirke et al.

Quality assurance in pathology in colorectal cancer screening and diagnosis: annotations of colorectal lesions. Virchows Arch 2011;458:21-30.

CRC cancer risk in HPS patients during follow up

39

Clinical analyses – Colorectal cancer risk

P A

R T

Chapter 2

40


41

Increased colorectal cancer risk during follow-up in patients with hyperplastic polyposis syndrome: a multicentre cohort study

K.S. Boparai E.M.H. Mathus-Vliegen

J.J. Koornstra F.M. Nagengast M. van Leerdam

C.J.M. van Noesel

M. Houben

A. Cats

L.P. van Hest

P. Fockens

E. Dekker

Gut. 2010 Aug;59(8):1094-100

Ch

ap

ter

Chapter 2

42

ABSTRACT

Background and aims: Patients with hyperplastic polyposis syndrome

(HPS) receive endoscopic surveillance to prevent malignant

progression of polyps. However, the optimal treatment and

surveillance protocol for these patients is unknown. The aim of this

study was to describe the clinical and pathological features of a large

HPS cohort during multiple years of endoscopic surveillance. Methods:

Databases were searched for HPS patients who were retrospectively

analysed. Endoscopy reports and histopathology reports were

collected to evaluate frequency of endoscopic surveillance and to

obtain information regarding polyp and CRC presence. Results: In 77

HPS patients, 1984 polyps were identified during a mean follow-up

period of 5.6 years (range:0.5-26.6). In 27(35%) patients CRC was

detected of which 22(28.5%) at initial endoscopy. CRC was detected

during surveillance in five patients (cumulative incidence: 6.5%) after a

median follow-up time of 1.3 years and a median interval of 11 months.

Of these interval CRCs, 4/5 were detected in diminutive serrated

polyps (range: 4-16mm). The cumulative risk of CRC under

surveillance was 7% at five years. At multivariate logistic regression,

an increasing number of hyperplastic polyps (odds ratio:1.05, p=0.013)

and serrated adenomas (odds ratio:1.09, p=0.048) was significantly

associated with CRC presence. Conclusions: HPS patients undergoing

endoscopic surveillance have an increased CRC risk. The number of

serrated polyps is positively correlated with the presence of CRC in

HPS, thus supporting a ‘serrated pathway’ to CRC. To prevent

malignant progression, adequate detection and removal of all polyps

seems advisable. If this is not feasible, surgical resection should be

considered.


43

INTRODUCTION

Colorectal cancer (CRC) ranks as the third most common

cause of cancer related death in the western world.1 A well known

mechanism describing CRC development is the adenoma-carcinoma

sequence which in the majority of cases is initiated by activation of the

Wnt signalling pathway.2, 3 Much information regarding this pathway

has been derived from polyposis syndromes such as familial

adenomatous polyposis (FAP) and MYH-associated polyposis (MAP).

In addition to this classical adenoma-carcinoma sequence, a proposed

“serrated neoplasia pathway”, describes the progression of serrated

polyps (i.e. hyperplastic polyps, sessile serrated adenomas and

traditional serrated adenomas) to CRC.4 It is proposed that this

possible alternative pathway is also associated with hyperplastic

polyposis syndrome (HPS). There are strong indications that mixed

pathways also exist in which both conventional adenomas and

serrated polyps are involved.5

Clinically, the condition HPS is characterized by the presence

of multiple hyperplastic polyps (HPs) spread throughout the colorectum

and is associated with an increased CRC-risk. Indeed, numerous

patients with CRC and concurrent HPS have been reported.6-12 While

previously the indicated management of HPS patients was unknown,

experts presently believe that these patients should undergo regular

endoscopic surveillance to prevent malignant progression of polyps.7, 13

However, the optimal treatment and surveillance protocol for HPS

patients is largely speculative. Therefore it seems possible that a

proportion of HPS patients may be insufficiently treated and

consequently be at risk of developing CRC under surveillance (interval

CRC).

Chapte

r 2

Chapter 2

44

The aim of this study was to describe the clinical and

pathological features of a large HPS cohort (n=77) during multiple

years of endoscopic surveillance. Furthermore, we assessed the

cumulative incidence and incidence rate of CRC during surveillance

and its association with the interval and frequency of surveillance

endoscopies. Finally, we analysed possible predictive variables that

may be associated with the occurrence of CRC in HPS.

PATIENTS AND METHODS

Study population

Databases of 7 medical centres in the Netherlands were searched for

patients satisfying the diagnostic criteria of HPS (i.e. at least five

histologically diagnosed HPs proximal to the sigmoid colon, of which 2

greater than 10mm in diameter, or more than 20 HPs distributed

throughout the colon) and undergoing endoscopic surveillance.8, 11

Owing to the common presence of both HPs and (sessile) serrated

adenomas (SAs) in HPS and the difficult histological differentiation

between these two groups, both HPs and SAs were used to fulfil the

criteria. 14-17 Of these patients, clinical data from May 1982 to June

2008 were retrospectively analysed. Adherence to the described

criteria was assessed by analysing endoscopy reports with

corresponding histopathology reports as well as histopathology reports

of colonic surgical resection specimens. This study was conducted in

accordance with the research code of our institutional medical ethical

committee on human experimentation, as well as in agreement with

the Helsinki Declaration of 1975, as revised in 1983. Patients with a

known germline APC mutation or bi-allelic MYH mutation were

excluded from the study.


45

Clinical characteristics

Demographic data of patients concerning age, sex and history

of CRC were ascertained. Endoscopy reports with corresponding

histopathology reports during follow-up were collected to evaluate the

duration, interval and frequency of endoscopic surveillance and to

derive information regarding the, number, size, distribution and

histology of polyps. If applicable, histopathology reports of surgical

colonic resection specimens were also used to obtain the above

mentioned polyp characteristics. Also, if genetic mutation analysis was

performed, these data were retrieved.

Polyps were classified as HP, serrated adenoma (SA), mixed

polyp (MP) or conventional adenoma. Because the distinction between

sessile serrated adenoma and traditional serrated adenoma had not

been made throughout the study period by each medical center and

because they are both considered to be precursor lesions in the

‘serrated pathway’, the category SAs comprised both types of

lesions.16 Regarding the number of polyps detected in this study, all

polyps were tallied once i.e. when a detected polyp was not removed

during endoscopy this polyp was not re-tallied at subsequent

endoscopies.

Information concerning the nature and reason of performed

colorectal surgery was obtained if applicable. Detailed information

regarding co-existent CRC and CRC incidence during surveillance was

examined by evaluating histopathology reports of colectomy resection

specimens and/or endoscopy reports. An interval CRC was defined as

a CRC detected after HPS diagnosis after at least two previous

endoscopies.

Chapte

r 2

Chapter 2

46

Statistical analysis

Statistical analyses were performed by using a statistical

software package (Statistical Package for the Social Sciences 15.0.1;

SPSS Inc, Chicago, Ill). The cumulative risk of developing CRC during

follow-up was analyzed by Kaplan-Meier survival analysis. Observation

time was measured from date of HPS diagnosis to the incidence of

carcinoma or end of the study period. Univariate binary logistic

regression was performed for chosen variables that may be associated

with the presence of CRC. For multivariate regression analyses, only

variables which showed an association (p<0.1) on univariate analysis

were used in a final multivariate model.

RESULTS

Patients

Data of 77 HPS patients from the period 1982-2008 were

retrospectively analysed in this study. Clinical characteristics of

patients are shown in table 1. The median age at diagnosis of HPS

was 56 years (range: 40-74). There were 42 males and 35 females.

Fifty-nine of 77 patients (77%) had >5 proximal HPs (of which 2 were

larger than 10mm) or >20 HPs spread throughout the colon. The other

17 patients had >5 proximal HPs/SAs (of which 2 were larger than

10mm) or >20 HPs/SAs spread throughout the colon. In 52/77 (68%)

patients, germline APC and MYH-mutation analysis was performed. In

all cases mutation analysis was negative except for one patient with a

mono-allelic MYH-mutation (Y165C) who had ≥ 15 adenomas. In all

patients harbouring ≥ 15 adenomas (n=5) mutation analysis was

performed. Main reasons for initial presentation were: colorectal polyps

detected elsewhere (n=23), a positive family history for CRC or

colorectal polyps (n=16), bloody stools/positive faecal occult blood test/


47

iron-deficiency anaemia (n=15), altered defecation pattern (n=8),

abdominal pain with or without altered bowel habits (n=5), polyps

detected at sigmoidoscopy screening programme (n=4) and personal

history of CRC (n=3).

Cumulatively, a median number of 15 HPs per patient were

found in this cohort. In 47 (61%) patients HPs ≥ 10mm were detected.

SAs and adenomas were identified in 52% and 69% of patients,

respectively. CRC was diagnosed in 27 (35%) patients: 22 (28.5%) at

initial endoscopy and 5 (6.5%) during follow-up.

A surgical colonic resection was performed in 33/77 (43%)

patients: 2 total colectomies, 13 subtotal colectomies, 8 hemi-

colectomies (6 left-sided) and 10 (recto)sigmoidal resections. Seven

patients underwent a surgical resection because of extensive

polyposis or due to difficult advancement of the endoscope during

examination resulting in incomplete visualization of the colon. The

other 26 patients underwent a colonic resection due to CRC diagnosis.

Consequently, during (a part of) follow-up 44 patients received

endoscopic surveillance of the intact colon; 17 patients of the

remaining segment proximal to the rectosigmoid colon; 15 patients of

the remaining distal colon segment and 2 patients did not receive

endoscopic surveillance after undergoing a proctocolectomy.

Chapte

r 2

Chapter 2

Nu

mb

er o

f ad

eno

ma

s

Nu

mb

er o

f MP

s

Nu

mb

er o

f SA

s

Nu

mb

er o

f HP

s

Tota

l poly

ps

- inte

rval C

RC

- at in

itial e

ndoscopy

Patie

nts

with

CR

C

Media

n n

um

ber o

f ad

en

om

as

in p

atie

nts

(rang

e)

Patie

nts

with

≥1

aden

om

a

Media

n n

um

ber o

f SA

s in

patie

nts

(ra

ng

e)

Patie

nts

with

≥1

SA

N

um

ber o

f pa

tients

with

an H

P ≥

10m

m

Media

n n

um

ber o

f pro

x. H

Ps

in p

atie

nts

(rang

e)

Media

n n

um

ber o

f HP

s

in p

atie

nts

(rang

e)

Media

n in

terv

al e

nd

osco

pie

s

in m

onth

s (ra

ng

e)

Mea

n F

U-tim

e a

fter H

PS

dia

gnosis

in

yrs

(rang

e)

Mea

n F

U-tim

e in

yrs

(ran

ge)

Media

n a

ge a

t dia

gnosis

(ran

ge

)

273

(14%

)

2 (0

.1%

)

302

(15%

)

140

7 (7

2%

)

198

4

5 (6

.5%

)

22 (2

8.5

%)

27 (3

5%

)

2 (0

-26)

53 (6

9%

)

1 (0

-24)

40 (5

2%

)

47 (6

1%

)

8 (1

-45)

15 (2

-53

)

11 (1

-96

)

4.0

(0.4

-21.0

)

5.6

(0.5

-26,6

)

56 (4

0-7

4)

All c

entre

s

(n=

77)

160

(14%

)

2 (0

.2%

)

259

(23%

)

705

(63%

)

112

4

4 (2

7%

)

11 (7

3%

)

15 (3

5%

)

2 (0

-26)

31 (7

2%

)

5 (0

-24)

32 (7

4%

)

25 (5

8%

)

7 (1

-30)

14 (2

-53

)

9 (1

-96)

3.2

(1.3

-6.1

)

4.4

(1.3

-9.3

)

55 (4

0-7

4)

Centre

1

(n=

43)

31 (1

1%

)

0

6 (2

%)

258

(87%

)

295

0

6 (1

00%

)

6 (7

5%

)

2 (0

-16)

6 (7

5%

)

0 (0

-6)

1 (1

3%

)

5 (6

3%

)

29 (5

-45

)

29 (1

1-7

3)

23 (5

-26

)

8.1

(1.1

-20.8

)

10.6

(2.9

-26.4

)

51 (4

2-6

9)

Centre

2

(n=

8)

46 (2

2%

)

0

9 (4

%)

161

(74%

)

216

1 (3

3%

)

2 (6

7%

)

3 (4

3%

)

6 (0

-14)

6 (8

6%

)

0 (0

-8)

2 (2

9%

)

5 (7

1%

)

10 (2

-21

)

25 (6

-45

)

7 (3

-63)

5.8

(1.1

- 11

.7)

6.4

(1.1

-12.3

)

56 (4

6-6

5)

Centre

3

(n=

7)

12 (1

4%

)

0

1 (1

%)

74 (8

5%

)

87

0

1 (1

00%

)

1 (1

7%

)

1 (0

-9)

3 (5

0%

)

0 (0

-1)

1 (1

7%

)

3 (5

0%

)

7 (2

-12)

10 (6

-27

)

6 (1

-17)

2.1

(1.2

-4-4

)

2.6

(1.2

-5.1

)

58 (5

6-6

7)

Centre

4

(n=

6)

Table

1. C

hara

cte

ristic

s o

f HP

S p

atie

nts

div

ided p

er c

entre

.

9 (8

%)

0

23 (2

5%

)

61 (6

7%

)

93

0

0

0

2 (0

-3)

4 (8

0%

)

1 (0

-20)

3 (6

0%

)

4 (8

0%

)

6 (2

-12)

12 (2

-22

)

8 (1

-24)

3.3

(1.9

-4.7

)

4.3

(3.0

-5.7

)

51 (4

3-7

2)

Centre

5

(n=

5)

8 (7

%)

0

0

109

(93%

)

117

0

0

0

1 (0

-5)

3 (6

0%

)

0

0

4 (8

0%

)

10 (4

-30

)

21 (9

-41

)

11 (3

-53

)

2.2

(1.5

-4.1

)

2.2

(1.5

-4.1

)

52 (4

5-6

1)

Centre

6

(n=

5)

7 (1

4%

)

0

4 (8

%)

39 (7

8%

)

50

0

2 (1

00%

)

2 (6

7%

)

0 (0

-7)

0 (0

-1)

0 (0

-1)

1 (3

3%

)

1 (3

3%

)

5 (5

-13)

16 (5

-18

)

13 (1

0-2

5)

3.2

(1.3

-6.1

)

4.4

(1.3

-9.3

)

57 (4

8-6

9)

Centre

7

(n=

3)

Clin

ical c

hara

cte

ristic

s o

f HP

S p

atie

nts

from

multip

le c

entre

s in

the N

eth

erla

nds


The mean follow-up period of patients was 5.6 years (range:

0.5 – 26.6) and from the point HPS was diagnosed this was 4.0 years

(range: 0.4 -21.0). During follow-up, the number of surveillance

endoscopies varied among patients. In the observed time period, 207

surveillance endoscopies were performed (median 3, range 0-11). One

patient was diagnosed with HPS based on the surgical resection

specimen and had not yet undergone surveillance endoscopies. The

median interval between surveillance endoscopies was 10 months

(range: 1-96).

Polyps

Polyp characteristics are outlined in table 1. In this study, 847/1407

(60%) HPs, 197/302 (65%) SSAs and 165/273 (60%) adenomas were

detected proximal to the sigmoid colon. The maximum size of HPs and

SAs was 30mm which were located in the ascending and transverse

colon, respectively. The largest adenoma detected in this cohort was

75mm, which was located in the ascending colon. Polyps were

detected during endoscopy with standard or high-resolution white-light

endoscopy. Narrow-band imaging was used in 22/294 (7%)

endoscopies in 22 patients.

CRC

Of the 77 HPS patients included in this study, 27 (35%) patients had

CRC (median age 56 years; range 36-75). In 14/27 (52%) of these

patients, CRC was located proximal to the sigmoid colon. One patient

had two separate synchronous CRCs, one proximally and one distally

located.

Chapte

r 2

Chapter 2

50

While CRC was diagnosed at initial colonoscopy in the majority

(22/27) of HPS patients, in five patients (cumulative incidence: 6.5%)

with a median age of 58 years (range 49-68) CRC was detected during

surveillance after the diagnosis HPS was made (mean follow-up time

5.6 yrs) without any prior history of CRC. The median follow-up time in

this group was 1.3 years (range: 0.4-6.7) with a median of 3

endoscopies (range: 2-4). Clinical and histological data of these

patients are summarized in table 2. During a total of 294.6 person

years of follow-up, this corresponds with a CRC- incidence rate during

surveillance of 17 per 1000 person-years. In four of the five (80%)

patients, CRC was detected during a planned endoscopy and was

located within a HP (3/4) or a SA (1/4) without causing clinical

symptoms. The median size of these polyps was 10mm (range: 4-

16mm). One patient (patient 2) presented with weight loss and fatigue

after a surveillance interval of more than 3 years. At endoscopy a large

CRC was detected. In four of five patients, CRC was located

proximally to the sigmoid colon. The median interval between

surveillance endoscopies in patients with an interval CRC was 11

months (range: 3-43) compared to 10 months (range: 1-96) in HPS

patients without an interval CRC (ns). The median interval between the

last surveillance endoscopy and CRC detection in patients with interval

carcinomas was also 11 months (range: 4-43). The calculated

cumulative risk of CRC in HPS during surveillance was 7% at five

years (figure 1). When analysing the cumulative risk separately for

patients with an intact colon and for patients with a surgical colonic

resection, the 5-year cumulative risk was 6% and 4% respectively.


5

4

3

2

1

Patie

nt

48

48

58

67

49

Age

F

M

M

F

M

Sex

3

2

4

3

2

Num

ber o

f end

oscopie

s

untill C

RC

Desce

ndin

g c

olo

n

Tra

nsvers

e c

olo

n

Rectu

m

Ascen

din

g

colo

n

Ascen

din

g

colo

n

Locatio

n

CR

C

not s

tate

d

(Tis

N0M

O )

10m

m

(T1N

0M

0)

4m

m

(Tis

N0M

0)

95m

m

(T3N

0M

0)

16m

m

(T3N

0M

0)

Siz

e C

RC

(T

NM

)

HP

HP

HP

non

e

SA

Imm

edia

te

adja

cen

t poly

ps

15.6

4.3

36.4

80.4

11.4

Tim

e (m

onth

s)

betw

ee

n H

PS

dia

gnosis

and

CR

C d

ete

c

10.1

4.3

11.6

44.2

7.7

Tim

e (m

onth

s)

betw

ee

n la

st

end

oscopy a

nd

dia

gnosis

CR

C

Caecum

Caecum

Sig

moid

(sub

tota

l cole

cto

my)

Tra

nsvers

e c

olo

n

Caecum

Most p

roxim

al

intu

batio

n p

oin

t la

st e

nd

oscopy

Ta

ble

2: C

hara

cte

ristic

s o

f HP

S p

atie

nts

in w

hic

h a

n in

terv

al c

arc

inom

a w

as d

ete

cte

d.

Multip

le p

oly

ps th

roug

ho

ut th

e

colo

rectu

m., w

hic

h w

ere

only

dia

gnostic

ally

bio

psie

d

Multip

le p

oly

ps d

ete

cte

d, o

f whic

h

11 p

oly

ps w

ere

rem

ove

d. M

ultip

le

poly

ps re

main

ed in

situ

All v

isib

le re

cto

sig

moid

al p

oly

ps

rem

oved.

Multip

le p

oly

ps th

roug

ho

ut th

e

colo

rectu

m. P

roce

dure

com

plic

ate

d

by p

erfo

ratio

n (a

borte

d). M

ultip

le

poly

ps re

main

ed in

situ

. Pa

tien

t re

turn

ed w

ith s

ym

pto

ms a

fter 4

4.2

m

on

ths.

>20 p

oly

ps w

hic

h w

ere

only

dia

gnostic

ally

bio

psie

d.

Abno

rmalitie

s a

nd tre

atm

en

t durin

g la

st e

ndoscop

y

befo

re d

iagn

osis

CR

C

Chapte

r 2

Chapter 2

52

To analyse an association with CRC in HPS, univariate logistic

regression was performed for 8 independent variables: age; sex;

number of HPs; number of SAs; number of adenomas; largest HP;

largest SA and largest adenoma (table 3). At univariate logistic

regression, the number of HPs and the number of SAs were

associated with CRC (p<0.1). At subsequent multivariate logistic

regression the number of HPs (p=0.013) and the number of SAs

(p=0.048) were significantly associated with CRC with corresponding

odds ratios of 1.05 (95%-CI: 1.01-1.10) and 1.09 (95%-CI: 1.00-1.19)

respectively.

Prognostic variables (univariate analysis)

Odds ratio 95%-CI p-value

Age (per year) 1.04 0.98-1.11 0.162

Male sex 0.67 0.26-1.72 0.408

Number of HPs (per polyp) 1.05 1.01-1.09 0.018*

Number of SAs ( per polyp) 1.08 0.99-1.17 0.076*

Number of adenomas (per polyp) 1.01 0.92-1.11 0.870

Largest HP (per mm) 0.98 0.91-1.06 0.611

Largest SA (per mm) 1.03 0.97-1.10 0.302

Largest adenoma (per mm) 0.99 0.96-1.04 0.887

Prognostic variables (multivariate analysis)

Odds ratio 95%-CI p-value

Number of HPs (per polyp) 1.05 1.01-1.10 0.013*

Number of SAs ( per polyp) 1.09 1.00-1.19 0.048*

Table 3. Results of univariate and multivariate logistic regression analysis: independent prognostic variables and corresponding odds ratios for the presence of colorectal cancer in hyperplastic polyposis syndrome. * Statistically significant p-value for univariate analysis: p< 0.1 and for multivariate analysis: p< 0.05


53

Figure 1. Cumulative proportion of HPS patients with colorectal cancer during surveillance

DISCUSSION

This multicentre cohort study showed that in a total of 27/77 (35%)

HPS patients, CRC was detected. Interestingly, CRC was detected in

5/77 (6.5%) patients during surveillance of which four CRCs within a

diminutive serrated polyp (HP or SA) resulting in a cumulative risk of

CRC under endoscopic surveillance of 7% in 5 years. This is

substantial considering that the lifetime risk of developing CRC in the

general population is estimated to be 6%.18 Of these patients with

interval CRCs, two CRCs were detected within a year (table 2: 11.4

and 4.3 months) after the diagnosis HPS was made and after two

previous endoscopies. The high frequency of endoscopies in a short

time period suggest that these patients were probably still in an

Chapte

r 2

Chapter 2

54

orientating treatment phase when CRC was detected. If surveillance

was defined as endoscopies performed after HPS diagnosis after at

least one year follow-up, the cumulative risk under surveillance would

be 4% at five years.

Although different management protocols have recently been

advised, thus far no uniform and adequately substantiated

management protocol exists for the endoscopic management of HPS

patients. Consequently, lack of clarity exists regarding the

recommended surveillance interval and which polyps to remove.

Recent studies recommend surveillance intervals ranging from one to

three years and concerning the removal of polyps, advice varies from

removal of only proximally located polyps to complete removal of all

polyps >5mm.7, 13

This absence of a standardized treatment protocol may also be

associated with the incidence of interval CRCs in this retrospective

multicentre study dating back to 1982. Possible explanations for the

incidence of interval CRCs could be that previously an association

between HPS and CRC was not made or that only proximal and/or

larger lesions were considered clinically significant, resulting in

incomplete removal of polyps. Also when considering the relatively

short median interval between endoscopies (median interval:

11months), it is likely that the interval CRCs were also present at prior

surveillance endoscopies but were not removed. This was indeed the

case for two of five of these patients who underwent incomplete

removal of all detected polyps during the last surveillance endoscopy

before CRC diagnosis (table 2: patient 2 and 4), underlining the

importance of comprehensive polyp removal during surveillance

Conversely, in three of five patients (patients 1, 3 and 5) all

detected polyps were biopsied or removed at previous endoscopy. A


55

possible explanation for this contrary finding could be that these CRCs

originating in serrated polyps were simply missed. This could possibly

be due to the multiplicity of polyps seen in HPS patients resulting in a

sub-optimal overview of all colorectal polyps. Alternatively, typical HPs

and (sessile) serrated adenomas seldom exceed 10mm in size,

suggesting that most polyps in HPS are diminutive.19-25 It has been

shown that the miss-rate of polyps <10mm can be as high as 23%.26

This could explain why these relatively small CRCs originating in

diminutive serrated polyps were not detected at previous endoscopy.

Nevertheless, considering their small size and the unknown

progression rate in HPS, it can not be excluded that these CRCs,

originating in serrated polyps (4/5) developed since the last

endoscopy. A previous retrospective polyp study of consecutive

patients with an average risk for CRC showed that the estimated

growth rates of HPs and SAs (both sessile- and tradional) compared to

conventional adenomas were similar or significantly higher.27

Moreover, a recent case report described the progression of a sessile

serrated adenoma to carcinoma within 8 months.28 In this respect, it is

conceivable that in HPS a subset of serrated polyps have an increased

progression rate leading to CRC. This is an interesting point

considering that the risk of high grade dysplasia or even invasive

cancer in diminutive lesions (<10mm) has been shown to be <2%. 29-31

The finding of CRC within a small serrated polyp in 4/5 (80%) interval

carcinomas suggests that in HPS small polyps have a greater

malignant potential than in the general population.

When considering the management of HPS patients, this study

suggests that the absence of a clear treatment protocol plays a role in

the presence of CRCs during surveillance. Considering that CRCs

detected in this study were as small as 4mm (detected in a HP: patient

Chapte

r 2

Chapter 2

56

3), removal of all polyps seems advisable but needs to be

prospectively assessed. Although these recommendations seem of

importance in preventing malignant progression in HPS, practical

difficulties may present when trying to comply with these guidelines in

a clinical setting. Firstly, besides being small, detection of HPs and

SAs is also complicated by their predominantly flat shape,

unremarkable colour and mucus coating which possibly increases

polyp miss rates.19, 32 Secondly, removal of all detected polyps during

endoscopic surveillance sessions in HPS patients with a large quantity

of polyps can be time-consuming and unfeasible, especially when

endoscopic mucosal resection (EMR) is indicated for predominantly

flat shaped serrated polyps.

With regard to polyp detection, previous randomized controlled

trials demonstrated that chromoendoscopy and narrow-band imaging

(NBI) increased the detection of HPs.33-38 Although not formally

investigated, these techniques could in this respect also be of value for

the detection of serrated polyps in HPS. Concerning polyp removal,

the multiplicity of polyps and the use of EMR can indeed lead to

increased duration of endoscopic procedures in HPS patients. In this

respect, it is important that these endoscopies are performed by

endoscopists experienced in EMR for complete, prompt and safe polyp

removal and that allowances are made for sufficient procedure time.

Annual surveillance by an experienced endoscopist specialized in

HPS, having advanced imaging techniques available such as

chromoendoscopy and NBI seems therefore advisable. Alternatively,

when complete endoscopic removal of all polyps is not feasible,

surgical colonic resection should seriously be considered since these

patients have an increased risk of malignant progression of polyps.


57

In this study, at multivariate logistic regression, an increasing

number of HPs and SAs was significantly associated with CRC

presence (OR of 1.05 and 1.09 respectively per polyp). In other words,

the CRC risk will increase by 5% and 9% respectively with each

additional HP or SA. Concordantly, results from previous literature

reports strongly suggest that SAs in particular play a role in a ‘serrated

pathway’ leading to CRC in HPS.19, 39-41 A possible explanation for the

significant association between HPs and CRC in this study could be

that HPs and SAs (primarily sessile serrated adenomas) are

histologically hard to distinguish, leading to incorrect differentiation and

misdiagnosis of these serrated polyps. Indeed, it has recently been

shown that even at re-evaluation the interobserver agreement for the

differentiation of serrated polyps remains only moderate (к=0.55).14, 15,

42 Nevertheless, the significant association between serrated polyps

and CRC in this study supports the hypothesis of a ‘serrated pathway’

leading to CRC in HPS (figure 2).

Figure 2. Endoscopic image of a serrated adenoma (10mm, ascending colon) detected in a patient with hyperplastic polyposis syndrome (A). At microscopy a focus of adenocarcinoma was seen within the serrated adenoma (B and C).

In conclusion, HPS is associated with an increased personal

CRC risk, even under endoscopic surveillance. Considering that these

advanced lesions were detected in polyps as small as 4mm (median:

Chapte

r 2

Chapter 2

58

10mm), which were not recognized as such, all polyps in HPS seem at

risk of representing advanced lesions warranting removal of all polyps.

However, the miss rate of polyps <10mm (which represents the

majority of polyps in HPS) has been shown to be as high as 23% with

standard white-light endoscopy suggesting that a considerable number

of polyps in HPS are missed. 26 Advanced endoscopic imaging

techniques such as chromoendoscopy and NBI may in this respect be

of additional value for the detection of polyps in HPS. Alternatively, if

endoscopically unfeasible, preventive colonic resection should be

considered. An increasing number of serrated polyps are associated

with the presence of CRC in HPS, supporting the theory of a ‘serrated

pathway’ leading to CRC. Future prospective data from large HPS

cohorts, undergoing a standardized treatment protocol are required to

further enhance our knowledge with regard to the rate of polyp

progression in these patients and to determine the optimal treatment

and surveillance protocol for these patients.


59

REFERENCES 1. Jemal A, Thun MJ, Ries LA, Howe HL, Weir HK, Center MM, Ward E,


2. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61:759-767.


4. Makinen MJ. Colorectal serrated adenocarcinoma. Histopathology 2007;50:131-150.

5. Boparai KS, Dekker E, van ES, Polak MM, Bartelsman JF, Mathus-Vliegen EM, Keller JJ, van Noesel CJ. Hyperplastic polyps and sessile serrated adenomas as a phenotypic expression of MYH-associated polyposis. Gastroenterology 2008;135:2014-2018.





Chapte

r 2

Chapter 2

60




13. East JE, Saunders BP, Jass JR. Sporadic and syndromic hyperplastic polyps and serrated adenomas of the colon: classification, molecular genetics, natural history, and clinical management. Gastroenterol Clin North Am 2008;37:25-46, v.





18. Winawer S, Fletcher R, Rex D, Bond J, Burt R, Ferrucci J, Ganiats T, Levin T, Woolf S, Johnson D, Kirk L, Litin S, Simmang C. Colorectal cancer screening and surveillance: clinical guidelines and rationale-Update based on new evidence. Gastroenterology 2003;124:544-560.



61


21. Estrada RG, Spjut HJ. Hyperplastic polyps of the large bowel. Am J Surg Pathol 1980;4:127-133.

22. Hayashi T, Yatani R, Apostol J, Stemmermann GN. Pathogenesis of hyperplastic polyps of the colon: a hypothesis based on ultrastructure and in vitro cell kinetics. Gastroenterology 1974;66:347-356.

23. Imperiale TF, Wagner DR, Lin CY, Larkin GN, Rogge JD, Ransohoff DF. Results of screening colonoscopy among persons 40 to 49 years of age. N Engl J Med 2002;346:1781-1785.

24. Johnson DA, Gurney MS, Volpe RJ, Jones DM, VanNess MM, Chobanian SJ, Avalos JC, Buck JL, Kooyman G, Cattau EL, Jr. A prospective study of the prevalence of colonic neoplasms in asymptomatic patients with an age-related risk. Am J Gastroenterol 1990;85:969-974.

25. Lieberman DA, Prindiville S, Weiss DG, Willett W. Risk factors for advanced colonic neoplasia and hyperplastic polyps in asymptomatic individuals. JAMA 2003;290:2959-2967.

26. van Rijn JC, Reitsma JB, Stoker J, Bossuyt PM, van Deventer SJ, Dekker E. Polyp miss rate determined by tandem colonoscopy: a systematic review. Am J Gastroenterol 2006;101:343-350.

27. Lazarus R, Junttila OE, Karttunen TJ, Makinen MJ. The risk of metachronous neoplasia in patients with serrated adenoma. Am J Clin Pathol 2005;123:349-359.


29. Butterly LF, Chase MP, Pohl H, Fiarman GS. Prevalence of clinically important histology in small adenomas. Clin Gastroenterol Hepatol 2006;4:343-348.

30. East JE, Suzuki N, Saunders BP. Comparison of magnified pit pattern interpretation with narrow band imaging versus chromoendoscopy for diminutive colonic polyps: a pilot study. Gastrointest Endosc 2007;66:310-316.

Chapte

r 2

Chapter 2

62

31. Sano Y, Ikematsu H, Fu KI, Emura F, Katagiri A, Horimatsu T, Kaneko K, Soetikno R, Yoshida S. Meshed capillary vessels by use of narrow-band imaging for differential diagnosis of small colorectal polyps. Gastrointest Endosc 2008.

32. Yano T, Sano Y, Iwasaki J, Fu KI, Yoshino T, Kato S, Mera K, Ochiai A, Fujii T, Yoshida S. Distribution and prevalence of colorectal hyperplastic polyps using magnifying pan-mucosal chromoendoscopy and its relationship with synchronous colorectal cancer: prospective study. J Gastroenterol Hepatol 2005;20:1572-1577.

33. Kiesslich R, von BM, Hahn M, Hermann G, Jung M. Chromoendoscopy with indigocarmine improves the detection of adenomatous and nonadenomatous lesions in the colon. Endoscopy 2001;33:1001-1006.

34. Lecomte T, Cellier C, Meatchi T, Barbier JP, Cugnenc PH, Jian R, Laurent-Puig P, Landi B. Chromoendoscopic colonoscopy for detecting preneoplastic lesions in hereditary nonpolyposis colorectal cancer syndrome. Clin Gastroenterol Hepatol 2005;3:897-902.

35. Lee JH, Kim JW, Cho YK, Sohn CI, Jeon WK, Kim BI, Cho EY. Detection of colorectal adenomas by routine chromoendoscopy with indigocarmine. Am J Gastroenterol 2003;98:1284-1288.

36. Ratiu N, Gelbmann C, Rath HR, Herfarth H, Kullmann F, Scholmerich J, Messmann H. Chromoendoscopy with indigo carmine in flexible sigmoidoscopy screening: does it improve the detection of adenomas in the distal colon and rectum? J Gastrointestin Liver Dis 2007;16:153-156.

37. Adler A, Aschenbeck J, Yenerim T, Mayr M, Aminalai A, Drossel R, Schroder A, Scheel M, Wiedenmann B, Rosch T. Narrow-Band Versus White-Light High Definition Television Endoscopic Imaging for Screening Colonoscopy: A Prospective Randomized Trial. Gastroenterology 2008.

38. Kaltenbach T, Friedland S, Soetikno R. A randomised tandem colonoscopy trial of narrow band imaging versus white light examination to compare neoplasia miss rates. Gut 2008;57:1406-1412.

39. Jass JR, Baker K, Zlobec I, Higuchi T, Barker M, Buchanan D, Young J. Advanced colorectal polyps with the molecular and morphological features of serrated polyps and adenomas: concept of a 'fusion' pathway to colorectal cancer. Histopathology 2006;49:121-131.


63


41. Minoo P, Baker K, Goswami R, Chong G, Foulkes WD, Ruszkiewicz AR, Barker M, Buchanan D, Young J, Jass JR. Extensive DNA methylation in normal colorectal mucosa in hyperplastic polyposis. Gut 2006;55:1467-1474.


Chapte

r 2

Chapter 2

64

Increased colorectal cancer risk in first degree relatives of patients with hyperplastic polyposis syndrome

K.S. Boparai J.B. Reitsma

V. Lemmens

T.A.M. van Os E.M.H. Mathus-Vliegen

J.J. Koornstra

F.M. Nagengast

L.P. van Hest

J.J. Keller

E. Dekker

Gut. 2010 Sep;59(9):1222-5

Ch

ap

ter

Chapter 3

66

ABSTRACT Introduction Hyperplastic polyposis syndrome (HPS) is characterized by the presence of multiple colorectal hyperplastic polyps and is associated with an increased colorectal cancer (CRC) risk. For first-degree relatives of HPS patients (FDRs) this has not been adequately quantified. Reliable evidence concerning the magnitude of a possible excess risk is necessary to determine whether preventive measures, like screening colonoscopies, in FDRs are justified. Aims and methods We analyzed the incidence rate of CRC in FDRs and compared this with the general population through person-year analysis after adjustment for demographic characteristics. Population-based incidence data from the Eindhoven Cancer Registry during the period 1970-2006 were used to compare observed numbers of CRC cases in FDRs with expected numbers based on the incidence in the general population. Results A total of 347 FDRs (41% male) from 57 pedigrees were included, contributing 11.053 person-years of follow-up. During the study period, a total of 27 CRC cases occurred among FDRs compared to 5 expected CRC cases (p<0.001). The relative risk of CRC in FDRs compared to the general population was 5.4 (95%-CI: 3.7-7.8). Four FDRs satisfied the criteria for HPS. Based on the estimated HPS prevalence of 1:3000 in the general population the projected relative risk of HPS in FDRs was 39 (95%-CI: 13-121). Conclusions FDRs of HPS patients have an increased risk for both CRC and HPS compared to the general population. Hence, as long as no genetic substrate has been identified, screening colonoscopies for FDRs seem justified but this needs to be prospectively evaluated.

CRC cancer risk in first-degree relatives of HPS patients

Introduction

Hyperplastic polyposis syndrome (HPS) is a condition characterized by

the presence of multiple hyperplastic polyps (HPs) spread throughout

the colon and is associated with an increased colorectal cancer risk. 1-5

Because of this increased risk of malignant progression, HPS patients

undergo endoscopic surveillance with removal of polyps or surgical

colonic resection. Although an increased CRC risk for these patients

has been established, it is uncertain whether an increased risk of CRC

and/or HPS for first-degree relatives (FDRs) exists and consequently

whether preventive measures, like screening colonoscopies, should be

performed in this group.

Although HPS was initially considered to be non-familial,

previously published case series report that up to 50% of HPS patients

have a FDR with CRC. 2, 6 In addition, HPS presence has been

described in multiple family members.2, 3, 7-9 Based on these reports, a

yet unidentified underlying genetic defect seems to play a role in at

least some HPS cases.

Concordantly, previous studies have shown that HPS is caused in a

small proportion of patients by a germline mutation in the MUTYH

gene resulting in a phenotype of multiple conventional adenomas and

serrated polyps.2, 10 However, in the overall majority it is unclear what

proportion of FDRs are at risk of developing CRC and/or HPS.

Furthermore, the possible mode of inheritance is unknown. Families

have been reported previously from which both an autosomal

recessive and autosomal dominant inheritance could be considered.

2, 6, 8, 9 Thus, to adequately inform FDRs regarding their risk of CRC

and/or HPS, reliable evidence concerning the magnitude of this excess

Chapte

r 3

Chapter 3

68

risk is necessary. The aim of this study was to estimate the incidence

rate of CRC and HPS in FDRs of HPS patients and to compare this

with the general population so as to determine whether preventive

measures such as screening colonoscopies in this group are justified.

Methods

Besides HPs, HPS patients often have multiple sessile serrated

adenomas (SSAs), traditional serrated adenomas and conventional

adenomas. HPs and SSAs have been shown to be histologically very

similar and difficult to differentiate microscopically with only moderate

concordance.11-14 For this reason HPS was defined as at least five

histologically diagnosed HPs and/or SSAs proximal to the sigmoid

colon, of which 2 greater than 10mm in diameter, or more than 20 HPs

and/or SSAs distributed throughout the colon. HPS patients (probands)

from 4 medical centres in the Netherlands were included when

satisfying the above mentioned diagnostic criteria for HPS. Of these

patients, family history data were obtained through face to face- and

telephone interviews or by examining data from the departments of

Clinical Genetics. Patients with a known germline APC mutation or bi-

allelic MUTYH mutation were excluded from the study. This study was

conducted in accordance with the research code of our institutional

medical ethical committee on human experimentation, as well as in

agreement with the Helsinki Declaration of 1975, as revised in 1983.

Risk assessment for CRC was performed by including person-

years at risk for CRC from 1 January 1970 until 1 January 2009

(censory date). FDRs were considered at risk from birth until date of

CRC diagnosis, date of death or the censory date. It was presumed

that from the time the proband developed CRC FDRs would have a


69

higher chance of receiving endoscopic screening for CRC which could

cause a bias when comparing with the general population. For this

reason, person-years at risk also ended at the date of CRC diagnosis

in the proband if applicable.

Person-years at risk were stratified according to 5-year age

group, gender and calendar year using SAS software, version 9.1

(SAS Institute Inc, Cary, NC). Similarly stratified population-based data

from the Eindhoven Cancer Registry were used to compare the

incidence of CRC in FDRs with that of the general population.15 The

expected CRC incidence in FDRs was calculated by applying the age-,

gender- and calendar-specific incidence rates of the general

population to the composition and follow-up years of the FDR cohort.

The observed versus expected number of CRC cases were formally

compared by calculating a relative risk and 95% confidence interval

(CI). This relative risk is calculated by taking the ratio of observed to

expected number of cases and its confidence interval by assuming a

Poisson distribution of cases. Two-sided P-values <0.05 were

considered statistically significant.

RESULTS

First-degree relatives (FDRs)

In this study a total of 347 FDRs (142 male) from 57 pedigrees were

included, contributing 11.053 person-years of follow-up (figure 1). The

median age at end of follow-up was 60 years (interquartile range: 44-

71). These FDRs consisted of 165 (48%) siblings, 100 (35%) parents

and 82 (17%) children. In total, 27 FDRs, consisting of 14 parents, 12

siblings and 1 child were excluded because (i) they died before 1

January 1970 (n=17) or (ii) information regarding that individual was

unknown (n=10).

Chapte

r 3

Chapter 3

70

Figure 1. Flow diagram

Colorectal cancer

During the study period, a total of 27 (8%) CRC cases occurred among

FDRs. The median age of CRC occurrence was 62 years (interquartile

range: 57-78) and 15 (56%) were of the male gender (table 1). The

relative risk of CRC in FDRs compared to the general population was

5.4 (95%-CI: 3.7-7.8). One additional CRC case occurred outside the

study period (before 1970) and was therefore not included. There were

no excluded CRC cases in FDRs which developed after CRC

diagnosis in the proband. Of the 27 FDRs with CRC, 4 probands

developed CRC at a later stage. In total, 16/57 (28%) probands were

diagnosed with CRC during the study period.


71

Table 1. Risk analysis of colorectal cancer (CRC) in first-degree relatives

of HPS patients compared with population-based data from the Eindhoven Cancer

Registry

Group Person-

years

Observed

CRCs

Expected

CRCs

RR

(observed/e

xpected)

95%-CI

Males 4736 15 2.3 6.5 3.9 -10.8

Females 6317 12 2.6 4.6 2.6 - 8.0

Combined 11053 27 5.0 5.4 3.7 -7.8

The difference in CRC risk between men and women within the FDR

group was not statistically different (RR: 1.9, 95%-CI: 0.9-4.1, Pearson

Chi-Square test: p=0.089). Also when comparing this between siblings

(brothers and sisters) and non-siblings (parents and children) within

the FDR group, no significant difference was seen (RR: 1.0, 95%-CI:

0.4-2.5, Pearson Chi-Square test: p=0.96). Other malignancies

recorded in the study period included breast cancer (n=10), gastric

cancer (n=2), ovarian cancer (n=2) and others (n=12).

Polyps

Endoscopies were performed in 65/347 (19%) of FDRs. Reasons for

endoscopy were not recorded. In this group, 35/65 (54%) individuals

had colorectal lesions from whom 24 the histology was known. In 7

FDRs multiple histologically confirmed HPs (≥ 5) were identified at a

median age of 58 years (range: 50-75) of which 6 had HPs proximal to

the rectosigmoid colon (table 2). Four of these FDRs from 4 different

pedigrees satisfied the criteria for HPS. Based on the estimated HPS

prevalence of 1:3000 in the general population, the projected relative

risk of HPS in FDRs would be 39 (95%-CI: 13-121). In FDRs with

CRC, 3/27 (11%) of cases had multiple HPs (two had HPS) compared

Chapte

r 3

Chapter 3

72

to 4/320 (1%) non-CRC cases (Fisher’s Exact Test: p=0.012). In 3

other FDRs, multiple polyps were detected of which the histology was

unknown. The difference in risk of having multiple HPs between men

and women within the FDR group was not statistically different (RR:

1.6, 95%-CI: 0.4-7.4, Fisher’s exact test: p=0.71). Also when

comparing this between siblings and non-siblings within the FDR group

no significant difference was seen (RR: 3.1, 95%-CI: 0.6-16.1, Fisher’s

exact test: p=0.252).

Table 2. First degree relatives (FDR) of HPS patients with multiple (≥ 5)

hyperplastic polyps (HPs).

* Patients satisfying the criteria for HPS.

FDR Relation Age at

endoscopy

Number of

HPs

Location Colorectal

cancer

(location)

1 mother 75 6 Distal colon No

2 sister 59 6 Pancolonic Yes (right)

3 brother 50 9 Rectosigmoid No

4 father 66 >10* Pancolonic Yes (right)

5 sister 56 >40* Pancolonic Yes (right)

6 brother 58 >10* Pancolonic No

7 brother 58 23* Distal colon No


73

Discussion

This retrospective study describes the largest series of FDRs of HPS

patients in which the presence of CRC and polyps was assessed and

is, to our knowledge, the first to quantify the relative risk of CRC and/or

HPS in FDRs. Our results showed that FDRs of HPS patients have an

increased risk for both CRC and HPS compared to the general

population warranting screening colonoscopies for this group.

A limitation of this study is that study data were collected in a

retrospective manner and based on medical charts and self-reported

information about family history. An important question therefore is

whether our approach could have lead to an over- or underestimation

of the incidence of CRC in FDRs. An overestimation of CRC risk may

cause stress16, unneeded referrals for genetic counseling and possible

unnecessary endoscopic procedures or surgery.17, 18 However,

previous studies evaluating the accuracy of patient reporting of familial

CRC by comparing patient family history reports with cancer registries,

showed that the specificity (i.e. the proportion of CRC-negatives which

are correctly identified as not having CRC) was 92-99%. These

findings imply that patients seldom incorrectly report CRC-negative

FDRs as having CRC, which would lead to an overestimation of CRC-

incidence in FDRs. Moreover, the sensitivity (i.e. the proportion of

CRC-positives which are correctly identified as having CRC) was 53-

86%. In other words, patients tend to under-report the incidence of

CRC in FDRs, leading to underestimation of CRC-incidence in

FDRs.19-21 Furthermore, the majority of probands included in this study

were symptomatic patients and thus represent a selected patient

population. These patients may have a more aggressive form of HPS

and also a higher risk of (familial) CRC than other unidentified

asymptomatic HPS cases. Therefore, our findings concerning their

Chapte

r 3

Chapter 3

74

FDRs can not by default be extrapolated to all (including unidentified)

HPS cases. However, considering that the aim of our study was to

analyze the risk of CRC and polyps in FDRs of all identified HPS

patients, we believe our findings are relevant for the management of

HPS patients and their relatives in a clinical setting. Finally, in this

study, 19% of FDRs received an endoscopy during follow-up for which

the reason was unknown. It was unknown at what rate endoscopies

were performed in the general population during the study period.

However, considering that FDR follow-up time ended at the date of

CRC diagnosis (if applicable) in the proband, we believe that the

amount of performed screening endoscopies for familial CRC in FDRs

will be comparable to the general population. Similarly, screening

endoscopies for HPS in FDRs have only recently been proposed by

some authorities and is to this date not standard practice of care.2, 6, 22

For this reason it is also unlikely that FDRs in this study received more

screening endoscopies for HPS than the general population.

In probands (i.e. HPS patients) a higher CRC incidence was

observed (16/57: 28%) during the study period compared to FDRs

(27/347: 8%). This finding was expected considering that not all FDRs

also had HPS. However, of the 4 FDRs with HPS, 2 (50%) had CRC

suggesting that the presence of multiple serrated polyps is associated

with CRC. These findings are concordant with a previous large HPS

cohort study which showed that the number of serrated polyps was

positively correlated with the risk of CRC.23

In a previous flexible sigmoidoscopy screening study performed

in a large cohort of asymptomatic individuals (n=40.673), the true

prevalence of HPS was estimated to be 1:3000 after subsequent

colonoscopies.24 Colorectal polyps, particularly HPs, do not

necessarily cause symptoms and thus could be left undiagnosed in


75

individuals if an endoscopy is not performed. In this study only 19% of

FDRs received an endoscopy. Consequently, although our study

concluded that FDRs have an increased risk of having HPS, an

underestimation of the true prevalence of HPS in FDRs seems likely.

In addition, of the FDRs with colorectal polyps the histology was

unknown in 11/35 cases. Of these individuals five were reported to

have multiple polyps of unknown histology. It seems possible that

these individuals could potentially have HPS too but this could not be

verified.

Alternatively, because the HPS prevalence in the general population

was based on a previous screening sigmoidoscopy study, a degree of

uncertainty exists regarding this estimation. This uncertainty decreases

the validity of our projected relative risk of HPS in FDRs.

With regard to the mode of inheritance, our study did not show

a significant difference in CRC incidence between siblings and non-

siblings (RR: 1.0, 95%-CI: 0.4-2.5, Pearson Chi-Square test: p=0.96).

These findings make it difficult to postulate a preference for either a

vertical or a horizontal transmission. Also concerning polyp incidence

in FDRs, although 3/4 (75%) of FDRs with HPS were siblings, these

numbers are too few to make any conclusions about the mode of

inheritance. These results are in concordance with previously reported

FDRs with HPS (table 3: 6 siblings vs 7 non-siblings). Alternatively, the

presence of FDRs with an intermediate phenotype of <10 HPs,

suggest that a co-dominant mode of inheritance, involvement of

several low penetrance genes, high risk genes with reduced

penetrance or even environmental factors may play a role.

Chapte

r 3

Chapter 3

76

Author

Family

member

(age yrs)

Histology

(detected/biopsied) Location HPS Comments

Chow et al.2

(2 families) Sister

1 (78)

HPs (70/33)

Adenomas (8/8) Prox. colon Yes <10mm

Son

1

(not stated)

Adenomas (4/4)

HP (1/1) Pancolonic No

Son2 (53) HPs (21/21) Dist. colon Yes <10mm

Son2 (51)

HPs (50-100/30)

Adenoma (1/1) Pancolonic Yes <10mm

Lage et al.6

(3 families) Brother

1 (67) HPs/SAs (19/19) Dist. colon Yes 3-25mm, CRC

Brother1 (69) Adenomas (12/4) Prox. colon No 3-7mm

Daughter2 (26) HPs (63/63) Distal colon Yes 2-5mm

Son3 (42)

HPs (50/4)

Adenoma (1/1) Distal colon Yes 2-3mm

Rashid et

al.9

(I family)

Brother1 (67) HPs (>20/>20) Not stated Yes

Son1 (42)

HPs (>20/>20)

Adenoma (1/1) Not stated Yes

Jeevaratnam

et al. 8 (1

family)

Sister1 (73)

HPs (multiple/3)

SAs (multiple/5) Pancolonic Yes

Largest 40mm,

CRC

Brother1 (55) HPs (4/4) Not stated No

3 HPs > 1cm,

CRC

Son1 (43)

HPs (9/9)

Mixed polyp (1/1) Pancolonic No <10mm

Table 3. Previous literature data concerning first-degree relatives with multiple colorectal polyps of known histology (>3 polyps). HP: hyperplastic polyp. SA: serrated adenoma. HPS: hyperplastic polyposis syndrome. CRC: colorectal cancer.

Regarding the management of FDRs, as long as no underlying

genetic cause has been identified, screening colonoscopies seem

justified for this group. Interestingly, of the 27 FDRs with CRC, only 4

probands had CRC, suggesting that screening colonoscopies should


77

be performed in all FDRs, independent of CRC presence in the

proband. The appropriate age at which screening colonoscopies

should be commenced is somewhat speculative. We advise

commencement at the age of 35 years or 5 years younger than the

lowest incidence age of HPS reported in the family. Subsequent

surveillance colonoscopies are advised at 6 year intervals with shorter

intervals when polyps are detected. However, future large prospective

screening studies in FDRs are required to further evaluate the

incidence of CRC and HPS and the optimal screening programme in

this group.

Chapte

r 3

Chapter 3

78

REFERENCES 1. Burt RW, Jass J. Hyperplastic polyposis. In: Hamilton SR and Aaltonen

LA, eds. World Health Organisation Classification of Tumours Pathology and Genetics. Berlin: Springer-Verlag, 2000:135-136.



4. Leggett BA, Devereaux B, Biden K, Searle J, Young J, Jass J. Hyperplastic polyposis: association with colorectal cancer. Am J Surg Pathol 2001;25:177-184.



7. Jass JR, Cottier DS, Pokos V, Parry S, Winship IM. Mixed epithelial polyps in association with hereditary non-polyposis colorectal cancer providing an alternative pathway of cancer histogenesis. Pathology 1997;29:28-33.



10. Boparai KS, Dekker E, van ES, Polak MM, Bartelsman JF, Mathus-Vliegen EM, Keller JJ, van Noesel CJ. Hyperplastic polyps and sessile


79

serrated adenomas as a phenotypic expression of MYH-associated polyposis. Gastroenterology 2008;135:2014-2018.





15. van Steenbergen LN, Lemmens VE, Louwman MJ, Straathof JW, Coebergh JW. Increasing incidence and decreasing mortality of colorectal cancer due to marked cohort effects in southern Netherlands. Eur J Cancer Prev 2009;18:145-152.

16. Douglas FS, O'Dair LC, Robinson M, Evans DG, Lynch SA. The accuracy of diagnoses as reported in families with cancer: a retrospective study. J Med Genet 1999;36:309-312.

17. Fry A, Campbell H, Gudmunsdottir H, Rush R, Porteous M, Gorman D, Cull A. GPs' views on their role in cancer genetics services and current practice. Fam Pract 1999;16:468-474.

18. Sweet KM, Bradley TL, Westman JA. Identification and referral of families at high risk for cancer susceptibility. J Clin Oncol 2002;20:528-537.

19. Aitken J, Bain C, Ward M, Siskind V, MacLennan R. How accurate is self-reported family history of colorectal cancer? Am J Epidemiol 1995;141:863-871.

20. Kerber RA, Slattery ML. Comparison of self-reported and database-linked family history of cancer data in a case-control study. Am J Epidemiol 1997;146:244-248.

Chapte

r 3

Chapter 3

80

21. Mitchell RJ, Brewster D, Campbell H, Porteous ME, Wyllie AH, Bird CC, Dunlop MG. Accuracy of reporting of family history of colorectal cancer. Gut 2004;53:291-295.

22. Young J, Jenkins M, Parry S, Young B, Nancarrow D, English D, Giles G, Jass J. Serrated pathway colorectal cancer in the population: genetic consideration. Gut 2007;56:1453-1459.

23. Boparai KS, Mathus-Vliegen EM, Koornstra JJ, Nagengast FM, van LM, van Noesel CJ, Houben M, Cats A, van Hest LP, Fockens P, Dekker E. Increased colorectal cancer risk during follow-up in patients with hyperplastic polyposis syndrome: a multicentre cohort study. Gut 2009.

24. Lockett MJ, Atkin W.S. Hyperplastic polyposis: prevalence and cancer risk. 48 ed. 2001.

Association between serrated polyps and MYH-associated polyposis

81

Molecular analyses – Etiology and colorectal cancer pathways

P A

R T

Chapter 4

82


83

Hyperplastic polyps and sessile serrated adenomas as phenotypic expression of MYH-associated polyposis (MAP)

K.S. Boparai E. Dekker

S. van Eeden

M.M. Polak

J.F.W.M. Bartelsman

E.M.H. Mathus –Vliegen

J.J. Keller

C.J.M. van Noesel

Gastroenterology. 2008 Dec;135(6):2014-8

Ch

ap

ter

Chapter 4

84

ABSTRACT

Background and aims: MYH-associated polyposis (MAP) is a disorder

caused by a bi-allelic germline MYH mutation, characterised by

multiple colorectal adenomas. These adenomas typically harbour

G:C→T:A transversions in the APC and K-ras genes caused by MYH

deficiency. Occasional hyperplastic polyps (HPs) have been described

in MAP patients but a causal relationship has never been investigated.

We examined the presence of HPs and sessile serrated adenomas

(SSAs) in 17 MAP patients and studied the occurrence of G:C→T:A

transversions in the APC and K-ras gene in these polyps. Methods:

MAP patients were analysed for the presence of HPs/SSAs. APC-

mutation cluster region and K-ras codon 12 mutation analysis was

performed in adenomas(n=22), HPs(n=63) and SSAs(n=10) from

these patients and from a control group of sporadic adenomas(n=17),

HPs(n=24) and SSAs(n=17). Results: HPs/SSAs were detected in

8/17(47%) of MAP patients of which 3(18%) met the criteria for

hyperplastic polyposis syndrome (HPS). APC mutations were only

detected in adenomas and comprised exclusively G:C→T:A

transversions. K-ras mutations were detected in 51/73(70%)

HPs/SSAs in MAP patients, compared to 7/41(17%) in sporadic

HPs/SSAs in the control group (p<0.0001). In HPs/SSAs, 48/51(94%)

of K-ras mutations exhibited G:C→T:A transversions, compared to

2/7(29%) of sporadic HPs/SSAs in the control group (p<0.0001).

Conclusions: HPs and SSAs are a common finding in MAP patients.

The detection of almost exclusively G:C→T:A transversions in the K-

ras gene of HPs/SSAs strongly suggests that these polyps are

causally related to MYH deficiency. This implies that distinct pathways,

i.e. APC-gene related in adenomas and non-related in HPS/SSAs,

appear to be operational in MAP.


85

Background and aims

Familial adenomatous polyposis (FAP) is an autosomal dominant

disorder characterised by the development of hundreds of colorectal

adenomas and eventually colorectal cancer (CRC) at young age. FAP

is caused by a germline mutation in the APC tumor suppressor gene.

A milder form of FAP, known as attenuated FAP (AFAP), is caused by

mutations in the extreme distal or proximal portion of the APC gene

resulting in fewer adenomas and an older age of onset. 1, 2

Recently, an autosomal recessive polyposis syndrome caused

by bi-allelic germline mutations in the mutY human homologue (MYH)

gene has been identified, known as MYH associated polyposis (MAP).

MYH is a DNA glycosylase that plays a key role in base excision repair

(BER) of 8-oxoG:A mismatches caused by reactive oxygen species.3

Deficiency of this pathway results in somatic G:C→T:A transversions

that are indeed found in the APC and K-ras genes in adenomas of

these patients.4-6 Clinically, MAP resembles AFAP with an average age

of onset around the mid 50’s and often fewer than 100 adenomas,

predominantly in the proximal colon. Polyps have been reported to be

mainly tubular adenomas, some tubulo-villous adenomas and

occasional hyperplastic polyps (HPs). 6-9

Chow et al reported one patient with a bi-allelic MYH mutation

and multiple HPs in addition to adenomatous polyposis.10 Here we

describe 17 MAP patients of which 8 (47%) harboured HPs and sessile

serrated adenomas (SSAs) in addition to conventional adenomas. We

provide evidence that the HPs and SSAs in MAP patients are not rare

and are causally associated with the MYH deficiency, reflected by

G:C→ T:A transversions in K-ras mutated genes of these polyps.

Chapte

r 4

Chapter 4

86

Materials and Methods

Patients and specimens

For this study, our cohort of 17 patients with a bi-allelic MYH mutation,

receiving treatment at the endoscopy department of the Academic

Medical Center (Amsterdam) from 21-7-1988 to

12-3-2008, was analysed for the presence of HPs or SSAs. MAP

patients with HPs/SSAs were arbitrarily classified in to two groups:

patients with multiple HPs and SSAs (≥10); or patients with occasional

HPs and SSAs (<10).

Polyp characteristics were recorded retrospectively from

previous colonoscopy reports or the gross description of the resection

specimens. All polyps were blindly re-evaluated and diagnosed

separately by two experienced pathologists as HP, SSA or adenoma.

SSA was defined by irregular crypts with architectural distortion,

serration and dilation extending to the base of the crypts that often

display boot- or T-shaped branching, and abnormal proliferation and

maturation with mature goblet or foveolar cells at the base of the

crypts.11 In case of disagreement, consensus was reached during a

multi-headed microscope session. This study was conducted in

accordance with the research code of our institutional medical ethical

committee on human experimentation, as well as in agreement with

the Helsinki Declaration of 1975, as revised in 1983.

Somatic mutation analysis

Epithelial cells from HPs (n=63), SSAs (n=10) and adenomas (n=22)

were microdissected and DNA was isolated as described previously.12

In addition, DNA was isolated from sporadic HPs (n=24) and SSAs

(n=17) of similar average size (median 3mm, range: 2-12mm), from a

randomly selected group of patients without a polyposis syndrome


87

(control group). Using previously described primers and assays, DNA

was analysed for mutations in the APC-mutation cluster region (codon

1250-1550), K-ras codon 12.13, 14 Detected mutations were confirmed

in a second experiment.

Statistics

Statistical analyses were performed by using a statistical software

package (Statistical Package for the Social Sciences 12.0.2; SPSS

Inc, Chicago, Ill). Somatic K-ras codon 12 and APC-mutation cluster

region (APC-MCR) configurations were compared with those of a

control panel using a two-sided Fisher exact test. A p-value of < 0.05

was considered statistically significant.

Results

From 17 patients with MAP, 8 (47%) unrelated patients were identified

having at least 1 HP and/or SSA. Of these 8 patients, 3 had >10 HPs

and/or SSAs (table 1). The median age was 50 (range 34-67) years.

Besides multiple adenomas (median 25, range: 3-39), a total of 145

HPs and 19 SSAs were identified from biopsies and polypectomy

specimens (figure 1). The median size of detected HPs and SSAs was

3mm (range: 2-9 mm).

Chapte

r 4

Chapter 4

88

Table 1. Histologically confirmed hyperplastic polyps and sessile serrated adenomas in 8 patients with a bi-allelic MYH mutation. HP: hyperplastic polyp; SSA: sessile serrated adenoma; TC: total colectomy; STC: subtotal colectomy; RC: right colectomy 1Biopsied polyps

A (hemi)colectomy was performed in 6/8 patients, due to concerns that

their polyps were too numerous to guarantee adequate colonoscopic

surveillance, resulting in the detection of most polyps in the remaining

distal colon or rectum. In all 3 patients with multiple HPs and/or SSAs,

more polyps (>30) were seen than biopsied (figure 2).

Patient Age

(yrs) Sex Resection

HPs1

analyzed/total

SSAs1

analyzed/total

Median size HPs/SSAs

(range mm)

MAP patients with multiple (≥10) hyperplastic polyps/sessile serrated

adenomas

1 67 F TC 24/106 8/17 4(2-6)/4(2-6)

2 46 M None 13/13 1/1 2 (2-3)/3

3 67 F RC 14/14 0/0 2(2-3)

MAP patients with occasional (<10) hyperplastic polyps/sessile serrated

adenomas

4 50 M None 2/2 1/1 4(2-5)/4

5 49 F STC 3/3 0/0 2(2-8)

6 47 M STC 4/4 0/0 5(2-9)

7 79 F STC 1/1 0/0 3

8 34 F TC 2/2 0/0 2

Total 50 63/145 10/19 3 (2-9)


89

Somatic APC and K-ras mutations

From the patients with MAP, 22 adenomas, 63 HPs and 10 SSAs were

analysed for APC-MCR and K-ras codon 12 mutations. In adenomas,

APC mutations were found in 9/22 (41%) of polyps, and comprised

exclusively G:C →T:A transversions, whereas no G:C →T:A

transversions were found in APC mutated adenomas in the control

group (p<0.0001). K-ras mutations were found in 5/22 (23%) of

adenomas, also comprising exclusively G:C →T:A transversions.

In HPs and SSAs of MAP patients and of the control group, no

APC mutations were detected (table 2). K-ras mutations were detected

in 51/73 (70%) of HPs/SSAs in MAP patients, which was significantly

more than the frequency in sporadic HPs/SSAs (7/41:17%) in the

control group (p<0.0001). Furthermore, in K-ras mutated HPs/SSAs

from our patients with MAP 48/51(94%) of mutations comprised a G:

C→T: A transversion (figure 3), compared to 2/7 (29%) of mutations in

sporadic HPs/SSAs in the control group (p<0.0001).

Chapte

r 4

Chapter 4

90

Figure 1. Examples of the spectrum of lesions found in the colon: hyperplastic polyp (top panel), sessile serrated adenoma (middle panel) and tubular adenoma (bottom panel).


91

Table 2. Detected APC-mutation cluster region and K-ras codon 12 mutations in hyperplastic polyps (HP), sessile serrated adenomas (SSA) and adenomas (AD) and distribution of G:C→T:A transversions compared to a control panel. *Statistically significant p-value for adenomas in patients with MYH-associated polyposis (MAP) compared to adenomas in the control group **Statistically significant p-value for HPs/SSAs in patients with MAP compared to HPs/SSAs in the control group.

Discussion

MAP is an autosomal recessive polyposis syndrome, caused by a bi-

allelic germline MYH gene mutation. Similar to FAP, MAP is

characterised by the presence of multiple adenomas in the colorectum

and a high cancer risk.4, 5 Here, we show that HPs and SSAs can also

be considered a phenotypic expression of MAP, reflected by the

detection of HPs/SSAs in 8/17 (47%) of patients of which 3 (18%) also

met the criteria for hyperplastic polyposis syndrome (HPS).6, 9

Interestingly, in previous large series of patients with MAP, only

‘occasional’ HPs and no SSAs were described. The presence of

HPs/SSAs in combination with adenomas may thus be more common

than previously thought. A possible cause for this discrepancy may lie

Somatic

mutation

spectrum

Patients with MYH-associated

polyposis (n=8)

Control group P-value

Polyps AD

(n=22)

HP

(n=63)

SSA

(n=10)

AD

(n=17)

HP

(n=24)

SSA

(n=17)

APC mutation 9 0 0 7 0 0

G:C→T:A 9(100%) 0 0 0 0 0 <0.0001*

K-ras

mutation 5 45 6 4 5 2 <0.0001**

G:C→T:A 5(100%) 42(93%) 6(100%) 3(75%) 1 (20%) 1 (50%) <0.0001** Chapte

r 4

Chapter 4

92

in the fact that HPs/SSAs are more difficult to detect endoscopically

due to their often diminutive size and flat or sessile shape.

Furthermore, in patients with multiple adenomatous polyps like in

MAP, the finding of small hyperplastic-looking polyps at colonoscopy

may seem of little clinical interest and are consequentially not biopsied,

preventing documentation of HPs/SSAs as a phenotypic feature of

MAP.

Figure 2. Endoscopic picture of multiple hyperplastic polyps in a patient with MYH-associated polyposis.

HPS is a recently recognised condition, frequently linked with

CRC and defined as at least five HPs proximal to the sigmoid colon, of

which two are greater than 10mm in diameter, or more than 20 HPs

distributed throughout the colon.15-18 Besides multiple HPs, the

presence of SSAs, traditional serrated adenomas and conventional

adenomas are common findings in this condition.10, 19 The combination


93

of BRAF mutations and CPG-island methylation (CIMP), evolving in

SSAs, are considered to be the key mechanism in the ‘serrated

neoplasia pathway’ leading to CRC in these patients.20-23 In our cohort

of MAP patients, 3 unrelated patients also met the criteria for HPS.

Two of these bi-allelic MYH mutations carriers were compound

heterozygote (Y176C and P402L; Y165C and G382D) and one was

monozygote (G382D). Most HPs/SSAs from these patients were

detected in the distal colon and rectum and not proximally. This could

partly be due to the fact that 2/3 of patients had received a

(hemi)colectomy so that only the distal colon and rectum remained. It

has been suggested that proximal HPs/SSAs have more BRAF

mutations than distal HPs/SSAs and are more likely to develop CRC

and thus more clinically relevant than distal HPs/SSAs.17, 20 Other

studies however, have shown that distal HPs/SSAs have a similar

frequency of BRAF mutations as proximal HPs/SSAs.19, 22 In addition,

in the largest case series describing HPS patients, 6/10 of patients had

a left-sided CRC, indicating that distal HPs/SSAs are also of clinical

significance in HPS.10

Chapte

r 4

Chapter 4

94

Figure 3. Somatic G:C →T:A transversion in codon 12 of K-ras in a hyperplastic polyp of a patient with MAP (above) compared to a hyperplastic polyp in the control group (below).

Including our 3 cases, 4 patients with MAP also meeting the

criteria for HPS have now been described.10 In these patients,

coincident multiple adenomas (median number 37, range: 25-40) were

also present. However, bi-allelic MYH gene mutation with HPs in the

absence of adenomas has never been reported. Alternatively, reported

analysis on the germline MYH gene in HPS patients (n=38) revealed a

bi-allelic MYH mutation in only one (3%) patient with 40 adenomas

compared to a median adenoma count of only 2 (range 0-22) in the

other HPS patients.10 These collective data suggest that in HPS

patients MYH mutation analysis should only be considered when

multiple (for example: ≥25) adenomas are also present. Additional

studies are however required to further understand to what extent

MYH testing should be performed in patients with HPS.

The adenomas in our series of MAP patients contained APC-

MCR and K-ras codon 12 mutations. In HPs/SSAs of MAP patients, no


95

APC mutations were detected. However, in these patients we found

significantly higher rates of K-ras mutations in HPs (70%) and SSAs

(60%) as compared to those found in sporadic HPs (21%) and SSAs

(12%) of the control group as well as to reported K-ras mutation

frequencies in sporadic HPs (4-47%) 19, 23-31 and SSAs (0-8%). 20, 22, 23,

26

MYH deficiency characteristically results in somatic G:C→T:A

transversions in the APC and K-ras genes of adenomas.4-6 Indeed, all

APC-MCR mutations detected in adenomas of our MAP patients were

G:C→ T:A transversions, compared to none in the control group.

Accordingly, in K-ras codon 12 mutated HPs/SSAs of these patients

with MYH deficiency, significantly more G:C→T:A transversions (94%)

were found compared to only 29% in control group polyps. This latter

percentage is concordant with previous reports of K-ras mutations in

HPs from patients without a polyposis syndrome showing G:C→T:A

transversions in only 0-33% of polyps.24, 30, 32 Similarly, in previous

large case series of pancreatic carcinomas, having no known

association with MYH deficiency, G:C→T:A transversions were

observed in 6-29% of K-ras mutated carcinomas.33-35 This frequency

range seems to reflect a random statistical chance, considering that

there are 9 different single mutations possible in codon 12 of K-ras

leading to amino acid replacement, of which 2 (22%) result in a

G:C→T:A transversion (i.e. GGT→TGT or GTT) .

In conclusion, our findings suggest that HPs and SSAs are

causally related to bi-allelic MYH mutations. Distinct colonic polyposis

pathways thus seem to prevail in at least a proportion of patients with

MYH deficiency, i.e. a pathway leading to conventional adenomas with

APC and/or K-ras mutations and a separate, non APC-route leading to

HPs/SSAs with K-ras mutations.

Chapte

r 4

Chapter 4

96

REFERENCES 1. Fearnhead NS, Britton MP, Bodmer WF. The ABC of APC. Hum Mol

Genet 2001;10:721-733.

2. Lamlum H, Al TN, Jaeger E, Frayling I, Sieber O, Reza FB, Eckert M, Rowan A, Barclay E, Atkin W, Williams C, Gilbert J, Cheadle J, Bell J, Houlston R, Bodmer W, Sampson J, Tomlinson I. Germline APC variants in patients with multiple colorectal adenomas, with evidence for the particular importance of E1317Q. Hum Mol Genet 2000;9:2215-2221.

3. Slupska MM, Baikalov C, Luther WM, Chiang JH, Wei YF, Miller JH. Cloning and sequencing a human homolog (hMYH) of the Escherichia coli mutY gene whose function is required for the repair of oxidative DNA damage. J Bacteriol 1996;178:3885-3892.

4. Al-Tassan N, Chmiel NH, Maynard J, Fleming N, Livingston AL, Williams GT, Hodges AK, Davies DR, David SS, Sampson JR, Cheadle JP. Inherited variants of MYH associated with somatic G:C-->T:A mutations in colorectal tumors. Nat Genet 2002;30:227-232.

5. Jones S, Emmerson P, Maynard J, Best JM, Jordan S, Williams GT, Sampson JR, Cheadle JP. Biallelic germline mutations in MYH predispose to multiple colorectal adenoma and somatic G:C-->T:A mutations. Hum Mol Genet 2002;11:2961-2967.

6. Lipton L, Halford SE, Johnson V, Novelli MR, Jones A, Cummings C, Barclay E, Sieber O, Sadat A, Bisgaard ML, Hodgson SV, Aaltonen LA, Thomas HJ, Tomlinson IP. Carcinogenesis in MYH-associated polyposis follows a distinct genetic pathway. Cancer Res 2003;63:7595-7599.

7. Gismondi V, Meta M, Bonelli L, Radice P, Sala P, Bertario L, Viel A, Fornasarig M, Arrigoni A, Gentile M, Ponz de LM, Anselmi L, Mareni C, Bruzzi P, Varesco L. Prevalence of the Y165C, G382D and 1395delGGA germline mutations of the MYH gene in Italian patients with adenomatous polyposis coli and colorectal adenomas. Int J Cancer 2004;109:680-684.

8. Sampson JR, Dolwani S, Jones S, Eccles D, Ellis A, Evans DG, Frayling I, Jordan S, Maher ER, Mak T, Maynard J, Pigatto F, Shaw J, Cheadle JP. Autosomal recessive colorectal adenomatous polyposis due to inherited mutations of MYH. Lancet 2003;362:39-41.


97

9. Sieber OM, Lipton L, Crabtree M, Heinimann K, Fidalgo P, Phillips RK, Bisgaard ML, Orntoft TF, Aaltonen LA, Hodgson SV, Thomas HJ, Tomlinson IP. Multiple colorectal adenomas, classic adenomatous polyposis, and germ-line mutations in MYH. N Engl J Med 2003;348:791-799.



12. Nielsen M, Franken PF, Reinards TH, Weiss MM, Wagner A, van der KH, Kloosterman S, Houwing-Duistermaat JJ, Aalfs CM, Ausems MG, Brocker-Vriends AH, Gomez Garcia EB, Hoogerbrugge N, Menko FH, Sijmons RH, Verhoef S, Kuipers EJ, Morreau H, Breuning MH, Tops CM, Wijnen JT, Vasen HF, Fodde R, Hes FJ. Multiplicity in polyp count and extracolonic manifestations in 40 Dutch patients with MYH associated polyposis coli (MAP). J Med Genet 2005;42:e54.

13. de Leng WW, Keller JJ, Luiten S, Musler AR, Jansen M, Baas AF, de Rooij FW, Gille JJ, Menko FH, Offerhaus GJ, Weterman MA. STRAD in Peutz-Jeghers syndrome and sporadic cancers. J Clin Pathol 2005;58:1091-1095.

14. de Leng WW, Westerman AM, Weterman MA, de Rooij FW, Dekken HH, De Goeij AF, Gruber SB, Wilson JH, Offerhaus GJ, Giardiello FM, Keller JJ. Cyclooxygenase 2 expression and molecular alterations in Peutz-Jeghers hamartomas and carcinomas. Clin Cancer Res 2003;9:3065-3072.




Chapte

r 4

Chapter 4

98







24. Zauber P, Sabbath-Solitare M, Marotta S, Zauber A, Bishop T. Comparative molecular pathology of sporadic hyperplastic polyps and neoplastic lesions from the same individual. J Clin Pathol 2004;57:1084-1088.

25. Fogt F, Brien T, Brown CA, Hartmann CJ, Zimmerman RL, Odze RD. Genetic alterations in serrated adenomas: comparison to conventional adenomas and hyperplastic polyps. Hum Pathol 2002;33:87-91.

26. O'Brien MJ, Yang S, Mack C, Xu H, Huang CS, Mulcahy E, Amorosino M, Farraye FA. Comparison of microsatellite instability, CpG island methylation phenotype, BRAF and KRAS status in serrated polyps and traditional adenomas indicates separate pathways to distinct colorectal carcinoma end points. Am J Surg Pathol 2006;30:1491-1501.


99

27. Yang S, Farraye FA, Mack C, Posnik O, O'Brien MJ. BRAF and KRAS Mutations in hyperplastic polyps and serrated adenomas of the colorectum: relationship to histology and CpG island methylation status. Am J Surg Pathol 2004;28:1452-1459.

28. Jen J, Powell SM, Papadopoulos N, Smith KJ, Hamilton SR, Vogelstein B, Kinzler KW. Molecular determinants of dysplasia in colorectal lesions. Cancer Res 1994;54:5523-5526.

29. Otori K, Oda Y, Sugiyama K, Hasebe T, Mukai K, Fujii T, Tajiri H, Yoshida S, Fukushima S, Esumi H. High frequency of KRAS mutations in human colorectal hyperplastic polyps. Gut 1997;40:660-663.



32. Jen J, Powell SM, Papadopoulos N, Smith KJ, Hamilton SR, Vogelstein B, Kinzler KW. Molecular determinants of dysplasia in colorectal lesions. Cancer Res 1994;54:5523-5526.

33. Motojima K, Urano T, Nagata Y, Shiku H, Tsurifune T, Kanematsu T. Detection of point mutations in the Kirsten-ras oncogene provides evidence for the multicentricity of pancreatic carcinoma. Ann Surg 1993;217:138-143.

34. Rozenblum E, Schutte M, Goggins M, Hahn SA, Panzer S, Zahurak M, Goodman SN, Sohn TA, Hruban RH, Yeo CJ, Kern SE. Tumor-suppressive pathways in pancreatic carcinoma. Cancer Res 1997;57:1731-1734.

35. Song MM, Nio Y, Dong M, Tamura K, Furuse K, Tian YL, He SG, Shen K. Comparison of K-ras point mutations at codon 12 and p21 expression in pancreatic cancer between Japanese and Chinese patients. J Surg Oncol 2000;75:176-185.

Chapte

r 4


101

Serrated polyps and Lynch syndrome: are they associated?

K.S. Boparai

E. Kurpershoek M.M. Polak A.R. Musler

E.Dekker C.J.M. van Noesel

Submitted

Ch

ap

ter

Chapter 5

102

ABSTRACT

Background and aims: Adenomas and colorectal cancers (CRCs) of

Lynch syndrome patients display defective mismatch repair (MMR)

associated with microsatellite instability (MSI). Serrated polyps have

also been described, but previous studies investigating a causal

relationship with Lynch syndrome by MMR testing have not been

conclusive. Presence of a BRAF mutation is an alternative method to

distinguish Lynch-associated CRCs from sporadic CRCs and may thus

be of value to investigate Lynch association in serrated polyps. We

studied the occurrence of defective MMR and BRAF mutations in

serrated polyps occurring in 101 Lynch patients. Methods: Lynch

patients with >1 serrated polyp were included. In all polyps of these

patients, MLH1, MSH2, MSH6 and PMS2 protein expression was

evaluated. In addition, APC-mutation cluster region, KRAS (exon 2)

and BRAF (exon 15) mutation analysis was performed in adenomas

and serrated polyps from these patients and from a control group of

sporadic adenomas (n=17) and serrated polyps (n=42). Results: We

identified 10/101 Lynch patients with >1 serrated polyp of whom 2/10

had multiple (≥10) and 8/10 occasional (<10) serrated polyps. A total of

69 polyps were available for analysis: 32 conventional adenomas and

37 serrated polyps. In 13/32 (40%) conventional adenomas and 0/37

serrated polyps, loss of MMR protein expression was observed.

Overall, BRAF mutations were identified in 16/37 (43%) serrated

polyps in Lynch compared to 20/42 (48%) serrated polyps in the

control group (NS). In Lynch patients with occasional serrated polyps,

BRAF mutations were significantly lower (23%) than control group

serrated polyps (49%: p=0.04) Conclusions: Overall, serrated polyps

do not seem to be a product of the MSI pathway in Lynch syndrome

reflected by the absence of defective MMR in these polyps and a

similarly high frequency of BRAF mutations as in sporadic serrated

polyps. For occasional serrated polyps a causal relationship with

Lynch syndrome can not be excluded.


103

INTRODUCTION

It is generally accepted that conventional adenomas of the

colorectum are the main lesions with an undisputable malignant

potential. The classical model describing colorectal cancer (CRC)

development is the adenoma-carcinoma sequence associated with

activation of the Wnt signalling pathway and chromosomal

instability.(1;2) An alternative route to CRC, the microsatellite instability

(MSI) pathway, was discovered in patients with Lynch syndrome.(3)

Lynch syndrome is an autosomal dominant disorder associated with an

increased risk (25-70%) of developing CRC.(4-6) Precursors of CRC in

these patients are also conventional adenomas which are assumed to

progress more rapidly to CRC than their sporadic counterparts.(7) The

MSI pathway in these patients is initiated by a bi-allelic deletion or

inactivation of one of the mismatch repair genes (MLH1, MSH2, MSH6

or PMS2). The hallmark of Lynch-associated tumors is microsatellite

instability (MSI) and loss of immunostaining of the affected mismatch

repair protein. MSI is found in more than 95% of Lynch associated

CRCs as opposed to only 15% of sporadic CRCs.6, 7 CRCs from Lynch

patients harbour predominantly APC, KRAS and P53 mutations and

virtually never a BRAF mutation.(3;8-13) BRAF testing has therefore

been shown to be an ideal method to exclude Lynch associated

tumors from sporadic tumors.(14;15) Recently, a newly proposed

‘serrated neoplasia pathway’ has been described involving the

progression of serrated polyps to CRC through accumulation of

genetic mutations, namely BRAF mutations and CPG-island

methylation. Supporting clinicohistological and molecular evidence for

such a pathway has been derived primarily from patients with serrated

polyposis syndrome (SPS).(13;16-21)

Chapte

r 5

Chapter 5

104

In Lynch syndrome, serrated polyps (i.e. hyperplastic polyps,

HPs; sessile serrated adenomas, SSAs; and traditional serrated

adenomas, TSAs) have also been described.(7;22-27) In a number of

previous studies, an attempt was made to verify whether these

serrated polyps are causally related to Lynch syndrome, reflected by

the presence of defective mismatch repair (MSI) and/or loss of

immunostaining. These studies have thus far been inconclusive. Both

conventional adenomas (24-93%)(23;28-37) and HPs/hyperplastic

abberant crypt foci (0-100%)(26;29;35;38;39) express defective

mismatch repair in highly variable frequencies. To analyze whether

serrated polyps are causally related to Lynch syndrome, BRAF

mutation analysis may be a valuable adjunct to mismatch repair gene

testing. Sporadic serrated polyps have been shown to display high (70-

78%) levels of BRAF mutations.33 It seems presumable that, like CRCs

in Lynch, serrated polyps when causally related to Lynch syndrome,

will display BRAF mutations at a far lower frequency than their

sporadic counterparts.

In this study, we describe the frequency of serrated polyps

(HPs/SSAs/TSAs) in 101 patients with a genetically confirmed

diagnosis of Lynch syndrome. We show that, overall, serrated polyps

in Lynch syndrome lack defective mismatch repair (MSI and/or loss of

immunostaining) and harbour a similarly high frequency of BRAF

mutations as their sporadic counterparts, strongly suggesting that they

are not associated with the MSI-pathway. At sub-analysis, we show

that occasional serrated polyps in Lynch patients have significantly

lower levels of BRAF mutations than their sporadic counterparts.

Therefore, a causal relationship with the MSI-pathway can not be

excluded in these polyps.


105

MATERIALS AND METHODS

Patients and specimens

For this study, 102 patients known at our centre with Lynch syndrome

and an identified germline mutation in one of the MMR genes was

analysed for the presence of serrated polyps. Patients were included if

>1 serrated polyp was identified. These patients were then arbitrarily

classified in to two groups: patients with multiple serrated polyps (≥10);

or patients with occasional serrated polyps (2-10).

Polyp characteristics were recorded retrospectively from

previous colonoscopy reports or the gross description of the resection

specimens at histopathology. All polyps were blindly re-evaluated and

diagnosed by a single experienced pathologist (CvN) as HP, SSA,

TSA or adenoma based on the histological features on H&E

staining.(40-42) Lesions proximal from the caecum, ascending colon,

transverse colon and descending colon were regarded as proximal and

polyps from the sigmoid colon and rectum were regarded as distal. For

the purpose of comparison, a previously selected control group

consisting of sporadic HPs (n=24), SSAs (n=18) and conventional

adenomas (n=17) from patients without a suspicion for Lynch

syndrome was used to compare somatic mutations.This study was

conducted in accordance with the research code of our institutional

medical ethical committee on human experimentation, as well as in

agreement with the Helsinki Declaration of 1975, as revised in 1983.

Molecular analysis

Mutation analysis

Using previously described primers and assays, DNA was analysed for

mutations in the APC-mutation cluster region (APC-MCR), KRAS

(exon 2) and BRAF (exon 15) of all polyps.37, 38 Previously performed

Chapte

r 5

Chapter 5

106

mutation analysis of these genes in a randomly selected sporadic

polyps served as a control group. Detected mutations were confirmed

in a second experiment.

Immunohistochemistry

Immunohistochemistry was performed on all polyps of the included

Lynch patients. Unstained 5-µm sections were cut from paraffin blocks

and the slides were deparaffinized. Primary monoclonal antibodies

used were specific for MLH1(1:50 BD Pharmingen, San Diego, USA);

MSH2(1:100 Oncogene Research Prod., San Diego, USA);

MSH6(1:200 BD Transduction Lab., San Jose, USA); PMS2(1:250 BD

Transduction Lab., San Jose, USA). Slides were immersed in 0.3%

hydrogen peroxide in methanol for 20 minutes. Subsequently, antigen

retrieval was carried out by 10 minutes of boiling in 10mM Tris/1mM

EDTA (pH 9) followed by incubation with above mentioned diluted

primary antibodies during 1 hour at room temperature. Post-antibody

block (Immunologic) in PBS was performed followed by

implementation of an antipolyvalent HRP detection system

(Immunologic) to visualize antibody binding sites with 3.3’-

diaminobenzidine as a chromogen. Sections were counterstained with

haematoxylin. Stains for MLH1, MSH2, MSH6 and PMS2 were

considered negative when there was complete absence of nuclear

expression in all neoplastic cells. Negative staining in a part of a lesion

or in a single crypt was registered separately.

Microsatellite analysis

In a subset of Lynch patients, harbouring many serrated polyps and

conventional adenomas (>20), MSI analysis was performed.

Microsatellite status was determined using an international standard


107

panel of 5 microsatellite markers (D17S250, D2S123, D5S346, BAT25

and BAT26) using standard techniques. A high degree of microsatellite

instability (MSI-high) was defined as two (40%) or more unstable

markers, MSI-Low as one unstable marker, and microsatellite stable

(MSS) as no unstable markers.

Statistics



Inc, Chicago, Ill). Somatic mutations in polyps of Lynch patients were

compared with those of the control group using a two-sided Fisher

exact test. A p-value of < 0.05 was considered statistically significant.

RESULTS

From 101 Lynch syndrome patients with a known germline mutation,

10 (10%) patients were identified with >1 serrated polyp. Of these

patients, 2 had ≥10 serrated polyps (table 1). The median age was 62

years (range: 44-75) with a male: female ratio of 3:7. In 10 patients, a

total of 89 polyps were identified of which 69 were available for

analysis: 32 conventional adenomas (21 tubular adenomas and 11

tubulovillous adenomas) and 37 serrated polyps (32 HPs and 5 SSAs).

The median size of serrated polyps was 2mm (range: 1-10mm) and

4mm (range: 2-30mm) for conventional adenomas. Of 34/37 serrated

polyps location could be ascertained. Nineteen of 34 (51%) serrated

polyps were located proximal to the sigmoid colon. In 8/10 (80%)

patients, serrated polyps were identified proximal to the sigmoid colon.

In 1/11 tubulovillous adenomas (20mm) an adenocarcinoma was

identified (at surgery: T2N0M0). The other 10 tubulovillous adenomas

displayed low-grade dysplasia. In 6/22 tubular adenomas, features of

Chapte

r 5

Chapter 5

108

high-grade dysplasia were seen. In 1/10 patients a subtotal colectomy

had been performed previously because of an adenocarcinoma

(T3N0M0, tissue not available for analysis). Previously performed

germline mutation analysis in patient 2 due to the high number of

adenomas showed a mono-allelic mutation of the MUTYH gene.

Table 1: Number of histologically verified polyps and total number of detected polyps in Lynch patients.

Patient

Age

(yrs)

Germline

mutation

Sex

Adenomas

Analyzed/total

HPs

Analyzed/total

SSAs

analyzed/total

Location

serrated

polyps

Lynch patients with multiple (≥10) serrated polyps (=SPS)

1 44 MSH6 F 2/2 12/>30 0/0 Pancolonic

2 69 MSH6 M 14/14 1/10 2/2 Pancolonic

Lynch patients with occasional (<10) serrated polyps (≠SPS)

3 60 MLH1 M 5/5 2/2 0/0 Pancolonic

4 62 MSH6 M 1/1 2/2 1/1 Pancolonic

5 50 PMS2 F 0/0 2/2 0/0 Rectosigmoid

6 68 MLH1 F 5/5 4/4 0/0 Pancolonic

7 62 MLH1 F 1/1 2/2 1/1 Right-side

8 62 MSH2 M 2/2 3/3 0/0 Rectosigmoid

9 75 MSH2 F 1/1 3/3 0/0 Left-side

10 65 MSH2 F 1/1 1/1 2/2 Right-side

Total 12 32/32 32/>59 5/5


109

Somatic APC, KRAS and BRAF mutation analysis

A total of 32 adenomas, 32 HPs and 5 SSAs were analysed for

APC-MCR, KRAS exon 2 and BRAF exon 15 mutations. In adenomas,

APC mutations were found in 6/32 (19%) of polyps. No KRAS

mutations or BRAF mutations were detected in adenomas. No

significant differences were seen compared to the control group

regarding APC and BRAF mutations (table 2). More KRAS mutations

were identified in sporadic adenomas (4/17) of the control group

compared to Lynch adenomas (0/32: p=0.01). The adenocarcinoma

identified in a tubulovillous adenoma displayed an APC mutation. In

serrated polyps, no APC mutations were identified. KRAS mutations

were identified 6/37 (16%) serrated polyps vs 7/42 (17%) in the control

group. BRAF mutations were detected in 16/37 (43%) serrated polyps

in Lynch patients compared to 20/42 (48%) in the control group (n.s.).

There was no significant difference between proximal and distal

serrated polyp location regarding BRAF mutation status.

When comparing patients with multiple serrated polyps and

satisfying the criteria for SPS (n=2) to patients with occasional serrated

polyps (n=8), we identified significantly (p=0.002) more BRAF

mutations in SPS serrated polyps (11/15 =73%) than in occasional

serrated polyps (5/22=23%). After subdivision, comparison of BRAF

mutations showed that occasional serrated polyps in Lynch patients

harboured a significantly lower BRAF frequency (23%) than the control

group (49%: p=0.04). Comparison of SPS serrated polyps with control

group serrated polyps showed no significant differences regarding

mutations.

Chapte

r 5

Chapter 5

110

Table 2. Detected APC, KRAS and BRAF mutations in serrated polyps (HPs, SSAs) and adenomas (AD) compared to a control group. * significantly more KRAS mutations in sporadic adenomas than Lynch adenomas

Immunohistochemistry and microsatellite analysis

Loss of expression of one of the MMR proteins was observed in 13/33

(39%) conventional adenomas in Lynch patients which correlated in all

cases with the germline MMR mutation. There was no correlation

between histology (10 tubular adenomas and 3 tubulovillous

adenomas) and loss of MMR expression (p=0.259). Also when grade

of dyspasia was analyzed, no association was observed with MMR

expression (p=0.672). The adenocarcinoma identified within a

tubulovillous adenoma in a Lynch patient did not demonstrate loss of

MMR expression. None of the serrated polyps showed loss of

expression of one of the MMR proteins. MMR protein expression in the

crypts of the serrated polyps was comparable to that of crypts in

normal epithelium of the colorectum. Expression of all four MMR

proteins was observed in most nuclei in the base of the crypts whereas

few to none of the luminal nuclei expressed any of the MMR proteins.

Somatic mutation Patients with Lynch Control group P-value

Serrated polyps

(n=37)

AD

(n=32)

Serrated polyps

(n=42)

AD

(n=17)

APC mutation 0 6

(19%) 0

7

(41%) ns

BRAF mutation 16 (43%) 0 20 (49%) 0 ns

KRAS mutation 6 (16%) 0 7 (17%) 4

(24%) 0.01*


111

Microsatellite analysis was performed in 30 polyps of Lynch

patients 1 and 2, both with a germline mutation in MSH6 and ≥10

serrated polyps, also satisfying the clinical diagnosis of SPS. Fifteen

adenomas, 13 HPs and 2 SSAs were analyzed. In patient 1, both

adenomas (2/2) were MSI-high and showed corresponding loss of the

MSH6 protein at immunohistochemistry. All serrated polyps were MSS

in this patient. In patient 2, 0/14 adenomas (including the

adenocarcinoma) was MSI-H and none showed immunohistochemical

loss of the MSH-6 protein. All serrated polyps were microsatellite

stable in this patient.

DISCUSSION

In this comprehensive cohort study of Lynch syndrome patients with a

proven germline MMR gene mutation, we demonstrated that a small

proportion (2%) of patients harbor multiple serrated polyps in addition

to conventional adenomas, two of whom also satifying the criteria for

SPS. Using conventional immunohistochemistry and MSI analysis,

previous studies have been performed to analyze whether serrated

polyps are causally related to Lynch syndrome. Results from these

studies have however been inconclusive. Based on the principle that

BRAF mutation testing is a highly sensitive method to distinguish

sporadic CRCs (high frequency of mtuations) from Lynch-associated

CRCs (low frequency of mutations)(14;15;43), we demonstrated that

serrated polyps most likely are not associated with Lynch syndrome,

reflected by both a similarly high frequency of BRAF mutations as

sporadic serrated polyps and an absence of MMR deficiency in all of

these polyps.

Our study is the first to utilize an alternative molecular approach

involving mutation analysis (APC, KRAS and BRAF) in adjunct to

Chapte

r 5

Chapter 5

112

defective MMR testing to evaluate whether serrated polyps are

causally related to Lynch syndrome. In addition, our study

encompasses the largest analysis of serrated polyps (including SSAs)

from Lynch patients who have been unambiguously defined by a

germline mutation thus representing true Lynch syndrome patients.

Prior reports evaluating solely defective MMR in serrated polyps

defined Lynch syndrome in a broader sense: fulfilment of either genetic

or clinical (Amsterdam) criteria. However, correlation between the

Amsterdam criteria and a germline mutation are imperfect. Indeed, it

has been shown that families satisfying the Amsterdam criteria may

carry an alternative diagnosis such as Syndrome X or serrated

polyposis syndrome (SPS).(19;44)

In Lynch syndrome, carcinogenesis is traditionally considered

to proceed through an accelerated adenoma-carcinoma sequence

driven by MMR deficiency. However, in the literature not all

conventional adenomas in these patients have been shown to express

defective MMR (24-93%). We also found loss of expression of one of

the MMR proteins in only 13/33 (39%) conventional adenomas. It is

conceivable that the remaining adenomas without loss of expression of

one of the MMR proteins represent co-existing sporadic conventional

adenomas. Concordantly, sporadic conventional adenomas show

defective mismatch repair in far lower percentages: 0-3% of

adenomas.(30;45) Nevertheless, independent of Lynch-associaton,

removal of all conventional adenomas reduces the incidence of CRC

and is therefore standard practice of care in these patients.(46;47)

Serrated polyps, which were traditionally considered to be

benign lesions, have recently also been considered to be lesions with

malignant potential through a serrated CRC pathway. The early

genetic events of this route, as yet identified, are predominantly BRAF


113

mutations and a generally enhanced CPG-island methylation status of

multiple genes.(20;29;48-53) Additional combined clinicohistological

and molecular evidence supporting a serrated CRC pathway include

right-sided carcinomas identified within serrated polyps harboring

identical BRAF mutations as the serrated polyp component(17) and

increased incidence of serrated polyps in patients with sporadic

microsatellite-unstable CRCs.(20;48;54) Removal of these lesions, in

particular large, right-sided serrated polyps, is therefore clinically

important.(13;17) However, serrated polyps associated with the

accelerated MSI-pathway may represent present precursor lesions of

MSI-CRC which evolve even faster than in the serrated CRC pathway

alone. Investigation of an association between serrated polyps and

Lynch is therefore clinically relevant. Three previous studies reported

defective MMR in serrated polyps. In two studies, MSI was observed in

hyperplastic aberrant crypt foci (4/4:100%)(39) and HPs

(2/17:11%)(23). Finally, in one case report a Lynch patient was

described with a mixed HP/adenoma in contiguity with a CRC.(55) We

found no defective MMR in serrated polyps which is in concordance

with a number of prior studies also showing an absence of defective

MMR in serrated polyps.(22;26;38;56) In addition, the similarly high

frequency of BRAF mutations in serrated polyps as their sporadic

counterparts in this study further supports the notion that serrated

polyps are not causally related to the MSI carcinogenesis pathway in

Lynch syndrome.

It is possible that two oncogenic pathways, a MSI-pathway and

a serrated CRC pathway, co-exist within Lynch syndrome patients with

serrated polyps. However, considering that a large proportion of

serrated polyps in our study were BRAF-mutated and thus far no

CRCs with a BRAF mutation have been identified in Lynch patients, it

Chapte

r 5

Chapter 5

114

seems that the MSI pathway predominates over the serrated CRC

pathway because of a relatively higher frequency of conventional

adenomas in Lynch patients, favouring a stochastic process of

carcinogenesis. Alternatively, the serrated CRC pathway may

represent a slower route of carcinogenesis than the MSI pathway.

BRAF mutations are more commonly associated with serrated polyps

and the serrated CRC pathway than KRAS mutations. Nevertheless, a

serrated CRC pathway, skewed towards initial KRAS mutations in a

minority of serrated polyps can not be excluded.

Interestingly, after subdividing Lynch patients in SPS Lynch

patients with multiple serrated polyps and non-SPS Lynch patients with

occasional serrated polyps, we found that BRAF mutations were more

common in serrated polyps of Lynch-SPS patients: 11/15 (73%)

compared to 5/22 (23%) in non-SPS patients (p=0.002). Based on

these findings we conclude that serrated polyps in SPS Lynch patients

in particular are an expression of a serrated-CRC pathway.

Alternatively, only 23% of serrated polyps in non-SPS patients were

BRAF mutated. This proportion is significantly lower than control group

serrated polyps (49%: p=0.04). Considering this low frequency of

BRAF mutations in serrated polyps of non-SPS Lynch patients, a

causal relationship with the MSI-pathway can not be excluded despite

lack of defective MMR. Thus, these occasional serrated polyps, lacking

BRAF mutations, may follow an accelerated route to CRC via the MSI-

pathway.

SPS is a condition characterized by the presence of multiple

serrated polyps spread throughout the colon and is associated with an

increased CRC risk.(57-60) However, the heterogeneous phenotype of

SPS such as the presence of different polyp histologies, differences in

polyp localization and number of polyps, suggest that SPS can be


115

subdivided into separate (genetic) conditions. Two Lynch patients

(patients 1 and 2) harboured ≥10 colorectal serrated polyps. While

patient 1 satisfied the classical criteria for SPS with few classical

adenomas(16), patient 2 had less serrated polyps and more

conventional adenomas thus representing an intermediate SPS with a

mixed phenotype. MSI analysis in adenomas of patient 1 showed high

degree of MSI in all (2/2) adenomas as well as concordant lack of

MSH-6 expression, indicating that these adenomas are a manifestation

of Lynch syndrome. Interestingly, 0/14 adenomas of patient 2,

including the adenocarcinoma, were MSI-H. The lack of MSI in all

adenomas of this Lynch patient with a mixed SPS phenotype suggests

that these adenomas are not associated with Lynch syndrome. To

substantiate this, we selected a control group of conventional

adenomas (n=15) which were matched for size, location, histology and

grade of dysplasia from Lynch patients without serrated polyps to

compare MSI status and found MSI-H in 9/15 adenomas (p=0.016,

data not shown). Although less common, it is possible that the

adenomas in patient 2 are an expression of mono-allelic MUTYH

deficiency.(61) However, only 2/6 APC-mutated adenomas displayed

G:C→T:A transversions, which are typical for MUTYH-deficiency. No

KRAS mutations were identified in these adenomas. In the literature, a

subset of serrated polyps has also been described to be causally

related to bi-allelic MUTYH-deficiency.(62) This seems unlikely in

patient 2 considering that of the mutated serrated polyps, 2 were

BRAF mutated and only one KRAS mutated (although it did have a

G:C→ T:A conversion). Another explanation could be that these

adenomas are in fact part of a mixed subtype of SPS. It remains

however difficult to verify this hypothesis considering that no hallmark

genetic characteristics of SPS have been identified. Alternatively,

Chapte

r 5

Chapter 5

116

these adenomas could simply be sporadic adenomas. However,

considering the multitude of adenomas, this seems unlikely.

Based on our observations, we conclude that, overall, serrated

polyps play an insignificant role in the MSI carcinogenesis pathway in

Lynch syndrome. A causal relationship between occasional serrated

polyps and the MSI-pathway can not be excluded. BRAF-mutated

serrated polyps may represent components of a co-existent serrated

CRC pathway. The MSI pathway of carcinogenesis seems however to

predominate over the serrated CRC pathway in these patients

reflected by the described absence of BRAF mutations in Lynch-

associated CRCs.


117

REFERENCES (1) Fearon ER, Vogelstein B. A genetic model for colorectal

tumorigenesis. Cell 1990;61(5):759-67.

(2) Vogelstein B, Fearon ER, Hamilton SR et al. Genetic alterations during colorectal-tumor development. N Engl J Med 1988;319(9):525-32.

(3) Aaltonen LA, Peltomaki P, Leach FS et al. Clues to the pathogenesis of familial colorectal cancer. Science 1993;260(5109):812-6.

(4) Hendriks YM, Wagner A, Morreau H et al. Cancer risk in hereditary nonpolyposis colorectal cancer due to MSH6 mutations: impact on counseling and surveillance. Gastroenterology 2004;127(1):17-25.

(5) Quehenberger F, Vasen HF, van Houwelingen HC. Risk of colorectal and endometrial cancer for carriers of mutations of the hMLH1 and hMSH2 gene: correction for ascertainment. J Med Genet 2005;42(6):491-6.

(6) Plaschke J, Engel C, Kruger S et al. Lower incidence of colorectal cancer and later age of disease onset in 27 families with pathogenic MSH6 germline mutations compared with families with MLH1 or MSH2 mutations: the German Hereditary Nonpolyposis Colorectal Cancer Consortium. J Clin Oncol 2004;22(22):4486-94.

(7) Lindgren G, Liljegren A, Jaramillo E et al. Adenoma prevalence and cancer risk in familial non-polyposis colorectal cancer. Gut 2002;50(2):228-34.

(8) Fujiwara T, Stolker JM, Watanabe T et al. Accumulated clonal genetic alterations in familial and sporadic colorectal carcinomas with widespread instability in microsatellite sequences. Am J Pathol 1998;153(4):1063-78.

(9) Huang J, Papadopoulos N, McKinley AJ et al. APC mutations in colorectal tumors with mismatch repair deficiency. Proc Natl Acad Sci U S A 1996;93(17):9049-54.

(10) Konishi M, Kikuchi-Yanoshita R, Tanaka K et al. Molecular nature of colon tumors in hereditary nonpolyposis colon cancer, familial polyposis, and sporadic colon cancer. Gastroenterology 1996;111(2):307-17.

Chapte

r 5

Chapter 5

118

(11) Losi L, Ponz de LM, Jiricny J et al. K-ras and p53 mutations in hereditary non-polyposis colorectal cancers. Int J Cancer 1997;74(1):94-6.

(12) Tomlinson I, Ilyas M, Johnson V et al. A comparison of the genetic pathways involved in the pathogenesis of three types of colorectal cancer. J Pathol 1998;184(2):148-52.

(13) Kambara T, Simms LA, Whitehall VL et al. BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum. Gut 2004;53(8):1137-44.

(14) McGivern A, Wynter CV, Whitehall VL et al. Promoter hypermethylation frequency and BRAF mutations distinguish hereditary non-polyposis colon cancer from sporadic MSI-H colon cancer. Fam Cancer 2004;3(2):101-7.

(15) Domingo E, Laiho P, Ollikainen M et al. BRAF screening as a low-cost effective strategy for simplifying HNPCC genetic testing. J Med Genet 2004;41(9):664-8.

(16) Boparai KS, Mathus-Vliegen EM, Koornstra JJ et al. Increased colorectal cancer risk during follow-up in patients with hyperplastic polyposis syndrome: a multicentre cohort study. Gut 2010;59(8):1094-100.

(17) Boparai KS, Dekker E, Polak MM et al. A serrated colorectal cancer pathway predominates over the classic WNT pathway in patients with hyperplastic polyposis syndrome. Am J Pathol 2011;178(6):2700-7.

(18) Chan TL, Zhao W, Leung SY et al. BRAF and KRAS mutations in colorectal hyperplastic polyps and serrated adenomas. Cancer Res 2003;63(16):4878-81.

(19) Jeevaratnam P, Cottier DS, Browett PJ et al. Familial giant hyperplastic polyposis predisposing to colorectal cancer: a new hereditary bowel cancer syndrome. J Pathol 1996;179(1):20-5.

(20) Makinen MJ, George SM, Jernvall P et al. Colorectal carcinoma associated with serrated adenoma--prevalence, histological features, and prognosis. J Pathol 2001;193(3):286-94.

(21) Nosho K, Kure S, Irahara N et al. A prospective cohort study shows unique epigenetic, genetic, and prognostic features of synchronous colorectal cancers. Gastroenterology 2009;137(5):1609-20.


119

(22) Andersen SH, Lykke E, Folker MB et al. Sessile serrated polyps of the colorectum are rare in patients with Lynch syndrome and in familial colorectal cancer families. Fam Cancer 2008;7(2):157-62.

(23) Iino H, Simms L, Young J et al. DNA microsatellite instability and mismatch repair protein loss in adenomas presenting in hereditary non-polyposis colorectal cancer. Gut 2000;47(1):37-42.

(24) Jarrar AM, Church JM, Fay S et al. Is the phenotype mixed or mistaken? Hereditary nonpolyposis colorectal cancer and hyperplastic polyposis syndrome. Dis Colon Rectum 2009;52(12):1949-55.

(25) Liljegren A, Lindblom A, Rotstein S et al. Prevalence and incidence of hyperplastic polyps and adenomas in familial colorectal cancer: correlation between the two types of colon polyps. Gut 2003;52(8):1140-7.

(26) Rijcken FE, van der Sluis T, Hollema H et al. Hyperplastic polyps in hereditary nonpolyposis colorectal cancer. Am J Gastroenterol 2003;98(10):2306-11.

(27) Walsh MD, Buchanan DD, Walters R et al. Analysis of families with Lynch syndrome complicated by advanced serrated neoplasia: the importance of pathology review and pedigree analysis. Fam Cancer 2009;8(4):313-23.

(28) Rijcken FE, Hollema H, Kleibeuker JH. Proximal adenomas in hereditary non-polyposis colorectal cancer are prone to rapid malignant transformation. Gut 2002;50(3):382-6.

(29) Iino H, Jass JR, Simms LA et al. DNA microsatellite instability in hyperplastic polyps, serrated adenomas, and mixed polyps: a mild mutator pathway for colorectal cancer? J Clin Pathol 1999;52(1):5-9.

(30) Aaltonen LA, Peltomaki P, Mecklin JP et al. Replication errors in benign and malignant tumors from hereditary nonpolyposis colorectal cancer patients. Cancer Res 1994;54(7):1645-8.

(31) Jacoby RF, Marshall DJ, Kailas S et al. Genetic instability associated with adenoma to carcinoma progression in hereditary nonpolyposis colon cancer. Gastroenterology 1995;109(1):73-82.

(32) Konishi M, Kikuchi-Yanoshita R, Tanaka K et al. Molecular nature of colon tumors in hereditary nonpolyposis colon cancer, familial polyposis, and sporadic colon cancer. Gastroenterology 1996;111(2):307-17.

Chapte

r 5

Chapter 5

120

(33) Akiyama Y, Iwanaga R, Saitoh K et al. Transforming growth factor beta type II receptor gene mutations in adenomas from hereditary nonpolyposis colorectal cancer. Gastroenterology 1997;112(1):33-9.

(34) Sasaki S, Masaki T, Umetani N et al. Microsatellite instability is associated with the macroscopic configuration of neoplasms in patients with multiple colorectal adenomas. Jpn J Clin Oncol 1998;28(7):427-30.

(35) Pino MS, Mino-Kenudson M, Wildemore BM et al. Deficient DNA mismatch repair is common in Lynch syndrome-associated colorectal adenomas. J Mol Diagn 2009;11(3):238-47.

(36) Muller A, Beckmann C, Westphal G et al. Prevalence of the mismatch-repair-deficient phenotype in colonic adenomas arising in HNPCC patients: results of a 5-year follow-up study. Int J Colorectal Dis 2006;21(7):632-41.

(37) Giuffre G, Muller A, Brodegger T et al. Microsatellite analysis of hereditary nonpolyposis colorectal cancer-associated colorectal adenomas by laser-assisted microdissection: correlation with mismatch repair protein expression provides new insights in early steps of tumorigenesis. J Mol Diagn 2005;7(2):160-70.

(38) Halvarsson B, Lindblom A, Johansson L et al. Loss of mismatch repair protein immunostaining in colorectal adenomas from patients with hereditary nonpolyposis colorectal cancer. Mod Pathol 2005;18(8):1095-101.

(39) Pedroni M, Sala E, Scarselli A et al. Microsatellite instability and mismatch-repair protein expression in hereditary and sporadic colorectal carcinogenesis. Cancer Res 2001;61(3):896-9.

(40) Hamilton SR, Vogelstein B, Kudo S et al. World Health Organization Classification of Tumours, Pathology and Genetics of Tumours of the Digestive System. Lyon, France: IARC Press, 2000: 104-19.

(41) Jass JR, Baker K, Zlobec I et al. Advanced colorectal polyps with the molecular and morphological features of serrated polyps and adenomas: concept of a 'fusion' pathway to colorectal cancer. Histopathology 2006;49(2):121-31.

(42) Snover DC, Jass JR, Fenoglio-Preiser C et al. Serrated polyps of the large intestine: a morphologic and molecular review of an evolving concept. Am J Clin Pathol 2005;124(3):380-91.


121

(43) Loughrey MB, Waring PM, Tan A et al. Incorporation of somatic BRAF mutation testing into an algorithm for the investigation of hereditary non-polyposis colorectal cancer. Fam Cancer 2007;6(3):301-10.

(44) Lindor NM, Rabe K, Petersen GM et al. Lower cancer incidence in Amsterdam-I criteria families without mismatch repair deficiency: familial colorectal cancer type X. JAMA 2005;293(16):1979-85.

(45) Young J, Leggett B, Gustafson C et al. Genomic instability occurs in colorectal carcinomas but not in adenomas. Hum Mutat 1993;2(5):351-4.

(46) Mecklin JP, Aarnio M, Laara E et al. Development of colorectal tumors in colonoscopic surveillance in Lynch syndrome. Gastroenterology 2007;133(4):1093-8.

(47) Winawer SJ, Zauber AG, Ho MN et al. Prevention of colorectal cancer by colonoscopic polypectomy. The National Polyp Study Workgroup. N Engl J Med 1993;329(27):1977-81.

(48) Hawkins NJ, Ward RL. Sporadic colorectal cancers with microsatellite instability and their possible origin in hyperplastic polyps and serrated adenomas. J Natl Cancer Inst 2001;93(17):1307-13.

(49) Jass JR, Biden KG, Cummings MC et al. Characterisation of a subtype of colorectal cancer combining features of the suppressor and mild mutator pathways. J Clin Pathol 1999;52(6):455-60.

(50) Jass JR, Iino H, Ruszkiewicz A et al. Neoplastic progression occurs through mutator pathways in hyperplastic polyposis of the colorectum. Gut 2000;47(1):43-9.

(51) Jass JR, Young J, Leggett BA. Hyperplastic polyps and DNA microsatellite unstable cancers of the colorectum. Histopathology 2000;37(4):295-301.

(52) Jass JR. Serrated route to colorectal cancer: back street or super highway? J Pathol 2001;193(3):283-5.

(53) Yao T, Nishiyama K, Oya M et al. Multiple 'serrated adenocarcinomas' of the colon with a cell lineage common to metaplastic polyp and serrated adenoma. Case report of a new subtype of colonic adenocarcinoma with gastric differentiation. J Pathol 2000;190(4):444-9.

Chapte

r 5

Chapter 5

122

(54) Goldstein NS, Bhanot P, Odish E et al. Hyperplastic-like colon polyps that preceded microsatellite-unstable adenocarcinomas. Am J Clin Pathol 2003;119(6):778-96.

(55) Jass JR, Cottier DS, Pokos V et al. Mixed epithelial polyps in association with hereditary non-polyposis colorectal cancer providing an alternative pathway of cancer histogenesis. Pathology 1997;29(1):28-33.

(56) Meijer TW, Hoogerbrugge N, Nagengast FM et al. In Lynch syndrome adenomas, loss of mismatch repair proteins is related to an enhanced lymphocytic response. Histopathology 2009;55(4):414-22.

(57) Hyman NH, Anderson P, Blasyk H. Hyperplastic polyposis and the risk of colorectal cancer. Dis Colon Rectum 2004;47(12):2101-4.

(58) Leggett BA, Devereaux B, Biden K et al. Hyperplastic polyposis: association with colorectal cancer. Am J Surg Pathol 2001;25(2):177-84.

(59) Rubio CA, Stemme S, Jaramillo E et al. Hyperplastic polyposis coli syndrome and colorectal carcinoma. Endoscopy 2006;38(3):266-70.

(60) Rashid A, Houlihan PS, Booker S et al. Phenotypic and molecular characteristics of hyperplastic polyposis. Gastroenterology 2000;119(2):323-32.

(61) Sieber OM, Lipton L, Crabtree M et al. Multiple colorectal adenomas, classic adenomatous polyposis, and germ-line mutations in MYH. N Engl J Med 2003;348(9):791-9.

(62) Boparai KS, Dekker E, van ES et al. Hyperplastic polyps and sessile serrated adenomas as a phenotypic expression of MYH-associated polyposis. Gastroenterology 2008;135(6):2014-8.

CRC pathways in patients with HPS

123

A serrated colorectal cancer pathway predominates over the classical WNT-pathway in patients with hyperplastic polyposis syndrome

Karam S. Boparai

Evelien Dekker

Mirjam M. Polak

Alex R. Musler

Susanne van Eeden

Carel J.M. van Noesel

American Journal of Pathology 2011 Jun;178(6):2700-7

Ch

ap

ter

Chapter 6

124

ABSTRACT

Background and aims: Hyperplastic polyposis syndrome (HPS) is characterized by the presence of multiple colorectal serrated polyps and is associated with an increased colorectal cancer (CRC) risk. The mixture of distinct precursor lesion types and malignancies in HPS provides a unique model to study the canonical pathway and a proposed serrated CRC-pathway in humans. Methods: To establish which CRC-pathways play a role in HPS and particularly to obtain new support for the serrated CRC pathway, we assessed the molecular characteristics of polyps(n=84) and CRCs(n=19) in 17 HPS patients as compared to control groups of various sporadic polyps(n=59) and sporadic, microsatellite-stable CRCs(n=16). Results: In both HPS and sporadic polyps, APC mutations were exclusively identified in adenomas whereas BRAF mutations were confined to serrated polyps. Six of 19(32%) HPS CRCs were identified within a serrated polyp. Mutation analysis performed in both the CRC and the serrated component of these lesions showed identical BRAF mutations. One HPS CRC was located within an adenoma, both components harboring an identical APC mutation. Overall, 10/19(53%) HPS CRCs carried a BRAF mutation, compared to none in control group CRCs (p=0.001). Six (60%) of the BRAF-mutated HPS CRCs were microsatellite-unstable (MSI-high) due to MLH1 methylation. Conclusion: Our findings provide novel supporting evidence for the existence of a predominant serrated CRC pathway in HPS, generating both microsatellite-stable and microsatellite-instable CRCs.


125

INTRODUCTION

Colorectal cancer (CRC) ranks as the second most common

cause of cancer-related death in the western world.1 The classical

model which describes CRC development is the adenoma-carcinoma

sequence associated with activation of the WNT signalling pathway.2, 3

This pathway is characterised by an initial, bi-allelic inactivation of the

adenomatous polyposis coli gene (APC) followed by mutations in key

oncogenes and tumor-suppressor genes, including KRAS, DCC and

TP53, resulting in adenoma initiation and progression to CRC. This

multi-step process of carcinogenesis has been elaborately studied and

much information has been derived from the familial adenomatous

polyposis (FAP) and MUTYH-associated polyposis (MAP) syndromes.

In addition to the adenoma-carcinoma sequence, an

alternative, microsatellite instability (MSI) pathway exists which is

characterized by deletion or inactivation of mismatch repair (MMR)

genes. Loss of one of the MMR genes occurs in 10-15 % of the

sporadic CRC, whereas 1-2% of CRCs with MMR gene loss is due to a

hereditary predisposition, i.e. in Lynch syndrome patients carrying a

mono-allelic MMR gene defect in the germline.

Recently a, “serrated neoplasia pathway”, has been proposed

which involves the progression of serrated polyps, i.e. hyperplastic

polyps (HPs), sessile serrated adenomas (SSAs) and/or traditional

serrated adenomas (TSAs), to CRC. The early genetic events of this

route, as yet identified, are BRAF or KRAS mutations and an

enhanced CPG-island methylation status of multiple genes.4-11 There is

evidence to suggest that a proportion of sporadic MSI CRCs originate

from serrated polyps as both these lesions commonly harbour

hypermethylated MLH1 in combination with BRAF mutations.12-16 In

addition, clinicohistological reports supporting a serrated CRC pathway

Chapte

r 6

Chapter 6

126

include CRCs in close vicinity of large hyperplastic polyps17, 18; CRCs

identified in mixed hyperplastic and adenomatous polyps19; and

increased incidence of serrated polyps in patients with sporadic

microsatellite-unstable CRCs.4, 10, 20 Currently however, proof of the

existence of a serrated CRC pathway, demonstrated by the combined

histological finding of a serrated polyp directly adjacent to a CRC and

concurrent molecular evidence for a sequential relationship has not

been delivered.

Hyperplastic Polyposis Syndrome (HPS) is a condition

characterized by the presence of multiple colorectal serrated polyps.

The genetic cause(s) of HPS is/are largely unknown. We recently

demonstrated that HPS can occur in the context of MAP21, but MUTYH

mutations seem to occur in only a small proportion of HPS patients.22

HPS is associated with an increased CRC-risk.5, 7, 19, 22-27 Previously

published case series report CRC at clinical presentation in up to 50%

of HPS patients and interval carcinomas, i.e. carcinomas occurring

after HPS diagnosis and during endoscopic surveillance, in up to 25%

of patients.24, 28 However, since HPS is a heterogeneous condition,

comprising serrated polyps of different categories i.e. HPs and SSAs

but also co-existent conventional adenomas22, 23, 28-30, it is uncertain

which polyps eventually lead to CRC in these patients and thus are

clinically relevant. In the case that the CRCs do originate from the

serrated polyps, HPS may prove to be a valuable model for studying

the serrated CRC pathway.

Within a cohort of 56 HPS patients, we identified 17 patients

with CRC. By combined histopathological and molecular analyses of

the polyps and CRCs in these patients, we obtained novel evidence for

a serrated CRC pathway in HPS which predominates over the

classical WNT pathway of carcinogenesis.


127

MATERIALS AND METHODS

Subjects

From a cohort of 56 HPS patients undergoing endoscopic

treatment/surveillance at the Academic Medical Centre in the

Netherlands, 21 HPS patients had CRC. From 17 of these patients

tissue was available (n=19) which was included in this study. HPS was

defined as at least five histologically diagnosed HPs and/or SSAs

proximal to the sigmoid colon, of which 2 greater than 10mm in

diameter, or more than 20 HPs and/or SSAs distributed throughout the

colon.28 Because both HPs and SSAs are common findings in HPS

and have been shown to be difficult to differentiate microscopically, all

serrated polyps were included in our criteria. 31-34 Patients with a

known germline APC mutation or a bi-allelic MUTYH mutation were

excluded from the study. The study was conducted in accordance with

the research code of our institutional medical ethical committee on

human experimentation, as well as in agreement with the Helsinki

Declaration of 1975, as revised in 1983.

Specimens

All retrieved CRCs (n=19) were formalin-fixed and paraffin-embedded.

H&E stained tissue sections were re-evaluated by two pathologists

(CvN, SvE). In addition, all CRCs were re-evaluated for the presence

of an adjacent serrated component and for features of a serrated

adenocarcinoma as claimed by others.10 A control group was selected

consisting of sporadic, microsatellite stable (MSS) CRCs (n=14) from

non-polyposis patients, matched for age, gender and CRC-location.

These CRCs were re-evaluated as described above.

In the case of a mutation identified in a CRC, ≥ 5 polyps in the closest

proximity of the CRC were selected and reviewed by a single

Chapte

r 6

Chapter 6

128

pathologist (CvN) who was blinded for patient characteristics and

original histological diagnosis. Polyps were classified as HP, SSA,

TSA, mixed polyp or conventional adenoma based on the histological

features on H&E staining.35-37 Polyps with a serrated morphology i.e.

HPs, SSAs, TSAs and mixed polyps were collectively designated as

‘serrated polyps’. A polyp control group was also selected consisting of

sporadic HPs (n=24), SSAs (n=18) and conventional adenomas (n=17)

from non-polyposis patients. For the purpose of analysis, lesions from

the caecum, ascending colon, transverse colon and descending colon

were regarded as proximal and those from the sigmoid colon and

rectum were regarded as distal.

Somatic mutation analysis

Epithelial cells from polyps and CRCs were microdissected and DNA

was isolated as described previously.38, 39 Using previously described

primers and assays, DNA was analysed for mutations in the APC-

mutation cluster region (APC-MCR), KRAS (exon 2), BRAF (exon 15)

and NRAS (exons 1 and 2).38, 39 In the case of a CRC in a polyp,

mutation analysis of TP53 (exons 4-10) was performed in an attempt

to assess whether these two components were clonally related. In

case of an identified genetic mutation in a CRC, mutation analysis of

surrounding polyps (≥5) was performed as described above. Detected

mutations were confirmed in a second independent experiment.

Microsatellite instability analysis

Microsatellite status of the CRCs of HPS patients was determined

using an international standard panel of 5 microsatellite markers

(D17S250, D2S123, D5S346, BAT25 and BAT26) using standard

techniques. A high degree of microsatellite instability (MSI-high) was


129

defined as two (40%) or more unstable markers, MSI-Low as one

unstable marker, and microsatellite stable (MSS) as no unstable

markers.

Immunohistochemistry

Immunohistochemistry was performed on CRCs and polyps of the

HPS patients. Unstained 5-µm sections were cut from paraffin blocks

and the slides were deparaffinized. Primary monoclonal antibodies

used were specific for MLH1(1:50 BD Pharmingen, San Diego, USA);

MSH2(1:100 Oncogene Research Prod., San Diego, USA);

MSH6(1:200 BD Transduction Lab., San Jose, USA); PMS2(1:250 BD

Transduction Lab., San Jose, USA); SMAD4(1:200 Santa Cruz, USA);

CTNNB1(1:10.000 BD Biosciences, San Diego, USA) and

TP53(1:2000 Neomarkers, Fremont, USA). Slides were immersed in

0.3% hydrogen peroxide in methanol for 20 minutes. Subsequently,

antigen retrieval was carried out by 10 minutes of boiling in 10mM

Tris/1mM EDTA (pH 9) followed by incubation with above mentioned

diluted primary antibodies during 1 hour at room temperature. Post-

antibody block (Immunologic) in PBS was performed followed by

implementation of an antipolyvalent HRP detection system

(Immunologic) to visualize antibody binding sites with 3.3’-

diaminobenzidine as a chromogen. Sections were counterstained with

haematoxylin.

Immunoreactivity for CTNNB1 (β-catenin) was regarded as

positive when strong nuclear staining was observed in >25% of the

cells. Stains for TP53 were regarded to be indicative of TP53

dysfunction or deletion when >75% of the lesional nuclei were

strongly positive or completely negative (absent staining). Stains for

Chapte

r 6

Chapter 6

130

SMAD4, MLH1, MSH2, MSH6 and PMS2 were considered negative

when there was complete absence of nuclear expression in all lesional

cells. Negative staining in a part of a lesion or in a single crypt was

registered separately.

Statistics



Inc, Chicago, Ill). Somatic mutations in CRCs and polyps of HPS

patients were compared with those of a control panel using a two-

sided Fisher exact test. A p-value of < 0.05 was considered statistically

significant.

RESULTS

Patients

The clinico-pathological features of the HPS patients are summarized

in table 1. The median age of this cohort of 17 HPS patients at CRC

diagnosis was 58 years (range: 41-75) with a male: female ratio of 8:9.

In all patients, germline APC and MUTYH-mutation analyses were

previously performed and found negative. A surgical colonic resection

was performed in 14/17 (82%) patients: 6 subtotal colectomies, 4

hemi-colectomies (2 right-sided) and 4 (recto)sigmoidal resections.

Histological evaluation of biopsies and surgical resection specimens of

these patients revealed a median of 16 HPs, 7 SSAs and 2

conventional adenomas per patient. All patients satisfied the criteria for

HPS defined by the World Health Organization (WHO).30


131

Mutation analysis in HPS polyps and control-group polyps

A total of 84 polyps, originating from 17 HPS patients with CRC, were

analysed for pathogenic mutations in the APC-mutation cluster region

(APC-MCR), KRAS (codons 12 and 13), BRAF (codon 600) and NRAS

(exons 1 and 2). The 84 HPS polyps consisted of 21 HPs, 38 SSAs, 3

TSAs, 2 mixed polyps (64 serrated polyps) and 20 conventional

adenomas. The control group of sporadic lesions (n=59) consisted of

24 HPs, 18 SSAs (42 serrated polyps) and 17 conventional adenomas

(table 2).

Molecular analysis, of both the HPS polyps and control group

polyps, showed APC-MCR mutations exclusively in the conventional

adenomas and BRAF mutations exclusively in the serrated polyps:

BRAF mutations were detected in 48/64 (75%) HPS serrated polyps,

whereas 20/42 (48%) of the control group serrated polyps harbored a

BRAF mutation (p=0.007). Also when evaluating HPS patients

individually, each patient harbored predominantly BRAF mutations in

their serrated polyps. In four patients, beside BRAF mutations a single

KRAS mutation was identified in a distal serrated polyp. No significant

difference in frequency of KRAS mutations in serrated polyps was

seen between groups (6% vs 17%; p=0.1), but in conventional

adenomas of HPS patients no KRAS mutations were detected,

compared to 4/17 (24%) conventional adenomas in the control group

(p=0.029). In none of the polyps KRAS and BRAF mutations were

found together.

Chapte

r 6

Chapter 6

132

Table 1. Clinico-pathological features of HPS patients with colorectal cancer G= gender, Location=location of carcinoma, AC=ascending colon, TC= transverse colon DC=descending colon

When location in the colon was analyzed, no association was

seen between the presence of a BRAF mutation and polyp location in

both HPS and in the control group (table 3). BRAF mutations were

observed in 36/46 (82%) proximal HPS serrated polyps and in 12/18

(67%) distal HPS serrated polyps. In HPS, KRAS mutated serrated

Case no. Age G CRC Location CRC Size (TNM) Adjacent polyps

1 59 F Caecum 56 mm (T3N0M0) -

2 41 F Rectosigmoid 25 mm (T2N0M0) -

3 75 M AC 50 mm (T3N0M0) -

4 58 M Rectosigmoid 4 mm (TisN0M0) HP

5 59 M AC < 20 mm (T1N0M0) SSA

6 68 F AC 95mm (T3N0M0) -

7 43 F DC 40mm (T4N1M0) -

8 49 F DC Not stated(TisN0M0) HP

9 54 M DC 50mm (T3N0M0) -

9 Rectosigmoid 20mm (T1N0M0) -

10 54 F AC 16mm (T1N0M0) SSA

11 56 F Rectosigmoid 65mm (T2N0M0) Adenoma

12 61 F Rectosigmoid

20mm (T1N0M0) -

13 52 M Rectosigmoid

40mm (T3N1M0) -

14 66 M AC 8 mm (T1N0M0) SSA

15 63 M AC 6 mm (T1N0M0) SSA

16 69 M TC 120mm (T3N1M1) -

17 68 F AC 12mm (TisNoM0) -

17 68 F Rectosigmoid 29mm (T2N0M0) -


133

polyps (4/15: 27%) were exclusively detected in the distal colon

(p=0.005).

Immunohistochemistry in polyps

In none of the 84 HPS polyps, loss of expression of the mismatch

repair genes (MLH1, MSH2, MSH6 and PMS2) or SMAD4 was

observed (not shown). In addition, none of these polyps showed

abnormal TP53 staining (either strong nuclear TP53 staining or

complete TP53 loss, not shown). In 9 conventional HPS adenomas,

we detected strong nuclear CTNNB1 staining (i.e. in >25% of lesional

cells, not shown). In 5 of these adenomas, an APC-MCR mutation was

identified. In four other APC-MCR mutated adenomas no abnormal

nuclear β-catenin staining was detected. Nuclear CTNNB1 was not

found in any of the serrated polyps of either group.

Table 2. Detected APC, KRAS and BRAF mutations in serrated polyps (HPs, SSAs, TSAs and mixed polyps) and adenomas (AD) compared to a control panel. *Statistically significant p-value for BRAF mutation frequency in serrated polyps of HPS patients compared to serrated polyps in the control group **Statistically significant p-value KRAS mutation frequency in adenomas of HPS patients compared to adenomas in the control group.

Somatic mutation Patients with HPS Control group P-value

Serrated polyps

(n=64) AD

(n=20) Serrated polyps

(n=42) AD

(n=17)

APC mutation 0 9

(45%) 0 7 (41%) Ns

BRAF mutation 48 (75%) 0 20 (48%) 0 0.007*

KRAS mutation 4 (6%) 0 7 (17%) 4 (24%) 0.029**

Chapte

r 6

Chapter 6

134

Histological characteristics of the colorectal carcinomas

Upon histological re-evaluation of CRCs, 6/19 (32%) HPS CRCs were

identified within/directly adjacent to a serrated polyp. In 1/19 (5%)

cases, the HPS CRC was identified within a conventional adenoma

(Fig.1). One patient had two synchronous CRCs. Although 6 CRCs

were identified within serrated polyps, no HPS CRCs displayed a

distinguishing morphology justifying the diagnosis serrated

adenocarcinoma.10

Mutation: BRAF mutation KRAS mutation

Group: HPS Control group HPS Control group

Location: Proximal Distal Proximal Distal Proximal Distal Proximal Distal

HP 8/13 (62) 5/8 (63) 1/2 (50) 9/22 (41) 0/13 (0) 3/8 (38) 0/2 (0) 5/22 (23)

SSA 25/30 (83) 5/8 (63) 10/18 (56) 0/1 (0) 0/30 (0) 1/8 (13) 2/17 (12) 0/1 (0)

TSA 1/1 (100) 2/2 (100) 0/0 0/0 0/1 (0) 0/2 (0) 0/0 0/0

MP 2/2 (100) 0/0 0/0 0/0 0/2 0/0 0/0 0/0

Table 3. Serrated polyp location and BRAF and KRAS mutation. MP=mixed polyp NOTE. All values are expressed as n (%).


135

Figure 1. Two colorectal carcinomas, one located within a serrated polyp (A and B, magnification 50x) and one within a villous adenoma (C, magnification 50x) of two HPS patients.

Mutation analysis in HPS CRCs and control-group CRCs

BRAF mutations were detected in 10/19 (53%) HPS CRCs, whereas

no BRAF mutations were detected in the CRCs of the control group

(p=0.001, table 4 and figure 2). All BRAF mutations involved the same

thymine to adenine transversion at nucleotide 1796, resulting in a

valine to glutamine substitution at codon 600 (V600E). We found no

Chapte

r 6

Chapter 6

136

NRAS mutations in the CRCs of either group. Of the 6 CRCs identified

within a serrated polyp, 5/6 (83%) carried the same BRAF mutation

(GTG → GAG) in both the polypous and tumor components (Table 5).

However, considering that these are hotspot mutations, these findings

do not prove a clonal relationship between these lesions. TP53 (exons

4-10) sequence analysis, performed on these 6 CRCs in order to

further explore the clonal relationship between the carcinomas and

precursor lesions, yielded no mutations.

Mutation

HPS carcinomas (n=19)

Carcinomas control group (n=14)

P-value

APC 2 (11%) 4 (29%) Ns

KRAS 1 (5%) 5 (36%) Ns

BRAF 10 (53%) 0 0.001*

NRAS 0 0 Ns

MSS 12 (63%) 14 NA

MSI-L 1 (5%) 0 NA

MSI-H 6 (32%) 0 NA

Table 4. Mutation spectrum of colorectal cancers in HPS patients compared to control-group CRCs. ns: not significant; NA: not applicable. *statistically significant p-value compared to control group CRC


137

Figure 2. Mutation profiles of HPS polyps/CRCs compared to sporadic control group polyps/CRCs. SPs = serrated polyps, Ads = conventional adenomas, CRCs = colorectal cancers

APC-MCR mutations were identified in 2/19 (11%) HPS CRCs

compared to 4/14 (29%) in the control group CRCs (not significant)

and 1/19 (5%) KRAS mutations were detected in HPS CRCs

compared to 5 (36%) in the control group (not significant). In one HPS

CRC (patient 6), both a BRAF mutation and an APC-MCR mutation

were detected. Interestingly, one of the HPS CRCs (patient 11),

harbouring an APC-MCR mutation (insertion 1364), was the single

HPS CRC found within a villous adenoma (Fig.1C). Subsequent APC-

MCR sequence analysis of the adenoma revealed the same mutation

(insertion 1364), establishing a clonal relationship between these

Chapte

r 6

Chapter 6

138

lesions. Accordingly, both the adenomatous and malignant

components displayed evident nuclear CTNNB1 staining (Fig.3).

When location in the colon was analysed, all BRAF-mutated

HPS CRCs were exclusively detected in the proximal colon (p=0.007).

No association was seen between location and KRAS mutations in the

control group or APC-mutations in both groups. The entire list of

molecular analyses on a per-carcinoma basis can be found as a

supplementary table online (http://ajp.amjpathol.org).

Table 5. Mutations in CRCs and adjacent polyps.

Carcinoma

Adjacent polyp

Mutation in CRC

Mutation in adjacent polyp

4 HP None None

5 SSA BRAF (GTG→GAG)

BRAF (GTG→GAG)

8 HP BRAF (GTG→GAG) BRAF (GTG→GAG)

10 SSA BRAF (GTG→GAG) BRAF (GTG→GAG)

11 Adenoma APC (insertion G 1354) APC (insertion G 1354)




139

Figure 3. Nuclear CTNNB1 expression in the adenocarcinoma and the adjacent adenoma of patient 11, both harbouring the same APC gene mutation (magnification 200x).

Genetic events detected immunohistochemically and by

microsatellite analysis in CRCs

In 6/19 (32%) HPS CRCs, loss of expression was seen of at least 1

mismatch repair (MMR) protein. In 6 CRCs, there was complete loss of

expression of both MLH1 and PMS2. All these 6 HPS CRCs were

microsatellite-unstable (MSI-high), were proximally located and

harboured a BRAF mutation. Two of the 6 MSI-high CRCs involved

combined serrated polyp-CRC lesions. In these lesions, the serrated

polyp components were microsatellite stable (MSS).

Strong nuclear β-catenin staining (>25%) was observed in 5/19

(26%) HPS CRCs. In one of these CRCs, an APC-MCR mutation was

identified and none of these was BRAF mutated. In all 5 of these HPS

CRCs, abnormal TP53 staining was observed (>75% nuclear staining

or complete absence), indicative of loss of functional TP53. Three

additional HPS CRCs displayed a perturbed TP53 status. In 2/19

(11%) CRCs loss of SMAD4, either focal or complete, was detected.

Chapte

r 6

Chapter 6

140

DISCUSSION

In this first comprehensive cohort study of HPS patients with

both CRCs and polyps, we demonstrated that the serrated polyps and

conventional adenomas of HPS patients not only morphologically

resemble their respective sporadic counterparts but also have similar

molecular profiles (table 3, figure 2). Interestingly, we identified a high

number of combined serrated polyp-CRC lesions which showed

identical BRAF mutations in both components, supporting the

existence of a serrated CRC pathway. Overall, we demonstrated that

both microsatellite-stable and –unstable CRCs in HPS predominantly

originate from the serrated polyps thus confirming that HPS patients

provide a valuable model to analyze the molecular characteristics of

the serrated CRC pathway.

In accordance with previous reports 13, 40, 41, the HPS serrated

polyps harboured significantly more BRAF mutations than those of the

control group (80 vs. 48%: p=0.001). The slight difference in the KRAS

mutation frequencies between both groups (7 vs. 17%) was not

statistically significant. Thus in HPS patients, BRAF mutations

correlate even stronger with serrated polyps than in non-HPS patients.

This contrasts with serrated polyps in MAP patients with a germline

MUTYH gene mutation, which contain significantly more KRAS

mutations (70%) than BRAF mutations (4%).21 Both BRAF and KRAS

have been identified as early or instigating events in the serrated

pathway. It has to be determined however, whether serrated lesions

with these mutually exclusive mutations are biologically equivalent and

have the same risk of developing to CRC.

The mutation profiles of the HPS CRCs were clearly more

similar to those found in the serrated polyps than in the conventional

adenomas (table 3 and figure 2). The profiles of the control group MSS


141

CRCs, as expected, were comparable to the profiles of the

conventional adenomas. As many as 10 of the included 19 HPS CRCs

were BRAF-mutated compared to none of the 14 control group CRCs

(p=0.001). Interestingly, all these BRAF-mutated CRCs were

proximally located. Previous molecular studies in HPS CRCs

encompassed small non-controlled series of selected cases.23, 42-44 and

a comparison between our results and these studies is not

straightforward owing to variation in patient selection criteria and

analysis of different or single genes. Overall however, BRAF mutations

were identified in 6/15 (40%) HPS-related CRC-cases described in the

assembled literature until now. We identified no BRAF mutations in the

control group of age-and sex-matched, MSS CRCs from non-polyposis

patients. Concordantly, BRAF mutations have been reported in only 5-

10% of sporadic MSS CRCs of non-polyposis patients (n>100).45-48

Considering that in the literature serrated polyps are suggested to be

precursor lesions of particularly MSI CRCs, we chose MSS

carcinomas in our control group in order to exclude MSI-CRCs from

potentially unrecognized HPS patients. However, this strategy is

arbitrary and eliminates sporadic MSI CRCs from non-HPS patients.

Hence, our statistically significant difference in BRAF mutation

frequency between HPS CRCs and control group CRCs is higher

compared to an unselected cohort of CRCs. We detected only one

KRAS mutation in a distally located HPS CRC, which corroborates two

previously published studies together analyzing a total of 15 HPS

CRCs.40, 41 To our knowledge, only one other study has described the

presence of a KRAS mutation in a single distally located HPS CRC,

supporting the notion that KRAS plays a minor role in the

carcinogenesis of HPS and is confined to the distal colon.23

Chapte

r 6

Chapter 6

142

In our study, 2 patients had synchronous CRCs. Theoretically,

a field effect may account for the development of synchronous

polyps/CRCs.49 In one patient (patient 9), no mutations were identified

in both CRCs. The other patient (patient 17) harboured one KRAS

mutation and one BRAF mutation respectively in each of the CRCs.

Hence, although a field effect associated with a serrated CRC pathway

may cause simultaneous CRCs with identical mutations, we were not

able to demonstrate this in patient 9 due to the lack of any mutations

and seems excluded in patient 17. Of note, clonal markers are

necessary to be able to demonstrate a true field effect. Such clonal

markers associated with the serrated pathway are currently not

available.

The histological characteristics of the HPS CRCs as a group,

either or not BRAF-mutated, were inconspicuous and not obviously

different from the control group CRCs. In particular, a serrated growth

pattern, as has been reported, 50, 51 was not apparent in our series of

HPS CRCs. We found a remarkable number of HPS CRCs (6/19:

32%) to be located within or directly adjacent to a serrated polyp (Fig.

1). In 5/6 of these combined lesions an identical hotspot mutation at

codon 600 (GTG→GAG) of BRAF was detected in both components.

These combined histological and molecular data are strongly

suggestive for a sequential relationship between serrated polyps and

CRC. A clonal relationship between the carcinomas and their assumed

precursor lesions could not be further substantiated due to lack of

appropriate clonal markers.

In the literature it has been shown that 90% of sporadic MSI-H

CRCs are caused by loss of MLH1 function as a result of methylation

of this mismatch repair gene. In these lesions, BRAF mutations, which

are associated with serrated polyps, are also a common finding


143

suggesting that the serrated pathway can generate MSI-H CRCs.13, 15

Interestingly, 6/19 HPS CRCs were MSI-H due to loss of MLH1 and

PMS2 and all 6 carried BRAF mutations. Additional analysis in these

CRCs also showed hypermethylation of MLH1 in all cases (data not

shown). These findings suggest a causal relationship between this

novel carcinogenic route and microsatellite-unstable CRCs. On the

other hand, our findings demonstrate that the serrated pathway, at

least in HPS, also generates MSS-CRCs.

Previous large cohort studies (>30 patients) have reported the

presence of at least one conventional adenoma in 69- 85% of HPS

patients and >5 conventional adenomas in 21-32% of cases.22, 23, 28-30

Concordantly, in our study APC-MCR mutations were detected in two

(13%) HPS CRCs. The identification of an HPS CRC (patient 11)

located within a conventional adenoma of the same clonal origin,

reflected by the identical APC-MCR mutation in both components, is

significant since this proves that the classical adenoma-carcinoma

pathway52 is also operational in HPS patients.

The apparent dominance of the serrated over the non-serrated

CRC pathway in HPS may either be due to a greater intrinsic risk of

tumour progression of the serrated polyps, or be simply a reflection of

the numerical prevalence of serrated polyps over adenomas. To our

knowledge, the only study that has addressed this issue in 2 HPS CRC

cases, reported no APC mutations.23 In that study however, patients

with >10 conventional adenomas were excluded. In our study, all

cases satisfied the WHO criteria for HPS in which the presence or

number of conventional adenomas is not an issue. Interestingly, in

HPS patient 11 with the classical CRC, a relative abundance of

conventional adenomas was observed (12 adenomas versus 17

serrated polyps), suggesting a stochastic rather than an intrinsically

Chapte

r 6

Chapter 6

144

biased process of carcinogenesis. Interestingly, in the other APC-

MCR-mutated HPS CRC (patient 6) also a BRAF mutation was

identified. Assuming that this CRC is of monoclonal origin, it may be

the outcome of an elusive ‘fusion’ pathway which combines

mechanisms associated with both conventional adenomas and

serrated polyps, as proposed previously.36

We observed 4 HPS CRCs with nuclear CTNNB1 staining,

associated with the WNT-pathway. Although in these CRCs, no APC-

MCR mutations or CTNNB1 exon 4-10 hotspot mutations were

identified, alternative mechanisms may be operational to activate the

WNT -pathway, e.g. methylation of the APC promotor regions and

MSI-related frameshift mutations in WNT pathway regulators like

AXIN2.53 In these HPS patients, a median of only 1 adenoma (range:

0-5) was identified, suggesting that the WNT-pathway indeed may be

involved at some stage of carcinogenesis via the serrated pathway.

Considering that no BRAF mutations or directly adjacent serrated

polyps were found, it seems unlikely that these CRCs are the outcome

of a proposed fusion pathway, but formally this can not be excluded.

Based on these observations, we conclude that distinct, APC-

and non-APC mediated CRC pathways are functional in HPS. A

serrated pathway, skewed towards initial BRAF mutations and

proximal localization, however seems to predominate, most likely due

to the numerical prevalence of serrated polyps in these patients. From

this it is inferred that all polyp types in HPS, should be considered

clinically relevant and be removed.


145

REFERENCES 1. Jemal A, Thun MJ, Ries LA, Howe HL, Weir HK, Center MM, Ward E,


2. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61:759-767.


4. Hawkins NJ, Ward RL. Sporadic colorectal cancers with microsatellite instability and their possible origin in hyperplastic polyps and serrated adenomas. J Natl Cancer Inst 2001;93:1307-1313.


6. Jass JR, Biden KG, Cummings MC, Simms LA, Walsh M, Schoch E, Meltzer SJ, Wright C, Searle J, Young J, Leggett BA. Characterisation of a subtype of colorectal cancer combining features of the suppressor and mild mutator pathways. J Clin Pathol 1999;52:455-460.


8. Jass JR, Young J, Leggett BA. Hyperplastic polyps and DNA microsatellite unstable cancers of the colorectum. Histopathology 2000;37:295-301.

9. Jass JR. Serrated route to colorectal cancer: back street or super highway? J Pathol 2001;193:283-285.

Chapte

r 6

Chapter 6

146

10. Makinen MJ, George SM, Jernvall P, Makela J, Vihko P, Karttunen TJ. Colorectal carcinoma associated with serrated adenoma--prevalence, histological features, and prognosis. J Pathol 2001;193:286-294.

11. Yao T, Nishiyama K, Oya M, Kouzuki T, Kajiwara M, Tsuneyoshi M. Multiple 'serrated adenocarcinomas' of the colon with a cell lineage common to metaplastic polyp and serrated adenoma. Case report of a new subtype of colonic adenocarcinoma with gastric differentiation. J Pathol 2000;190:444-449.

12. Jass JR. Serrated adenoma of the colorectum and the DNA-methylator phenotype. Nat Clin Pract Oncol 2005;2:398-405.


14. McGivern A, Wynter CV, Whitehall VL, Kambara T, Spring KJ, Walsh MD, Barker MA, Arnold S, Simms LA, Leggett BA, Young J, Jass JR. Promoter hypermethylation frequency and BRAF mutations distinguish hereditary non-polyposis colon cancer from sporadic MSI-H colon cancer. Fam Cancer 2004;3:101-107.

15. Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B, Velculescu VE. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature 2002;418:934.

16. Young J, Simms LA, Biden KG, Wynter C, Whitehall V, Karamatic R, George J, Goldblatt J, Walpole I, Robin SA, Borten MM, Stitz R, Searle J, McKeone D, Fraser L, Purdie DR, Podger K, Price R, Buttenshaw R, Walsh MD, Barker M, Leggett BA, Jass JR. Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings: parallel pathways of tumorigenesis. Am J Pathol 2001;159:2107-2116.

17. Azimuddin K, Stasik JJ, Khubchandani IT, Rosen L, Riether RD, Scarlatto M. Hyperplastic polyps: "more than meets the eye"? Report of sixteen cases. Dis Colon Rectum 2000;43:1309-1313.

18. Warner AS, Glick ME, Fogt F. Multiple large hyperplastic polyps of the colon coincident with adenocarcinoma. Am J Gastroenterol 1994;89:123-125.


147

19. Urbanski SJ, Kossakowska AE, Marcon N, Bruce WR. Mixed hyperplastic adenomatous polyps--an underdiagnosed entity. Report of a case of adenocarcinoma arising within a mixed hyperplastic adenomatous polyp. Am J Surg Pathol 1984;8:551-556.

20. Goldstein NS, Bhanot P, Odish E, Hunter S. Hyperplastic-like colon polyps that preceded microsatellite-unstable adenocarcinomas. Am J Clin Pathol 2003;119:778-796.

21. Boparai KS, Dekker E, van ES, Polak MM, Bartelsman JF, Mathus-Vliegen EM, Keller JJ, van Noesel CJ. Hyperplastic polyps and sessile serrated adenomas as a phenotypic expression of MYH-associated polyposis. Gastroenterology 2008;135:2014-2018.







28. Boparai KS, Mathus-Vliegen EM, Koornstra JJ, Nagengast FM, van LM, van Noesel CJ, Houben M, Cats A, van Hest LP, Fockens P, Dekker E. Increased colorectal cancer risk during follow-up in patients

Chapte

r 6

Chapter 6

148

with hyperplastic polyposis syndrome: a multicentre cohort study. Gut 2009.

29. Buchanan DD, Sweet K, Drini M, Jenkins MA, Win AK, Gattas M, Walsh MD, Clendenning M, McKeone D, Walters R, Roberts A, Young A, Hampel H, Hopper JL, Goldblatt J, George J, Suthers GK, Phillips K, Young GP, Chow E, Parry S, Woodall S, Tucker K, Muir A, Field M, Greening S, Gallinger S, Green J, Woods MO, Spaetgens R, de la Chapelle A, Macrae F, Walker NI, Jass JR, Young JP. Phenotypic diversity in patients with multiple serrated polyps: a genetics clinic study. Int J Colorectal Dis 2010;25:703-712.










149

38. de Leng WW, Keller JJ, Luiten S, Musler AR, Jansen M, Baas AF, de Rooij FW, Gille JJ, Menko FH, Offerhaus GJ, Weterman MA. STRAD in Peutz-Jeghers syndrome and sporadic cancers. J Clin Pathol 2005;58:1091-1095.

39. de Leng WW, Westerman AM, Weterman MA, de Rooij FW, Dekken HH, De Goeij AF, Gruber SB, Wilson JH, Offerhaus GJ, Giardiello FM, Keller JJ. Cyclooxygenase 2 expression and molecular alterations in Peutz-Jeghers hamartomas and carcinomas. Clin Cancer Res 2003;9:3065-3072.


41. Chan AO, Issa JP, Morris JS, Hamilton SR, Rashid A. Concordant CpG island methylation in hyperplastic polyposis. Am J Pathol 2002;160:529-536.




45. Samowitz WS, Sweeney C, Herrick J, Albertsen H, Levin TR, Murtaugh MA, Wolff RK, Slattery ML. Poor survival associated with the BRAF V600E mutation in microsatellite-stable colon cancers. Cancer Res 2005;65:6063-6069.

46. Barault L, Charon-Barra C, Jooste V, de la Vega MF, Martin L, Roignot P, Rat P, Bouvier AM, Laurent-Puig P, Faivre J, Chapusot C, Piard F. Hypermethylator phenotype in sporadic colon cancer: study on a population-based series of 582 cases. Cancer Res 2008;68:8541-8546.

Chapte

r 6

Chapter 6

150

47. Ogino S, Nosho K, Kirkner GJ, Kawasaki T, Meyerhardt JA, Loda M, Giovannucci EL, Fuchs CS. CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer. Gut 2009;58:90-96.

48. de VS, Weijenberg MP, Herman JG, Wouters KA, de Goeij AF, van den Brandt PA, de Bruine AP, van EM. MGMT and MLH1 promoter methylation versus APC, KRAS and BRAF gene mutations in colorectal cancer: indications for distinct pathways and sequence of events. Ann Oncol 2009;20:1216-1222.

49. Nosho K, Kure S, Irahara N, Shima K, Baba Y, Spiegelman D, Meyerhardt JA, Giovannucci EL, Fuchs CS, Ogino S. A prospective cohort study shows unique epigenetic, genetic, and prognostic features of synchronous colorectal cancers. Gastroenterology 2009;137:1609-1620.

50. Makinen JM, Makinen MJ, Karttunen TJ. Serrated adenocarcinoma of the rectum associated with perianal Paget's disease: a case report. Histopathology 2002;41:177-179.

51. Makinen MJ. Colorectal serrated adenocarcinoma. Histopathology 2007;50:131-150.


53. Thorstensen L, Lind GE, Lovig T, Diep CB, Meling GI, Rognum TO, Lothe RA. Genetic and epigenetic changes of components affecting the WNT pathway in colorectal carcinomas stratified by microsatellite instability. Neoplasia 2005;7:99-108.

NBI increases polyp detection in HPS

151

Endoscopic imaging – Detection and differentiation of polyps

P A

R T

Chapter 7

152


153

Increased polyp detection using narrow-band imaging compared to high-resolution endoscopy in patients with hyperplastic polyposis syndrome

Karam S. Boparai

Frank J.C. van den Broek

Susanne van Eeden

Paul Fockens

Evelien Dekker

Endoscopy. 2011 Aug;43(8):676-82

Ch

ap

ter

Chapter 7

154

SUMMARY

Background and study aims: Hyperplastic polyposis syndrome

(HPS) is associated with colorectal cancer and is characterized by

multiple hyperplastic polyps (HPs), sessile serrated adenomas (SSAs)

and adenomas. Narrow-band imaging (NBI) may improve the detection

of polyps in HPS. We aimed to compare polyp miss-rates of NBI

compared to high-resolution endoscopy (HRE).

Patients and Methods: This was a single center, randomized cross-

over study in which consecutive HPS patients underwent tandem

colonoscopy with HRE and NBI, in randomized order with removal of

all detected polyps.

Results: In 22 patients with HPS, 209 polyps were detected: 27

normal histology, 116 HPs, 42 SSAs and 24 adenomas. Within

patients assigned to HRE first (n=11) a total of 78 polyps was

detected; subsequent NBI added 44 polyps. In patients examined with

NBI first, 78 polyps were detected and subsequent HRE added 9.

Polyp miss-rates of HRE and NBI were 36% and 10% (OR 0.21; 0.09-

0.45). Flat polyp shape was independently associated with increased

miss-rate.

Conclusion: NBI significantly reduces polyp miss-rates in HPS

patients. We recommend using either NBI or chromoendoscopy for

colonoscopic surveillance of HPS patients with removal of all detected

polyps.


155

INTRODUCTION

Hyperplastic polyposis syndrome (HPS) is characterized by the

presence of multiple hyperplastic polyps (HPs) spread throughout the

colon and is associated with an increased colorectal cancer (CRC)

risk.[1-4] Besides HPs, sessile serrated adenomas (SSAs) and

conventional adenomas are common findings in this condition as well.

The presence of SSAs is even considered typical for HPS.[5-7]

Whereas sporadic HPs are traditionally considered to be low-

risk lesions, a novel serrated neoplasia pathway has been suggested

which describes the progression of serrated polyps (i.e. HPs, SSAs

and traditional serrated adenomas) to CRC through accumulation of

genetic mutations.[8-15] Molecular research strongly suggest that

serrated polyps are lesions which may lead to CRC with BRAF and

CPG-island methylator phenotype (CIMP).[16-20] In addition,

clinicohistological reports supporting a serrated neoplasia pathway

include CRCs in close vicinity of large hyperplastic polyps [21,22] and

CRCs identified in serrated polyps [23-25]. Therefore, serrated polyps

seem to represent direct premalignant lesions. SSAs have even been

recommended to be managed as conventional adenomas.[26]

Serrated polyps in HPS however, have been shown to have even

higher numbers of BRAF mutations and CIMP compared to sporadic

serrated polyps.[20,27] Moreover, HPS patients have far more

serrated polyps than people in the general population. This may (in

part) explain the increased risk of CRC in HPS patients. Numerous

HPS patients with CRC arising in a serrated polyp have been

reported.[23] Thus, detection and removal of serrated polyps seems

necessary to prevent CRC in patients with HPS.

Chapte

r 7

Chapter 7

156

In HPS patients however, SSAs and HPs are generally small

and flat.[28-30] These features are associated with polyp miss-rates of

up to 26% using standard colonoscopy.[31-33] Improved detection of

these polyps by using advanced endoscopic techniques seems

therefore desirable. Chromoendoscopy has previously been shown to

improve the detection of small and flat lesions, specifically HPs, in

patients undergoing surveillance colonoscopy.[32-36] However,

chromoendoscopy is a labour-intensive and time-consuming

technique. Narrow-band imaging (NBI) is an easier push-on-a-button

technique that enhances mucosal and vascular detail without the use

of dyes, and has proven to be superior to high-resolution endoscopy

(HRE) for the detection of sporadic HPs.[37,38] The aim of this

randomized trial was to compare NBI and HRE for the detection of

polyps in patients with HPS.


Patients

Between October 2007 and October 2008, consecutive HPS

patients were recruited for this study at the Academic Medical Center

in Amsterdam. A diagnosis of HPS is based on the following criteria: 1)

≥20 HPs found during previous colonoscopies; 2) ≥5 HPs proximal to

the sigmoid colon of which 2 were larger than 1cm; or 3) any HP

occurring proximal to sigmoid colon in an individual who has a first-

degree relative with HPS.[1,4] However, owing to the common

presence of both HPs and SSAs in HPS and the difficult histological

differentiation between these two groups, both HPs and SSAs were

used to fulfil the criteria. [26,39-41] Patients were excluded in case of

inflammatory bowel disease, severe coagulopathy, <18 years of age,


157

insufficient bowel preparation (<90% of colonic mucosa visible) and a

known germline APC mutation or bi-allelic MYH mutation. From

included patients informed consent was obtained and the study was

approved by our institutional review board.

Endoscopic equipment

For this study the Evis Lucera system (CV-260, Olympus Inc.,

Tokyo, Japan) and a high-resolution video colonoscope (CF-H260Z)

were used integrating HRE, NBI and optical magnification (100×). The

endoscopist could easily switch between the imaging modes by

pressing a button on the shaft of the endoscope. As only high-

resolution monitors were used, the high-definition signal of the system

was not utilized.

Study design and randomization

We used a cross-over study design with randomized order.

Consecutive patients underwent tandem colonoscopy with HRE and

NBI by the same endoscopist and the order of these techniques was

randomized (figure 1). After informed consent, block randomization

was performed by a single investigator by opening sealed opaque

envelopes (containing notes with ‘HRE’ or ‘NBI’ in a 1:1 ratio) once the

cecum was reached.

Chapte

r 7

Chapter 7

158

Figure 1. Flow diagram

Colonoscopic procedure

Patients were prepared with 4 liters polyethylene glycol solution

(Kleanprep, Norgine Inc., Amsterdam, Netherlands) and underwent

colonoscopy under conscious sedation with midazolam and fentanyl.

All procedures were performed by the same endoscopist (ED) who

was highly trained in NBI (>500 NBI colonoscopies).

The colonoscope was advanced to the cecum in the HRE mode

of the endoscope. No attention was paid to polyps during the insertion

phase. Cecal intubation was confirmed by identification of the

appendiceal orifice and ileocecal valve. After extensive rinsing and

suctioning of remaining stools, the level of bowel preparation was

determined as excellent (100% of colonic mucosa visible), good (90-


159

99%) or poor (<90%). Patients with poor bowel preparation were

excluded from this study.

Hereafter, each colonic segment (ascending, transverse,

descending and recto sigmoid colon) was examined twice, once with

HRE and once with NBI. The order of the two techniques was

determined by randomization. During withdrawal with the first

technique, each segment of the colon was meticulously inspected for

the presence of polyps. Of all detected polyps the size (estimated by

an opened biopsy forceps), location (colonic segment and distance

from the anus) and Paris classification were noted.[42] Hereafter, each

polyp was immediately removed by endoscopic mucosal resection or

biopsy removal (if <5mm) and sent for pathology in separate jars.

After first inspection and polyp clearance of each colonic

segment, the colonoscope was advanced again to the beginning of the

segment. The other imaging technique was then used for the second

inspection of the same segment. In case of indistinctive hepatic or

splenic flexures, a random biopsy was taken for reference of each

colonic segment. If additional polyps were detected during the second

examination, their size, location and Paris classification were noted

before removal.

Withdrawal times during the HRE and NBI examinations were

measured by using a stopwatch. Time for performing polypectomy was

not included in these withdrawal times. A maximum colonoscopy time

of 2 hours was set for the entire endoscopic procedure; otherwise the

procedure was too long and inconvenient for the patient. It was not

possible to blind the endoscopist to the imaging intervention, and,

logistically, it was not possible to have a different endoscopist perform

the second examination.

Chapte

r 7

Chapter 7

160

Histopathology

Resection specimens were evaluated by an expert GI

pathologist (SvE) who was blinded for endoscopic technique. Lesions

were classified as normal mucosa, HP, SSA, traditional serrated

adenoma, mixed polyp or conventional adenoma based on the

morphological features on H&E staining.[19,26,43] As previously

described by Torlakovic et al, SSAs were defined by architectural

distortion with irregular dilated crypts, including dilatation of the base of

the crypts that often have a boot, L or inverted T shape, serration

including at the base of the crypts and abnormal proliferation and

maturation with mature goblet or foveolar cells at the base of the

crypts.[44]

Outcome measures

Primary outcome measure was the polyp miss-rate of each technique,

defined as the number of polyps detected during the second inspection

divided by the total number of polyps detected during both

examinations. Additional exploratory sub-analyses were performed

regarding polyp location, -size and -shape.

Statistical analysis and sample size

Polyp miss-rates of NBI and HRE were compared by Chi-square

testing. Logistic regression analysis was used to evaluate associations

between polyp characteristics and polyp miss-rate (i.e. dependent

variable), using odds ratios (OR) plus 95%-conficence interval to

represent the strength of the association. Histopathology of each polyp

served as reference standard. The STARD statements were used for

reporting diagnostic test accuracy.[45] In addition the CONSORT

statements were used for reporting this randomized controlled trial.[46]


161

Previous research comparing NBI and HRE for adenoma

detection showed a 3.3-fold increase in detection of HPs with NBI.[47]

As the general polyp miss-rate is 22%, we hypothesized a 3.3-fold

decrease in miss-rate with NBI resulting in a polyp miss-rate of

6.7%.[31] To detect this difference in polyp miss-rate with a power of

80% and significance level of 5%, a total of 188 polyps were required.

In a previous analysis of HPS patients undergoing surveillance

endoscopies at our department we found a mean number of 9 polyps

per HPS patient, resulting in (188/9=) 22 patients for inclusion in this

trial.[48]

RESULTS

A total of 22 patients with HPS were randomized to tandem

colonoscopy with either HRE first (n=11) or NBI first (n=11). Patient

characteristics are demonstrated in table 1 and were comparable

between the randomization groups.

Chapte

r 7

Chapter 7

162

Demographics Randomization

HRE (n=11) NBI (n=11) P

Mean age, yrs (range) 58 (33-73) 62 (43-76) .331

Male 8 (73%) 4 (36%) .198

Personal history of high-grade neoplasia or CRC 6 (55%) 4 (36%) .670

Partial colectomy 5 (45%) 3 (27%) .659

Number of previous polyps (mean number per patient)

Hyperplastic polyps 18.1 11.3 .121

Sessile serrated adenomas 4.4 7.6 .183

Adenomas 2.9 4.2 .508

Bowel preparation

Excellent 10 (91%) 6 (55%) .149

Good 1 (9%) 5 (45%)

Examination time (minutes), mean (±SD)

First inspection 15.0 (3.7) 13.9 (3.8) .485

Second inspection 11.2 (3.6) 8.9 (2.3) .115

Table 1: demographics of patients randomized to HRE and NBI as first inspection

technique

Polyp miss-rates

High-resolution endoscopy: During HRE as first examination

technique, a total number of 78 polyps (mean size 6.0mm; range 2-15)

were detected. Histology demonstrated normal tissue in 8 polyps, HP

in 58, SSA in 5 and conventional adenoma in 7. Subsequent

inspection with NBI added 44 polyps (mean 6.1mm; 2-20) of which 4

with normal histology, 29 HPs, 8 SSAs and 3 conventional adenomas.

The corresponding overall polyp miss-rate of HRE hence was 36%

(95%-CI: 28-45).


163

Narrow-band imaging: During NBI as first examination

technique, a total number of 78 polyps (mean size 5.4mm; range 2-20)

were found (13 normal, 26 HP, 25 SSA, 14 adenomas). Subsequent

inspection with HRE added 9 polyps (mean 4.7mm; 2-10) of which 2

had normal histology, 3 were HP and 4 SSA. The corresponding

overall polyp miss-rate of NBI was 10% (95%-CI: 5.5-19).

The overall polyp miss-rate for NBI was significantly lower than

for HRE (OR 0.21; 95%-CI: 0.094-0.45; p<0.001). Table 2

demonstrates the polyp miss-rates for HPs, SSAs and adenomas

separately. Table 3 shows the polyp miss-rates for flat (Paris 0-IIa, 0-

IIb, 0-IIa+c) and protruded (Paris 0-Is, 0-Ip) lesions and for proximal

and distal colonic locations of polyps.

On multivariable logistic regression analysis, the use of NBI

was independently associated with a reduction in polyp miss-rate (OR

0.17; 95%-CI: 0.08-0.39), whereas flat macroscopic appearance was

associated with an increased miss-rate (OR 3.73; 1.72-8.05). Polyp

size, colonic location and histology were not associated with the miss-

rate.

Chapte

r 7

Chapter 7

164

HRE NBI P

Overall polyps

First inspection, n 78 78 .741

Second inspection, n 44 9 -

Miss-rate, % (95%-CI) 36 (28-45) 10 (5.5-19) <.001

Hyperplastic polyps



Miss-rate, % (95%-CI) 33 (24-44) 10 (3.6-26) .017

Sessile serrated adenomas



Miss-rate, % (95%-CI) 62 (36-82) 14 (5.5-31) .003

Adenomas



Miss-rate, % (95%-CI) 30 (11-60) 0 (0-22) .059

Table 2: polyp detection during the first and second inspection and polyp miss-rates among patients randomized to either HRE or NBI as first inspection technique, subdivided for histopathological outcome of polyps.


165

HRE NBI P

Flat polyps



Miss-rate, % (95%-CI) 49 (37-60) 12 (6.1-23) <.001

Protruded polyps



Miss-rate, % (95%-CI) 18 (9.8-31) 6.7 (1.9-21) .195

Proximal location



Miss-rate, % (95%-CI) 38 (26-52) 10 (4.4-21) .001

Distal location



Miss-rate, % (95%-CI) 35 (25-46) 11 (4.3-25) .011

Table 3: polyp detection during the first and second inspection and polyp miss-rates among patients randomized to either HRE or NBI as first inspection technique, subdivided for macroscopic appearance and colonic location of polyps.

DISCUSSION

Previous large prospective randomized trials comparing NBI with HRE

for adenoma detection showed that NBI was associated with an

increased sporadic HP detection rate, although adenoma detection

rates were equal.[47,49] Table 4 lists all randomized clinical trials

comparing NBI with standard white-light endoscopy for the detection of

HPs and non-adenomatous polyps. These studies showed that NBI

was particularly of value for the detection of HPs.

Chapte

r 7

Chapter 7

166

Hyperplastic polyp detection

Author Study design N WLE NBI p-value

Adler 2008[47] RCT: NBI vs WLE 401 Detected: 23 Detected: 56 <0.001

Adler 2009[49] RCT: NBI vs WLE 1256 Detected: 116 Detected: 146 0.03

Non-adenomatous polyp detection

Author Study design N WLE NBI p-value

Inoue 2008[50] RCT: NBI vs WLE 109 Detected: 12 Detected: 24 n.a.

Kaltenbach

2009[51]

Tandem design:

WLE-NBI 276

Miss rate:

12.6%

Miss rate:

10.1% n.s.

Table 4: Randomized clinical trials comparing the detection of hyperplastic polyps between narrow-band imaging (NBI) and white light endoscopy (WLE). N, number of patients; RCT, Randomized controlled trial; n.a., not analyzed; n.s., not significant.

This study demonstrated that NBI had a significantly lower

polyp miss-rate than HRE (10% versus 36%; OR 0.21; p<0.001) in

HPS patients harboring multiple serrated polyps. As in previous

studies, NBI did not prove of additional value for the detection of

adenomas. These findings could be regarded as disappointing.

However, whereas serrated polyps traditionally are considered to be

harmless lesions, especially when they are small, recent molecular

research in serrated polyps in HPS suggests these are high-risk

lesions leading to CRC. [11,16,52,53] Furthermore, a previous large

cohort study showed that 5/77 (7%) HPS patients developed CRC

despite endoscopic surveillance of which 4/5 were detected within

relatively small serrated polyps (range: 4-16mm).[54] This supports our

opinion that the increased detection of serrated polyps with NBI in HPS

is of clinical relevance.

Our study additionally showed that NBI is of particular value for the

detection of serrated polyps which are flat in shape (HRE miss-rate

49% vs. NBI miss-rate 12%; p<0.001). Previous studies demonstrated


167

that NBI did not detect more flat adenomas than HRE.[47,49,51,55] A

possible reason for this incongruence could be the fact that flat

adenomas are generally red in colour and therefore easier visible than

flat HPs and SSAs, which have the same colour as their surroundings

and are often covered by a layer of mucus. During NBI, serrated

polyps appear whiter in colour, thereby increasing the contrast

between the polyps and surrounding colonic tissue (figure 2).

Particularly these features may explain the higher miss-rate of serrated

polyps by HRE.

Figure 2. Examples of detected flat sessile serrated adenomas imaged with high-resolution endoscopy (A + C) and corresponding images with narrow-band imaging (B+D)

Several remarks regarding this study must be made before

making any firm recommendations based on our results. Adler et al

Chapte

r 7

Chapter 7

168

previously postulated that the level of experience with NBI may induce

a learning effect for improved recognition of polyps with HRE as well,

causing the difference in polyp detection between NBI and HRE to be

larger at the beginning of the learning curve.[56] However, in our study

the endoscopist had already performed more than 500 colonoscopies

with NBI, making a learning effect with regard to HRE unlikely.

Secondly, there was an unequal total number of detected polyps within

the randomization groups (a total of 122 polyps for HRE randomization

vs. 87 for NBI). These numbers approximate the true (detected and

undetected) number of baseline polyps in each group. This difference

was difficult to overcome considering the fact that both a patient with

>5 proximal HPs as well as a patient with more than 30 HPs satisfied

the criteria for HPS. The large variance of the number of polyps

between HPS patients will easily lead to unequal numbers of polyps

after randomization of only 22 patients. Due to this expected unequal

distribution of baseline polyp numbers, we chose a cross-over study

design which compares the percentage of missed polyps (i.e. polyp

miss rates) with each modality independent of the total number of

baseline polyps in each group instead of comparing the total number of

detected polyps (i.e. detection rates). Contrary to polyp miss-rates,

polyp detection rates are also dependent on the total number of

baseline polyps present in each group. Detection rate analysis is

therefore only possible when baseline polyp numbers are equal

between groups. For this reason detection rate analysis in the setting

of our study would have been unsuitable. If we would have compared

polyp detection rates at first inspection, one would expect a bias in

favour of the group with more baseline polyps. In our study that would

have resulted in a bias in favour of HRE (n=122) and against NBI

(n=87). This bias also explains the equal number of detected polyps at


169

first inspection with HRE and NBI while after second inspection polyp

miss-rates with HRE were higher. Finally, this pilot study was

performed by a single experienced endoscopist who was unblinded to

the imaging intervention. Although comparison of investigation times

between randomization groups (both >6 minutes per inspection)

showed no significant differences at first and second inspection, a bias

by the endoscopist in favour of NBI can not be excluded. Nevertheless,

the significantly large difference in miss-rates between NBI and HRE

for the detection of polyps in HPS warrants confirmation in a

subsequent multi-centre study involving more patients and different

endoscopists.

With regard to the management of HPS patients, considering

that in these patients CRCs as small as 4mm have been described,

removal of all polyps ≥3mm seems indicated in any case, but this

surveillance strategy needs to be prospectively assessed. Concerning

polyps <3mm, a previous study analyzing the differentiation of polyps

in HPS showed that 20/35 polyps <3mm were high-risk sessile

serrated adenomas (9/35) and conventional adenomas (11/35).[48]

Differentiating these diminutive premalignant polyps from HPs with NBI

by means of polyp colour differentiation (lighter than the surrounding

mucosa is unsuspicious; darker than or the same as the surrounding

mucosa is suspicious) rendered a sensitivity of 95% for sessile

serrated adenomas and conventional adenomas (diagnostic accuracy:

78%). For this reason removal of polyps darker than or the same as

the surrounding mucosa may be sufficient for polyps of this size.

Previous randomized studies have shown that

chromoendoscopy increases the detection of sporadic HPs in non-

HPS patients compared to standard white-light endoscopy.[57-60] Our

Chapte

r 7

Chapter 7

170

study with NBI (“electronic chromoendoscopy”), which allows the

endoscopist to switch between modalities at a switch of the button,

showed similar results for the detection of serrated polyps in HPS

patients. Although the value of chromoendoscopy in HPS has not

formally been investigated, this cheap and readily available technique

seems to be a valid alternative for the endoscopic surveillance of HPS

patients.

In summary, this pilot study demonstrated that NBI is

associated with a reduced polyp miss-rate when compared to HRE in

patients with HPS. These findings suggest that all polyps in patients

with HPS need to be resected during colonoscopic surveillance, which

should be done using either NBI or chromoendoscopy.


171

REFERENCES 1 Hyman NH, Anderson P, Blasyk H. Hyperplastic polyposis and the risk

of colorectal cancer. Dis Colon Rectum 2004; 47: 2101-2104

2 Leggett BA, Devereaux B, Biden K, Searle J, Young J, Jass J. Hyperplastic polyposis: association with colorectal cancer. Am J Surg Pathol 2001; 25: 177-184

3 Rubio CA, Stemme S, Jaramillo E, Lindblom A. Hyperplastic polyposis coli syndrome and colorectal carcinoma. Endoscopy 2006; 38: 266-270

4 Rashid A, Houlihan PS, Booker S, Petersen GM, Giardiello FM, Hamilton SR. Phenotypic and molecular characteristics of hyperplastic polyposis. Gastroenterology 2000; 119: 323-332

5 Chow E, Lipton L, Lynch E, D'Souza R, Aragona C, Hodgkin L et al. Hyperplastic polyposis syndrome: phenotypic presentations and the role of MBD4 and MYH. Gastroenterology 2006; 131: 30-39

6 Ferrandez A, Samowitz W, DiSario JA, Burt RW. Phenotypic characteristics and risk of cancer development in hyperplastic polyposis: case series and literature review. Am J Gastroenterol 2004; 99: 2012-2018

7 Torlakovic E, Snover DC. Serrated adenomatous polyposis in humans. Gastroenterology 1996; 110: 748-755

8 Hawkins NJ, Ward RL. Sporadic colorectal cancers with microsatellite instability and their possible origin in hyperplastic polyps and serrated adenomas. J Natl Cancer Inst 2001; 93: 1307-1313

9 Iino H, Jass JR, Simms LA, Young J, Leggett B, Ajioka Y et al. DNA microsatellite instability in hyperplastic polyps, serrated adenomas, and mixed polyps: a mild mutator pathway for colorectal cancer? J Clin Pathol 1999; 52: 5-9

10 Jass JR, Biden KG, Cummings MC, Simms LA, Walsh M, Schoch E et al. Characterisation of a subtype of colorectal cancer combining features of the suppressor and mild mutator pathways. J Clin Pathol 1999; 52: 455-460

11 Jass JR, Iino H, Ruszkiewicz A, Painter D, Solomon MJ, Koorey DJ et al. Neoplastic progression occurs through mutator pathways in hyperplastic polyposis of the colorectum. Gut 2000; 47: 43-49

Chapte

r 7

Chapter 7

172

12 Jass JR, Young J, Leggett BA. Hyperplastic polyps and DNA microsatellite unstable cancers of the colorectum. Histopathology 2000; 37: 295-301

13 Jass JR. Serrated route to colorectal cancer: back street or super highway? J Pathol 2001; 193: 283-285

14 Makinen MJ, George SM, Jernvall P, Makela J, Vihko P, Karttunen TJ. Colorectal carcinoma associated with serrated adenoma--prevalence, histological features, and prognosis. J Pathol 2001; 193: 286-294

15 Yao T, Nishiyama K, Oya M, Kouzuki T, Kajiwara M, Tsuneyoshi M. Multiple 'serrated adenocarcinomas' of the colon with a cell lineage common to metaplastic polyp and serrated adenoma. Case report of a new subtype of colonic adenocarcinoma with gastric differentiation. J Pathol 2000; 190: 444-449

16 Kambara T, Simms LA, Whitehall VL, Spring KJ, Wynter CV, Walsh MD et al. BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum. Gut 2004; 53: 1137-1144

17 Minoo P, Baker K, Goswami R, Chong G, Foulkes WD, Ruszkiewicz AR et al. Extensive DNA methylation in normal colorectal mucosa in hyperplastic polyposis. Gut 2006; 55: 1467-1474

18 Spring KJ, Zhao ZZ, Karamatic R, Walsh MD, Whitehall VL, Pike T et al. High prevalence of sessile serrated adenomas with BRAF mutations: a prospective study of patients undergoing colonoscopy. Gastroenterology 2006; 131: 1400-1407

19 Jass JR, Baker K, Zlobec I, Higuchi T, Barker M, Buchanan D et al. Advanced colorectal polyps with the molecular and morphological features of serrated polyps and adenomas: concept of a 'fusion' pathway to colorectal cancer. Histopathology 2006; 49: 121-131

20 Beach R, Chan AO, Wu TT, White JA, Morris JS, Lunagomez S et al. BRAF mutations in aberrant crypt foci and hyperplastic polyposis. Am J Pathol 2005; 166: 1069-1075

21 Azimuddin K, Stasik JJ, Khubchandani IT, Rosen L, Riether RD, Scarlatto M. Hyperplastic polyps: "more than meets the eye"? Report of sixteen cases. Dis Colon Rectum 2000; 43: 1309-1313

22 Warner AS, Glick ME, Fogt F. Multiple large hyperplastic polyps of the colon coincident with adenocarcinoma. Am J Gastroenterol 1994; 89: 123-125


173

23 Boparai KS, Mathus-Vliegen EM, Koornstra JJ, Nagengast FM, van LM, van Noesel CJ et al. Increased colorectal cancer risk during follow-up in patients with hyperplastic polyposis syndrome: a multicentre cohort study. Gut 2010; 59: 1094-1100

24 Oono Y, Fu K, Nakamura H, Iriguchi Y, Yamamura A, Tomino Y et al. Progression of a Sessile Serrated Adenoma to an Early Invasive Cancer Within 8 Months. Dig Dis Sci 2008;

25 Urbanski SJ, Kossakowska AE, Marcon N, Bruce WR. Mixed hyperplastic adenomatous polyps--an underdiagnosed entity. Report of a case of adenocarcinoma arising within a mixed hyperplastic adenomatous polyp. Am J Surg Pathol 1984; 8: 551-556

26 Snover DC, Jass JR, Fenoglio-Preiser C, Batts KP. Serrated polyps of the large intestine: a morphologic and molecular review of an evolving concept. Am J Clin Pathol 2005; 124: 380-391

27 Chan TL, Zhao W, Leung SY, Yuen ST. BRAF and KRAS mutations in colorectal hyperplastic polyps and serrated adenomas. Cancer Res 2003; 63: 4878-4881

28 Jaramillo E, Watanabe M, Rubio C, Slezak P. Small colorectal serrated adenomas: endoscopic findings. Endoscopy 1997; 29: 1-3

29 Matsumoto T, Mizuno M, Shimizu M, Manabe T, Iida M, Fujishima M. Serrated adenoma of the colorectum: colonoscopic and histologic features. Gastrointest Endosc 1999; 49: 736-742

30 Rubio CA, Jaramillo E. Flat serrated adenomas of the colorectal mucosa. Jpn J Cancer Res 1996; 87: 305-309

31 van Rijn JC, Reitsma JB, Stoker J, Bossuyt PM, van Deventer SJ, Dekker E. Polyp miss rate determined by tandem colonoscopy: a systematic review. Am J Gastroenterol 2006; 101: 343-350

32 Brooker JC, Saunders BP, Shah SG, Thapar CJ, Thomas HJ, Atkin WS et al. Total colonic dye-spray increases the detection of diminutive adenomas during routine colonoscopy: a randomized controlled trial. Gastrointest Endosc 2002; 56: 333-338

33 Hurlstone DP, Cross SS, Slater R, Sanders DS, Brown S. Detecting diminutive colorectal lesions at colonoscopy: a randomised controlled trial of pan-colonic versus targeted chromoscopy. Gut 2004; 53: 376-380

Chapte

r 7

Chapter 7

174

34 Lapalus MG, Helbert T, Napoleon B, Rey JF, Houcke P, Ponchon T. Does chromoendoscopy with structure enhancement improve the colonoscopic adenoma detection rate? Endoscopy 2006; 38: 444-448

35 Le RM, Coron E, Parlier D, Nguyen JM, Canard JM, Alamdari A et al. High resolution colonoscopy with chromoscopy versus standard colonoscopy for the detection of colonic neoplasia: a randomized study. Clin Gastroenterol Hepatol 2006; 4: 349-354

36 Park SY, Lee SK, Kim BC, Han J, Kim JH, Cheon JH et al. Efficacy of chromoendoscopy with indigocarmine for the detection of ascending colon and cecum lesions. Scand J Gastroenterol 2008; 43: 878-885

37 Adler A, Pohl H, Papanikolaou IS, bou-Rebyeh H, Schachschal G, Veltzke-Schlieker W et al. A prospective randomised study on narrow-band imaging versus conventional colonoscopy for adenoma detection: does narrow-band imaging induce a learning effect? Gut 2008; 57: 59-64

38 Aschenbeck J, Adler A, Yenerim T, Mayr M, Aminalai A, Drossel R et al. Narrow-Band Versus White-Light HDTV Endoscopic Imaging for Screening Colonoscopy: A Prospective Randomized Trial. Gastroenterology 2008;

39 Farris AB, Misdraji J, Srivastava A, Muzikansky A, Deshpande V, Lauwers GY et al. Sessile serrated adenoma: challenging discrimination from other serrated colonic polyps. Am J Surg Pathol 2008; 32: 30-35

40 Sandmeier D, Seelentag W, Bouzourene H. Serrated polyps of the colorectum: is sessile serrated adenoma distinguishable from hyperplastic polyp in a daily practice? Virchows Arch 2007; 450: 613-618

41 Torlakovic E, Skovlund E, Snover DC, Torlakovic G, Nesland JM. Morphologic reappraisal of serrated colorectal polyps. Am J Surg Pathol 2003; 27: 65-81

42 Endoscopic Classification Review Group. Update on the paris classification of superficial neoplastic lesions in the digestive tract. Endoscopy 2005; 37: 570-578

43 Hamilton SR, Vogelstein B, Kudo S et al. World Health Organization Classification of Tumours, Pathology and Genetics of Tumours of the Digestive System.Lyon, France: IARC Press, 2000: 104-119


175

44 Torlakovic EE, Gomez JD, Driman DK, Parfitt JR, Wang C, Benerjee T et al. Sessile Serrated Adenoma (SSA) vs. Traditional Serrated Adenoma (TSA). Am J Surg Pathol 2008; 32: 21-29

45 Simel DL, Rennie D, Bossuyt PM. The STARD statement for reporting diagnostic accuracy studies: application to the history and physical examination. J Gen Intern Med 2008; 23: 768-774

46 Schulz KF, Altman DG, Moher D. CONSORT 2010 statement: updated guidelines for reporting parallel group randomised trials. PLoS Med 2010; 7: e1000251

47 Adler A, Pohl H, Papanikolaou IS, Abou-Rebyeh H, Schachschal G, Veltzke-Schlieker W et al. A prospective randomised study on narrow-band imaging versus conventional colonoscopy for adenoma detection: does narrow-band imaging induce a learning effect? Gut 2008; 57: 59-64

48 Boparai KS, van den Broek FJ, van ES, Fockens P, Dekker E. Hyperplastic polyposis syndrome: a pilot study for the differentiation of polyps using high resolution endoscopy, autofluorescence imaging and narrow-band imaging. Gastrointest Endosc 2009;

49 Adler A, Aschenbeck J, Yenerim T, Mayr M, Aminalai A, Drossel R et al. Narrow-Band Versus White-Light High Definition Television Endoscopic Imaging for Screening Colonoscopy: A Prospective Randomized Trial. Gastroenterology 2008;

50 Inoue T, Murano M, Murano N, Kuramoto T, Kawakami K, Abe Y et al. Comparative study of conventional colonoscopy and pan-colonic narrow-band imaging system in the detection of neoplastic colonic polyps: a randomized, controlled trial. J Gastroenterol 2008; 43: 45-50

51 Kaltenbach T, Friedland S, Soetikno R. A randomised tandem colonoscopy trial of narrow band imaging versus white light examination to compare neoplasia miss rates. Gut 2008; 57: 1406-1412

52 Beach R, Chan AO, Wu TT, White JA, Morris JS, Lunagomez S et al. BRAF mutations in aberrant crypt foci and hyperplastic polyposis. Am J Pathol 2005; 166: 1069-1075

53 Chan AO, Issa JP, Morris JS, Hamilton SR, Rashid A. Concordant CpG island methylation in hyperplastic polyposis. Am J Pathol 2002; 160: 529-536

Chapte

r 7

Chapter 7

176

54 Boparai KS, Mathus-Vliegen EM, Koornstra JJ, Nagengast FM, van LM, van Noesel CJ et al. Increased colorectal cancer risk during follow-up in patients with hyperplastic polyposis syndrome: a multicentre cohort study. Gut 2009;

55 Paggi S, Radaelli F, Amato A, Meucci G, Mandelli G, Imperiali G et al. The impact of narrow band imaging in screening colonoscopy: a randomized controlled trial. Clin Gastroenterol Hepatol 2009; 7: 1049-1054

56 Adler A. Narrow Band Imaging (NBI) Influences the Learning Curve for Conventional Endoscopy - Final Results of a Prospective Randomized Study in the Detection of Colorectal Adenomas [abstract]. Gastrointest Endosc 2007; 65: AB116

57 Brooker JC, Saunders BP, Shah SG, Thapar CJ, Thomas HJ, Atkin WS et al. Total colonic dye-spray increases the detection of diminutive adenomas during routine colonoscopy: a randomized controlled trial. Gastrointest Endosc 2002; 56: 333-338

58 Hurlstone DP, Cross SS, Slater R, Sanders DS, Brown S. Detecting diminutive colorectal lesions at colonoscopy: a randomised controlled trial of pan-colonic versus targeted chromoscopy. Gut 2004; 53: 376-380

59 Lapalus MG, Helbert T, Napoleon B, Rey JF, Houcke P, Ponchon T. Does chromoendoscopy with structure enhancement improve the colonoscopic adenoma detection rate? Endoscopy 2006; 38: 444-448

60 Le RM, Coron E, Parlier D, Nguyen JM, Canard JM, Alamdari A et al. High resolution colonoscopy with chromoscopy versus standard colonoscopy for the detection of colonic neoplasia: a randomized study. Clin Gastroenterol Hepatol 2006; 4: 349-354

Hyperplastic polyposis syndrome: a pilot study for the differentiation of polyps using high resolution endoscopy, autofluorescence imaging and narrow-band imaging

K.S. Boparai

F.J.C. van den Broek

S. van Eeden

P. Fockens

E. Dekker

Gastrointestinal Endoscopy 2009 Nov;70(5):947-55

Ch

ap

ter

Chapter 8

ABSTRACT

Background: Endoscopic differentiation and removal of potentially

premalignant sessile serrated adenomas (SSAs) may be an important

step in preventing colorectal cancer (CRC) development in

hyperplastic polyposis syndrome (HPS).

Objective: To assess the value of high resolution endoscopy (HRE),

autofluorescence imaging (AFI) and narrow-band imaging (NBI) for

differentiating polyps in HPS.

Design: A prospective polyp series.

Setting: Single tertiary referral center.

Patients and Interventions: 7 patients with HPS underwent

colonoscopy using endoscopic trimodal imaging (ETMI), which

incorporates HRE, AFI and NBI in one system. All detected polyps

were analysed with AFI for colour and with NBI for Kudo pit pattern

and vascular pattern intensity (VPI).

Main outcome measurements: The accuracy, sensitivity and

specificity of AFI and NBI in differentiating detected polyps were

determined by using histology as a gold standard.

Results: A total of 19 hyperplastic polyps (HPs), 32 SSAs and 15

adenomas were detected. For differentiating SSAs from HPs, AFI-

colour, Kudo pit pattern and VPI resulted in a diagnostic accuracy of

55%, 55% and 52% respectively. For differentiating adenomas from

HPs, this was 65%, 94% and 90% respectively. Macroscopically, the

combination of size ≥3mm and proximal location resulted in the highest

accuracy (76%) for differentiating SSAs from HPs.

Limitations: Small sample size.

Conclusion: Endoscopic differentiation between HPs and SSAs using

ETMI proved unsatisfactory. Differentiation of adenomas from HPs

was well possible with NBI but not with AFI.


179

INTRODUCTION

Hyperplastic polyposis syndrome (HPS) is a recently

recognised condition characterised by the presence of multiple (>30)

hyperplastic polyps (HPs) spread throughout the colon and has

frequently been linked with colorectal cancer (CRC).1-4 Besides

multiple HPs, serrated adenomas are frequently seen in HPS as well.

1-8 In fact, the co-existence of sessile serrated adenomas (SSAs) has

been considered by some as a characterizing feature of this condition.9

Molecular research in SSAs strongly suggests that these

polyps are precursor lesions which may lead to CRC.10-13 Accordingly,

authorities recommend that SSAs should be endoscopically managed

like conventional adenomas.14 In this respect, endoscopic

differentiation of SSAs from HPs and removal of SSAs may be an

important step in preventing cancer development in HPS. However,

HPs and SSAs, being both often small in size and sessile/flat in shape,

are similar in appearance and therefore difficult to distinguish from

each other when using standard endoscopy (figure 1).15-18

Novel endoscopic imaging techniques may aid in the

differentiation of conventional adenomas and HPs with high

accuracy.19-22 Autofluorescence imaging (AFI) facilitates differentiation

of adenomas from non-adenomatous polyps based on different

fluorescence emission spectra.23-25 The use of AFI in differentiating

polyps in HPS patients has not been described before. Narrow band

imaging (NBI) utilizes short wavelength visible light to provide

improved details of the mucosal pit pattern and microvasculature. Pit

pattern analysis by applying the Kudo classification has shown to be a

reliable approach for distinguishing adenomas from non-adenomatous

polyps and has also been used to describe serrated adenomas.26-31 Chapte

r 8

Chapter 8

180

Figure 1: Similar endoscopic appearance of a hyperplastic polyp (left) and a sessile serrated adenoma (right) using conventional white light endoscopy with corresponding haemotoxylin and eosin stains below

However, at the times of these studies the diagnosis SSA was

not yet in practice and Kudo pit patterns for serrated adenomas varied

considerably from II to as high as IV.27, 28 In addition, the assessment

of microvasculature and vascular pattern intensity (VPI) with NBI is

believed to be a relatively easy method for differentiating adenomas

from non-adenomatous polyps, but has not been evaluated in HPS.20,

29, 30, 32-34

The aim of this study was to assess the diagnostic accuracy of

high resolution endoscopy (HRE), AFI and NBI for the differentiation of

polyps in patients with HPS.


181


Study population

This study was conducted at the Academic Medical Center

Amsterdam and was approved by the local medical ethics committee.

Consecutive patients with HPS were invited to participate when

fulfilling the criteria for HPS in accordance with the World Health

Organisation (WHO): (1) at least five histologically confirmed HPs

proximal to the sigmoid colon, of which two are greater than 10mm in

diameter, or (2) more than 30 HPs distributed throughout the colon.35

Patients <18 years or patients with severe coagulopathy or insufficient

bowel cleansing were excluded from this study.

Endoscopic equipment

All procedures were performed with the endoscopic tri-modal

imaging (ETMI) system, which integrates HRE, AFI and NBI into one

unit (XCV-260 HP, Olympus Inc., Tokyo, Japan). The endoscope

(XCF-H240FZL) is equipped with a movable lens for optical

magnification (up to 100×) and two high-quality charge coupled

devices: one for HRE/NBI and one for AFI. The light source used in

this system (XCLV-260HP) was of the type “sequential RGB-

illumination”. The ETMI specifications have previously been described

in detail.36, 37 During colonoscopy, the endoscopist could easily switch

between the three imaging modalities by pressing a button on the shaft

of the endoscope. A high resolution monitor was used for all

procedures.

Chapte

r 8

Chapter 8

182

Colonoscopy procedure

Patients were prepared with 4-6 L polyethylene glycol solution

(Kleanprep; Norgine GmbH, Marburg, Germany) and underwent

colonoscopy under conscious sedation with midazolam and/or

fentanyl. The colonoscope was advanced until cecal intubation was

confirmed by identification of the appendiceal orifice and ileocecal

valve. Upon reaching the cecum, the level of bowel preparation was

determined as good (100% of the mucosa visible), moderate (90-

100%) or poor (<90%) after rinsing and suctioning.

During withdrawal of the endoscope, HRE was used to detect

colonic polyps. All detected polyps were assessed for size (open

biopsy forceps: 8mm), shape (Paris classification) and location.38

Subsequently, each polyp was assessed with AFI for polyp colour:

green, ambiguous or purple. Purple and ambiguous colours were

considered suspicious for adenoma and green was considered non-

suspicious for adenoma. Hereafter, the Kudo pit pattern (I-V) was

assessed with NBI.31 Kudo pit pattern III-V were considered suspicious

for adenoma, while pit pattern I-II were non-suspicious. Still images

(BMP-format) with all modalities were acquired after which the polyp

was resected and harvested for histopathology. All procedures and

instant assessments with AFI and NBI were performed by one

experienced endoscopist (ED), who has performed >2,500

colonoscopies and >50 ETMI colonoscopies.

In addition, representative NBI images of all detected lesions

were later assessed for vascular pattern intensity (VPI) as described

by East et al.20 For this purpose, all sharp high-quality images were

displayed in a random order to the same endoscopist who was blinded

for final histopathology. Images were directly displayed on a personal


183

computer in standard format (3.2 × 2.4 inch; 200 pixels/inch) without

any post-processing. The VPI was scored as lighter (weak), the same

(normal) or darker than (strong) the surrounding mucosa. Strong VPI

was considered suspicious for adenoma, whereas weak/normal VPI

was considered non-suspicious.

Reference standard

All polyp specimens were blindly evaluated by a gastrointestinal

pathologist (SvE). Lesions were classified as HP, SSA, traditional

serrated adenoma (TSA), mixed polyp or conventional adenoma based

on the morphological features on H&E staining which was used as

reference standard.13, 14, 39

Statistical analysis

SSAs were regarded as adenomatous polyps, owing to their

premalignant potential.10-14 Consequently, it was examined whether

these polyps were suspicious on AFI (ambiguous/purple) and/or NBI-

VPI (strong). In addition, NBI pit patterns of SSAs were expected to be

comparable to HPs (Kudo II) as these polyps are described to be

microscopically difficult to distinguish from each other.14, 18, 40, 40 TSAs,

mixed polyps and conventional adenomas were expected to be

suspicious on AFI and NBI as these polyps harbour neoplastic

changes of the epithelium.

The sensitivity, specificity, and diagnostic accuracy plus 95%-

confidence interval (95%-CI, using the Wilson procedure without

correction for continuity) for differentiating SSAs, TSAs, mixed polyps

and conventional adenomas (i.e. adenomatous group) from HPs were

determined for each modality by comparing the endoscopic diagnosis Chapte

r 8

Chapter 8

184

to final histopathology, which served as reference standard. Lesions

histologically diagnosed as normal mucosa were excluded from the

analysis. The size, location and shape of all lesions were summarized

and compared between the different polyps using the Chi-Square test,

Fisher’s exact test, Kruskal-Wallis test or Mann-Whitney U test when

appropriate. To make statistical comparisons and to calculate the 95%-

CI, it is necessary to assume that results for individual polyps

constitute statistically independent observations, even when there may

have been more than one polyp assessed in individual patients. A p-

value less than 0.05 from a single test was considered statistically

significant, but it is recognized that there was multiple testing of

outcome data arising from individual polyps. Examining the nominal p-

values in light of correction for multiple testing using the method of

correction of Bonferroni, it is suggested that only those nominal p-

values less than 0.01 will retain significance after correction. The

uncorrected p-values are presented with the warning that p-values

between 0.01 and 0.05 should be considered as provisional. For

reporting the results of this study, the STARD guidelines were used.41

Results

From January 2005 to July 2006, 7 patients (5 male) who met

the criteria for HPS, underwent colonoscopy with ETMI. The median

age of all patients was 55.8 (range 54-71) years. At colonoscopy all

patients had good to moderate bowel preparation. A total of 66 polyps

(19 HPs, 32 SSAs and 15 tubular adenomas) were detected as well as

10 additional lesions displaying normal mucosa on histology (excluded

from the analysis). Macroscopic polyp characteristics are summarized

in table 1. Overall, SSAs were larger than HPs (p<0.001, Mann-


185

Whitney U test) and adenomas (p<0.001, Mann-Whitney U test). There

was no significant difference in size between HPs and adenomas.

Concerning the differentiation of SSAs from HPs, the odds ratio

for predicting a polyp to be SSA was 2.3 (95%-CI: 1.2-4.4) for size (per

mm increase), 4.9 (1.3-18.0) for flat shape and 3.9 (1.1-14.7) for

proximal location. The combination of size ≥3mm and proximal location

yielded the largest differential value (p<0.0001) between SSAs (21/32:

66%) and HPs (1/19: 5%) with a corresponding odds ratio of 34.4 (4.0-

292). The sensitivity and specificity of this combination for

differentiating SSAs from HPs would be 66% (95%-CI: 48-80%) and

95% (75-99%) respectively. The overall diagnostic accuracy would be

76% (62-87%).

Autofluorescence imaging

With AFI, 10/19 (53%) HPs displayed a green colour versus

14/32 (44%) SSAs and 3/15 (20%) adenomas (p=0.142). The

sensitivity, specificity and diagnostic accuracy of AFI for discriminating

SSAs from HPs based on colour were 56% (95%-CI: 39-72%), 53%

(32-73%) and 55% (41-68%) respectively. Differentiation with AFI

between adenomas and HPs had a sensitivity, specificity and accuracy

of 80% (55-93%), 53% (32-73%) and 65% (48-79%).

Chapte

r 8

Chapter 8

186

Polyp

characteristics

All polyps

(n=66)

HP

(n=19)

SSA

(n=32)

Adenoma

(n=15)

P-

value

Median size mm

(interquartile range) 2 (2-5) 2 (1-2) 3 (2-8) 2 (1-3) 0.001*

Location 0.013**

Proximal colon 51 (77%) 11 (58%) 27 (84%) 13 (87%)

Distal colon 6 (9%) 1 (5%) 3 (9%) 2 (13%)

Rectum 9 (14%) 7 (37%) 2 (7%) 0 (0%)

Shape 0.048**

0-Ip 0 0 0 0

0-Is 18 (38%) 9 (47%) 5 (16%) 4 (27%)

0-II 48 (62%) 10 (53%) 27 (84%) 11 (73%)

Table 1: Clinicopathological characteristics of all detected polyps in 7 patients with hyperplastic polyposis syndrome. Proximal colon = cecum, ascending and transverse colon; distal colon = descending colon and sigmoid colon. *Kruskal-Wallis test (only SSAs differed significantly from HPs and adenomas on additional testing with the Mann-Whitney U test). **Pearson Chi-Square test. Note that Bonferroni correction for multiple testing removes statistical significance except where p<0.01 in this table.

Narrow band imaging

The sensitivity, specificity and diagnostic accuracy of the Kudo

classification with NBI for differentiation of SSAs from HPs were 28%

(95%-CI: 16-33%), 100% (83-100%) and 55% (41-68%). For

differentiating adenomas from HPs, the obtained sensitivity, specificity

and diagnostic accuracy were 87% (81-98%), 100% (83-100%) and

94% (62-96%) (table 2). During the subsequent NBI image evaluation,

the VPI was assessed for 14 HPs, 28 SSAs and 15 adenomas. Images

of the other detected polyps could not be included for analysis

because VPI assessment was not possible (e.g. blurry images). The

sensitivity, specificity and diagnostic accuracy of VPI for differentiating

SSAs from HPs, were 36% (21-54%), 86% (6-96%) and 52% (38-67%)

respectively. When adenomas were compared with HPs the sensitivity,


187

specificity and diagnostic accuracy were 93% (74-96%), 86% (60-96%)

and 90% (70-98%).

NBI HP (n=19) SSA (n=32) Adenoma (n=15) P-value

Pit pattern <0.0001*

Kudo I-II 19 (100%) 23 (72%) 2 (13%)

Kudo III-V 0 9 (28%) 13 (87%)

NBI HP (n=14) SSA (n=28) Adenoma (n=15) P-value

VPI <0.0001*

Weak/normal 12 (86%) 18(64%) 1 (7%)

Strong 2 (14%) 10 (36%) 14 (93%)

Table 2: Kudo pit pattern classification and vascular pattern intensity (VPI) assessment of all detected polyps with NBI. *p-value for adenomas compared to HPs and SSAs.

Table 3 lists the sensitivities, specificities and diagnostic accuracies of

the different endoscopic modalities for the grouped differentiation of

both high-risk SSAs and conventional adenomas from low-risk HPs.

Table 3: Sensitivity, specificity and diagnostic accuracy of all endoscopic modalities for differentiating sessile serrated adenomas and conventional adenomas (high risk) from hyperplastic polyps (low risk) in patients with hyperplastic polyposis syndrome.

SSA/adenoma vs. HP

Modality AFI NBI-pit pattern NBI-VPI

Sensitivity

(95%-CI)

30/47: 64%

(50-76%)

22/47: 47%

(33-61%)

24/43: 56%

(41-70%)

Specificity

(95%-CI)

10/19: 53%

(32-73%)

19/19: 100%

(83-100%)

12/14: 86%

(60-96%)

Diagnostic accuracy

(95%-CI)

40/66: 61%

(49-72%)

41/66:62%

(50-73%)

36/57: 63%

(50-74%)

Chapte

r 8

Chapter 8

188

DISCUSSION

In the present study, SSAs were significantly larger and more

often proximally located than HPs but the latter finding has its nominal

significance removed by Bonferroni correction for multiple testing of

data. This is in concordance with recent comparative studies, in which

sporadic SSAs were also found to be larger than sporadic HPs and

preferentially located in the right colon whereas HPs were more often

found distally.12, 18, 42 Interestingly, in our study the combination of size

≥3mm (median size of SSAs) and proximal location, showed a highly

significant difference between SSAs and HPs but with a corresponding

diagnostic accuracy of only 76%.

This pilot study demonstrated that the diagnostic accuracy of

AFI was unsatisfactory for differentiating SSAs from HPs (accuracy

55%). The presence of epithelial dysplasia and hypervascularisation

within adenomatous polyps are considered to be responsible for their

different fluorescence emission spectra when compared to non-

adenomatous polyps and hence lead to a different colour on AFI.24, 43

Although epithelial dysplasia has occasionally been described in

advanced SSAs progressing to carcinomas 44, SSAs generally lack

these histopathological features as confirmed by the present study

(figure 2).


189

Figure 2 (left column). Green (A), ambiguous (B) and purple (C) coloured sessile serrated adenomas using autofluorescence imaging (AFI). Figure 3 (right column). Variation of pit-pattern characteristics in sessile serrated adenomas using narrow-band imaging (NBI): Kudo I (A), Kudo II (B), Kudo IIIL (C)

Therefore, as expected a-priori, AFI appeared insufficient to

distinguish HPs from SSAs. Furthermore, the diagnostic accuracy of

AFI for differentiating adenomas from HPs was also insufficient (65%).

Chapte

r 8

Chapter 8

190

This is in accordance with a previous study evaluating AFI for

differentiation of neoplastic and non-neoplastic lesions in the colon, in

which a similar diagnostic accuracy (68%) was obtained.45

Kudo pit pattern analysis with NBI demonstrated an equally

insufficient diagnostic accuracy for differentiation of SSAs from HPs as

AFI (55%). SSAs are microscopically defined as polyps with irregular

crypts displaying dilatation, branching and exaggerated serration,

especially at the base of the crypts. However, despite these defined

crypt characteristics, microscopic differentiation of SSAs from HPs

remains difficult.14, 18, 40, 42 NBI pit pattern analysis, which describes the

orifices of crypts, showed similar (Kudo II) phenotypes in HPs and

SSAs, confirming that crypt characteristics of these polyps are not only

microscopically but also endoscopically difficult to use for

differentiation purposes (figure 3).

As HPs and SSAs mainly differ in histological anatomy at the

base of the crypts, it was therefore expected a-priori that the pit pattern

of these polyps, which is assessed at the luminal site of the crypts,

was comparable. Differentiation of adenomas from HPs however was

very well possible with NBI (accuracy 94%). This corresponds with

previous studies in which the diagnostic accuracy of NBI for

differentiation of adenomas from non-neoplastic polyps ranged from

77-99%.19-21, 29, 30, 32, 33, 46

Previous use of VPI in sporadic polyps suggested that this

method has a comparable diagnostic accuracy in differentiating

adenomas from non-adenomatous polyps as pit pattern analysis.20, 29,

46 Based on the principle of increased vascularization, adenomatous

polyps would have a stronger VPI and thus have a darker colour when

viewed with NBI. 19, 20, 29, 30, 32-34, 46 Owing to the presumed premalignant

potential of SSAs, we examined whether a darker colour due to


191

hypervascularization could be observed in these polyps. Overall only

10/28 (36%) of SSAs displayed a darker colour than the surrounding

mucosa, resulting consequently in a low diagnostic accuracy and

insufficient differential value (figure 4).

Figure 4: Weak (A), normal (B) and strong (C) vascular pattern intensity (VPI) displayed in three different sessile serrated adenomas using narrow-band imaging (NBI).

NBI-VPI analysis did however show a similarly high diagnostic

accuracy as NBI-pit pattern for differentiating adenomas from HPs.

These high diagnostic accuracies observed with NBI (pit-pattern and

Chapte

r 8

Chapter 8

192

VPI) are interesting considering that the median size of all polyps in

this study was only 2mm. This corresponds with results from previous

studies evaluating NBI for differentiating diminutive (<10mm) polyps. 20,

47-49

This study was performed in HPS patients in a tertiary referral

center by a single endoscopist specialized in HPS. This could explain

the diminutive size of polyps detected in these patients as they

undergo annual surveillance endoscopies with removal of most polyps,

leaving only small ones in situ. However, typical HPs seldom exceed

5mm in size and in a recent prospective study of unselected

consecutive patients undergoing colonoscopy, 83% of detected SSAs

were ≤10mm and 36% were ≤5mm.12, 50-55 These findings suggest that

the predominant polyps in HPS, i.e. HPs and SSAs, are typically small.

Nevertheless, HPS is associated with a significantly increased risk of

developing CRC.5 Also in our personal (unpublished) experience of

annual HPS surveillance, intra-mucosal carcinomas have been

detected in serrated polyps as small as 4mm. Therefore, unlike in the

general population, diminutive polyps should be considered clinically

relevant in HPS.

A possible limitation of this study is that only HPS patients were

selected for endoscopic differentiation of polyps using ETMI. Thus, our

results for differentiating polyps can not by default be extrapolated to

polyps in non-HPS patients, as these are not necessarily identical.

Furthermore, one may question the generalizability of this pilot study

since the sample size of SSAs was relatively small. However, as the

accuracies of AFI, NBI-pit pattern and NBI-VPI were all far from

acceptable (52-55%) in differentiating SSAs from HPs, and the upper

limit of the 95%-confidence interval of these accuracies was 68% at

best, a larger sample size is unlikely to alter the conclusion of this pilot


193

study. Moreover, during real-time polyp assessment, AFI was used

first after which an assessment with NBI was performed. This may

cause AFI-colour to influence the subsequent NBI assessment and

thus cause bias. However, when differentiating adenomas from HPs,

NBI results were markedly better than prior AFI results, suggesting that

bias due to the order of assessment was minimal. Finally, in this study

diminutive lesions displaying normal mucosa on histology (n=10) were

excluded from analysis. When these lesions were grouped with HPs at

additional analysis, diagnostic accuracies for the different endoscopic

modalities remained largely unchanged with a maximum difference of

5% (range: 1-5%).

In summary, the diagnostic accuracy of AFI, NBI-pit pattern and

NBI-VPI proved unsatisfactory for differentiating SSAs from HPs in

patients with HPS. Differentiation of conventional adenomas from HPs

was well possible using both NBI-pit pattern and NBI-VPI. Proximal

colonic location combined with a size ≥ 3mm proved to be the most

valuable for differentiating SSAs from HPs. Nevertheless, the

diagnostic accuracy of this combination appears still too low for clinical

use (76%).

Chapte

r 8

Chapter 8

194

REFERENCES 1. Hyman NH, Anderson P, Blasyk H. Hyperplastic polyposis and the

risk of colorectal cancer. Dis Colon Rectum 2004;47:2101-2104.






7. Ferrandez A, Samowitz W, DiSario JA, Burt RW. Phenotypic characteristics and risk of cancer development in hyperplastic polyposis: case series and literature review. Am J Gastroenterol 2004;99:2012-2018.




195






15. Jaramillo E, Watanabe M, Rubio C, Slezak P. Small colorectal serrated adenomas: endoscopic findings. Endoscopy 1997;29:1-3.

16. Matsumoto T, Mizuno M, Shimizu M, Manabe T, Iida M, Fujishima M. Serrated adenoma of the colorectum: colonoscopic and histologic features. Gastrointest Endosc 1999;49:736-742.

17. Rubio CA, Jaramillo E. Flat serrated adenomas of the colorectal mucosa. Jpn J Cancer Res 1996;87:305-309.


19. Chiu HM, Chang CY, Chen CC, Lee YC, Wu MS, Lin JT, Shun CT, Wang HP. A prospective comparative study of narrow-band imaging, chromoendoscopy, and conventional colonoscopy in the diagnosis of colorectal neoplasia. Gut 2007;56:373-379. C

hapte

r 8

Chapter 8

196

20. East JE, Suzuki N, Saunders BP. Comparison of magnified pit pattern interpretation with narrow band imaging versus chromoendoscopy for diminutive colonic polyps: a pilot study. Gastrointest Endosc 2007;66:310-316.

21. Hirata M, Tanaka S, Oka S, Kaneko I, Yoshida S, Yoshihara M, Chayama K. Magnifying endoscopy with narrow band imaging for diagnosis of colorectal tumors. Gastrointest Endosc 2007;65:988-995.

22. McCallum AL, Jenkins JT, Gillen D, Molloy RG. Evaluation of autofluorescence colonoscopy for the detection and diagnosis of colonic polyps. Gastrointest Endosc 2008;68:283-290.

23. DaCosta RS, Andersson H, Cirocco M, Marcon NE, Wilson BC. Autofluorescence characterisation of isolated whole crypts and primary cultured human epithelial cells from normal, hyperplastic, and adenomatous colonic mucosa. J Clin Pathol 2005;58:766-774.

24. Haringsma J, Tytgat GN, Yano H, Iishi H, Tatsuta M, Ogihara T, Watanabe H, Sato N, Marcon N, Wilson BC, Cline RW. Autofluorescence endoscopy: feasibility of detection of GI neoplasms unapparent to white light endoscopy with an evolving technology. Gastrointest Endosc 2001;53:642-650.

25. Wang TD, Van DJ, Crawford JM, Preisinger EA, Wang Y, Feld MS. Fluorescence endoscopic imaging of human colonic adenomas. Gastroenterology 1996;111:1182-1191.

26. Kudo S, Rubio CA, Teixeira CR, Kashida H, Kogure E. Pit pattern in colorectal neoplasia: endoscopic magnifying view. Endoscopy 2001;33:367-373.

27. Morita T, Tamura S, Miyazaki J, Higashidani Y, Onishi S. Evaluation of endoscopic and histopathological features of serrated adenoma of the colon. Endoscopy 2001;33:761-765.

28. Oka S, Tanaka S, Hiyama T, Ito M, Kitadai Y, Yoshihara M, Haruma K, Chayama K. Clinicopathologic and endoscopic features of colorectal serrated adenoma: differences between polypoid and superficial types. Gastrointest Endosc 2004;59:213-219.

29. Tischendorf JJ, Wasmuth HE, Koch A, Hecker H, Trautwein C, Winograd R. Value of magnifying chromoendoscopy and narrow band imaging (NBI) in classifying colorectal polyps: a prospective controlled study. Endoscopy 2007;39:1092-1096.


197

30. Machida H, Sano Y, Hamamoto Y, Muto M, Kozu T, Tajiri H, Yoshida S. Narrow-band imaging in the diagnosis of colorectal mucosal lesions: a pilot study. Endoscopy 2004;36:1094-1098.

31. Kudo S, Rubio CA, Teixeira CR, Kashida H, Kogure E. Pit pattern in colorectal neoplasia: endoscopic magnifying view. Endoscopy 2001;33:367-373.

32. Hirata M, Tanaka S, Oka S, Kaneko I, Yoshida S, Yoshihara M, Chayama K. Evaluation of microvessels in colorectal tumors by narrow band imaging magnification. Gastrointest Endosc 2007;66:945-952.

33. Rastogi A, Bansal A, Wani S, Callahan P, McGregor DH, Cherian R, Sharma P. Narrow-band imaging colonoscopy-a pilot feasibility study for the detection of polyps and correlation of surface patterns with polyp histologic diagnosis. Gastrointest Endosc 2007.

34. Konerding MA, Fait E, Gaumann A. 3D microvascular architecture of pre-cancerous lesions and invasive carcinomas of the colon. Br J Cancer 2001;84:1354-1362.


36. Curvers WL, Singh R, Song LM, Wolfsen HC, Ragunath K, Wang K, Wallace MB, Fockens P, Bergman JJ. Endoscopic tri-modal imaging for detection of early neoplasia in Barrett's oesophagus: a multi-centre feasibility study using high-resolution endoscopy, autofluorescence imaging and narrow band imaging incorporated in one endoscopy system. Gut 2008;57:167-172.

37. van den Broek FJ, Fockens P, van ES, Reitsma JB, Hardwick JC, Stokkers PC, Dekker E. Endoscopic tri-modal imaging for surveillance in ulcerative colitis: randomised comparison of high-resolution endoscopy and autofluorescence imaging for neoplasia detection; and evaluation of narrow-band imaging for classification of lesions. Gut 2008;57:1083-1089.

38. Endoscopic Classification Review Group. Update on the paris classification of superficial neoplastic lesions in the digestive tract. Endoscopy 2005;37:570-578.

Chapte

r 8

Chapter 8

198



41. Simel DL, Rennie D, Bossuyt PM. The STARD statement for reporting diagnostic accuracy studies: application to the history and physical examination. J Gen Intern Med 2008;23:768-774.


43. Georgakoudi I, Jacobson BC, Van DJ, Backman V, Wallace MB, Muller MG, Zhang Q, Badizadegan K, Sun D, Thomas GA, Perelman LT, Feld MS. Fluorescence, reflectance, and light-scattering spectroscopy for evaluating dysplasia in patients with Barrett's esophagus. Gastroenterology 2001;120:1620-1629.

44. Goldstein NS. Small colonic microsatellite unstable adenocarcinomas and high-grade epithelial dysplasias in sessile serrated adenoma polypectomy specimens: a study of eight cases. Am J Clin Pathol 2006;125:132-145.

45. F.J.C.van den Broek, Curvers WL, Hardwick JC, Fockens P, Dekker E. Interobserver variability and accuracy of colonic pit patterns by narrow band imaging and the additional value of autofluorescence characteristics. 65 ed. Gastrointestinal Endoscopy: 2007:AB349.

46. Su MY, Hsu CM, Ho YP, Chen PC, Lin CJ, Chiu CT. Comparative study of conventional colonoscopy, chromoendoscopy, and narrow-band imaging systems in differential diagnosis of neoplastic and nonneoplastic colonic polyps. Am J Gastroenterol 2006;101:2711-2716

47. East JE, Suzuki N, Bassett P, Stavrinidis M, Thomas HJ, Guenther T, Tekkis PP, Saunders BP. Narrow band imaging with magnification for the characterization of small and diminutive colonic polyps: pit pattern and vascular pattern intensity. Endoscopy 2008;40:811-817.

48. Rogart JN, Jain D, Siddiqui UD, Oren T, Lim J, Jamidar P, Aslanian H. Narrow-band imaging without high magnification to differentiate


199

polyps during real-time colonoscopy: improvement with experience. Gastrointest Endosc 2008;68:1136-1145.

49. Sano Y, Ikematsu H, Fu KI, Emura F, Katagiri A, Horimatsu T, Kaneko K, Soetikno R, Yoshida S. Meshed capillary vessels by use of narrow-band imaging for differential diagnosis of small colorectal polyps. Gastrointest Endosc 2008.


51. Estrada RG, Spjut HJ. Hyperplastic polyps of the large bowel. Am J Surg Pathol 1980;4:127-133.

52. Hayashi T, Yatani R, Apostol J, Stemmermann GN. Pathogenesis of hyperplastic polyps of the colon: a hypothesis based on ultrastructure and in vitro cell kinetics. Gastroenterology 1974;66:347-356.

53. Imperiale TF, Wagner DR, Lin CY, Larkin GN, Rogge JD, Ransohoff DF. Results of screening colonoscopy among persons 40 to 49 years of age. N Engl J Med 2002;346:1781-1785.

54. Johnson DA, Gurney MS, Volpe RJ, Jones DM, VanNess MM, Chobanian SJ, Avalos JC, Buck JL, Kooyman G, Cattau EL, Jr. A prospective study of the prevalence of colonic neoplasms in asymptomatic patients with an age-related risk. Am J Gastroenterol 1990;85:969-974.

55. Lieberman DA, Prindiville S, Weiss DG, Willett W. Risk factors for advanced colonic neoplasia and hyperplastic polyps in asymptomatic individuals. JAMA 2003;290:2959-2967.

Chapte

r 8


201

Summary & Future Perspectives

Summary and future perspectives

203

SUMMARY

Colorectal cancer (CRC) is a condition associated with a high mortality

rate. Previous molecular research performed in patients with FAP and

Lynch syndrome has revealed that conventional adenomas are pre-

malignant lesions that can progress to CRC. It has been shown that

cancer development in these adenomas can follow different molecular

pathways involving seperate oncogenes and tumor-suppressor genes.

Recently, a novel CRC pathway has been suggested involving

histologically different polyps namely serrated polyps. In these serrated

polyps, alternative molecular processes are suggested to lead to CRC.

In this thesis we investigated a proposed ‘serrated neoplasia pathway’.

To this aim, patients with hyperplastic polyposis syndrome (HPS)

harboring multiple serrated polyps but also conventional adenomas

represented a unique demographic opportunity for clinical and

molecular analysis of serrated polyps and their association with CRC.

Clinical analyses – Colorectal cancer risk

In chapter 2 of this thesis we describe the clinical and pathological

features of a large multi-centre HPS cohort during multiple years of

unprotocollized endoscopic surveillance. We showed that one third of

HPS patients presented with co-existent CRC and in an additional 7%

of patients CRCs were identified despite endoscopic surveillance

(interval-CRCs). These findings are substantial considering that the

lifetime risk of developing CRC in the general population is estimated

to be only 6%. Interestingly, at multivariate logistic regression, serrated

polyps (i.e. HPs and SSLs) and not conventional adenomas were

significantly associated with CRC presence thus supporting the

existence of a predominant ‘serrated pathway’ to CRC in these

Sum

mary

& f

utu

re p

ers

pectives

204

patients. Considering that CRCs were detected in polyps as small as

4mm during endoscopic surveillance, all polyps in HPS seem at risk of

representing advanced lesions warranting removal of all polyps (at

least polyps ≥3mm) at annual surveillance endoscopies. If this is not

feasible, surgical resection should be considered.

Although an increased CRC risk for HPS patients has been

established based on previous cohort studies, it was yet unknown

whether first-degree relatives (FDRs) have an increased risk of CRC

and/or HPS. Consequently, the need for preventive measures, like

screening colonoscopies, in this group was doubtful. In chapter 3 we

performed a retrospective study involving, at the time, the largest

described cohort of FDRs in which we analyzed the incidence rate of

CRC and HPS. This incidence we subsequently compared with the

general population through person-year analysis. This way we were

able to quantify the suggested increased CRC incidence in the

literature by means of a relative risk. Our results showed that FDRs of

HPS patients have an increased risk for both CRC (RR: 3.7-7.8) and

HPS (RR: 13-121) compared to the general population warranting

screening colonoscopies for this whole group as long as no genetic

substrate is identified which can help us indentify FDRs with a high

risk.

Molecular analyses – Etiology and colorectal cancer pathways

Although an underlying genetic disorder seems likely, thus far no

genetic causes of HPS have been identified. However, previous case

series of patients with a known genetic disorder have reported

individuals with multiple serrated polyps. In chapter 4 we investigated

the presence of serrated polyps in MYH-associated polyposis (MAP)

and analysed whether these polyps were causally related to MAP.


205

Phenotypically, MAP polyps encompass conventional adenomas and

very rarely serrated polyps. Conversely, at re-evaluation of all polyps in

our cohort of MAP patients we found that almost half of all patients had

at least one serrated polyp and 18% had ≥ 10 serrated polyps. At

subsequent molecular analysis, we found serrated polyps to be

causally related to MYH-deficiency, reflected by the presence of

almost exclusively G:C→T:A transversions in the KRAS gene of these

lesions. This implies that distinct pathways, i.e. APC-gene related in

adenomas and non-related in serrated polyps, appear to be

operational in MAP.

Similar to patients with MAP, detected polyps in patients with

Lynch syndrome, who have a germline defect in one of the MMR

genes, invariably represent conventional adenomas. However, recent

studies have also described serrated polyps in Lynch patients.

Subsequent molecular studies evaluating whether these polyps are

causally related to Lynch syndrome by means of

immunohistochemistry have however been inconclusive. In chapter 5

the presence of serrated polyps in a large cohort of genetically proven

Lynch patients was analysed. To examine whether serrated polyps are

causally related to Lynch we utilized an alternative molecular approach

involving mutation analysis (APC, KRAS and BRAF) in adjunct to

defective MMR testing. The possible finding of an association between

Lynch and serrated polyps could imply that these lesions follow an

accelerated route to CRC. Based on the principle that Lynch CRCs are

not BRAF mutated, BRAF analysis is method to distinguish Lynch-

associated CRCs from sporadic CRCs. We hypothesised that a high

frequency of BRAF mutations in serrated polyps, comparable to

sporadic control group serrated polyps, would indicate that these

polyps are not Lynch-associated. In this study, we demonstrated that

Sum

mary

& f

utu

re p

ers

pectives

206

serrated polyps in Lynch-HPS patients (>10 serrated polyps) are not

associated with Lynch due to the high frequency of BRAF mutations in

these polyps. Alternatively, in Lynch patients with occasional serrated

polyps (<10), serrated polyps had significantly lower levels of BRAF

mutations than control group polyps. It can be surmised that

considering this low frequency of BRAF mutations in serrated polyps of

non-HPS Lynch patients, a causal relationship with the MSI-pathway

can not be excluded despite lack of defective MMR. These polyps

associated with the accelerated MSI-pathway may represent precursor

lesions of MSI-CRC which evolve even faster than in the serrated CRC

pathway alone.

Because of the unique mixture of different precursor lesion

types in HPS, it was yet unknown which polyps lead to CRC in HPS

and thus which polyps are clinically relevant. In chapter 6 we

evaluated which pathways are operational in HPS by studying the

histological and molecular characteristics of all available CRCs in a

large cohort of HPS patients. We found in this comprehensive cohort

study of HPS patients a high number of combined serrated polyp-CRC

lesions which showed identical BRAF mutations in both components,

supporting the existence of a serrated CRC pathway. Overall, we

demonstrated that both microsatellite-stable and –unstable CRCs in

HPS predominantly originate from serrated polyps, reflected by a high

percentage of BRAF mutations in these CRCs, but also from

conventional adenomas. The predominance of a serrated CRC

pathway over the classical Wnt-pathway seems due to the numerical

prevalence of serrated polyps in HPS patients. From this it is inferred

that all polyp types in HPS, should be considered clinically relevant.

Furthermore, we showed at molecular analysis of lesions in the same

cohort that CRCs evolving through the serrated CRC pathway were


207

predominantly right-sided. Considering that CRC in HPS can be as

small as 4mm, it seems advisable to remove at least all polyps ≥3mm

and especially proximally located serrated polyps.

Endoscopic imaging – Detection and differentiation of polyps

Due to the increased risk of malignant progression of HPS polyps,

optimal endoscopic detection, differentiation and removal of in

particular high-risk polyps is necessary to prevent CRC development in

these patients. Chapter 7 showed that NBI significantly reduces polyp

miss-rates in HPS. To evaluate for which polyps NBI was particularly

of value, we compared miss-rates for different polyp histologies and

shapes. NBI was especially of value for the detection of flat serrated

polyps as opposed to conventional adenomas. Considering the

predominance of a serrated CRC pathway in HPS, our findings are

clinically relevant and support the implementation of NBI for the

endoscopic surveillance of HPS patients.

Chapter 8 evaluates the value of ETMI (high-resolution

endoscopy, NBI and autofluorescence imaging) for the real-time

endoscopic differentiation of polyps in HPS. None of the three

modalities rendered a sufficient diagnostic accuracy for the

differentiation of SSLs from HPs. Differentiation of adenomas from

HPs however was well possible with NBI but not with AFI. Based on

these findings, we conclude that ETMI offers insufficient diagnostic

tools to differentiate between high-risk SSLs from relatively ‘innocuous’

HPs but is of value for the differentiation of conventional adenomas

from HPs.

Sum

mary

& f

utu

re p

ers

pectives

208

FUTURE PERSPECTIVES

Research in this thesis provides additional supporting evidence for a

serrated CRC pathway in humans. This pathway is particularly

predominant in patients with HPS who have a high-risk of malignant

progression, even under unprotocollized endoscopic surveillance.

Currently, no uniform and adequately substantiated management

protocol exists for these patients. Prospective management studies,

based on previous clinical and molecular research, will hopefully

provide us with data with which we can construct a safe and effective

management protocol for these patients. In this light, adequate

endoscopic detection as well as real-time differentiation and removal of

only high-risk polyps in HPS would strongly increase management

efficiency. Regarding polyp detection, future multi-centre studies need

to be performed to evaluate whether NBI indeed increases polyp

detection in HPS. Concerning real-time differentiation, results from

recent studies analyzing polyp differentiation using NBI, AFI and even

endomicroscopy have been disappointing, especially regarding SSAs.

Future endoscopic techniques with which molecular characteristics

such as mutation- and methylation status (“molecular imaging”) can be

evaluated real-time during endoscopy may be of value in this respect.

Thus far, HPS patients have been mostly identified when

symptomatic (the reason for the endoscopy), and a large proportion of

them have a co-incident CRC at the time of diagnosis. With the recent

development of whole-genome sequencing, genome-wide association

studies are currently being performed in order to unravel the genetic

make-up and inheritance pattern of HPS patients. Based on these

findings a reclassification of HPS may be possible so as to identify

subtypes that have a higher risk of CRC development (e.g. patients

with a higher number of polyps or larger polyps) or a more dominant


209

inheritance pattern. Based on these findings we will hopefully be able

to recognize which individuals need to receive (more frequent)

screening colonoscopies. In addition, causative target genes may be

identified which in turn can be treated with specific gene-inhibitors.

Until then, prospective screening studies should be performed in first-

degree relatives of HPS patients to adequately assess the risk of HPS

in these individuals.

Sum

mary

& f

utu

re p

ers

pectives

211

Samenvatting

& Toekomstperspectief

Samenvatting & toekomstperspectief

213

Colorectaal carcinoom (CRC) is een aandoening die geassocieerd is

met een hoge mortaliteit. Voorgaande moleculaire studies bij patiënten

met FAP en Lynch syndroom toonden aan dat conventionele

adenomen pre-maligne lesies zijn die zich kunnen ontwikkelen tot

CRC. Progressie tot kanker in deze adenomen kan volgens

verschillende moleculaire routes verlopen waarbij verschillende

oncogenen en tumor-suppresor genen een rol spelen. Recentelijk is

een nieuwe CRC route voorgesteld waarbij niet adenomen maar

serrated poliepen een rol spelen. In deze serrated poliepen treden

alternatieve moleculaire veranderingen plaats welke leiden tot CRC. In

dit proefschrift onderzochten wij de voorgestelde “serrated neoplasia

pathway”. Hiervoor maakten wij gebruik van patiënten met

hyperplastische polyposis syndroom (HPS). HPS patiënten hebben

multipele serrated poliepen maar ook conventionele adenomen en

vormen hierom een unieke groep voor klinische en moleculaire

analyse van serrated poliepen en hun relatie met CRC.

Klinisch onderzoek – Colorectaal kanker risico

In hoofdstuk 2 van dit proefschrift beschreven wij de klinische en

pathologische eigenschappen van een groot multi-center HPS cohort

na meerdere jaren ongeprotocoliseerd endoscopische surveillance. Wij

toonden aan dat een derde van de HPS patiënten zich al presenteerde

met gelijktijdig CRC. Daarnaast werd er bij nog 7% van de HPS

patiënten CRC ontdekt, ondanks endoscopische surveillance (interval-

CRCs). Deze bevindingen zijn substantieel aangezien het geschatte

levensrisico op CRC in de algehele bevolking slechts 6% is. Bij

multivariaat logistische regressie bleken serrated poliepen en niet

adenomen significant geassocieerd te zijn met de aanwezigheid van

Sam

envattin

g &

toekom

stp

ers

pectief

214

CRC. Deze bevindingen ondersteunen de aanwezigheid van een

overheersend ‘serrated CRC pathway’ in HPS patiënten. Aangezien

carcinomen tijdens endoscopische surveillance ook werden ontdekt in

poliepen van slechts 4mm, lijken alle poliepen pre-maligne lesies te

kunnen zijn waardoor verwijdering van deze poliepen (in ieder geval

poliepen ≥3mm) tijdens jaarlijke endoscopische surveillance

geïndiceerd lijkt. Indien dit niet mogelijk is, dient chirurgische resectie

overwogen te worden.

In hoofdstuk 3 hebben wij een retrospectieve studie verricht,

betreffende het grootste cohort eerste-graads familieleden van HPS

patiënten waarbij we de incidentie van CRC en HPS onderzochten.

Deze incidenties werden vervolgens vergeleken met de algemene

bevolking door middel van persoonsjaar analyse. Zo konden wij het

gesuggereerde verhoogd familiair risico kwantificeren met een relatief

risico. Onze resultaten toonden een verhoogd relatief risico voor zowel

CRC (RR: 3.7-7.8) als HPS (RR: 13-121) in vergelijking met de

algemene bevolking. Hierom zijn screening coloscopieën voor deze

hele groep geïndiceerd zolang er geen genetische markers bekend

zijn die ons kunnen helpen hoog-risico individuen te identificeren.

Moleculaire analyses – etiologie en colorectaal kanker routes

Hoewel een onderliggend genetische aandoening aannemelijk is, is er

tot nog toe geen genetische oorzaak voor HPS ontdekt. Er zijn echter

studies beschreven waarbij patiënten met een bekende genetische

aandoening multipele serrated poliepen hadden.

In hoofdstuk 4 onderzochten wij de aanwezigheid van serrated

poliepen in MYH-associated polyposis (MAP) en of deze poliepen

causaal gerelateerd zijn met MAP. Poliepen in MAP bestaan

voornamelijk uit conventionele adenomen en zeer zelden uit serrated


215

poliepen. Bij herevaluatie van alle poliepen in ons cohort MAP

patiënten vonden wij echter dat bijna de helft van de patiënten

minstens 1 serrated poliep had en dat 18% ≥ 10 serrated poliepen

had. Bij moleculair onderzoek zagen wij dat serrated poliepen causaal

gerelateerd waren met MYH-deficiëntie doordat gevonden mutaties in

deze poliepen bijna exclusief uit specifieke G:C→T:A transversies in

het KRAS gen bestonden. Dit suggereert dat verschillende pathways

operatief zijn in MAP, namelijk APC-gen gerelateerd in adenomen en

niet-APC gerelateerd in serrated poliepen.

Vergelijkbaar met MAP bestaan poliepen in patiënten met

Lynch syndroom die een kiembaan mutatie hebben in een van deze

MMR genen voornamelijk uit conventionele adenomen. In voorgaande

studies zijn er echter ook serrated poliepen beschreven bij deze

patiënten. Hierop volgende moleculaire studies, die met behulp van

immunohistochemie onderzochten of serrated poliepen causaal

gerelateerd zijn met Lynch syndroom, waren niet conclusief.

In hoofdstuk 5 werd de aanwezigheid van serrated poliepen in het

grootste cohort genetisch bewezen Lynch syndroom patiënten

beschreven. Om een causaal verband te onderzoeken maakten wij

naast immunohistochemie gebruik van mutatie analyse (APC, KRAS

en BRAF). Een associatie tussen Lynch en serrated poliepen zou

kunnen wijzen op een versnelde CRC route in deze poliepen.

Aangezien Lynch-geassocieerde CRCs zelden BRAF gemuteerd zijn,

is BRAF analyse een manier om onderscheid te maken tussen Lynch-

geassocieerde CRCs en sporadische CRCs. Wij veronderstelden dat

een hoge frequentie BRAF mutaties in serrated poliepen, vergelijkbaar

met die van een controle groep sporadische serrated poliepen, erop

zou wijzen dat deze poliepen niet geassocieerd zijn met Lynch. Wij

toonden aan dat serrated poliepen in patiënten met Lynch-HPS (≥10

Sam

envattin

g &

toekom

stp

ers

pectief

216

serrated poliepen) niet geassocieerd zijn met Lynch vanwege de hoge

frequentie BRAF mutaties in deze poliepen. In tegenstelling, serrated

poliepen van Lynch patiënten met <10 serrated poliepen hadden

signicant lagere frequenties BRAF mutaties dan in de controle groep.

Gezien deze lage frequentie BRAF mutaties in patiënten met weinig

serrated poliepen, kan een causaal verband tussen deze poliepen en

de MSI-pathway niet worden uitgesloten. MSI-pathway geassocieerde

serrated poliepen zouden precursor lesies kunnen zijn die zich nog

sneller ontwikkelen tot CRC dan serrated poliepen die alleen

geassocieerd zijn met de serrated CRC pathway. Vanwege de unieke

diversiteit aan precursor lesies in HPS was het tot nog toe onduidelijk

welke poliepen zich tot CRC ontwikkelen in HPS en welke poliepen

derhalve klinisch significant zijn. In hoofdstuk 6 onderzochten wij

welke pathways operationeel zijn in HPS. Hiervoor onderzochten wij

de histologische en moleculaire eigenschappen van alle beschikbare

CRCs in een groot cohort HPS patiënten. Wij leverden nieuw

ondersteunend bewijs voor een serrated CRC pathway door in

combinatie (serrated poliep–CRC) lesies, identieke BRAF mutaties

aan te tonen in beide componenten. Tevens toonden wij aan dat

microsatteliet stabiele en –instabiele CRCs in HPS zich voornamelijk

ontwikkelen via een serrated CRC pathway en in mindere mate via de

Wnt-pathway aangezien de meerderheid van de CRCs een BRAF

mutatie hadden. De overheersing van een serrated CRC pathway ten

opzichte van de klassieke Wnt-pathway lijkt te verklaren door een

numerieke meerderheid aan serrated poliepen in HPS. Hierom lijken

alle histologische subtypes klinisch relevant te zijn. CRCs die zich

ontwikkelden via de serrated CRC pathway waren voornamelijk

rechtszijdig. Aangezien CRCs in HPS tot 4mm klein zijn beschreven


217

lijkt resectie van alle poliepen ≥3mm en voornamelijk proximaal

gelocaliseerde serrated poliepen geïndiceerd.

Endoscopische imaging – Detectie en differentiatie van poliepen

Gezien het verhoogde risico op maligne progressie van HPS poliepen

is endoscopische detectie, differentiatie en resectie van in het

bijzonder hoog-risico poliepen noodzakelijk om CRC ontwikkeling te

voorkomen. Hoofdstuk 7 demonstreerde dat NBI het percentage

gemiste poliepen significant vermindert in HPS in vergelijking met

high-resolution endoscopie (wit-licht). Om te evalueren welke poliepen

minder gemist werden met NBI, vergeleken wij miss-rates voor

verschillende histologieën en vormen. NBI was vooral van

toegevoegde waarde voor de detectie van vlakke serrated poliepen en

niet voor de detectie van adenomen. Aangezien de serrated CRC

pathway domineert in HPS patiënten, zijn deze bevindingen van

klinisch belang en ondersteunen zij het gebruik van NBI voor de

endscopische surveillance van HPS patiënten. In hoofdstuk 8

beschreven we het gebruik van ETMI (high-resolution endoscopie, NBI

en autofluorescentie imaging) voor ‘real-time’ endoscopische

differentiatie van poliepen in HPS. Geen van de modaliteiten toonden

voldoende diagnostische accuratesse om hoog risico sessile serrated

lesions van relatief ‘onschuldige’ HPs te onderscheiden. NBI (en niet

AFI) was echter wel waardevol voor de differentiatie tussen

hyperplastische poliepen en adenomen. S

am

envattin

g &

toekom

stp

ers

pectief

Acknowledgements

219

Acknowledgements

Acknowledgements

221

ACKNOWLEDGEMENTS

Het laatste hoofdstuk van dit proefschrift, maar misschien ook wel het

belangrijkste hoofdstuk van dit proefschrift. Niet omdat het voor

sommigen van u ook meteen het enige is dat u zult lezen, (uiteraard

neem ik u hiervoor absoluut niet kwalijk) maar meer omdat ik hier de

kans krijg om alle mensen persoonlijk te bedanken die hebben

bijgedragen aan de totstandkoming van dit boekje.

Allereerst mijn twee directe begeleiders die van het begin tot het eind

mij hebben bijgestaan: Prof. dr. C.J.M. van Noesel en Dr. E. Dekker.

Where molecular meets clinical. Beste Carel, jij hebt me bijgebracht

wat moleculaire wetenschap is. Jouw open en rustige houding als ook

je positiviteit zijn enorm inspirerend geweest. Een ‘walk of despair’

richting jouw kamer transformeerde vaak tot een ‘walk of triumph’ de

kamer uit. Jij bent het voorbeeld van een academicus die hoogstaande

wetenschap met plezier bedrijft. Bedankt voor je onvoorwaardelijke

steun, laagdrempeligheid en de vrijheid die je me hebt gegeven. Beste

Evelien, jouw visie, enthousiasme en vrolijkheid zijn de fundering van

dit proefschrift. Hoewel het volgens mij onmogelijk is voor elk gewoon

mens om jou bij te benen, weet ik dat je dit ons allen niet kwalijk neemt

en ons indien nodig een zetje (of twee) in de rug zal geven als we te

ver achterblijven. Bedankt voor de kans die je me hebt gegeven om

met jou aan dit onderzoeksproject te werken. Ik hoop dat we in de

toekomst nog veel samen kunnen brainstormen en mooi onderzoek

kunnen verrichten.

222

Prof. dr. E.M.H. Mathus-Vliegen, beste Lisbeth, als gevestigd MDL-arts

met zeer veel kennis en ervaring omtrent erfelijke polyposis

syndromen, zijn jouw inhoudelijke adviezen en commentaar van groot

belang geweest bij dit onderzoeksproject. Bedankt voor de altijd

plezierige samenwerking.

Prof. dr. P. Fockens, beste Paul, in het begin was je nog wat plagerig

als het om serrated poliepen ging. Jouw suggestie om mijn eerste

artikel naar de “Journal of serrated polyps of zo..” te sturen zal ik dan

ook zeker niet vergeten (het blad bestaat nog niet overigens)! Bedankt

voor je belangrijke bijdrage aan dit boekje en met name bij de imaging

studies.

Dr. J.B. Reitsma, beste Hans, statistiek is ingewikkeld. Maar daarmee

is de kous niet af. Statistiek is verwarrend. Statistiek is frustrerend.

Maar statitistiek is ook van essentieel belang om de waarde van

onderzoeksresultaten te kwantificeren. Veel dank voor al je hulp met

de analyses, in het bijzonder hoofdstuk 3.

Beste mede-auteurs die nog niet werden vernoemd in dit dankwoord.

Hartelijk dank voor jullie bijdrage en kritische verbeteringen aan de

stukken van dit proefschrift: J.F.W.M. Bartelsman, A. Cats, S. van

Eeden, L.P. van Hest, M. Houben, J.J. Keller, J.J. Koornstra, E.

Kurpershoek, M. van Leerdam, V. Lemmens, T.A.M. van Os.

Geachte leden van leescommissie: Prof. dr. F. Baas, Prof. dr. W.A.

Bemelman, Prof. dr. G.A. Meijer, Dr. F.M. Nagengast en Prof. dr.

G.J.A. Offerhaus. Veel dank voor uw bereidheid dit proefschrift op zijn

wetenschappelijke waarde te beoordelen. Dear Dr. J.E. East, it is an

Acknowledgements

223

honour for me that you were willing to critically review my thesis and

that you will be present during my defence.

Moleculair analysten van het MD-Lab: Mirjam, Alex, Mireille en Jitske.

Zonder al jullie hulp was dit boekje nooit tot stand gekomen. Mirjam, jij

bent mijn lab guru geweest gedurende mijn tijd bij jullie. Veel dank

voor alle tijd die je in mij hebt gestoken en al je geduld bij de vele

mislukte proefjes maar vooral voor je opgewektheid, je energie en je

humor. Alex, bedankt voor al je hulp bij de immuno’s en het flinterdun

snijden van piepkleine poliepjes. Jij bent niet alleen de koning op de

woensdag! Mireille, bedankt voor je hulp bij de MSI en methylatie

analyses zonder jou was het een hopeloze onderneming geweest.

Jitske, jij bent een allrounder waar ik vaak met vragen terecht kon –

bedankt hiervoor en de mooie woordenwisselingen..;) !!

Mijn dank aan mijn collega-artsonderzoekers van de Tytgatsuite

waarmee ik het geluk had om een kamer te delen: Anne, Simone en

Rogier jullie zijn pionier-Tytgatters en zullen dat altijd blijven. Latere

versterkingen: Denny, bedankt voor het mooie up-to-date

dierennieuws; Koen en Thomas, jullie hebben het concept

kantoorgrappen tot een hoger niveau getild; Boney, veel succes met je

eind-examen (na de school-onderzoeken natuurlijk). Jullie zijn allen

dierbare collega’s geworden en ik weet zeker dat we ook in de

toekomst met veel plezier vele jaren samen zullen werken. Alle andere

collega onderzoekers: Wouter, Bart, Renée, Wout (dude), David, Joep,

Annikki, Charlotte, Noor, Tessa, Willemijn, Susan, Esmerij, Wietske,

Sjoerd, Claire, Babette, Olivia, Femme en Emma, bedankt voor de

gezellige tijd samen!

224

Collega’s van B1-245. Het was zoals jullie weten de eerste dagen

behoorlijk wennen op de kamer. De stilte was oorverdovend en een

speld had geen kans om onomgemerkt op de zacht maar oud ex-

bestuurstapijt te landen. Maar gelukkig kon uiteindelijk hard getik

afgewisseld worden met overleg, goede (en slechte) verhalen, koffie

pauzes en een sporadisch youtube filmpje. Bedankt voor een

fantastische tijd samen roomies. Persoonlijk bericht voor jullie:

Frans van der Pants, bedankt voor ons vele overleg, gezelligheid en

hulp bij hoofdstuk 7 en 8. Ik weet zeker dat je een koningschirurg zal

worden! Roos Poe, ontzettende tofferd met je Apple laptop én je

‘gewone’ PC. Ik ben benieuwd welk boek je nu weer aan het lezen

bent. Freddepet, nog steeds als laatste naar huis? Lorenzio, bedankt

voor je mooie verhalen over het mooie leven van Spaanse stieren en

hoe ze uiteindelijk Spaanse worst worden. Nadine, veel succes met je

promotie! Teaco en Yark, met jullie was er gelukkig wel iets frequenter

een kort intermezzo in te lassen.

Yark, jij bent de nieuwe HPS/SPS koning. Ik vond het erg gezellig met

je samen te werken en wens je veel succes met de Eclipse, de

PROTECT en alle andere lopende en toekomstige studies. Hopelijk

kunnen we geregeld brainstormen over nieuwe studies en lopende

zaken onder het genot van een biertje.

Christine Cohen, jij hebt het overzicht, jij kent de patiënten en jij weet

wie aan welke studies deelneemt. Bedankt voor al je hulp, positiviteit

en enthousiasme de afgelopen jaren!

Lisbeth Mathus-Vliegen, Ellen de la Mar en Monique Dijselhof. Om de

nodige onderzoekscentjes te verdienen heb ik het eerste half jaar met

Acknowledgements

225

veel plezier met jullie samengewerkt aan een fase-2 studie betreffende

een afslankmiddel bij obese patienten. Hoewel het inhoudelijk vrij

weinig met poliepen te maken had, was deze tijd met jullie erg

bijzonder waarbij we veel plezier hebben gehad samen. Om een paar

episodes te noemen: spookrijden in Genève, de altijd verloren bril van

Ellen etc. Laten we vaak blijven afspreken.

Mijn paranimfen: Laurien, jij hebt al vanaf mijn eerste dag als

onderzoeker gezeurd dat je mijn paranimf wilde zijn. Daar kon ik niet

om heen en stiekem zou ik dat ook niet willen. Je bent mij erg

dierbaar! Patrick, huisarts living the good life, bedankt voor je

onvoorwaardelijke vriendschap en we gaan zeker binnenkort de

Himalaya trek doen samen.

Al mijn vrienden waarvan sommigen nog steeds niet weten waar ik

onderzoek naar heb gedaan. Bedankt voor de goede tijden samen, de

afleiding, ontspanning en jullie vriendschap. Biram old man, you

deserve a special mention. Thank you for being my best friend. You

like to explore different paths of life but I am confident that you will

eventually make your own path. Don’t stop making lemonade!

My family, extended family and close friends in India and dispersed

around the globe: although we are far away from each other, I luckily

get to see you regularly. Despite the distances we will always remain

close!

226

The Boparai family:

My parents, Ma and Pa, without you I wouldn’t be where I am today.

There are so many things to thank you for but most importantly I thank

you for being the best parents I could ever wish for.

Neelu, you snaggle tooth-vulture, thank you for being an ever caring

elder sister. I am glad you have Ed, Kiran and little Tej to take care of

you a little too.

Mohini (monu), little-big sister, world-traveller and carnivore of the

highest order, I hope we will still be laughing at the same jokes when

we are old wrinkled prunes.

Acknowledgements

227

CURRICULUM VITAE

Karam Boparai was born in Purmerend, the Netherlands, on the 31st

January 1981. Here he attended high school at the Jan van Egmond

College. After graduating in 1999 he ventured to Delft to study

Mechanical Engineering. After realizing that this was not his calling, he

promptly decided to study Medicine at the University of Amsterdam

(UVA). During his studies he grew increasingly interested in the field of

Gastroenterology and Hepatology. This interest led him to Dr. Evelien

Dekker and Prof. dr. Carel van Noesel at the Academic Medical Center

in Amsterdam with whom he performed a research project regarding

serrated polyps and their association with MYH-associated polyposis.

After graduating from Medical school in March 2007 he joined the

department of Gastroenterology and Hepatology to continue both

clinical and molecular research concerning the Serrated Neoplasia

Pathway under supervision of Dr. Evelien Dekker, Prof. dr. Carel van

Noesel, Prof. dr. E.M.H. Mathus-Vliegen and Prof. dr. P. Fockens

which led to this dissertation. He was (co)applicant of the successful

grant proposal to the KWF which will further focus on the clinical

management of patients with serrated polyposis syndrome. Since

October 2010, Karam is doing his Internal Medicine residency at the

Onze Lieve Vrouwe Gasthuis in Amsterdam. He will commence his

training as Gastroenterologist at the Onze Lieve Vrouwe Gasthuis and

the Academic Medical Center from October 2012.

Curriculum Vitae

UvA-DARE (Digital Academic Repository) The …Histological polyp types Traditionally, colorectal...

Documents

Transcript of UvA-DARE (Digital Academic Repository) The …Histological polyp types Traditionally, colorectal...