I

STUDY OF TUMOR HETEROGENEITY IN COLORECTAL

CANCER BASED ON PROTEIC EXPRESSION

AND GENE MUTATION

ŞI MUTAŢIILOR GENICE

THESIS ADVISOR

PROFESSOR, PhD MIHAI CRUCE

OVIDIU MIRCEA ZLATIAN

CRAIOVA

2010

PhD Student

OVIDIU MIRCEA ZLATIAN

UNIVERSITY OF MEDICINE AND PHARMACY

OF CRAIOVA

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INTRODUCTION

Colorectal cancer (CRC) is the second cause of mortality by cancer in Europe and has a

high incidence, concordant with prolonging the life expectancy.

A major challenge in understanding the colorectal cancer is the characterization of

phenotypic and genotypic changes of the neoplastic cells. The main obstacle is the haotic

evolution of the neoplastic process, which is also the main cause of the failure of therapy, due to

the fact that sooner or later cellular chemoresistant clones are selected. One of the consequences

of this type of evolution is the divergence of daughter cells characteristics which induce the

tumor heterogeneity phenomen.

By definition the intratumor heterogeneity supposes the onset of differences between

neoplastic cells during tumor development. It was proved the existence of cell subpopulations for

every histological type and location of the cancer, both in humans and in experimental disease in

animals.

The tumor heterogeneity has a theoretical importance in understanding the global

evolution of neoplastic process, but also in establishing the histological and molecular

phenotypes and further in assignment and conduction of therapy. Cell subpopulations

therapeutically targeted are contigous both spatially and as origin and act as ecosystems which

manifest characteristics transcending those of individual cells.

In order to understand the development and evolution of such systems a fusion of

methods of population biology and those of cell biology is needed, for the purpose of

deciphering the fundamental causes which underlie the onset of heterogeneity and the common

denominator of these populations. The study of the general phenomen of tumor heterogeneity

suppose the investigation of the causal relations between phenotype and genotype.

In order to experimentally study the tumor heterogeneity I proposed first to examine the

epidemiology of colorectal cancer and the risk factors involved in colorectal oncogenesis, the

morphopathology of colorectal cancer and the molecular mechanisms involved in oncogenesis

and tumor progression.

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I established as first experimental objective the statistical evaluation of prevalence of

colorectal cancer in the Oltenia area and determination of the possible correlations between

clinical and pathological parameters of the patients with colorectal cancer.

A second objective is the examination of the phenotypic expression of tumor

heterogeneity by an immunohistochemical study of the proteins expressed by neoplastic cells

from the colorectal tumors.

The correlation of observed phenotypic heterogeneity with the genotypic heterogeneity

which determines it was the third objective of the present thesis which proposes the detection of

some mutations of the Kras and p53 genes by molecular methods, due to their important role in

tumor initiation and progression.

Keywords: colorectal cancer, immunohistochemistry, tumor heterogeneity, Kras and

p53 mutations.

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I. STATE OF KNOWLEDGE

In Chapter I – The epidemiology and risk factors of the colorectal cancer I presented

the geographical distribution of CRC I presented the geographical distribution of the CRC

prevalence, which is raised in developed countries, and also that of CRC mortality, increased in

countries with low or medium economical level, due to the poor access to performant diagnosis

and therapy services (Coleman, Quaresma, 2008). I showed the time variations of these

variables, reffering to Romania (Zavoral, Suchanek et al, 2009). Then I described the risk factors

for colorectal cancer, both those which belong to the patient (age, intestinal inflammatory

diseases, race, ethnicity, genetic disorders), as well as belonging to environment (alimentation,

obesity, smoking status, alcohol consumption, diabetes, infectious factors).

In Chapter 2 – Morphopathology of colorectal cancer I described the anatomy and

normal structure of the colon and rectum, from the macroscopic and microscopic perspective. I

presented the main premalignant lesions of the colorectal mucosa and I showed the

morphological steps of the transition from the normal mucosa to colorectal benign tumors. I

summarized the main colorectal malignant tumors, with a short macroscopic and microscopic

description. Then I presented the main immunohistochemical markers used to phenotype and

evaluate the prognosis of colorectal tumors. Finally, I analyzed the natural evolution of

colorectal cancer, the local (invasion) and at dinstance (metastazing) extension. .

În Chapter 3 – The colorectal cancer genetics I described the most important genetic

phenomena which determine the initiation and progression of CRC, by presenting the main

mechanisms which underlie the genomic instability, which induce the evolution of colorectal cancer

by facilitating the acquisition of mutations associated with malignant phenotype. I presented the main

tumor suppressing genes affected by mutations, with the observation of the high number of genes

mutated in CRC, but for most of them the mutations frequency is small, which implies an enormous

colorectal cancer heterogeneity, which refelects the heterogeneity of clinical aspects. Then I showed

some modifications of tumor cell biology, like activation of growth factors pathways or alterations in

regulatory mechanisms of the stem cells. Finally I discussed the applications of colorectal cancer‟s

molecular biology data in detection, prognosis and therapy.

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II. OWN CONTRIBUTIONS

CHAPTER 4. CLINICAL AND STATISTICAL STUDY OF THE CASES

WITH COLORECTAL MALIGNANT AND BENIGN TUMORS

I performed a statistical anlysis for the purpose of evaluating the prevalence of CRC in

the Oltenia area and correlation with clinical and pathological parameters of the patients.

BIOLOGICAL MATERIAL

I studied 788 patients with malignant and benign colorectal tumors, from whom were

preleved biopsies in clinical or internal medicine clinics of the Regional Clinical Emergency

Hospital of Craiova, Romania, between the years 2003-2009. By removing the patients with

incomplete date, I selecten for the study 650 patients, from whom 606 had malignant tumors

(334 men and 272 women).

METHODS

By descriptive statistics I discovered correlations between patient age, tumor location,

macroscopic aspect, grade and histological type, which were verified by correlative statistic, and

those with a high semnification were more deeply analyzed. As statistical methods I used the

simple Pearson correlation and that based on rank (Spearman). In order to detect differences

between groups of patients I used the Student t-test and for nominal data I utilized non-

parametric tests (CHI squared and Fischer‟s exact test for small groups).

The statistical calculations and graphics were executed using the software packages SPSS

17 (SPSS Inc., Chicago, IL, USA) and Microsoft Excel 2010.

RESULTS AND DISCUSSION

Regarding the location of tumors, CRC had a high incidence in sigmoid colon (217 cases

– 35,81%) and rectum (268 cases – 44,22%). I observed an increased incidence in advanced

ages, as the mean age was 64,77 years, without significant sex differences (p=0,619).

I also noticed thatfor the first colic segments (from cec to descendent colon) the incidence

of CRC is higher in women, and the situation is inverse for sigmoid and rect, the report

women/men is 0,85 for cec and 1,72 for rect (Figure 1), the association between patients sex and

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rectal location is statistically significant. The tumor location was also associated with the

differentiation grade (p=0,0650).

Figure 1. The distribution of the studied cases by tumor location and patients’s sex

The patients‟s age significantly differ according to localization of tumors (ANOVA test‟s

p=0,190), the difference between colonic and rectal tumors is significant.

Vârsta pacienţilor diferă semnificativ în funcţie de localizarea tumorilor (test ANOVA,

p=0,190), diferenţa între tumorile cu localizare colonică şi rectală fiind semnificativă (p = 0,015).

Most colorectal carcinoma (68,48%) had a moderate grade of differentiation. I

demonstrated that the histopathological type of papillary carcinoma is strongly associated with

well-differentiated grade (CHI square‟s p<0,0001), and that the infiltrative and stenosant forms

are almost always associated with intestinal wall invasion (invaded in 132 from the 138 cases

with these macroscopic forms, CHI square‟s p<0,0001) and of organs in the neighbourhood (28

from 138 cases, CHI square‟s p = 0,0256 <0,05). Also the invasion of the organs was present in

23,08% of the poorly differentiated carcinoma.

The logistic regression showed a significant influence (p<0,05) of tumor location (B=-

0,233) and grade (B=+0,302) on the presence of lymphnodes invasion, results similar with other

studies (Chou, Row et al, 2010). In our study ande statistic modeling had a rate of correct

prediction of 65%, not very high but which justify using the statistical model for estimating

lymphnodes invasion.

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CHAPTER 5. HISTOCHEMICAL AND IMMUNOHISTOCHEMICAL

UNVEILING OF TUMOR HETEROGENEITY IN COLORECTAL CANCER

Immunohistochemistry plays an important role in differentiating tumor types, assessment

of agresiveness and recognizing of metastasis origin. Although the molecular analysis are

increasingly used, many therapeutical protocols are still based on histological types and

immunohistochemical phenotypes. From these reasons I performed a histological study, a

necessary step in every research regarding the characterization of tumor heterogeneity.

BIOLOGICAL MATERIAL

The patients were randomly selected from our cases with a histopathological diagnosis of

colorectal cancer, namely 50 cases: 27 men (54%) and 23 women (46%), with a mean age of

59,7 years (41-79 years). For each case clinical and pathological informations were recorded:

age, sex, tumor location, histological type and tumor grade.

METHODS

The samples were obtained from surgery, were formalin-fixed and included in paraffin

using the classic protocol. Afterwards, the blocks were cutted at the microtome in sections 3-5

µm thick.

The histochemical and immunohistochemical study was performed in Pathological

Anatomy laboratory from the national “Victor Babes: Institute from Bucharest, Romania.

Histochemical methods. The sections were stained with haematoxiline-eosine and were

examined using the Zeiss Axiostar Plus microscope and the Olympus camera, resolution 5 MP.

Immunohistochemical methods (IHC). For the IHC study we used the sections which

were sheeted on glass slides coveredwith polilysine. The slides were processed using the three

stages method Avidin - Biotin Peroxidase (ABC) (HSU 1981). We used as IHC markers:

cytokeratins (CK7, CK20), mucins (MUC1, MUC2), Ki-67, PCNA (Proliferaating Cells Nuclear

Antigen), P53, KRAS (Kirsten-RAS), BCL2, PTEN (Phosphatase and Tensine homologue) ,

EGFR (Epidermal growth factor receptor.

RESULTS AND DISCUSSION

Hematoxiline-eosine stain permitted the assessment of microscopical grade of the

studied adenocarcinoma: well (figure 2A), moderated and poorly differentiated (figure 2B), the

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most frequent grade was G2. Histologically, most adenocarcinoma haven‟t special

characteristics, 9 were mucinous, 8 coloid and 1 with signet-ring cells.

A B Figure 2. A. Well differentiated adenocarcinoma with infiltration in submucosa,

adiacent to normal mucosa. HE stain, 100x. B Poorly differentiated colonic adenocarcinoma –

solid area of tumor cells without glandular differentiation. HE stain, 100x.

IHC detection of cytokeratins detected the expression of cytokeratin 20 in most cases

(80%) with a focal pattern in tumoral cells, and of CK7 in 8 cases with diffuse pattern (figure

3A). 78% of adenocarcinoma presented the CK20+/CK7- phenotype, other studies identified this

phenotype in 65,85%. The phenotype CK20-/CK7+ was identified in 5 cases (10%) on the

proximal colon, in the literature being reported the absence of this phenotype in distal colon.

Also in my study I found 3 cases (15,78%) with expression of both cytokeratins (CK20+/CK7+),

distally located, other studies of adenocarcinomas reporting his presence in 22% of the cases.

Expression of mucins (MUC 1 and (MUC2) in normal colorectal mucosa has the

pattern MUC2+/MUC1-, phenotype present in only 18%of the cases, which shows the frequency

of alterations of mucin expression in adenocarcinoma. We also identify the following patterns:

MUC1+/MUC2- in 38% cases, MUC1+/MUC2+ in 36% cases and MUC1-/MUC2- in 8% cases

(figure 3B). Procentul ridicat de expresie al proteinei MUC1 (74%) este frecvent întâlnit în

carcinoamele colorectale.

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A B Figure 3. A. Expression of citokeratin 20 in a well differentiated colorectal adenocarcinoma

(IHC stain, 100X). Focal positivity in tumor cells and difuse in normal epithelial cells. B. Expression

of MUC2 mucin in a case of moderated differentiated adenocarcinoma (IHC stain, 200X). The

positivity is present in cytoplasm and membrane of the tumor cells.

Ki-67 expression was detected in all the studied cases with indexes between 10 and 95%

(figure 4A), with increased intratumoral heterogeneity, with great differences between indexes on the

same section. I observed a big number of Ki-67 positive cells in the tumoral glands. It couldn‟t be

established a statistical significant relationship between positivity index for Ki-67 and histological

grade (ANOVA analysis for groups with differentiation G1, G2 and G3, p=0,014, < 0,05).

PCNA expression was also present in all cases with various degrees of intratumoral

heterogeneity, both at the level of whole tumor as well as in the malignant glands (figure 4B). the mean

index of PCNA was 15-30% in well differentiated adenocarcinoma, 25-60% in moderated

differentiated and 50-90% in poorly differentiated. It was observed a significant correlation between

PCNA index and differentiation grade (ANOVA analysis for the three grades, p=0,085, >0,05).

A B Figure 4. A. Expression of Ki-67 in a case of moderated differentiated adenocarcinoma

(IHC stain, 100X). Positivity index:70% of the tumor cells. B. Expression of PCNA in a moderated fifferentiated adenocarcinoma, positivity index: 70%.

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P53 expression was observed in 43 cases (86%), the positivity index varied between 5

and 80%, previous studies (Bousserouel , Kauntz et al, 2010) reported between 30% and 60,6%

positive cases. P53 supraexpression wasn‟t identified in normal tissue adiacent to the tumor. The

positivity was relative homogenous in tumoral mass (figure 5), in sections at different levels the

indexes were similar (low intratumoral heterogeneity).

In the interpretation of these results we must account for that immunohistochemically

detected supraexpression of P53 protein means an accumulation in the cells. But not all mutations of

p53 gene lead to his accumulation, so it is possible the existence of false negative results.

Absence of tumor positivity for P53 was correlated with poor differentiation (p<0,001), as

other studies found (Carneiro, Ramalho et al, 2006). The supraexpression of P53 was associated with

raised mean indexes of PCNA and Ki-67 (Student t-test, p=0,0042 and p=0,0025, respectively),

suggested that the P53 protein accumulated in cells which already had a growth advantage by

mutation which increase the proliferation rate.

By correlating these results with those of molecular analysis, we observed that the

positivity for P53 wss present in all cases in which were investigated the p53 gene‟s

polymorphisms. 5 of the 6 samples with positivity index above 50% had the Pro/Pro allele

(present in 50% of the studied cases, Fisher‟s p = 0,017). We consider that this associations is

due mainly to high frequency of Pro/Pro allele in colorectal cancer patients, rather than a link of

this polymorphism with the immunohistochemical expression of P53.

A B

Figure 5. Colon adenocarcinomas marked for P53 (IHC stain, 100X). A. Well

differentiated adenocarcinoma, P53 index: 80% of tumor cells, with adiacent negative normal

tissue. B. Moderately differentiated adenocarcinoma, P53 index:70% of tumor cells.

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The KRAS protein has a positivity index between 5 and 95%. The immune rewaction

was considered positive when at least 30% of the tumor cells were marked. The expression of

KRAS protein was identified to 26 (52%) of the cases (figure 6A), most indexes were over 50%.

These percents are close to those reported by similar studies (Cejas, Lopez-Gomez et al, 2009).

The Kras expression was signicantly raised in poorly differentiated adenocarcinoma (6 cases of

9, p=0,037), possibly due to their rapid replication and increased chance to accumulate

oncogenic mutations.

By correlating Kras mutations detected by molecular methods (33,33% cases) with

KRAS expression (52 % cases) it can be observed that in 3 (75%) of the 4 cases with KRAS

mutations, the expression was increased with a positivity index over 70%. Other studies of this

relation showed a significant increase of Kras expression in the cells with KRAS mutations

(p=0,010).

The high index of positivity for KRAS and P53 in these patients suggests that the

mutations of these 2 genes are present in the majority of tumor cells (low intratumoral

heterogeneity), meaning that a single cell clone is responsible for tumor development. In order to

confirm this hypothesis are needed studies with both markers and analysis of double-marked

cells.

A B Figure 6. A. Well differentiated adenocarcinoma with positivity for KRAS protein (IHC

stain, 100X). KRAS positivity index: over 80% of the tumor cells. Next to the tumor it can be

observed a normal tissue negative for KRAS. B. Colon adenocarcinoma marked for BCL2 (IHC

stain, 100X). Positivity for KRAS in the cytoplasm of tumor cells and also in lymphocytes.

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The BCL2 expression was identified in a small number of cases (12%) and varied in

intensity. The positivity pattern was predominantly cytoplasmatic, more pregnant in the apical

region, although occasionally was identified perinuclear positivity (figure 6B). the marked tumor

cells were mostly located within the tumoral glands from the parabasal and superficial area of

tumors. Although the tumoral positivity was heterogenous, the lymphocytes were positive in all

sections examined (internal positive control).

Expression of nuclear protein PTEN was investigated in 12 cases. The 8 positive cases

(66,67%) were well differentiated adenocarcinomas and the 4 negative cases were 3 moderately

differentiated and 1 well differentiated adenocarcinomas. The normal colorectal epitelia adiacent

to tumor areas has a nuclear positivity for PTEN (figure 7A).

It was showed that PTEN supression is induced by RAS/ERK pathway, which explains

why from the 4 negative cases 3 were positive for KRAS.

S-a arătat că supresia PTEN este indusă prin calea RAS/ERK, astfel putându-se explica

faptul că dintre cele 4 cazuri negative pentru PTEN, 3 au fost pozitive pentru KRAS.

EGFR was identified in 21 cases (42%). The EGFR was also positive in blood vessels

(figure 7B) which demonstrates microangiogenesis in invasion front and tumoral glands.

A B Figure 7. A. Expression of PTEN in a moderately differentiated adenocarcinoma (IHC

stain, 100X), nuclear positivity both in tumor cells as well as in the stromal and inflammatory

cells(internal positive control). B. Expression of EGFR in a colorectal adenocarcinoma (IHC

stain, 100X). Staining is negative in tumor cells and positive in blood vessels.

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CHAPTER 6. DETECTION OF ACTIVATING MUTATIONS IN CODONS

12 AND 13 OF THE KRAS GENE INVOLVED IN COLORECTAL

PATOLOGY

Kras mutations occur in 30-45% of CRC patients and are restricted to certain codons. The

most frequent mutations are in the 12 and 13 codons from exon 2, rarely in codons 59 and 61

from exon 3 (Bousserouel, Kauntz et al, 2008; Bukhari, Niazi, 2010). We proposed to elucidate

the correlation between Kras mutations in 12 and 13 codons in patients with colorectal cancer

and some clinical and pathological characteristics.

The molecular analysis of Kras and p53 mutations by PCR-RFLP method was performed

in the Molecular Genetics Laboratory from “Victor Babeş” National Institute, Bucharest,

Romania.

BIOLOGICAL MATERIAL

The molecular tests were performed on bioptic samples obtained by colonoscopy from 12

patients with colorectal adenocarcinoma (test lot) and 12 patients with other inflammatory colic

disorders (control lot), formalin fixed and included in paraffin blocks. We tried to match the age

and sex structure of the control lot with that of the test lot (p coefficients > 0,05) in order to

extract valid conclusions from small lots.

METHODS

Genomic DNA extraction used the QIAamp DNA Mini Kit (QIAGEN). For every

sample we used 10-20 sections 5 μm thick. The evaluation of DNA quantity and purity was

performed by ultraviolet spectrophotometry (figure 8).

The PCR-RFLP assay was performed according to literature recommendations. PCR

reactions were performed in a final volume of 25 μL, containing 1X reaction buffer,

deoxinucleotide triphosphate (dATP, dCTP, dGTP, dTTP) 0,2 mM, magnesium chloride 1,5

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mM, primers 1 μM and 1,5 units “DNA Taq polymerase” (Promega). All PCR reactions were

performed using the thermocycler Gene Amp PCR System 9700 (ABI).

Enzymatic restriction reaction can detect the point mutations which create or destroy a

recognition situs for restriction enzymes. The result is a restriction fragment lengh polymorphism

(RFLP).

In order to analyze Kras mutations in codons 12 and 13 we performed enzymatic

restriction with the enzymes Mva I (Microccocus varians) and Hae III (Haemophilus

aegypticus).

Separation of restriction products was performed by submerse electrophoresis in agar

gel 2-4% i TBE at/cm, during 30-60 minutes.The migrated nucleic acids were visualized in UV

light after staining with ethidium bromide.

RESULTS AND DISCUSSION

Spectrophotometry showed DNA genomic concentrations above 1200 ng/ µl in all

cases, and values of the fraction Abs. 260 / Abs. 280 between 1,70 and 2,06 (figure 8).

Figure 8. Spectrofotometric evaluation of purity and concentration of DNA extracts

obtained with QIAamp Tissue Kit (Qiagen) from formalin fixed and paraffin included tumoral

tissues. Sample 1 (patient D.M , male 65 years, diagnosed with colonic adenocarcinoma).

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The PCR-RFLP method for Kras’s codon 12 mutations showed: a DNA fragment of

106 bp corresponding to the normal allele, without mutations in codon 12 („wild type”) and a

135 bb fragment which corresponds to mutant allele, which couldn‟t be cleaved by the restriction

enzyme because codon 12 mutation modified the recognition situs for MVA I enzyme (figure 9).

Figure 9. Agar gel electrophoresis in TBE 1X on agar gel of products obtained by PCR-

RFLP analysis for detecting mutations in Kras gene. 1 and 3: samples with mutation in codon

12; 2: sample without mutation in codon 12; 4: k- Kras 12 negative control sample; 5: k+ Kras

12 positive control sample; M: 100 bp DNA Ladder (Fermentas); N: unrestricted PCR

amplicon; R: PCR amplicon after enzymatic restriction with Mva I enzyme (Fermentas).

By PCR-RFLP analysis 4 (33,33%) of the 12 colorectal adenomas analyzed presenting an

activating mutation in the codon 12 of the Kras gene, compared with one sample (8,33%) in the

control lot, percentages which enframes between those from literature (35-55%).

The Kras mutations were more frequent in proximal colon (33,33% compared with

16,67%), but due to the small number of cases we can‟t prove the significance. The wild

phenotype was more frequent in females compared with males.

After performing the PCR-RFLP analysis for detecting mutations in codon 13 of the

Kras gene, for all analyzed samples we obtained 3 DNA fragments of 85, 48 and 29 bp

respectively, corresponding to the wild-type allele, without mutation. If it would be present a

mutation in codon 13 we obtained just two DNA fragments, one of 74bp and the other of

85 bp (figure 10).

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Figure 10. 1, 2, 3: samples without mutation in codon 13 of the Kras gene; 4: k- Kras 13

negative control sample; 5,6: k+ Kras 13 positive control sample; M1: 25 bp DNA Step Ladder

(Promega); M2: GeneRuler 50 bp DNA Ladder (Fermentas); N: unrestricted PCR amplicon; R:

PCR amplicon after enzymatic restriction with Hae III enzyme (Fermentas).

In our study none of the 12 analyzed samples presented mutation in codon 13 of the

Kras gene. Taking into account the small number of analyzed samples and the frequency

between 7 and 10% of these mutations in CRC patients (Andreyev, Norman et al, 2001), our

result is plausible.

Regarding the prognostic importance of Kras mutations in CRC patients, it has been

shown that the glycin-valin mutation in codon 12 significantly decreases the surviving rate,

regardless of the therapy. There are controversies about association between Kras mutations

and prognosis in patients with metastases, which haven‟t been treated with anti -EGFR

antibodies, as some studies reports a significant correlation with unfavourable prognosis

(Cejas, Lopez-Gomez et al, 2006).

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CHAPTER 7. EVALUATION OF SOME POLYMORPHIC VARIANTS OF

THE P53 GENE IN COLORECTAL PATHOLOGY

The tumor suppressor p53 gene, located on 17p13 chromosome, is one of the most

frequently mutated genes in all human cancers. A lot of studies reported the presence of somatic

or ereditary mutations of p53 gene in CRC, their frequency reaching 45%. It is largely believed

that in half of human cancers the p53 gene is affected by somatic mutations (Markowitz,

Bertagnolli et al, 2009).

The genetic polymorphisms, especially uninucleotidic (Single Base Polymorphisms -

SNP) were associated with predisposition to several cancers, but our understanding of their

implication in pathogeny of colorectal cancer is still at the beginning. In p53 gene were

identified several such polymorphisms, both in coding and non-coding regions. We proposed to

study the distribution of some of these polymorphic variants in patients with colorectal cancer.

7.1 EVALUATION OF CODON 72 POLYMORPHISMS OF THE P53

GENE

SNP de la codonul 72 (Arg72Pro) este un polimorfism cunoscut de mult şi frecvent

întâlnit în cancerul colorectal. Alela Arg72 inhibă apoptoza, probabil datorită localizării

mitocondriale şi a fost asociată cu creşterea riscului de dezvoltare a tumorilor (Katkoori, Jia et al,

2007).

În acest studiu ne-am propus să studiem polimorfismele la nivelul codonului 72 al genei

p53 prin RFLP-PCR la lotul de pacienţi cu CRC comparativ cu lotul martor, pentru a studia

posibila asociere dintre aceste polimorfisme şi riscul de dezvoltare a CRC şi de asemenea pentru

a evalua corelaţia lor cu alţi factori clinico-demografici.

BIOLOGICAL MATERIAL

Am luat în studiu aceleaşi blocuri de parafină folosite pentru evaluarea mutaţiilor Kras,

provenite de la două loturi de pacienţi: 12 pacienţi cu CRC (lot test) şi 12 pacienţi cu afecţiuni

colice inflamatorii (lot martor).

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METHODS

Din fiecare bloc de parafină s-au realizat secţiuni de 10 μm, care au fost colectate în

tuburi de 1,5 ml. Dintre acestea am folosit două secţiuni pentru extracţia ADN.

Extracţia ADN genomic din ţesuturile înglobate în blocuri de parafină s-a efectuat după

un protocol preluat din literatură (Shi, Cote et al, 2009), constând în deparafinare, tratarea în

două bai de xilen şi două de alcool absolut, proteoliză şi separarea ADN prin precipitare.

Cantitatea şi calitatea ADN-ului rezultat au fost analizate spectrofotometric.

Reacţia PCR a folosit primeri cu specificitate pentru codonul 72 din exonul 4. Reacţia a

fost realizată într-un volum final de 25 μL care conţinea 50 ng ADN genomic, 1X PCR buffer

(Biotools) cu 2 mM MgCl2, 0,4 μM din fiecare primer (Genescript), 50 μM dNTP (Biotools) şi

0,5 U ADN polimerază.

Reacţia de restricţie enzimatică pentru analiza mutaţiilor din codonul 72 al genei p53,

produsele de amplificare PCR de 279 pb a fost restricţionat cu enzima BstUI (1 U la 37°C timp

de 16 ore)

RESULTS AND DISCUSSION

În cazul polimorfismului p53 Arg72Pro, alela sălbatică Arg /Arg a produs două benzi

(160 şi 119 pb), varianta Pro / Pro a fost identificată printr-o bandă unică (279 bp) şi varianta

heterozigotă Pro/Arg a produs trei benzi (279, 160, şi 119 pb) (figura 11).

Figura 11. Analiza RFLP a polimorfismului Arg72Pro al p53, prin folosirea enzimei

BstUI. Legenda: M: ladder alelic 100 pb, 1-6: produşi de digestie; Arg/Arg sălbatic este clivat de

BstUI rezultând două fragmente de 119 şi 160 pb, alela mutantă Pro/Pro produce un fragment

de 279 pb. 1 şi 6: forma heterozigotă Arg/Pro. 2 şi 3: forma mutantă. 4 şi 5: forma sălbatică.

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From our study resulted that the frequency of wild-type allele Arg/Arg for SNP Arg72Pro

was 41,66% in test lot and 33,33% in control lot, and the mutant Pro/Pro allele had a double

frequency in test lot. It can be seen a correlation (risk ratio: 2, p=0,249), sustained by previous

studies which reported a significant association of the mutant Pro/Pro phenotype with risk for

colorectal cancer. By correlating the p53 polymorphisms with clinical and pathological

characteristics, we observed that the Pro/Pro allele is associated with tumor location but not with

other variables.

Another result which can be of great importance is significantly increased frequency of

the Arg/Arg allele (risk ratio: 2,5, p=0,084) compared with Pro/Pro. This phenomenon was

associated with exposure to hypoxia or ultraviolet radiation. Another possible cause is the diet

and therefore, indirectly, the lifestyle.

We also detected a possible association between the frequency of Pro/Pro mutation and

distal location (p=0,242), but not with other variables (age, sex, habitate), results similar with

other studies (Bojesen şi Nordestgaard, 2008).

These correlations should be verified in the future on bigger lots, in the aim of obtaining

results usefull for determining the agresiveness of colorectal cancer.

The causal semnification of associations between any genetic polymorphisms with

colorectal cancer remains unveiled, especially for some SNP located in uncoding or unknown

regions of the genome.

The mutations of Kras and p53 genes haven‟t necessarily impact on their function and

for this reason we tried to correlate the presence of detected Kras mutations in codon 12 with

p53 polymorphisms in codon 72. from the 4 cases with mutations in codon 12 of the 12 of Kras

gene, two (50%) presented the Arg/Pro phenotype in codon 72 of p53, one case Arg/Arg and

one case Pro/Pro phenotype. Both cases with Kras muations associated with Arg/Pro phenotype

were males over 72 years of age. If we account for the increased frequency of Kras mutations

observed and of the phenotype Arg/Pro in advanced ages, is little probable an association

between age and mutational status of the two genes (Fisher‟s p=0,595).

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7.2 EVALUATION OF VAL217MET-RS35163653 AND GLY120ALA-

RS35993958 POLYMORPHISMS OF THE P53 GENE

In this study I demonstrated the utility of qRT-PCR method for detection of p53

mutations in DNA from paired samples: feces and biopsy. We used the TaqMan (Applied

Biosystems) probes for detecting of two p53 polymorphisms (Val217Met - rs35163653,

Gly120Ala - rs35993958), with little known clinical significance and unknown role in colorectal

cancer. The investigations of these polymorphisms were performed in Molecular Genetics

Laboratory from university of medicine and pharmacy of Craiova.

BIOLOGICAL MATERIAL

The studied patients had ages between 32 and 84 years with a mean of 68,17; the report

between females and males was 19:34 (42,19% women and 57,81% males). From the 53

patients, 5 were diagnosed with benigne tumors, one with Crohn disease, two with malignant

suspicion and the rest with colorectal neoplasms.

All samples were evaluated by a pathologist in order to establish the histological type,

differentiation grade and tumoral stage. From all patients biopsies were gathered from tumor, in

49 cases were also sampled feces for analyzing the fecal DNA.

METHODS

Isolation of genomic DNA from the tissue samples we used the Wizard® Genomic DNA

Purification Kit (Promega, Madison, WI), and for isolation of total DNA from feces the

QIAamp® Stool Mini Kit (Qiagen).

Quantification and purity determination of the isolated DNA was performed by

spectrophotometric method, with Eppendorf Biophotometer.

PCR reaction was performed in a final volume of 25 μL. We used TaqMan® Gene

Expression Master Mix and TaqMan® SNP Genotyping Assays (Applied Biosystems, Foster

City, CA), specific for every polymorphism studied. TaqMan Gene Expression Master Mix

contains AmpliTaq Gold UP (Ultra Pure) DNA polymerase, deoxiribonucleotide triphosphates

(dNTPs), ROX™ passive reference, as well as buffer components optimized for sensibility,

precision, specificity and reproductibility.

The cycling was performed with the system Real-Time Corbet RotorGene 6200 HRM.

The TaqMan Gene Expression Master Mix protocol indicates the execution of just 40 cycles in

21

the 1 minute attachement/extension, after that the amplification curves haven‟t entered in the flat

phase. Accordingly, we gradually increased the number of amplification cycles and the time of

the attachment/extension step, and we found optimal values of 50 cycles and 90 seconds.

The qRT-PCR was realized with TaqMan (Applied Biosystems) probes marked with

specific fluorescent dyes.

RESULTS AND DISCUSSION

After spectrophotometric check of extracted DNA purity we performed the qRT-PCR

reaction. The obtained data were processed (figure 12) using Rotor-Gene 6000 Series Software

1.7 (Build 75), copyright 2000-2006 Corbett Research. In table 1 it is presented the correlation

between observed fluorescent signals and allele sequency.

Table 1. Correlation between observed fluorescent signals and allele sequency

Fluorescence Result

Only VIC dye fluorescence Homozigosity for allele 1

Only 6FAM dye fluorescence Homozigosity for allele 2

Fluorescent signals for both dyes Heterozigosity for alleles 1 and 2

Figure 12. Data obtained by qRT-PCR and processed with specialized software.

For SNP rs 35993958, in 5 samples of fecal DNA the fluorescent signal was absent on

both alleles and for another 5 samples the signal was present only on allele 1 (homozigosity). For

22

all other paired samples the fluorescent signal was present on both alleles (heterozigosity). From

the unpaired ones, 6 were heterozigous and one homozigous.

For SNP rs 5163653, 4 samples of fecal DNA the fluorescent signal was absent on both

alleles, with the observation that in one samples the signal was absent on both probes, which

suggests the poor quality of this type of DNA.

For the other samples of genomic DNA the fluorescent signal was present only on the

allele 1 (homozigosity).

Our study detected a mutant phenotype of p53 gene in SNP Gly120Ala in 5 cases

(9,43%). The patients were mostly (80%) aged over 55 years, 60% were females and 40% had

tumors located on nthe right colon. All analyzed cases were homozygous for SNP Val217Met

phenotype.

We can conclude that the p53 gene has an important role in development of the colorectal

cancer due to its expression on both allelic variants for SNP rs35993958 and expression only on

allele 1 for SNP rs35163653.

Because the fluorescent signal was present for a significantly high number of probes for

genomic DNA isolated from feces, it can be sustained the use of this testing in screening

programs of colorectal cancer (Itzkowitz, Jandorf et al, 2007; Tomlinson, Webb et al, 2008).

This test is accessible for patients due to high compliance.

23

FINAL CONCLUSIONS

The clinical and statistical study showed a correlation between patient‟s sex and tumor

location on the right or left colon, and also between local invasion and well differentiated

adenocarcinoma (p=0,0426). By statistical regression and modeling was demonstrated a

high influence of location (Odds Ratio = 0,792) and tumor grade (Odds Ratio = 1,353) on

invasion of the lymph nodes, fact with practical importance in diagnosis and therapy.

The expression profiles of cytokeratins and mucins are modified in colorectal cancer,

compared with those of the normal mucosa, fact which demonstrate their usefulness in

defining tumoral phenotypes which can be used in prognosis and therapy. The cell

proliferation markers, Ki-67 and PCNA, were positive with an index between 10 and

95% in all cases. The expression of tumor suppressing protein P53 was identified in 86%

of cases, and that of KRAS oncoprotein in 52%.

The intratumoral fenotipic heterogeneity was raised for proliferation markers with high

index differences, as oncoproteins showed an homogenous expression in tumor mass, the

heterogeneity was expressed only intertumorally. That‟s why we recommend the use of

proliferation markers in order to asses the intratumor heterogeneity and of tumor

suppressor or oncogenic proteins for the intertumor heterogeneity.

We ascertained a positive correlation with statistical significance (p=0,037) between

expression of KRAS protein and activating mutations of Kras gene.

By PCR-RFLP method we demonstrated the higher prevalence of activating mutations in

codon 12 of the Kras gene in colorectal cancer patients (33,33%) compared with control

lot (8,33%), with a high frequency for tumors on proximal colon. We didn‟t detect

mutations in codon 13 of the Kras gene in patients from the test and control lots.

We used the qRT-PCR to identify two polymorphic variants of the p53 gene (SNP

rs35993958 and rs35163653) in genomic DNA extracted from feces, and we detected a

mutant phenotype in SNP Gly120Ala in 9,43% of the cases studied, but all cases were

were homozygote for SNP Val217Met.

The statistical correlation of the results of immunohistochemical study with the gene

mutations detected by molecular methods permitted us to establish a link between

mutation-dependent genotipic heterogeneity and phenotypic heterogeneity which depends

on extratumoral factors.

24

SELECTIVE BIBLIOGRAPHY

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mutations in patients with colorectal cancer: the „RASCAL II‟ study. Br J Cancer 2001;85: 692–

696.

Bojesen S.E., Nordestgaard B.G. The common germline Arg72Pro polymorphism of p53

and increased longevity. Genetics and Molecular Research 2008;9:2:651-660.

Bousserouel S., Kauntz H., Gosse F., et al. Identification of gene expression profiles

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Bukhari M.H., Niazi S., Ghani R. et al. Detection of DNA by scraping the formalin-fixed

paraffin-embedded tissue. Proc Inst Mech Eng H 2008;222:999-1003.

Carneiro F.P., Ramalho L.N., Britto-Garcia S., Ribeiro-Silva A., Zucoloto S. Immunohistochemical expression of p16, p53, and p63 in colorectal adenomas and

adenocarcinomas. Dis Colon Rectum 2006;49:588-94.

Cejas P., Lopez-Gomez M., Aguayo C., Madero R., de Castro Carpeno J. et al. KRAS

Mutations in Primary Colorectal Cancer Tumors and Related Metastases: A Potential Role in

Prediction of Lung Metastasis. PLoS ONE 2009;4(12):e8199. doi:

10.1371/journal.pone.0008199.

Chou J.F., Row D., Gonen M., Liu Y.H., Schrag D., Weiser M.R. Clinical and pathologic

factors that predict lymph node yield from surgical specimens in colorectal cancer: a population-

based study. Cancer 2010;116:2560-70.

Coleman M.P., Quaresma M, Berrino F. et al. Cancer survival in five continents: a

worldwide population-based study (CONCORD). Lancet Oncol 2008;9(8):730-756.

Itzkowitz S.H., Jandorf L., Brand R., Rabeneck L., Schroy P.C., III, Sontag, S., Johnson

D., Skoletsky J., Durkee K., Markowitz S., Shuber A. Improved fecal DNA test for colorectal

cancer screening. Clin Gastroenterol Hepatol 2007;5(1):111-117 available from: PM:17161655.

Katkoori V.R., Jia X., Shanmugam C., Wan W. et al. Prognostic significance of p53 codon

72 polymorphism differs with race in colorectal adenocarcinoma. Clin. Cancer Res.

2009;15:2406-2416.

Markowitz S.D., Bertagnolli M.M. Molecular origins of cancer: Molecular basis of

colorectal cancer. N Engl J Med 2009;361:2449-60.

Shi S.R., Cote R.J., Wu L. et al. DNA extraction from archival formalin-fixed, paraffin-

embedded tissue sections based on the antigen retrieval principle: heating under the influence of

pH. J Histochem Cytochem 2002;50:1005-11.

Su J., Zheng J. Use of tumor proliferation marker ki-67 and PCNA in surgical pathology.

Zhonghua Bing Li Xue Za Zhi 2009;38:568-71.

Tomlinson I.P., Webb E., Carvajal-Carmona L., et al. A genome-wide association study

identifies colorectal cancer susceptibility loci on chromosomes 10p14 and 8q23.3. Nat Genet

2008;40:623-30.

Zavoral M., Suchanek S., Zavada F., Dusek L., Muzik J., Seifert B., Fric P. Colorectal

cancer screening in Europe. World J Gastroenterol 2009;15(47):5907-5915.

25

CURRICULUM VITAE

Name: ZLATIAN

Surname: OVIDIU

Date and place of birth: 24.05.1976, Craiova, Dolj

Citizenship: Romanian

Marital status: married

Actual workplace and position: Assistant, Department of Bacteriology-Virology-Parasitology,

Faculty of General Medicine, University of Medicine and Pharmacy of Craiova.

Scientific title: Assistant Professor

Education:

Institution Period Grade or diplomas

School no. 9, Craiova 1982-1990

„Carol I”, Highschool

Craiova, profile chemistry-

biology

1990-1994

Faculty of General Medicine,

University of Medicine and

Pharmacy of Craiova,

promotion 2000

1994-2000 General medicine

physician

Science Project: “Molecular mechanisms of mitochondrial maladies with neurological

manifestations”, coordinated by Professor PhD Mihai Cruce.

Medical professional experience:

Period: Place: Institution: Position:

1.01.2001 - 31.12.2001 Craiova Spitalul Clinic Nr. 1 Medic stagiar

1.01.2002 - 31.12.2006 Craiova

Laborator clinic şi

microbiologie,

Spitalul Clinic Nr. 1

Medic rezident,

Medicină de laborator

01.03.2007 până în

prezent Craiova

SC Omnimed

Laborator SRL

Medic specialist,

Medicină de laborator

Didactic professional experience:

Perioada: Localitatea: Instituţia: Funcţia:

1.03.2007 till

present Craiova

Faculty of General Medicine,

University of Medicine and

Pharmacy of Craiova, promotion

2000

Assistant

Professor

26

PUBLICATIONS

Articles in extenso, in national CNCSIS journals

Zlatian O., Jalbă C.S., Roşu Lucica, Cruce M. Tumor heterogeneity in colorectal cancer:

immunohistochemical study. Annals of the Romanian Society for Cell Biology, 2011;XV:1

(in press).

Zlatian O., Jalbă C.S., Ioana M., Cimpoeru Alina, Yousaf Sajjad, Cioboata Elena, Pintilie B,

Amelia Bârcă, Cruce M, Roşu Lucica. Evaluation of activating mutations in codons 12 and

13 from exon 2 of kras oncogene in patients with colorectal adenocarcinoma. Annals of the

Romanian Society for Cell Biology, 2010;XIV:2:114-120.

Jalbă C.S. , Jalbă B.A., Vladoi Anca-Daniela, Ioana M., Zlatian O., Cruce M. Correlations

between vascular endothelial growth factor expression and colorectal cancer. Annals of the

Romanian Society for Cell Biology, 2010;XIV:2:99-105.

Roşu Lucica, Puiu Ileana, Zlatian O., Bejenaru L., Călina Daniela, Katramados M.

Imunodeficienţa – Factor favorizant al parazitozelor intestinale la copii cu infecţie gripală.

Craiova Medicală, 2005;VII:2:146-150.

Roşu Lucica, Zamfirescu Luiza, Zlatian O., Puiu Ileana, Ştefan Lorena, Boroghină Adela.

Aspecte etiologice in otitele medii supurate. Factori de risc asociaţi. Medicina Modernă,

2005; XII:3:138-140.

Manolescu Mirela, Mixich Rodica, Ungureanu Anca, Zlatian O. Incidenţa infecţiilor cu

Mycobacterium Tuberculosis în Spitalul Clinic de Urgenţă Craiova în perioada 1990-2004.

Medicina Modernă, 2005;XII:5:229-231.

Cruce M., Ardelean A., Zlatian O. Remodelarea matricei extracelulare şi inducţia

fenotipului celular. Analele Societăţii Române de Biologie Celulară, 2004;IX:2:59-67.

Cruce M., Stănoiu B., Ardelean A., Zlatian O. Semnalizarea prin receptorii serin/treonin

kinazici ai TGF- β. În: Căi si reţele de semnalizare celulară, Editura Aius, Craiova,

2004;vol3: pag.24-31.

Cruce M., Stănoiu B., Pirici D., Ardelean A., Zlatian O. Semnalizarea dependentă de

proteoliza reglată. În: Căi si reţele de semnalizare celulară, Editura Aius, Craiova, 2004, 93-

97.

Roşu Lucica, Petrică Cristina, Stoicescu Irina, Zlatian O., Lazaroniu G. Chlamidia,

Mycoplasma şi Ureaplasma, musafiri nepoftiţi în sfera urogenitală. Medicina Modernă, 2004;

11;1:20-24.

Ardelean A., Cruce M., Stănoiu B., Buteică Elena, Zlatian O. Remodelarea matricei

extracelulare si migraţia celulelor crestelor neurale. Analele Societăţii Române de Biologie

Celulară, 2003;VIII:2:51-58.

Stănoiu B., Ardelean A., Buteică Elena, Pisoschi Cătălina, Baniţă Monica, Zlatian O., Cruce

M. Angiogeneza si remodelarea matricei extracelulare. Analele Societăţii Române de

Biologie Celulară, 2003;VIII:1:62-75.

Cruce M., Stănoiu B., Ardelean A., Baniţă Monica, Buteică Elena, Pirici D., Zlatian O.

Remodelarea mucoasei colorectale si cancerul colorectal. Analele Societăţii Române de

Biologie Celulară, 2003;VIII:1:82-88.

27

Studies published in abstract books at national or international conferences:

Zlatian O., Pleşea E., Roşu Lucica, Pirici D., Cruce M. Modificări ale profilului

imunohistochimic în neoplasmul de colon. A XXXIX-a Conferinţă Naţională de Imunologie,

Călimăneşti-Căciulata, Craiova, Romania, 24-26 septembrie 2009.

Roşu Lucica, Siloşi Isabela, Rogoz Suzana, Petrescu F., Rogoveanu I., Biciuşcă V., Zlatian

O., Ilie Andreea. TNF-alpha în hepatitele virale B şi C. . A XXXIX-a Conferinţă Naţională

de Imunologie, Călimăneşti-Căciulata, Craiova, Romania , 24-26 septembrie 2009.

Zlatian O., Cruce M., Pirici D., Ioana M. Detecţia deleţiilor/mutaţiilor genei p53 în

adenoamele colorectale prin FISH. Al IX-lea Congres National al Societăţii Române de

Morfologie, Craiova, Romania , 28-31 mai 2008.

Roşu Lucica, Zlatian O., Rosu Alexandra, Godeanu Lucia Statie, Comişel Sorina, Cara Lia,

Nania Daniela, Picu Mihaela. Rolul markerilor imunologici în managementul bolnavilor cu

infecţie HIV..A XII-a Conferinţă Naţională de Microbiologie cu participare internaţională,

Sibiu, România, 30.10-1.11 2008.

Zlatian O., Roşu Lucica, Manolescu Mirela, Mixich Rodica, Ilie Andreea, Porumbiţă

Cătălina. Aportul investigaţiilor bacteriologice în confirmarea diagnosticului clinic de

tuberculoză renală. A 3-a Conferinţă a ALMR cu participare internaţională, Iasi, Romania

27-30 iunie 2007.

Ioana M., Zlatian O., Stănoiu B., Buteică Elena, Ciobanu Felicia, Cruce M., Mixich F.

Diagnosticul molecular al pacienţilor cu cancer colorectal ereditar non-polipozic (HNPCC).

Zilele UMF din Craiova, Romania, ed. 36, 9-11 iunie 2006.

Zlatian O., Ioana M., Buteică Elena, Hertzog Zorica, Stănoiu B., Cruce M. Evaluarea prin

PCR a potenţialului de transformare malignă a zonelor displazice ale mucoasei colorectale.

Zilele UMF din Craiova, Romania, ed. 36, 9-11 iunie 2006.

Dena Bianca, Manolescu Mirela, Ungureanu Anca, Roşu Lucica, Zlatian O., Puiu

Alexandra. Enterococcus antibiotics resistance – present and perspective. 29th Balkan

Medical Week, Golden Sands, Varna, Bulgaria, 28-30 september, 2006.