Research Article Histological Characterization of Biliary...

Research ArticleHistological Characterization of Biliary Intraepithelial Neoplasiawith respect to Pancreatic Intraepithelial Neoplasia

Yasunori Sato1 Kenichi Harada1 Motoko Sasaki1 and Yasuni Nakanuma12

1 Department of Human Pathology Kanazawa University Graduate School of Medicine 13-1 Takara-machiKanazawa 920-8640 Japan

2Department of Pathology Shizuoka Cancer Center 1007 Shimonagakubo Nagaizumi-cho Sunto-gun Shizuoka 411-8777 Japan

Correspondence should be addressed to Yasuni Nakanuma nakanumastaffkanazawa-uacjp

Received 20 January 2014 Revised 19 March 2014 Accepted 19 March 2014 Published 10 April 2014

Academic Editor Masakazu Yamamoto

Copyright copy 2014 Yasunori Sato et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Biliary intraepithelial neoplasia (BilIN) is a precursor lesion of hilarperihilar and extrahepatic cholangiocarcinoma BilINrepresents the process of multistep cholangiocarcinogenesis and is the biliary counterpart of pancreatic intraepithelial neoplasia(PanIN)This study was performed to clarify the histological characteristics of BilIN in relation to PanIN Using paraffin-embeddedtissue sections of surgically resected specimens of cholangiocarcinoma associatedwith BilIN and pancreatic ductal adenocarcinomaassociated with PanIN immunohistochemical staining was performed using primary antibodies againstMUC1MUC2MUC5ACcyclin D1 p21 p53 and S100P For mucin staining Alcian blue pH 25 was used Most of the molecules examined here showedsimilar expression patterns in BilIN and PanIN in which their expression tended to increase along with the increase in atypia ofthe epithelial lesions Significant differences were observed in the increase in mucin production and the expression of S100P inPanIN-1 and the expression of p53 in PanIN-3 when compared with those in BilIN of a corresponding gradeThese results suggestthat cholangiocarcinoma and pancreatic ductal adenocarcinoma share at least in part a common carcinogenic process and furtherconfirm that BilIN can be regarded as the biliary counterpart of PanIN

1 Introduction

Cholangiocarcinoma that arises under conditions of chronicbiliary diseases such as hepatolithiasis often undergoes themultistep carcinogenesis process [1] Biliary intraepithelialneoplasia (BilIN) is known as a premalignant lesion ofcholangiocarcinoma that represents the multistep cholan-giocarcinogenesis [2] The classification is applicable to flatatypical epithelial lesions in the intrahepatic large bile ductsand the extrahepatic bile ducts and it is also applied tolesions in the gallbladder according to the current WorldHealth Organization (WHO) classification for tumors of thedigestive system [3]

BilIN is a concept that is proposed based on the morpho-logical resemblance to pancreatic intraepithelial neoplasia(PanIN) Similar to PanIN BilIN is classified into three gradesaccording to the degree of cytological and architecturalatypia BilIN-1 (low-grade lesions) BilIN-2 (intermediate-grade lesions) and BilIN-3 (high-grade lesions carcinoma

in situ) Using the BilIN classification there is increasingevidence that molecular and genetic alterations accumulateduring the progression of BilIN to cholangiocarcinoma [4ndash7]

Since the biliary tract and pancreas share a commondevelopmental process as well as morphological character-istics as duct systems it is plausible that some biliary andpancreatic diseases show similar pathological features andbiological behaviors [8] Indeed our recent comparativeanalysis showed that hilar cholangiocarcinoma and ductaladenocarcinoma of the pancreas share many clinicopatho-logical features [9] In addition we showed that intraductalpapillary neoplasm of the bile duct (IPNB) and intraductalpapillarymucinous neoplasm (IPMN) of the pancreas as wellas mucinous cystic neoplasm (MCN) of the biliary tract andpancreas exhibit similar immunohistochemical phenotypessuggesting a common carcinogenic process of the tumors[10] where all these tumors were classified as premalignantlesions according to the current WHO classification

Hindawi Publishing CorporationInternational Journal of HepatologyVolume 2014 Article ID 678260 7 pageshttpdxdoiorg1011552014678260

2 International Journal of Hepatology

Table 1 Primary antibodies used for immunohistochemical analysis

Antigen Clone Company Dilution Antigen retrievalMUC1 DF3 Toray Fuji Bionics (Tokyo Japan) 1 50 MWMUC2 Ccp58 Novocastra (Newcastle UK) 1 100 MWMUC5AC CLH2 Novocastra 1 200 MWCytokeratin 20 Ks 208 DakoCytomation (Glostrup Denmark) 1 50 MWCyclin D1 SP4 Nichirei (Tokyo Japan) Prediluted MWlowast

p21 EPR3993 Abcam (Cambridge UK) 1 100 MWp53 DO-7 DakoCytomation 1 100 MWS100P EPR6143 Abcam 1 100 MWMW microwaving in 10 nmolL citrate buffer (pH 60) for 20 minutes MWlowast microwaving in tris-ethylenediaminetetraacetic acid buffer (pH 90) for 20minutes

As far as the histological characteristics of BilIN andPanIN are concerned previous studies have examined theirfeatures individually and detailed data on comparative anal-ysis of BilIN and PanIN are lacking This study was thereforeconducted to clarify the histological characteristics of BilINwith respect to PanIN

2 Materials and Methods

21 Tissue Preparation Hepatolithiatic livers associated withperihilar cholangiocarcinoma were used as a model of mul-tistep cholangiocarcinogenesis A total of 25 hepatolithiaticlivers with cholangiocarcinoma and a total of 22 pancre-atic specimens with pancreatic ductal adenocarcinoma wereretrieved from the files of our laboratory and affiliatedhospitals The patients were selected during the periodbetween 1997 and 2007 All cases were surgically resectedand all liver and pancreatic specimens were histologicallyaccompanied by BilIN and PanIN respectively In all cases ofcholangiocarcinoma the main part of the tumor was locatedin hilar or perihilar region of the liver and they appearedto arise from the intrahepatic large bile ducts or the rightor left hepatic bile duct Most cholangiocarcinoma casesshowed macroscopic features of mass-forming type andorintraductal growth type Foci of BilIN were microscopicallylocated in the intrahepatic large bile ducts and the hepaticbile ducts and they were not observed in the septal andinterlobular bile ducts The mean age and sex distribution(male-female ratio) of the patients were 62 years and 11 14for the liver specimens and 68 years and 12 10 for thepancreatic specimens respectively The samples were fixedin 10 neutral formalin and embedded in paraffin Then4-120583m-thick paraffin-embedded sections were prepared Onerepresentative section from each case was used

22 Histochemistry and Immunohistochemistry Alcian blue(at pH 25) was used for mucin staining Immunostainingwas performedusing the sectionswith the primary antibodieslisted inTable 1 After the blocking of endogenous peroxidasethe sections were incubated in protein block solution (Dako-Cytomation Glostrup Denmark) They were then incubatedovernight at 4∘C with each of the primary antibodies Theirsources optimal dilution and antigen retrieval methods

are shown in Table 1 They were treated with secondaryantibodies conjugated to a peroxidase-labeled polymer usingthe HISTOFINE system (Nichirei Tokyo Japan) Colordevelopment was performed using 331015840-diaminobenzidinetetrahydrochloride and the sections were lightly counter-stained with hematoxylin Negative controls consisted ofsubstitution of the primary antibodies with nonimmuneserum and were consistently negative

23 Histological Assessment Semiquantitative analysis of thestained sections was performed Staining intensity was evalu-ated in a high-power field for the neoplastic and nonneoplas-tic epithelia of the bile ducts and pancreatic ducts From thesections of 25 liver specimens and 22 pancreatic specimensfoci of interest were selected The number of foci examinedwas as follows nonneoplastic large bile duct 14 BilIN-1 17BilIN-23 24 invasive carcinoma (cholangiocarcinoma) 50nonneoplastic pancreatic duct 13 PanIN-1 22 PanIN-23 15invasive carcinoma (pancreatic ductal adenocarcinoma) 44

For mucin staining with Alcian blue (pH 25) the signalintensity in the cytoplasm andor on the luminal surface ofthe epithelial cells was evaluated using the following gradingsystem 1+ (mild) 2+ (moderate) and 3+ (marked) Thecytoplasmic andor luminal immunostaining of MUC1 andthe cytoplasmic immunostaining of MUC2 and MUC5ACwere graded as follows 0 (negative) 1+ (mild to moderate)and 2+ (marked) For evaluation of the nuclear staining ofcyclin D1 p21 p53 and S100P the percentage of positivenuclei to the total number of nuclei of the epithelial cells wascalculated and it was graded as follows 0 (negative) 1+ (notexceeding 10) and 2+ (more than 10) For p53 nuclearstaining only the proportion of intensely positive nuclei wasscored

24 Statistics Statistical significance was determined usingthe Mann-Whitney 119880-test A 119875 value less than 005 wasaccepted as the level of statistical significance

3 Results and Discussion

Morphological appearances such as loss of nuclear polarityincreased nucleus-to-cytoplasm ratio nuclear hyperchroma-sia and architectural atypia were basically similar between

International Journal of Hepatology 3

BilIN-1 PanIN-1A PanIN-1B

BilIN-3 PanIN-3

Figure 1 Histology of biliary intraepithelial neoplasia (BilIN) and pancreatic intraepithelial neoplasia (PanIN) Representative images ofBilIN-1 and BilIN-3 and PanIN-1A PanIN-1B and PanIN-3 are shown Hematoxylin and eosin staining Original magnifications times400

the corresponding grades of BilIN and PanIN which wereobserved in sections stained with hematoxylin and eosin(Figure 1)

Mucin staining with Alcian blue (pH 25) showed thatboth BilIN and PanIN frequently had cytoplasmic andorluminal surface mucin (Figure 2) According to the grade ofBilIN and PanIN PanIN-1 tended to have more abundantcytoplasmic mucin than BilIN-1 and the results of semi-quantitative analysis confirmed this tendency (Figure 3) Theabundant mucin expression in PanIN-1 is consistent with thedefinition of PanIN-1 in which the lesion is composed oftall columnar cells with basally located nuclei and abundantsupranuclear mucin [11]

The immunohistochemical expression of MUC1 wasincreased along with the increase in the grade of BilINand PanIN and no significant difference in its expressionstatus was observed between BilIN and PanIN (Figures 2and 3) Similarly the expression of MUC5AC was frequentlyobserved in all grades of both BilIN and PanIN (Figures 2and 3) The results of the expression status of MUC1 andMUC5AC in BilIN were almost identical to those in ourprevious report [4]

Focal immunohistochemical expression of MUC2 wasobserved in several foci of BilIN whereas MUC2 positivitywas exceptional in PanIN (Figures 2 and 3) Although theexpression of CK20 was typically negative in both BilINand PanIN in this study (data not shown) BilIN is notinfrequently associated with metaplastic change of intestinaltype while intestinal-type PanIN is generally not found[12 13] These observations may explain the focal MUC2expression in BilIN rather than in PanIN

The results of immunostaining of MUC1 MUC2MUC5AC and CK20 for BilIN and PanIN in this studyare summarized in Table 2 For comparison the resultsof our previous comparative analysis that examined theimmunohistochemical characteristics of IPNB IPMN ofthe pancreas hepatic MCN and pancreatic MCN [10] are

also shown in Table 2 It is noteworthy that all of thesepremalignant lesions show similar immunoprofiles to eachother between the biliary tract and pancreas supportingthe concept that BilIN IPNB and hepatic MCN are biliarycounterparts of PanIN IPMN and pancreatic MCNrespectively

As for the expression of cell cycle-related molecules theimmunohistochemical expression of cyclin D1 and p21 wasabsent or focal in nonneoplastic epithelium of the bile ductsand the pancreatic ductsThey were occasionally observed inBilIN-1 and PanIN-1 and more frequently in BilIN-23 andPanIN-23 (Figures 2 and 3) in which the frequency of theexpression of cyclin D1 and p21 in BilIN in this study wascomparable to that in our previous report [5] Semiquanti-tative analysis showed that there was no significant differencein their expression status between BilIN and PanIN

The expression of p53 was not observed in nonneoplasticepithelium of the bile ducts and the pancreatic ducts aswell as in BilIN-12 and PanIN-12 By contrast BilIN-3 andPanIN-3 occasionally showed the expression of p53 and itsfrequency was significantly higher in PanIN-3 than in BilIN-3 (Figures 2 and 3) Because the process of carcinogenesisis often complicated by inflammatory changes in the biliarytract the molecular alterations may be more complex inBilIN due to cholangitis than those seen in PanIN wherethe influence of inflammation is usually insignificant in thedevelopment of pancreatic cancer In fact our recent studyshowed that the detection rate of KRAS mutation in BilINwas not as high as that seen in PanIN [6] Therefore itis predicted that factors other than genetic alterations mayalso affect the process of the development of BilIN andcholangiocarcinoma

S100P is a molecule that is highly expressed by perihilarand extrahepatic cholangiocarcinoma as well as pancreaticductal adenocarcinoma [9 14] In this study the expression ofS100P was frequently observed in both BilIN and PanIN of allgrades (Figure 2) Semiquantitative analysis showed that its


Table 2 Immunoprofiles of premalignant lesions of the biliary tract and pancreas

Intraepithelial neoplasia Intraductal papillary neoplasm Mucinous cystic neoplasmBilIN PanIN IPNB IPMN Hepatic MCN Pancreatic MCN

MUC1 + + + + + +MUC2 + minus + + minus minus

MUC5AC ++ ++ ++ ++ ++ ++CK20 minus minus + + minus minus

The results of comparative analysis for biliary and pancreatic neoplasms in the present study and our previous report (10) are summarized minus likely absent+ occasionally present ++ usually present BilIN biliary intraepithelial neoplasia CK cytokeratin IPMN intraductal papillary mucinous neoplasm IPNBintraductal papillary neoplasm of the bile duct MCN mucinous cystic neoplasm PanIN pancreatic intraepithelial neoplasia

Alc

ian

blue

BilIN PanIN

MU

C1M

UC2

MU

C5A

C

BilIN-1

BilIN-3

BilIN-2

BilIN-1

PanIN-1

PanIN-2

PanIN-1

PanIN-3

Cycl

in D

1p2

1p5

3S1

00P

BilIN PanIN

BilIN-1

BilIN-3

BilIN-3

BilIN-2

PanIN-3

PanIN-2

PanIN-3

PanIN-1

Figure 2 Representative images of histochemical and immunohistochemical stainingThe results ofmucin stainingwith Alcian blue (pH 25)and immunostaining of MUC1 MUC2 MUC5AC cyclin D1 p21 p53 and S100P for biliary intraepithelial neoplasia (BilIN) and pancreaticintraepithelial neoplasia (PanIN) are shown Original magnifications times400

expression was significantly high in PanIN-1 compared withthat in BilIN-1 although both BilIN-1 and PanIN-1 exhibiteda high frequency of S100P expression (Figure 3)

Most of the molecules examined in this study showedsimilar expression patterns in BilIN and PanIN There weresignificant differences in the increase in mucin productionand the expression of S100P in PanIN-1 and the expressionof p53 in PanIN-3 when compared with those in BilIN ofcorresponding grade

The immunohistochemical expression of MUC1 cyclinD1 p21 p53 and S100P tended to be increased in invasive

foci of cholangiocarcinoma and pancreatic ductal adenocar-cinomawhen compared to those in BilIN-23 and PanIN-23respectively (Figure 3)These results were consistent with theconcept of multistep carcinogenesis

4 Conclusions

BilIN and PanIN showed similar histological and immuno-histochemical features with several exceptions These resultssuggest that cholangiocarcinoma and pancreatic ductal ade-nocarcinoma share at least in part a common carcinogenic


Alc

ian

blue

Alc

ian

blue

BilIN-1

BilIN-23

Foci ()0 20 40 60 80 100

Non-neoplastic

Non-neoplastic

PanIN-1

PanIN-23

Foci ()0 20 40 60 80 100

lowast

BilIN PanIN

Invasive Invasive

3+

1+

2+

3+

1+

2+

MU

C1

MU

C1

0 20 40 60 80 100 0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

MU

C2

MU

C2

0 20 40 60 80 100 0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

MU

C5AC

MU

C5AC

0 20 40 60 80 100 0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

Cycli

n D

1

Cycli

n D

1

Foci ()0 20 40 60 80 100

Foci ()0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

0

2+

1+

0

2+

1+

Figure 3 Continued


p21

0 20 40 60 80 100

p21

0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN PanINp5

3

0 20 40 60 80 100

p53

0 20 40 60 80 100

lowast

lowast

Non-neoplastic

PanIN-12

PanIN-3

Invasive

BilIN-12

BilIN-3

Non-neoplastic

Invasive

S100

P

Foci ()0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

S100

P

Foci ()0 20 40 60 80 100

lowastBilIN-1

BilIN-23

Non-neoplastic

Invasive

0

2+

1+

0

2+

1+

Figure 3 Semiquantitative analysis of the results of histochemical and immunohistochemical staining The analysis was performed asdescribed in Section 2 for the lesions of nonneoplastic epithelium of the bile ducts and the pancreatic ducts biliary intraepithelial neoplasia(BilIN) pancreatic intraepithelial neoplasia (PanIN) and invasive carcinoma lowast119875 lt 005 versus the results of BilIN of correspondinghistological grade or cholangiocarcinoma

process and further confirm that BilIN can be regarded as thebiliary counterpart of PanIN

Abbreviations

BilIN Biliary intraepithelial neoplasiaCK CytokeratinIPMN Intraductal papillary mucinous neoplasmIPNB Intraductal papillary neoplasm of the bile ductMCN Mucinous cystic neoplasmPanIN Pancreatic intraepithelial neoplasiaWHO World Health Organization

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] YNakanumaM Sasaki Y Sato et al ldquoMultistep carcinogenesisof perihilar cholangiocarcinoma arising in the intrahepatic

large bile ductsrdquo World Journal of Hepatology vol 1 no 1 pp35ndash42 2009

[2] Y Zen N V Adsay K Bardadin et al ldquoBiliary intraepithelialneoplasia an international interobserver agreement study andproposal for diagnostic criteriardquoModern Pathology vol 20 no6 pp 701ndash709 2007

[3] J Albores-Saavedra N V Adsay J M Crawford et al ldquoCarci-noma of the gallbladder and extrahepatic bile ductsrdquo in WHOClassification of Tumors of the Digestive System F T BosmanF Carneiro R H Hruban and N DTheise Eds pp 266ndash273IARCWorldHealthOrganization of Tumors Lyon France 4thedition 2010

[4] Y Zen M Sasaki T Fujii et al ldquoDifferent expression patternsof mucin core proteins and cytokeratins during intrahepaticcholangiocarcinogenesis from biliary intraepithelial neopla-sia and intraductal papillary neoplasm of the bile ductmdashanimmunohistochemical study of 110 cases of hepatolithiasisrdquoJournal of Hepatology vol 44 no 2 pp 350ndash358 2006

[5] Y Nakanishi Y Zen S Kondo T Itoh K Itatsu and YNakanuma ldquoExpression of cell cycle-related molecules in bil-iary premalignant lesions biliary intraepithelial neoplasia and


biliary intraductal papillary neoplasmrdquo Human Pathology vol39 no 8 pp 1153ndash1161 2008

[6] M Hsu M Sasaki S Igarashi Y Sato and Y NakanumaldquoKRAS and GNAS mutations and p53 overexpression in biliaryintraepithelial neoplasia and intrahepatic cholangiocarcino-masrdquo Cancer vol 119 no 9 pp 1669ndash1674 2013

[7] Y Sato M Sasaki K Harada et al ldquoPathological diagnosis offlat epithelial lesions of the biliary tract with emphasis on biliaryintraepithelial neoplasiardquo Journal of Gastroenterology vol 49no 1 pp 64ndash72 2014

[8] Y Nakanuma K Harada M Sasaki and Y Sato ldquoProposalof a new disease concept ldquobiliary diseases with pancreaticcounterpartsrdquo Anatomical and pathological basesrdquo Histologyand Histopathology vol 29 no 1 pp 1ndash10 2014

[9] C Gandou K Harada Y Sato et al ldquoHilar cholangiocar-cinoma and pancreatic ductal adenocarcinoma share simi-lar histopathologies immunophenotypes and development-related moleculesrdquoHuman Pathology vol 44 no 5 pp 811ndash8212013

[10] T Matsubara Y Sato M Sasaki et al ldquoImmunohistochemicalcharacteristics andmalignant progression of hepatic cystic neo-plasms in comparison with pancreatic counterpartsrdquo HumanPathology vol 43 no 12 pp 2177ndash2186 2012

[11] R H Hruban G Kloppel P Boffetta et al ldquoDuctal adenocar-cinoma of the pancreasrdquo in WHO Classification of Tumors ofthe Digestive System F T Bosman F Carneiro R H Hrubanand N D Theise Eds pp 281ndash291 IARC World HealthOrganization of Tumors Lyon France 4th edition 2010

[12] Y Sato K Harada M Sasaki and Y Nakanuma ldquoHistologicalcharacteristics of biliary intraepithelial neoplasia-3 and intraep-ithelial spread of cholangiocarcinomardquo Virchows Archiv vol462 no 4 pp 421ndash427 2013

[13] J Albores-Saavedra J Wu T Crook R H Amirkhan LJones and R H Hruban ldquoIntestinal and oncocytic variantsof pancreatic intraepithelial neoplasia A morphological andimmunohistochemical studyrdquo Annals of Diagnostic Pathologyvol 9 no 2 pp 69ndash76 2005

[14] Y Sato K HaradaM Sasaki and Y Nakanuma ldquoClinicopatho-logical significance of S100 protein expression in cholangiocar-cinomardquo Journal of Gastroenterology andHepatology vol 28 no8 pp 1422ndash1429 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Behavioural Neurology

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Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Primary antibodies used for immunohistochemical analysis

Antigen Clone Company Dilution Antigen retrievalMUC1 DF3 Toray Fuji Bionics (Tokyo Japan) 1 50 MWMUC2 Ccp58 Novocastra (Newcastle UK) 1 100 MWMUC5AC CLH2 Novocastra 1 200 MWCytokeratin 20 Ks 208 DakoCytomation (Glostrup Denmark) 1 50 MWCyclin D1 SP4 Nichirei (Tokyo Japan) Prediluted MWlowast

p21 EPR3993 Abcam (Cambridge UK) 1 100 MWp53 DO-7 DakoCytomation 1 100 MWS100P EPR6143 Abcam 1 100 MWMW microwaving in 10 nmolL citrate buffer (pH 60) for 20 minutes MWlowast microwaving in tris-ethylenediaminetetraacetic acid buffer (pH 90) for 20minutes

As far as the histological characteristics of BilIN andPanIN are concerned previous studies have examined theirfeatures individually and detailed data on comparative anal-ysis of BilIN and PanIN are lacking This study was thereforeconducted to clarify the histological characteristics of BilINwith respect to PanIN

2 Materials and Methods

21 Tissue Preparation Hepatolithiatic livers associated withperihilar cholangiocarcinoma were used as a model of mul-tistep cholangiocarcinogenesis A total of 25 hepatolithiaticlivers with cholangiocarcinoma and a total of 22 pancre-atic specimens with pancreatic ductal adenocarcinoma wereretrieved from the files of our laboratory and affiliatedhospitals The patients were selected during the periodbetween 1997 and 2007 All cases were surgically resectedand all liver and pancreatic specimens were histologicallyaccompanied by BilIN and PanIN respectively In all cases ofcholangiocarcinoma the main part of the tumor was locatedin hilar or perihilar region of the liver and they appearedto arise from the intrahepatic large bile ducts or the rightor left hepatic bile duct Most cholangiocarcinoma casesshowed macroscopic features of mass-forming type andorintraductal growth type Foci of BilIN were microscopicallylocated in the intrahepatic large bile ducts and the hepaticbile ducts and they were not observed in the septal andinterlobular bile ducts The mean age and sex distribution(male-female ratio) of the patients were 62 years and 11 14for the liver specimens and 68 years and 12 10 for thepancreatic specimens respectively The samples were fixedin 10 neutral formalin and embedded in paraffin Then4-120583m-thick paraffin-embedded sections were prepared Onerepresentative section from each case was used

22 Histochemistry and Immunohistochemistry Alcian blue(at pH 25) was used for mucin staining Immunostainingwas performedusing the sectionswith the primary antibodieslisted inTable 1 After the blocking of endogenous peroxidasethe sections were incubated in protein block solution (Dako-Cytomation Glostrup Denmark) They were then incubatedovernight at 4∘C with each of the primary antibodies Theirsources optimal dilution and antigen retrieval methods

are shown in Table 1 They were treated with secondaryantibodies conjugated to a peroxidase-labeled polymer usingthe HISTOFINE system (Nichirei Tokyo Japan) Colordevelopment was performed using 331015840-diaminobenzidinetetrahydrochloride and the sections were lightly counter-stained with hematoxylin Negative controls consisted ofsubstitution of the primary antibodies with nonimmuneserum and were consistently negative

23 Histological Assessment Semiquantitative analysis of thestained sections was performed Staining intensity was evalu-ated in a high-power field for the neoplastic and nonneoplas-tic epithelia of the bile ducts and pancreatic ducts From thesections of 25 liver specimens and 22 pancreatic specimensfoci of interest were selected The number of foci examinedwas as follows nonneoplastic large bile duct 14 BilIN-1 17BilIN-23 24 invasive carcinoma (cholangiocarcinoma) 50nonneoplastic pancreatic duct 13 PanIN-1 22 PanIN-23 15invasive carcinoma (pancreatic ductal adenocarcinoma) 44

For mucin staining with Alcian blue (pH 25) the signalintensity in the cytoplasm andor on the luminal surface ofthe epithelial cells was evaluated using the following gradingsystem 1+ (mild) 2+ (moderate) and 3+ (marked) Thecytoplasmic andor luminal immunostaining of MUC1 andthe cytoplasmic immunostaining of MUC2 and MUC5ACwere graded as follows 0 (negative) 1+ (mild to moderate)and 2+ (marked) For evaluation of the nuclear staining ofcyclin D1 p21 p53 and S100P the percentage of positivenuclei to the total number of nuclei of the epithelial cells wascalculated and it was graded as follows 0 (negative) 1+ (notexceeding 10) and 2+ (more than 10) For p53 nuclearstaining only the proportion of intensely positive nuclei wasscored

24 Statistics Statistical significance was determined usingthe Mann-Whitney 119880-test A 119875 value less than 005 wasaccepted as the level of statistical significance

3 Results and Discussion

Morphological appearances such as loss of nuclear polarityincreased nucleus-to-cytoplasm ratio nuclear hyperchroma-sia and architectural atypia were basically similar between



BilIN-3 PanIN-3

















Alc

ian

blue

BilIN PanIN

MU

C1M

UC2

MU

C5A

C

BilIN-1

BilIN-3

BilIN-2

BilIN-1

PanIN-1

PanIN-2

PanIN-1

PanIN-3

Cycl

in D

1p2

1p5

3S1

00P

BilIN PanIN

BilIN-1

BilIN-3

BilIN-3

BilIN-2

PanIN-3

PanIN-2

PanIN-3

PanIN-1






4 Conclusions



Alc

ian

blue

Alc

ian

blue

BilIN-1

BilIN-23

Foci ()0 20 40 60 80 100

Non-neoplastic

Non-neoplastic

PanIN-1

PanIN-23

Foci ()0 20 40 60 80 100

lowast

BilIN PanIN

Invasive Invasive

3+

1+

2+

3+

1+

2+

MU

C1

MU

C1

0 20 40 60 80 100 0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

MU

C2

MU

C2

0 20 40 60 80 100 0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

MU

C5AC

MU

C5AC

0 20 40 60 80 100 0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

Cycli

n D

1

Cycli

n D

1

Foci ()0 20 40 60 80 100

Foci ()0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

0

2+

1+

0

2+

1+

Figure 3 Continued


p21

0 20 40 60 80 100

p21

0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN PanINp5

3

0 20 40 60 80 100

p53

0 20 40 60 80 100

lowast

lowast

Non-neoplastic

PanIN-12

PanIN-3

Invasive

BilIN-12

BilIN-3

Non-neoplastic

Invasive

S100

P

Foci ()0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

S100

P

Foci ()0 20 40 60 80 100

lowastBilIN-1

BilIN-23

Non-neoplastic

Invasive

0

2+

1+

0

2+

1+



Abbreviations




References























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















BilIN-3 PanIN-3

















Alc

ian

blue

BilIN PanIN

MU

C1M

UC2

MU

C5A

C

BilIN-1

BilIN-3

BilIN-2

BilIN-1

PanIN-1

PanIN-2

PanIN-1

PanIN-3

Cycl

in D

1p2

1p5

3S1

00P

BilIN PanIN

BilIN-1

BilIN-3

BilIN-3

BilIN-2

PanIN-3

PanIN-2

PanIN-3

PanIN-1






4 Conclusions



Alc

ian

blue

Alc

ian

blue

BilIN-1

BilIN-23

Foci ()0 20 40 60 80 100

Non-neoplastic

Non-neoplastic

PanIN-1

PanIN-23

Foci ()0 20 40 60 80 100

lowast

BilIN PanIN

Invasive Invasive

3+

1+

2+

3+

1+

2+

MU

C1

MU

C1

0 20 40 60 80 100 0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

MU

C2

MU

C2

0 20 40 60 80 100 0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

MU

C5AC

MU

C5AC

0 20 40 60 80 100 0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

Cycli

n D

1

Cycli

n D

1

Foci ()0 20 40 60 80 100

Foci ()0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

0

2+

1+

0

2+

1+

Figure 3 Continued


p21

0 20 40 60 80 100

p21

0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN PanINp5

3

0 20 40 60 80 100

p53

0 20 40 60 80 100

lowast

lowast

Non-neoplastic

PanIN-12

PanIN-3

Invasive

BilIN-12

BilIN-3

Non-neoplastic

Invasive

S100

P

Foci ()0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

S100

P

Foci ()0 20 40 60 80 100

lowastBilIN-1

BilIN-23

Non-neoplastic

Invasive

0

2+

1+

0

2+

1+



Abbreviations




References























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Alc

ian

blue

BilIN PanIN

MU

C1M

UC2

MU

C5A

C

BilIN-1

BilIN-3

BilIN-2

BilIN-1

PanIN-1

PanIN-2

PanIN-1

PanIN-3

Cycl

in D

1p2

1p5

3S1

00P

BilIN PanIN

BilIN-1

BilIN-3

BilIN-3

BilIN-2

PanIN-3

PanIN-2

PanIN-3

PanIN-1






4 Conclusions



Alc

ian

blue

Alc

ian

blue

BilIN-1

BilIN-23

Foci ()0 20 40 60 80 100

Non-neoplastic

Non-neoplastic

PanIN-1

PanIN-23

Foci ()0 20 40 60 80 100

lowast

BilIN PanIN

Invasive Invasive

3+

1+

2+

3+

1+

2+

MU

C1

MU

C1

0 20 40 60 80 100 0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

MU

C2

MU

C2

0 20 40 60 80 100 0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

MU

C5AC

MU

C5AC

0 20 40 60 80 100 0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

Cycli

n D

1

Cycli

n D

1

Foci ()0 20 40 60 80 100

Foci ()0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

0

2+

1+

0

2+

1+

Figure 3 Continued


p21

0 20 40 60 80 100

p21

0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN PanINp5

3

0 20 40 60 80 100

p53

0 20 40 60 80 100

lowast

lowast

Non-neoplastic

PanIN-12

PanIN-3

Invasive

BilIN-12

BilIN-3

Non-neoplastic

Invasive

S100

P

Foci ()0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

S100

P

Foci ()0 20 40 60 80 100

lowastBilIN-1

BilIN-23

Non-neoplastic

Invasive

0

2+

1+

0

2+

1+



Abbreviations




References























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Alc

ian

blue

Alc

ian

blue

BilIN-1

BilIN-23

Foci ()0 20 40 60 80 100

Non-neoplastic

Non-neoplastic

PanIN-1

PanIN-23

Foci ()0 20 40 60 80 100

lowast

BilIN PanIN

Invasive Invasive

3+

1+

2+

3+

1+

2+

MU

C1

MU

C1

0 20 40 60 80 100 0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

MU

C2

MU

C2

0 20 40 60 80 100 0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

MU

C5AC

MU

C5AC

0 20 40 60 80 100 0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

Cycli

n D

1

Cycli

n D

1

Foci ()0 20 40 60 80 100

Foci ()0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

0

2+

1+

0

2+

1+

Figure 3 Continued


p21

0 20 40 60 80 100

p21

0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN PanINp5

3

0 20 40 60 80 100

p53

0 20 40 60 80 100

lowast

lowast

Non-neoplastic

PanIN-12

PanIN-3

Invasive

BilIN-12

BilIN-3

Non-neoplastic

Invasive

S100

P

Foci ()0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

S100

P

Foci ()0 20 40 60 80 100

lowastBilIN-1

BilIN-23

Non-neoplastic

Invasive

0

2+

1+

0

2+

1+



Abbreviations




References























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















p21

0 20 40 60 80 100

p21

0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN-1

BilIN-23

Non-neoplastic

Invasive

BilIN PanINp5

3

0 20 40 60 80 100

p53

0 20 40 60 80 100

lowast

lowast

Non-neoplastic

PanIN-12

PanIN-3

Invasive

BilIN-12

BilIN-3

Non-neoplastic

Invasive

S100

P

Foci ()0 20 40 60 80 100

BilIN-1

BilIN-23

Non-neoplastic

Invasive

S100

P

Foci ()0 20 40 60 80 100

lowastBilIN-1

BilIN-23

Non-neoplastic

Invasive

0

2+

1+

0

2+

1+



Abbreviations




References























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Histological Characterization of Biliary...

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