2008 Summer Meeting - Leeds - The Pathological Society of Great ...

O9Development and characterisation of a new breast cancer cellline of basal phenotypeV Speirs 1 , CA Green 1 , RH Partanen 1 , AM Shaaban 2 , AM Hanby 11 Leeds Institute of Molecular Medicine, University of Leeds, 2 St James'sInstitute of Oncology, St James's University Hospital, LeedsBreast cancer is heterogeneous and current cell line models cannot adequatelyreflect all the phenotypic and genotypic changes observed clinically. During ourroutine development of short term primary cultures of breast cancer epithelialcells we observed spontaneous immortalisation of one of these cultures after 12months in vitro. These cells, termed LGI1T, were isolated in 2002 from arecurrent breast tumour from an elderly patient who received tamoxifen asprimary therapy and grew as a monolayer with cobblestone morphology. Theprimary tumour was classified as a lymph node, ER and PR negativesquamous cell carcinoma but expressed ER. Phenotypic characterisation byimmunofluorescence revealed expression of basal cytokeratins CK5/6 andCK14, the hormone receptors ER1 and ER2 but not ER or HER2 or theluminal markers CK18 and CK19, which is consistent with basal breast cancer.As with the primary tumour, LGI1T cells remained unresponsive to tamoxifenin vitro, however there was some evidence of hypersensitivity at low doses(>10 - 10M). Telomerase activity was assessed by a PCR-based ELISA andremained high in LGI1T cells which is indicative of escape from senescence. Insummary we have developed and characterised a new breast cancer cell line ofbasal phenotype. As there is currently considerable interest in understanding thebiology of basal breast cancer this cell line represents a powerful new tool forinvestigating this further.O11Combining Proteomics and Genomics to Identify Biomarkersfor Colorectal CancerMDe Wit 1 , CR Jimenez 2 , B Carvalho 1 ,SPiersma 2 , R Lamerichs 3 ,GA Meijer 1 , RJA Fijneman 1,21 Department of Pathology VU University Medical Centre, Amsterdam, TheNetherlands, 2 Department of Medical Oncology, VU University MedicalCentre, Amsterdam, The Netherlands, 3 Philips Research, Eindhoven, TheNetherlandsIntroduction and Aim: Detection of colorectal cancer (CRC) at an early stage ofdisease is a realistic approach to reduce cancer death. The aim of this study is toidentify biomarkers that discriminate low-risk colon adenomas from high-riskadenomas and CRC and that can be applied for molecular imaging. Bymicroarray expression analysis, we obtained a list of genes of which mRNAlevels are increased in CRC compared to low-risk adenomas. Genes encodingproteins with extracellular domains are the most promising candidates formolecular imaging. Therefore we set out to identify cell surface proteins.Methods: CRC cell lines were cultured until 70-80% confluency and incubatedwith Biotin to biotinylate cell surface proteins, which were isolated from thewhole cell lysate. Protein mixtures were fractionated by gradient 1D SDS-PAGE and further processed for in-depth proteomics analysis by liquidchromatographyfollowed by tandem mass spectrometry (LC-MS/MS).Results: A total of 1046 proteins were identified upon analysis of threebiological replicates of colo 205 cells, 563 of which were reproducibly detectedin all three samples. Integration of the cell-surface proteomics data with themicroarray expression data showed that 98 of the 1046 proteins matched withgenes that showed mRNA overexpression in CRC compared to low-riskadenomas.Conclusion: These preliminary data indicate that this strategy resulted in thesuccessful identification of cell surface biomarkers. Currently we are extendingthe experiments to other CRC cell lines and the results of these experimentsshall be presented.O10Methylation Pattern of High Risk Flat Adenomas in CRCQJM Voorham 1 , B Carvalho 1 , AJ Spiertz 2 , NCT Van Grieken 1 ,SDerks 2 , H Grabsch 3 , B Rembacken 4 ,AP De Bruïne 2 ,M Van Engeland 2 , GA Meijer 1 .1 Dept of Pathology, VU University Medical Centre, Amsterdam, TheNetherlands, 2 Dept of Pathology, University Maastricht, Maastricht, TheNetherlands, 3 Pathology and Tumour Biology, Leeds Institute ofMolecular Medicine, University of Leeds, UK, 4 Centre for DigestiveDiseases, Leeds General Infirmary, Leeds, UKIntroductionFlat colorectal adenomas are considered to have a different molecularpathogenesis than polypoid-shaped lesions and are associated with moreaggressive clinical behaviour. In CRC development methylation is an earlyevent, but little is known about promoter hypermethylation in flat lesions.AimTo analyze the methylation status of 16 CRC related genes in flat adenomas andcompare this to established methylation patterns of polypoid lesions.Materials and methods44 FFPE flat adenomas, (classified according to the Paris classification) and 11flat carcinomas were used. Promoter methylation status of 16 genes (O 6 MGMT,hMLH1, APC, p14 ARF , p16 INK4A , RASSF1A, RASSF2A, GATA-4, GATA-5,CHFR, NEUROG1, IGF2, HLTF, CACNA1G, RUNX3 and SOCS1) wasstudied by methylation-specific PCR.ResultsThe methylation frequency of these genes in flat adenomas was comparable tothat observed in polypoid adenomas, except for CHFR (P=0.02) and GATA-5(P=0.03) which where significantly less frequently methylated in flat adenomas.In the panel of flat carcinomas the promoter regions of GATA-4 (73%) andGATA-5 (100%) were most frequently methylated. Similar methylationpatterns were found for flat and polypoid carcinomas, except for CHFR(P=0.03), which shows less methylation in flat carcinomas.ConclusionFor these 16 genes methylation status was similar for flat and polypoid lesions,except for lower promoter methylation for GATA-5 and CHFR in flatadenomas and for CHFR in flat carcinomas. This is consistent with methylationbeing an early event in pathogenesis of both flat and polypoid colorectalcarcinoma.O12Chromosomal aberrations and APC promoter methylation insporadic and coeliac disease related small intestinaladenocarcinomasB Diosdado 1 , TE Buffart 1 ,MTijssen 1 , BJ Bolijn 1 , B Ylstra 1 ,B Carvalho 1 , R Watkins 2 ,FLewis 2 , ID Nagtegaal 3 ,CJJ Mulder 4 ,K Maude 5 , HI Grabsch 2 ,PQuirke 2 , P Howdle 5 , GA Meijer 11 Department of Pathology, VU University Medical Centre, Amsterdam,The Netherlands, 2 Pathology and Tumour Biology, Leeds Institute ofMolecular Medicine, University of Leeds, 3 Department of Pathology,University Medical Centre Nijmegen, Nijmegen, The Netherlands,4 Department of Gastroenterology, VU University Medical Centre,Amsterdam, The Netherlands, 5 Section of Medicine, Surgery andAnaesthesia, Leeds Institute of Molecular Medicine, University of LeedsBackground: Small intestinal adenocarcinoma (SIAC) is a rare malignancyaccounting for less than 2% of all gastrointestinal cancers. The molecularmechanisms involved in SIAC pathogenesis are not completely characterized.Coeliac disease (CD) is a chronic disorder caused by intolerance to gluten ingenetically predisposed individuals. CD patients show 80-fold increased risk ofdeveloping SIAC. Despite this evidence, DNA copy number and methylationchanges of CD-related to non-CD SIAC have not been compared.Aim: To determine the patterns of DNA copy number alterations in non-CDand CD-related SIAC.Methods: Array Comparative Genome Hybridization, microsatellite instabilityand APC promoter hypermethylation status were determined in 35 non-CD and15 CD-related SIAC.Results: Gains of 7p22-p22, 7q22-q34, 8q24, 20q11-q13 overlapped betweenCD-related and non-CD SIAC. CD-related SIAC showed frequent (>15%)losses of 5q15-q23 and gains of 5p13-p15, 7p36, 9q34, 13q12-q13 and 20p13-20q11, whereas non-CD-related SIAC showed frequent losses of 4q12-q35. Tofurther investigate the association between 5q15-q23 loss and the APC gene, wedetermined the methylation status of the APC promoter region. 58% of the CDrelatedand 32% of the non-CD SIAC showed APC promoter hypermethylation(p=0.009).Conclusions: this is the largest study comparing CD-related and non-CD SIAC,which show similar patterns of chromosomal aberrations, however, CD-relatedSIAC present increased frequency of 5q15-q23 losses, APC promoterhypermethylation and decreased frequency of 4q12-q35 losses.80 Summer Meeting (194 th ) 1–4 July 2008 Scientific Programme