O9Development and characterisation <strong>of</strong> a new breast cancer cellline <strong>of</strong> basal phenotypeV Speirs 1 , CA Green 1 , RH Partanen 1 , AM Shaaban 2 , AM Hanby 11 <strong>Leeds</strong> Institute <strong>of</strong> Molecular Medicine, University <strong>of</strong> <strong>Leeds</strong>, 2 St James'sInstitute <strong>of</strong> Oncology, St James's University Hospital, <strong>Leeds</strong>Breast cancer is heterogeneous and current cell line models cannot adequatelyreflect all the phenotypic and genotypic changes observed clinically. During ourroutine development <strong>of</strong> short term primary cultures <strong>of</strong> breast cancer epithelialcells we observed spontaneous immortalisation <strong>of</strong> one <strong>of</strong> these cultures after 12months in vitro. <strong>The</strong>se 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. <strong>The</strong>primary tumour was classified as a lymph node, ER and PR negativesquamous cell carcinoma but expressed ER. Phenotypic characterisation byimmun<strong>of</strong>luorescence revealed expression <strong>of</strong> 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 <strong>of</strong> hypersensitivity at low doses(>10 - 10M). Telomerase activity was assessed by a PCR-based ELISA andremained high in LGI1T cells which is indicative <strong>of</strong> escape from senescence. Insummary we have developed and characterised a new breast cancer cell line <strong>of</strong>basal phenotype. As there is currently considerable interest in understanding thebiology <strong>of</strong> 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 <strong>of</strong> Pathology VU University Medical Centre, Amsterdam, <strong>The</strong>Netherlands, 2 Department <strong>of</strong> Medical Oncology, VU University MedicalCentre, Amsterdam, <strong>The</strong> Netherlands, 3 Philips Research, Eindhoven, <strong>The</strong>NetherlandsIntroduction and Aim: Detection <strong>of</strong> colorectal cancer (CRC) at an early stage <strong>of</strong>disease is a realistic approach to reduce cancer death. <strong>The</strong> aim <strong>of</strong> 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 <strong>of</strong> genes <strong>of</strong> which mRNAlevels are increased in CRC compared to low-risk adenomas. Genes encodingproteins with extracellular domains are the most promising candidates formolecular imaging. <strong>The</strong>refore 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 <strong>of</strong> 1046 proteins were identified upon analysis <strong>of</strong> threebiological replicates <strong>of</strong> colo 205 cells, 563 <strong>of</strong> which were reproducibly detectedin all three samples. Integration <strong>of</strong> the cell-surface proteomics data with themicroarray expression data showed that 98 <strong>of</strong> the 1046 proteins matched withgenes that showed mRNA overexpression in CRC compared to low-riskadenomas.Conclusion: <strong>The</strong>se preliminary data indicate that this strategy resulted in thesuccessful identification <strong>of</strong> cell surface biomarkers. Currently we are extendingthe experiments to other CRC cell lines and the results <strong>of</strong> these experimentsshall be presented.O10Methylation Pattern <strong>of</strong> 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 <strong>of</strong> Pathology, VU University Medical Centre, Amsterdam, <strong>The</strong>Netherlands, 2 Dept <strong>of</strong> Pathology, University Maastricht, Maastricht, <strong>The</strong>Netherlands, 3 Pathology and Tumour Biology, <strong>Leeds</strong> Institute <strong>of</strong>Molecular Medicine, University <strong>of</strong> <strong>Leeds</strong>, UK, 4 Centre for DigestiveDiseases, <strong>Leeds</strong> General Infirmary, <strong>Leeds</strong>, 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 <strong>of</strong> 16 CRC related genes in flat adenomas andcompare this to established methylation patterns <strong>of</strong> polypoid lesions.Materials and methods44 FFPE flat adenomas, (classified according to the Paris classification) and 11flat carcinomas were used. Promoter methylation status <strong>of</strong> 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.Results<strong>The</strong> methylation frequency <strong>of</strong> 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 <strong>of</strong> flat carcinomas the promoter regions <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> Pathology, VU University Medical Centre, Amsterdam,<strong>The</strong> Netherlands, 2 Pathology and Tumour Biology, <strong>Leeds</strong> Institute <strong>of</strong>Molecular Medicine, University <strong>of</strong> <strong>Leeds</strong>, 3 Department <strong>of</strong> Pathology,University Medical Centre Nijmegen, Nijmegen, <strong>The</strong> Netherlands,4 Department <strong>of</strong> Gastroenterology, VU University Medical Centre,Amsterdam, <strong>The</strong> Netherlands, 5 Section <strong>of</strong> Medicine, Surgery andAnaesthesia, <strong>Leeds</strong> Institute <strong>of</strong> Molecular Medicine, University <strong>of</strong> <strong>Leeds</strong>Background: Small intestinal adenocarcinoma (SIAC) is a rare malignancyaccounting for less than 2% <strong>of</strong> all gastrointestinal cancers. <strong>The</strong> 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 <strong>of</strong>developing SIAC. Despite this evidence, DNA copy number and methylationchanges <strong>of</strong> CD-related to non-CD SIAC have not been compared.Aim: To determine the patterns <strong>of</strong> 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 <strong>of</strong> 7p22-p22, 7q22-q34, 8q24, 20q11-q13 overlapped betweenCD-related and non-CD SIAC. CD-related SIAC showed frequent (>15%)losses <strong>of</strong> 5q15-q23 and gains <strong>of</strong> 5p13-p15, 7p36, 9q34, 13q12-q13 and 20p13-20q11, whereas non-CD-related SIAC showed frequent losses <strong>of</strong> 4q12-q35. T<strong>of</strong>urther investigate the association between 5q15-q23 loss and the APC gene, wedetermined the methylation status <strong>of</strong> the APC promoter region. 58% <strong>of</strong> the CDrelatedand 32% <strong>of</strong> 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 <strong>of</strong> chromosomal aberrations, however, CD-relatedSIAC present increased frequency <strong>of</strong> 5q15-q23 losses, APC promoterhypermethylation and decreased frequency <strong>of</strong> 4q12-q35 losses.80 <strong>Summer</strong> <strong>Meeting</strong> (194 th ) 1–4 July <strong>2008</strong> Scientific Programme
O13K-Ras and B-Raf mutations in the MRC CR08 FOCUS TrialSRichman 1 , P Chambers 1 , F Elliott 1 ,CDaly 1 ,MBraun 1 ,J Barrett 1 , G Taylor 1 ,PQuirke 1 , M Seymour 1 , and MRC FocusInvestigators 21 <strong>Leeds</strong> Institute <strong>of</strong> Molecular Medicine, 2 Medical Research Council,LondonEpidermal Growth Factor Receptor (EGFR) activation by ligand binding, withsubsequent activation <strong>of</strong> Ras/Raf/MAPK pathways, is important for survival,proliferation, angiogenesis and metastasis in some cancers. Monoclonalantibodies to EGFR are used in colorectal cancer (CRC), but tumours withactivating KRas mutations may be resistant to EGFR inhibition, due todownstream activation <strong>of</strong> the Ras/Raf pathway. In FOCUS, advanced CRCpatients were randomised to 5FU, 5FU/irinotecan or 5FU/oxaliplatin. Wepreviously showed that benefit from irinotecan/oxaliplatin was associated withtopoisomerase-1 (Topo1) expression, assessed immunohistochemically. Weinvestigated whether K-Ras (or BRaf) status is also predictive <strong>of</strong> benefit fromthese drugs.KRas/BRaf pyrosequencing and Topo1 immunohistochemistry were performedin 801 patients. <strong>The</strong>re was no correlation between Topo1 expression andRas/Raf mutations. Mutation rates were consistent with previous literature:KRas(codons 12,13, 61)BRaf (V600E)EitherMutation 336 (41.9%) 60 (7.5%) 392 (48.9%)No Mutation 449 (56.1%) 729 (91.0%) 406 (50.7%)No Result 16 (2.0%) 12 (1.5%) 3 (0.4%)<strong>The</strong> impact <strong>of</strong> oxaliplatin/irinotecan on Progression-Free and Overall Survivalwere analysed according to Ras/Raf status. No association was found: wild-typeand mutant patients are equally likely to benefit from these drugs (interactionp=0.4-0.8).KRas/BRaf mutations are independent <strong>of</strong> Topo1 and are not predictivebiomarkers for irinotecan/oxaliplatin. FOCUS3 will prospectively assess Topo1and KRas to select optimum combinations <strong>of</strong> anti-EGFR andirinotecan/oxaliplatin therapy.O15<strong>The</strong> Prognosis <strong>of</strong> Oesophageal Carcinoma Depends on theNumber <strong>of</strong> Lymph Nodes Examined in the ResectionSpecimenCP Twine 1 , WG Lewis 1 , A Casbard 2 ,MA Morgan 1 ,DChan 1 ,GWB Clark 1 , T Havard 3 ,TD Crosby 4 ,SA Roberts 1 ,GT Williams 51 University Hospital <strong>of</strong> Wales, Cardiff, 2 Wales Cancer Trials Unit, Cardiff3 Royal Glamorgan Hospital, Llantrisant, 4 Velindre Hospital, Cardiff,5 School <strong>of</strong> Medicine, Cardiff University<strong>The</strong> prognosis in surgically resected oesophageal carcinoma (OC) is dependenton the number <strong>of</strong> regional lymph nodes involved. In colorectal cancer prognosisis also dependent on the number <strong>of</strong> lymph nodes examined in the resectionspecimen. We have investigated whether this phenomenon occurs in OC.237 consecutive patients undergoing oesophagectomy for OC (median age 61yr, 184 male, 189 adenocarcinoma, 42 squamous carcinoma, 123 followingneoadjuvant chemotherapy) by a regional network were studied. Data obtainedfrom routinely generated pathology reports were analysed in relation to patientsurvival.17% <strong>of</strong> tumours were pT1, 14% pT2, 61% pT3 and 8% pT4. <strong>The</strong> median lymphnode count (LNC) was 12 (range 1-38). LNC correlated strongly withoutcome; a plateau was reached after a count <strong>of</strong> 10. <strong>The</strong> median, 2 year and 5year survival was 42 months, 52% and 29% respectively when 10 nodeswere examined (n=149), P=0.005. In 105 patients classified as pN0 thecumulative 2 yr survival was 67% when 10 nodes were examined. On forward conditionalmultivariate Cox regression analysis LNC was independently associated withsurvival (HR 0.95, 95% CI 0.91-0.99, P=0.01), as was pT stage and the absolutenumber <strong>of</strong> positive lymph nodes. <strong>The</strong> effect <strong>of</strong> LNC was independent <strong>of</strong>neoadjuvant chemotherapy.<strong>The</strong>se results demonstrate the importance <strong>of</strong> careful pathological examinationand lymph node retrieval in OC resections. At least 10 nodes should beexamined to designate an OC as pN0.O14Patient Survival According to the Quality <strong>of</strong> Colonic CancerSurgery: Time for Action?NP West 1 , EJA Morris 2 ,ORotimi 3 , A Cairns 3 ,PJ Finan 4 ,PQuirke 11 Pathology & Tumour Biology, <strong>Leeds</strong> Institute <strong>of</strong> Molecular Medicine,<strong>Leeds</strong>, UK, 2 Cancer Epidemiology Group, Centre for Epidemiology &Biostatistics, Northern & Yorkshire Cancer Registry Information Services,St. James's University Hospital, <strong>Leeds</strong>, UK, 3 Gastrointestinal Pathology,<strong>Leeds</strong> Teaching Hospitals Trust, <strong>Leeds</strong>, UK, 4 John Goligher ColorectalUnit, <strong>Leeds</strong> General Infirmary, <strong>Leeds</strong>, UKWe have previously shown that the quality <strong>of</strong> colonic cancer surgery varieswidely when retrospectively assessing the plane <strong>of</strong> mesocolic dissection. Untilnow its relationship to patient survival has remained unknown.400 colonic cancer resections performed between 1997-2002, which hadadequate photographic images, were collected and studied. Cases were gradedas having surgery in the mesocolic plane, intramesocolic plane or muscularispropria plane. All cases were assessed by two independent observers withagreement in 85.5% <strong>of</strong> cases. Additionally, tissue morphometry was performedon the cross sectional images from 252 cases using the Leica QWin imageanalyzer. Patients were retrospectively followed up for a period <strong>of</strong> five years todetermine overall survival.32% <strong>of</strong> cases were resected in the mesocolic plane, 44% in theintramesocolic and 24% in the muscularis propria plane. Better quality surgeryremoved more tissue around the tumour (p=0.0003), with a greater distance tothe resection margins (p
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