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

PL5Identification of Key Breast Cancer PhenotypesAR Green 1 , JM Garibaldi 2 ,DSoria 2 , F Ambrogi 3 ,DPowe 1 ,ERakha 1 , GR Ball 4 ,PLisboa 5 , T Etchells 5 , P Boracchi 3 ,E Biganzoli 3 , IO Ellis 1 .1 Pathology, School of Molecular Medical Sciences, Nottingham UniversityHospitals and University of Nottingham, 2 School of Computer Science,University of Nottingham, 3 Institute of Medical Statistics and Biometry,University of Milan, 4 School of Biomedical and Natural Sciences,Nottingham Trent University, 5 School of Computing and MathematicalSciences, Liverpool John Moores UniversityBreast cancer is a heterogeneous disease, of which several forms have beenidentified on the basis of their gene expression characteristics but translation ofthis into routine clinical practice remains elusive or prohibitively expensive.We have previously used protein expression characteristics to identifycomparable classes. In this study, we extend this approach and further definethe key criteria for class membership.Expression of twenty-five proteins, with known relevance to breast cancer,have been assessed in a series of 1,076 patients. This large data set has beenexamined by four alternative data clustering techniques: Hierarchical, K-means,Partitioning around medoids, Adaptive resonance theory. Concordance betweentechniques was used to elucidate 'core classes' of patients.A total of 663 (62%) of the 1076 patients were assigned to six core classes,while 413 (38%) patients were of mixed class. Three core classes correspond towell known clinical phenotypes (luminal A/B and HER2). Two classescorrespond to the well known basal phenotype, but exhibit a noveldifferentiation into two sub-groups. The last class appears to characterise anovel luminal subgroup.Key clinical phenotypes of breast cancer can be identified using standard,widely available immunocytochemistry technology. The main luminal andbasal breast cancer phenotypes appear to be heterogeneous, containing distinctsub-groups. The six clinical phenotypes determined in this study are a newluminal group, luminal-N, new basal sub-groups, basal p53 altered and basalp53 normal, as well as the well-established luminal A/B and HER2 groups.PL7Nanog, a putative stem cell gene, stimulates cell proliferationin colorectal cancer cell lines but is enriched in the CD133negative sub-population of tumour cells.S Sandhu 1 ,RSeth 1 , S Crook 1 , D Jackson 1 , MIlyas 1 .1 Department of Pathology, QMC, NUH, Nottingham.Introduction:Nanog is a transcription factor associated with maintenance ofembryonic stem cells. It is reported that Wnt signalling can stimulate Nanogexpression and since most colorectal cancers arise as a consequence of aberrantWnt signalling, it may represent a mechanism of maintaining stem cells in thesetumours. We sought to evaluate Nanog expression and function in colorectalcancer (CRC). Methods:24 human CRC cell lines were evaluated for Nanogexpression using real-time QPCR. In addition, Nanog expression was tested inCD133+ and CD133- sub-populations of the cell line SW480. Nanog functionwas tested using SiRNA followed by proliferation and apoptosis assays.Results: All 24 CRC cell lines showed Nanog expression. Variable expressionwith up to 4 orders of magnitude difference between the highest and the lowestexpressing cell line was found. HT29 was chosen for functional analysis. Geneknockdown resulted in an increase in cell number (p = 0.01) but there was nodifference in apoptotic activity. Quantitative analysis of CD133+ and CD133-sub-populations of SW480 showed that Nanog is enriched (approximately twofold)in the CD133- cells.Conclusions: Nanog expression is classically associated with ES cells althoughour data show that it is also expressed in CRC cell lines. In CRC, Nanog maydrive cell proliferation but it does not appear to influence apoptosis. Althoughpurportedly involved in stem cell maintenance, Nanog expression does notcorrelate with expression of CD133 (another stem cell marker) and the relativeroles of these two genes is unclear.PL6The potential role of 3’ alternate splicing events in theregulation of SEPT9 protein expressionGC Crossan 1 , N Pentland 1 , SEH Russell 1 ,PA Hall 1 .1 Queen's University Belfast, Belfast, Northern IrelandWe have shown that the 5’UTRs of SEPT9_v4 and v4* contribute to theregulation of SEPT9_i4 levels (Hum Mol Gen 2007:16:742-52). We have nowaddressed the role of the 3’UTR in regulating SEPT9 expression. Bioinformaticanalysis indicates that there are three 3’ ends encoding 3 carboxy termini (‘a’,‘b’ &‘c’) with two different 3’ UTRs (version 1 in ‘a’ & version 2 in ‘b’ &‘c’)due to non-consensus alternate splicing within exon 12. This was confirmed bya comprehensive set of RT-PCR experiments coupled with sequencing.Furthermore by RT-PCR we find differential expression of the 3 distinct 3’ends in a panel of samples and cell lines with the ‘a’ form being predominant inmalignant tumours. We hypothesise that the different 3’ UTRs might contributeto the translational efficiency of SEPT9 transcripts. One UTR is short (32 bpversion 2) while version 1 is 1938 bp and has considerable predicted secondarystructure as defined by the mFOLD algorithm (G = -816.10 ). Furtherbioinformatic analysis shows that the splicing events that create version 2 UTRremove a set of potential microRNA binding sites present in version 1 UTR.We have created luciferase reporter constructs where the luc gene has either theversion 1 or version 2 UTR, or mutants forms of the version 1 UTR. Usingthese tools we are investigating the role of sequences within the UTRs toregulate luciferase transgene expression.GPC received a Pathological Society Intercalated BSc AwardSummer Meeting (194 th ) 1–4 July 2008 Scientific Programme29