Integrated genomic analysis of 456 pancreatic ductal adenocarcinomas identified 32 recurrently mutated genes that aggregate into 10 pathways: KRAS, TGF-β, WNT, NOTCH, ROBO/SLIT signalling, G1/S transition, SWI-SNF, chromatin modification, DNA repair and RNA processing. Expression analysis defined 4 subtypes: (1) squamous; (2) pancreatic progenitor; (3) immunogenic; and (4) aberrantly differentiated endocrine exocrine (ADEX) that correlate with histopathological characteristics. Squamous tumours are enriched for TP53 and KDM6A mutations, upregulation of the TP63∆N transcriptional network, hypermethylation of pancreatic endodermal cell-fate determining genes and have a poor prognosis. Pancreatic progenitor tumours preferentially express genes involved in early pancreatic development (FOXA2/3, PDX1 and MNX1). ADEX tumours displayed upregulation of genes that regulate networks involved in KRAS activation, exocrine (NR5A2 and RBPJL), and endocrine differentiation (NEUROD1 and NKX2-2). Immunogenic tumours contained upregulated immune networks including pathways involved in acquired immune suppression. These data infer differences in the molecular evolution of pancreatic cancer subtypes and identify opportunities for therapeutic development.

Background and aims: Intraductal papillary mucinous tumours (IPMT) of the pancreas constitute a unique pathological entity with an overall incidence of associated invasive malignancy of 20%. The malignant potential of an individual IPMT cannot be accurately predicted. Preoperative estimation of the risk of associated invasive malignancy with IPMT would be of significant clinical benefit. As aberrations in cell cycle regulatory genes are associated with the progression of precursor pancreatic ductal lesions to invasive adenocarcinoma, we examined expression of key cell cycle regulatory genes in the cyclin D1/retinoblastoma pathway and the transforming growth factor β/Smad4 signalling pathway in a cohort of patients with surgically resected IPMT. Methods: Sections of formalin fixed paraffin embedded pancreatic tissue from a cohort of 18 patients with IPMT were examined using immunohistochemistry for protein expression of cell cycle regulatory genes p16INK4A , p21 CIP1, p27 KIP1, cyclin D1, pRb, and p53, as well as the cell signalling molecule Smad4. A comparison of expression levels was made between adenoma/borderline IPMT (10 patients) and intraductal papillary mucinous carcinoma (IPMC) (eight patients, four of whom harboured invasive carcinoma). Statistical analysis was performed using the χ 2 and Fisher's exact tests. Results: Aberrant expression of the proteins examined increased in frequency from adenoma/ borderline IPMT to IPMC. Specifically, there was a significantly greater incidence of loss of p16 INK4A expression in IPMC: 8/8 lesions (100%) compared with 1/10 (10%) adenoma/borderline IPMT (p<0.001). Similarly, loss of Smad4 expression was associated with IPMC: 3/8 (38%) versus adenoma/borderline IPMT 0/10 (p<0.03). Loss of Smad4 expression within the IPMT was the best marker for the presence of invasive carcinoma (p<0.001). Conclusions: These data indicate that loss of p16 INK4A and Smad4 expression occur more frequently in IPMC alone, or with associated invasive carcinoma, compared with adenoma/borderline IPMT. Aberrant protein expression of these cell cycle regulatory genes in IPMT and pancreatic intraepithelial neoplasia in the current model of pancreatic cancer progression suggest similarities in their development and may also represent the subsequent risk of invasive carcinoma.

The EMS1 and CCND1 genes at chromosome 11q13 are ampli®ed in about 15% of primary breast cancers but appear to confer dierent phenotypes in ER positive and ER negative tumours. Since there are no published data on EMS1 expression in large series of breast cancers we examined the relationship of EMS1 expression with EMS1 gene copy number and expression of mRNAs for cyclin D1 and ER. In a subset of 129 patients, where matched tumour RNA and DNA was available, EMS1 mRNA overexpression was associated predominantly with gene ampli®cation (P=0.0061), whereas cyclin D1 mRNA overexpression was not (P=0.3142). In a more extensive series of 351 breast cancers, there was no correlation between cyclin D1 and EMS1 expression in the EMS1 and cyclin D1 overexpressors (P=0.3503). Although an association between EMS1 mRNA expression and ER positivity was evident (P=0.0232), when the samples were divided into quartiles of EMS1 or cyclin D1 mRNA expression, the increase in the proportion of ER positive tumours in the ascending EMS1 mRNA quartiles was not statistically signi®cant (P=0.0951). In marked contrast there was a signi®cant stepwise increase in ER positivity in ascending quartiles of cyclin D1 mRNA (P=0.030). A potential explanation for this dierence was provided by the observation that in ER positive breast cancer cells oestradiol treatment resulted in increased cyclin D1 gene expression but was without eect on EMS1. The relationship between EMS1 expression and clinical outcome was examined in a subset of 234 patients with median follow-up of 74 months. High EMS1 expression was associated with age 450 years (P=0.0001), postmenopausal status (P=0.0008), lymph node negativity (P=0.019) and an apparent trend for worse prognosis in the ER negative subgroup. These data demonstrate that overexpression of EMS1 mRNA is largely due to EMS1 gene ampli®ca-tion, is independent of cyclin D1 and ER expression and, in contrast to cyclin D1, is not regulated by oestrogen. Independent overexpression of these genes may confer dierent phenotypes and disease outcomes in breast cancer as has been inferred from recent studies of EMS1 and CCND1 gene ampli®cation.

Background: Current biological therapies for breast cancer are tailored to steroid hormone (ER, PR) and HER2 receptor status. Understanding the biological basis of resistance to current targeted therapies and the identification of new potential therapeutic targets is an ongoing challenge. The PI3K pathway is altered in a high proportion of breast cancers and may be associated with specific phenotypes and therapeutic resistance. We undertook an integrative study of mutational, copy number and expression analysis of key regulators of the PI3K pathway in a cohort of 292 invasive breast cancer patients with known treatment outcomes.Material and Methods: Mutations in 3 "hotspots" in exons 9 and 20 of the PIK3CA gene were determined by sequencing and PIK3CA gene copy number by qPCR. PTEN loss and Akt activation i.e. pAkt (Ser473) were assessed by immunohistochemistry using tissue microarrays. Cores were scored by 2 independent observers blinded to clinicopathological data and patient outcome and a histoscore calculated by multiplying the percent positive cells and staining intensity in a range 0-3. Standard statistical tests were applied to assess relationship between the measured parameters and patient outcome.Results: Alterations identified included PIK3CA mutations (12/168 i.e. 7%), PIK3CA copy number gain (28/209 i.e. 14%), PTEN loss (73/258 i.e. 28%) and AKT activation (62/258 i.e. 24%). Overall at least one parameter was altered in 72% i.e. 139 of 193 assessable primary breast cancers. PI3K pathway activation was significantly associated with ER negative (P=0.0008) and PR negative (P=0.006) status, high tumor grade (P=0.032) and a "basal-like" phenotype (P=0.01), where 92% (25/27) of tumors had an altered pathway. In univariate analysis PI3K pathway aberrations were associated with death from breast cancer however this relationship was not maintained in multivariate analysis. No association was identified between an activated pathway and outcome in tamoxifen- or chemotherapy-treated patients.Discussion: We conclude that &gt;70% of breast cancers have an alteration in at least one component of the PI3K pathway and this might be exploited to therapeutic advantage especially in "basal-like" cancers. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2123.