Amber Johns

Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.

Hundreds of individual human cancer genome sequences are expected to be published in 2010, and thousands per year after that. The International Cancer Genome Consortium (ICGC) was launched with the aim of keeping track of the data relating to large-scale cancer genome studies of all major cancers in adults and children — a total of 50 different cancer types and/or subtypes. In this issue the ICGC team ( http://www.icgc.org ) spells out the policies and planning for the project. The International Cancer Genome Consortium (ICGC) was launched to coordinate large-scale cancer genome studies in tumours from 50 different cancer types and/or subtypes that are of clinical and societal importance across the globe. Systematic studies of more than 25,000 cancer genomes at the genomic, epigenomic and transcriptomic levels will reveal the repertoire of oncogenic mutations, uncover traces of the mutagenic influences, define clinically relevant subtypes for prognosis and therapeutic management, and enable the development of new cancer therapies.

Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

The analysis of T-cell antigens in long-term survivors of pancreatic ductal adenocarcinoma suggests that neoantigen immunogenicity and quality, not purely quantity, correlate with survival. A small percentage of patients with pancreatic cancer survive beyond five years, but the reason for their relative longevity remains uncertain. In this retrospective analysis, Vinod Balachandran et al. evaluate the immune mechanisms of long-term survival in human pancreatic cancer. The analysis shows that survival correlates with high mutation load in conjunction with increased infiltration of cytolytic T cells and polyclonal T-cell responses and that mutations at the tumour antigen MUC16 locus are enriched in long-term survivors. Additionally, patients with high predicted neoantigen–microbial cross-reactivity scores tended to live longest. The authors provide evidence that the quality rather than quantity of neoantigens determines survival. Pancreatic ductal adenocarcinoma is a lethal cancer with fewer than 7% of patients surviving past 5 years. T-cell immunity has been linked to the exceptional outcome of the few long-term survivors1,2, yet the relevant antigens remain unknown. Here we use genetic, immunohistochemical and transcriptional immunoprofiling, computational biophysics, and functional assays to identify T-cell antigens in long-term survivors of pancreatic cancer. Using whole-exome sequencing and in silico neoantigen prediction, we found that tumours with both the highest neoantigen number and the most abundant CD8+ T-cell infiltrates, but neither alone, stratified patients with the longest survival. Investigating the specific neoantigen qualities promoting T-cell activation in long-term survivors, we discovered that these individuals were enriched in neoantigen qualities defined by a fitness model, and neoantigens in the tumour antigen MUC16 (also known as CA125). A neoantigen quality fitness model conferring greater immunogenicity to neoantigens with differential presentation and homology to infectious disease-derived peptides identified long-term survivors in two independent datasets, whereas a neoantigen quantity model ascribing greater immunogenicity to increasing neoantigen number alone did not. We detected intratumoural and lasting circulating T-cell reactivity to both high-quality and MUC16 neoantigens in long-term survivors of pancreatic cancer, including clones with specificity to both high-quality neoantigens and predicted cross-reactive microbial epitopes, consistent with neoantigen molecular mimicry. Notably, we observed selective loss of high-quality and MUC16 neoantigenic clones on metastatic progression, suggesting neoantigen immunoediting. Our results identify neoantigens with unique qualities as T-cell targets in pancreatic ductal adenocarcinoma. More broadly, we identify neoantigen quality as a biomarker for immunogenic tumours that may guide the application of immunotherapies.

The diagnosis of pancreatic neuroendocrine tumours (PanNETs) is increasing owing to more sensitive detection methods, and this increase is creating challenges for clinical management. We performed whole-genome sequencing of 102 primary PanNETs and defined the genomic events that characterize their pathogenesis. Here we describe the mutational signatures they harbour, including a deficiency in G:C > T:A base excision repair due to inactivation of MUTYH, which encodes a DNA glycosylase. Clinically sporadic PanNETs contain a larger-than-expected proportion of germline mutations, including previously unreported mutations in the DNA repair genes MUTYH, CHEK2 and BRCA2. Together with mutations in MEN1 and VHL, these mutations occur in 17% of patients. Somatic mutations, including point mutations and gene fusions, were commonly found in genes involved in four main pathways: chromatin remodelling, DNA damage repair, activation of mTOR signalling (including previously undescribed EWSR1 gene fusions), and telomere maintenance. In addition, our gene expression analyses identified a subgroup of tumours associated with hypoxia and HIF signalling. The genomes of 102 primary pancreatic neuroendocrine tumours have been sequenced, revealing mutations in genes with functions such as chromatin remodelling, DNA damage repair, mTOR activation and telomere maintenance, and a greater-than-expected contribution from germ line mutations. Pancreatic neuroendocrine tumours (PanNETs) are the second most common epithelial neoplasm of the pancreas. Aldo Scarpa, Sean Grimmond and colleagues report whole-genome sequencing of 102 primary PanNETs and present analysis of their mutational signatures as part of the International Cancer Genome Consortium. They find frequent mutations in genes with functions that include chromatin remodelling, DNA damage repair, activation of mTOR signalling, and telomere maintenance. They also identify mutational signatures, including one resulting from inactivation of the DNA repair gene MUTYH, and report a larger than expected germline contribution to PanNET development.

Current adjuvant therapies for pancreatic cancer (PC) are inconsistently used and only modestly effective. Because a high proportion of patients who undergo resection for PC likely harbor occult metastatic disease, any adjuvant trials assessing therapies such as radiotherapy directed at locoregional disease are significantly underpowered. Stratification based on the probability (and volume) of residual locoregional disease could play an important role in the design of future clinical trials assessing adjuvant radiotherapy.We assessed the relationships between margin involvement, the proximity to operative resection margins and outcome in a cohort of 365 patients who underwent operative resection for PC.Microscopic involvement of a resection margin by tumor was associated with a poor prognosis. Stratifying the minimum clearance of resection margins by 0.5-mm increments demonstrated that although median survival was no different to clear margins based on these definitions, it was not until the resection margin was clear by more than 1.5 mm that optimal long-term survival was achieved.These data demonstrate that a margin clearance of more than 1.5 mm is important for long-term survival in a subgroup of patients. More aggressive therapeutic approaches that target locoregional disease such as radiotherapy may be beneficial in patients with close surgical margins. Stratification of patients for entry onto future clinical trials based on this criterion may identify those patients who benefit from adjuvant radiotherapy.

BackgroundCurrent staging methods for pancreatic cancer (PC) are inadequate, and biomarkers to aid clinical decision making are lacking. Despite the availability of the serum marker carbohydrate antigen 19.9 (CA19.9) for over two decades, its precise role in the management of PC is yet to be defined, and as a consequence, it is not widely used.MethodsWe assessed the relationship between perioperative serum CA19.9 levels, survival and adjuvant chemotherapeutic responsiveness in a cohort of 260 patients who underwent operative resection for PC.ResultsBy specifically assessing the subgroup of patients with detectable CA19.9, we identified potential utility at key clinical decision points. Low postoperative CA19.9 at 3 months (median survival 25.6 vs 14.8 months, P = 0.0052) and before adjuvant chemotherapy were independent prognostic factors. Patients with postoperative CA 19.9 levels >90 U/ml did not benefit from adjuvant chemotherapy (P = 0.7194) compared with those with a CA19.9 of ≤90 U/ml (median 26.0 vs 16.7 months, P = 0.0108). Normalization of CA19.9 within 6 months of resection was also an independent favorable prognostic factor (median 29.9 vs 14.8 months, P = 0.0004) and normal perioperative CA19.9 levels identified a good prognostic group, which was associated with a 5-year survival of 42%.ConclusionsPerioperative serum CA19.9 measurements are informative in patients with detectable CA19.9 (defined by serum levels of >5 U/ml) and have potential clinical utility in predicting outcome and response to adjuvant chemotherapy. Future clinical trials should prioritize incorporation of CA19.9 measurement at key decision points to prospectively validate these findings and facilitate implementation.

Fine-tuned manipulation of tumor tension and vasculature enhances response to chemotherapy and impairs metastatic spread in pancreatic cancer.

Personalized medicine strategies using genomic profiling are particularly pertinent for pancreas cancer. The Individualized Molecular Pancreatic Cancer Therapy (IMPaCT) trial was initially designed to exploit results from genome sequencing of pancreatic cancer under the auspices of the International Cancer Genome Consortium (ICGC) in Australia. Sequencing revealed small subsets of patients with aberrations in their tumor genome that could be targeted with currently available therapies.The pilot stage of the IMPaCT trial assessed the feasibility of acquiring suitable tumor specimens for molecular analysis and returning high-quality actionable genomic data within a clinically acceptable timeframe. We screened for three molecular targets: HER2 amplification; KRAS wild-type; and mutations in DNA damage repair pathways (BRCA1, BRCA2, PALB2, ATM).Tumor biopsy and archived tumor samples were collected from 93 patients and 76 were screened. To date 22 candidate cases have been identified: 14 KRAS wild-type, 5 cases of HER2 amplification, 2 mutations in BRCA2, and 1 ATM mutation. Median time from consent to the return of validated results was 21.5 days. An inability to obtain a biopsy or insufficient tumor content in the available specimen were common reasons for patient exclusion from molecular analysis while deteriorating performance status prohibited a number of patients from proceeding in the study.Documenting the feasibility of acquiring and screening biospecimens for actionable molecular targets in real time will aid other groups embarking on similar trials. Key elements include the need to better prescreen patients, screen more patients, and offer more attractive clinical trial options.

The importance of epigenetic modifications such as DNA methylation in tumorigenesis is increasingly being appreciated. To define the genome‐wide pattern of DNA methylation in pancreatic ductal adenocarcinomas (PDAC), we captured the methylation profiles of 167 untreated resected PDACs and compared them to a panel of 29 adjacent nontransformed pancreata using high‐density arrays. A total of 11,634 CpG sites associated with 3,522 genes were significantly differentially methylated (DM) in PDAC and were capable of segregating PDAC from non‐malignant pancreas, regardless of tumor cellularity. As expected, PDAC hypermethylation was most prevalent in the 5′ region of genes (including the proximal promoter, 5′UTR and CpG islands). Approximately 33% DM genes showed significant inverse correlation with mRNA expression levels. Pathway analysis revealed an enrichment of aberrantly methylated genes involved in key molecular mechanisms important to PDAC: TGF‐β, WNT, integrin signaling, cell adhesion, stellate cell activation and axon guidance. Given the recent discovery that SLIT‐ROBO mutations play a clinically important role in PDAC, the role of epigenetic perturbation of axon guidance was pursued in more detail. Bisulfite amplicon deep sequencing and qRT‐PCR expression analyses confirmed recurrent perturbation of axon guidance pathway genes SLIT2, SLIT3, ROBO1, ROBO3, ITGA2 and MET and suggests epigenetic suppression of SLIT‐ROBO signaling and up‐regulation of MET and ITGA2 expression. Hypomethylation of MET and ITGA2 correlated with high gene expression, which was associated with poor survival. These data suggest that aberrant methylation plays an important role in pancreatic carcinogenesis affecting core signaling pathways with potential implications for the disease pathophysiology and therapy.

Pancreatic cancer is molecularly diverse, with few effective therapies. Increased mutation burden and defective DNA repair are associated with response to immune checkpoint inhibitors in several other cancer types. We interrogated 385 pancreatic cancer genomes to define hypermutation and its causes. Mutational signatures inferring defects in DNA repair were enriched in those with the highest mutation burdens. Mismatch repair deficiency was identified in 1% of tumors harboring different mechanisms of somatic inactivation of MLH1 and MSH2. Defining mutation load in individual pancreatic cancers and the optimal assay for patient selection may inform clinical trial design for immunotherapy in pancreatic cancer. Pancreatic cancer is molecularly diverse, with few effective therapies. Increased mutation burden and defective DNA repair are associated with response to immune checkpoint inhibitors in several other cancer types. We interrogated 385 pancreatic cancer genomes to define hypermutation and its causes. Mutational signatures inferring defects in DNA repair were enriched in those with the highest mutation burdens. Mismatch repair deficiency was identified in 1% of tumors harboring different mechanisms of somatic inactivation of MLH1 and MSH2. Defining mutation load in individual pancreatic cancers and the optimal assay for patient selection may inform clinical trial design for immunotherapy in pancreatic cancer. Pancreatic ductal adenocarcinoma has a 5-year survival of <5%, with therapies offering only incremental benefit,1Vogelzang N.J. et al.J Clin Oncol. 2012; 30: 88-109Crossref PubMed Scopus (85) Google Scholar potentially due to the diversity of its genomic landscape.2Bailey P. et al.Nature. 2016; 531: 47-52Crossref PubMed Scopus (1973) Google Scholar, 3Biankin A.V. et al.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1379) Google Scholar, 4Waddell N. et al.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1466) Google Scholar Recent reports link high mutation burden with response to immune checkpoint inhibitors in several cancer types.5Le D.T. et al.N Engl J Med. 2015; 372: 2509-2520Crossref PubMed Scopus (6099) Google Scholar Defining tumors that are hypermutated with an increased mutation burden and understanding the underlying mechanisms in pancreatic cancer has the potential to advance therapeutic development, particularly for immunotherapeutic strategies. Whole genome sequencing (WGS, n = 180) and whole exome sequencing (n = 205) of 385 unselected predominantly sporadic pancreatic ductal adenocarcinoma (Supplementary Table 1) defined a mean mutation load of 1.8 and 1.1 mutation per megabase (Mb), respectively (Supplementary Table 2). Outlier analysis identified 20 tumors with the highest mutation burden (5.2%, 15 WGS and 5 exome) (Table 1 and Supplementary Figure 1A), 5 of which were considered extreme outliers and classified as hypermutated as they contained ≥12 somatic mutations/Mb, the defined threshold for hypermutation in colorectal cancer.6Cancer Genome Atlas NetworkNature. 2012; 487: 330-337Crossref PubMed Scopus (5894) Google Scholar Immunohistochemistry for mismatch repair (MMR) proteins (MSH2, MSH6, MLH1, and PMS2) identified 4 MMR-deficient tumors, all of which were hypermutated (n = 180, Figure 1).Table 1Clinical and Histologic Features and Proposed Etiology for Highly Mutated Pancreatic Ductal Adenocarcinoma Tumors (n = 20)Sample IDPersonal and family history of malignancyHistologyMutation load, mutations/MbIHC resultMSIsensor scoreKRAS mutationPredominant mutation signature (mutations/Mb)SV subtype (no. of events)Proposed etiologyHypermutation (extreme outliers) ICGC_0076aSample sequenced by WGS, other samples by exome sequencing.NoneMixed signet ring, mucinous and papillary adenocarcinoma38.55Absent MLH1 and PMS228.3p.G12VMMR (18.3)Scattered (131)MMR deficiency: >280 kb somatic homozygous deletion over MSH2. ICGC_0297aSample sequenced by WGS, other samples by exome sequencing.NoneUndifferentiated adenocarcinoma60.62Absent MSH2 and MSH627.33WTMMR (33.4)Scattered (75)MMR deficiency: Somatic MLH1 promoter hypermethylation. ICGC_0548aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, moderately differentiated30.13Absent MSH2 and MSH617.47WTMMR (16.6)Stable (49)MMR deficiency: >27 kb somatic inversion rearrangement disrupting MSH2. ICGC_0328aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma16.63Normal3.2p.G12DUnknown (11.9)Scattered (110)Cell line with signature: etiology unknown. ICGC_00901 FDR, father CRCDuctal adenocarcinoma, moderately differentiated12.9Absent MSH2 and MSH60.21p.G12CNANAMMR deficiency: somatic MSH2 splice site c.2006G>A.Highly mutated tumors ICGC_0054aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated6.52Normal0.01p.G12VHR deficiency (1.3)Unstable (310)HR deficiency: no germline or somatic cause found. ICGC_0290aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated6.54Not available0.07p.G12VHR deficiency (3.1)Unstable (558)HR deficiency: Germline BRCA2 mutation c.7180A>T, p.A2394*. Somatic CN-LOH. ICGC_0215aSample sequenced by WGS, other samples by exome sequencing.2 FDR lung cancer, 2 FDR prostate cancer. Previous CRC and melanomaDuctal adenocarcinoma, moderately differentiated6.27Normal0.01p.G12VHR deficiency (1.9)Scattered (111)HR deficiency: Germline ATM mutation c.7539_7540delAT, p.Y2514*. Somatic CN-LOH. ICGC_0324NoneDuctal adenocarcinoma, moderately differentiated6.24Normal0p.G12DNANAUndefined ICGC_0034aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated6.09Normal4.02p.G12DHR deficiency (3.4)Unstable (366)HR deficiency: Germline BRCA2 mutation c.5237_5238insT, p.N1747*. Somatic CN-LOH. ICGC_0131aSample sequenced by WGS, other samples by exome sequencing.Lung cancer after PCDuctal adenocarcinoma, moderately differentiated5.63Normal0p.G12DT>G at TT sites (3.0)Focal (147)T>G at TT sites signature: etiology potentially associated with DNA oxidation ICGC_0006aSample sequenced by WGS, other samples by exome sequencing.1 FDR, father lung cancerAdenocarcinoma arising from IPMN, moderately differentiated5.29Normal0.01p.G12DHR deficiency (1.2)Unstable (211)HR deficiency: Somatic BRCA2 c.5351dupA, p.N1784KfsTer3. Somatic CN-LOH. ICGC_0321aSample sequenced by WGS, other samples by exome sequencing.2 FDR, mother and cousin breast cancerDuctal adenocarcinoma, poorly differentiated4.79Not available0p.G12DHR deficiency (2.1)Unstable (286)HR deficiency: Germline BRCA2 c.6699delT, p.F2234LfsTer7. Somatic CN loss- 1 copy. ICGC_0309aSample sequenced by WGS, other samples by exome sequencing.NoneAdenocarcinoma arising from IPMN, moderately differentiated4.74Normal0.03p.G12VT>G at TT sites (3.1)Unstable (232)T>G at TT sites signature: etiology potentially associated with DNA oxidation ICGC_0005aSample sequenced by WGS, other samples by exome sequencing.1 FDR, mother CRCDuctal adenocarcinoma, poorly differentiated4.72Not available1p.G12VHR deficiency (1.1)Focal (95)HR deficiency: No germline or somatic cause found. ICGC_0016aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated4.61Normal3.03p.G12VHR deficiency (1.7)Unstable (447)HR deficiency: potentially linked to Somatic RPA1 c.273G>T, p.R91S ICGC_00461 FDR, brother PCDuctal adenocarcinoma, poorly differentiated4.3Normal0p.Q61HNANAUndefined GARV_0668aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated4.3Not available2.19p.G12VHR deficiency (1.6)Unstable (464)HR deficiency: Germline BRCA2 c.7068_7069delTC, p.L2357VfsTer2. Somatic CN loss - 1 copy. ICGC_0291NoneDuctal adenocarcinoma, well differentiated3.84Not available0.03p.G12RNANAHR deficiency: Somatic BRCA2 c.7283T>A, p.L2428*. ICGC_0256NoneDuctal adenocarcinoma, poorly differentiated3.72Not available0.06p.G12DNANAUndefinedCRC, colorectal cancer; FDR, first-degree relative; IHC, immunohistochemistry; IPMN, intraductal papillary mucinous neoplasm; CN-LOH, copy neutral loss of heterozygosity; CN, copy number; PC, pancreatic cancer; NA, not applicable to exome data.a Sample sequenced by WGS, other samples by exome sequencing. Open table in a new tab CRC, colorectal cancer; FDR, first-degree relative; IHC, immunohistochemistry; IPMN, intraductal papillary mucinous neoplasm; CN-LOH, copy neutral loss of heterozygosity; CN, copy number; PC, pancreatic cancer; NA, not applicable to exome data. KRAS mutation status and histopathologic characteristics have been associated with MMR-deficient pancreatic tumors.7Goggins M. et al.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar Of the 4 MMR-deficient tumors in our cohort, 2 were KRAS wild-type; 3 had undifferentiated to moderately differentiated histology and one had a signet-ring component. These features were not predictive of MMR deficiency in our cohort, as 11 additional non−MMR-deficient tumors had a signet-ring cell component or colloid morphology, and 131 of 347 assessable tumors had poorly or undifferentiated histology. Mutational signature analysis can detect MMR deficiency indirectly based on the pattern of somatic mutations.8Alexandrov L.B. et al.Nature. 2013; 500: 415-421Crossref PubMed Scopus (6213) Google Scholar An MMR-deficient signature dominated the MMR-deficient tumors (with WGS), and was minimal in MMR intact tumors (Supplementary Figure 1). In addition, microsatellite instability (MSI), a hallmark of MMR deficiency in colorectal cancer, was detected in all three MMR deficient tumors with WGS using MSIsensor9Niu B. Ye K. et al.Bioinformatics. 2014; 30: 1015-1016Crossref PubMed Scopus (294) Google Scholar (Supplementary Table 2). MSI was not identified for the fourth MMR deficient sample potentially due to the reduced number of microsatellite loci in exome data. The underlying causes of MMR deficiency in the 4 cases were private somatic events. For 2 cases, MSH2 was disrupted by different structural rearrangements, 1 case contained a missense MSH2 mutation and the last, methylation of the MLH1 promoter (Figure 1). The missense mutation caused an MSH2 splice acceptor site mutation that alters the same nucleotide results in a pathogenic skipping of exon 13 in germline studies.10Thompson B.A. et al.Nat Genet. 2014; 46: 107-115Crossref PubMed Scopus (346) Google Scholar Hypermethylation of the MLH1 promoter is the predominant mechanism of MSI in sporadic colon cancer.11Boland C.R. et al.Gastroenterology. 2010; 138: 2073-2087 e3Abstract Full Text Full Text PDF PubMed Scopus (1359) Google Scholar The remaining hypermutated tumor contained an intact MMR pathway, and was a cell line (ATCC, CRL-2551) with an unidentified mutational signature, therefore the high mutation burden in this sample may be the result of long-term cell culture. The 15 samples (11 WGS and 4 exome) identified in the outlier analysis with high mutation burden, but not hypermutated (∼4 to 12 mutations/Mb) contained no evidence of MMR deficiency. Mutational signature analysis of the WGS samples indicated homologous recombination (HR) repair deficiency as the most substantial (range, 1.0–3.4 mutations/Mb) contributor to the mutation burden for 8 WGS mutation load outlier tumors. In support of a HR defect4Waddell N. et al.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1466) Google Scholar; 7 of these tumors contained high levels of genomic instability with >200 structural variants and mutations in genes involved in HR were present for 6 of 8 cases (Supplementary Table 2). In addition, 1 case that had undergone exome sequencing had a somatic BRCA2 nonsense mutation that likely contributed to HR deficiency in this case. A mutational signature associated with T>G mutations at TT sites previously described in other cancers, including esophageal cancer12Nones K. Waddell N. Wayte N. et al.Nat Commun. 2014; : 5Google Scholar was the major contributor (>3 mutations/Mb) in 2 samples. For these 2 and the remaining 4 cases, no potential causative event could be identified. Although germline defects in MMR genes are well reported in pancreatic cancer13Grant R.C. Selander I. et al.Gastroenterology. 2015; 148: 556-564Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar in our cohort, they did not contribute to MMR deficiency even in those with familial pancreatic cancer or a personal or family history of Lynch-related tumors. A germline truncating variant was detected in PMS2 in 1 case, but did not have loss of the second allele, had normal immunohistochemistry staining and did not display a MMR mutational signature (Supplementary Table 2). MMR deficiency is important in the evolution in a small, but meaningful proportion of pancreatic cancers with a prevalence of 1% (4 of 385) in our cohort. This is consistent with recent studies using the Bethesda polymerase chain reaction panel,14Laghi L. et al.PLoS One. 2012; 7: e46002Crossref PubMed Scopus (55) Google Scholar and with previous estimates of MSI prevalence of 2%−3%.15Nakata B. et al.Clin Cancer Res. 2002; 8: 2536-2540PubMed Google Scholar However, in tumors with low epithelial content that underwent exome sequencing, the sensitivity of somatic mutation detection is reduced, which will affect mutation burden and signature analysis. While cognizant of small numbers, immunohistochemistry was the most accurate in defining MMR due to multiple genomic mechanisms of MMR gene inactivation. Multiple methods to define MMR deficiency may be required for clinical trials that aim to recruit MMR-deficient participants to assess the potential efficacy of checkpoint inhibitors or other therapies in pancreatic cancer. Homologous recombination-deficient tumors, and those with a novel signature seen in esophageal cancer had an increased mutation burden, and need further evaluation as potential patient selection markers for clinical trials of checkpoint inhibitor and other therapies that target tumors with a high mutation burden. The authors would like to thank Cathy Axford, Deborah Gwynne, Mary-Anne Brancato, Clare Watson, Michelle Thomas, Gerard Hammond, and Doug Stetner for central coordination of the Australian Pancreatic Cancer Genome Initiative, data management, and quality control; Mona Martyn-Smith, Lisa Braatvedt, Henry Tang, Virginia Papangelis, and Maria Beilin for biospecimen acquisition; and Sonia Grimaldi and Giada Bonizzato of the ARC-Net Biobank for biospecimen acquisition. For a full list of contributors see Australian Pancreatic Cancer Genome Initiative: http://www.pancreaticcancer.net.au/apgi/collaborators. The cohort consisted of 385 patients with histologically verified pancreatic exocrine carcinoma, prospectively recruited between 2006 and 2013 through the Australian Pancreatic Cancer Genome Initiative (www.pancreaticcancer.net.au) as part of the International Cancer Genome Consortium.1Hudson T.J. et al.Nature. 2010; 464: 993-998Crossref PubMed Scopus (1689) Google Scholar Ethical approval was granted at all treating institutions and individual patients provided informed consent upon entry to the study. The clinicopathologic information for the cohort is described in (Supplementary Table 1), and the global mutation profile has previously been reported for some of these tumors (Supplementary Table 2). Tumor and normal DNA were extracted after histologic review from fresh frozen tissue samples collected at the time of surgical resection or biopsy, as described previously.2Biankin A.V. et al.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1513) Google Scholar Tumor cellularity was determined from single-nucleotide polymorphism array data using qpure.3Song S. et al.PLoS One. 2012; 7: e45835Crossref PubMed Scopus (85) Google Scholar Tumors with epithelial content ≥40% underwent WGS lower cellularity tumors underwent whole exome sequencing. DNA from patient-derived pancreas cell lines and matched normal was also extracted. Exome and WGS were performed using paired 100-bp reads on the Illumina HiSeq 2000, as described previously.2Biankin A.V. et al.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1513) Google Scholar, 4Waddell N. et al.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1686) Google Scholar Regions of germline and somatic copy number change were detected using Illumina SNP BeadChips with GAP.5Popova T. et al.Genome Biol. 2009; 10 (R128−R128)Crossref PubMed Scopus (151) Google Scholar Somatic structural variants were identified from WGS reads using the qSV tool.4Waddell N. et al.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1686) Google Scholar, 6Patch A.M. et al.Nature. 2015; 521: 489-494Crossref PubMed Scopus (930) Google Scholar Single nucleotide variants were called using 2 variant callers: qSNP7Kassahn K.S. et al.PLoS One. 2013; 8: e74380Crossref PubMed Scopus (52) Google Scholar and GATK.8McKenna A. et al.Genome Res. 2010; 20: 1297-1303Crossref PubMed Scopus (14755) Google Scholar Mutations identified by both callers or, those that were unique to a caller but verified by an orthogonal sequencing approach, were considered high confidence and used in all subsequent analyses. Small indels (<200 bp) were identified using Pindel9Ye K. et al.Bioinformatics. 2009; 25: 2865-2871Crossref PubMed Scopus (1391) Google Scholar and each indel was visually inspected in the Integrative Genome Browser. The distribution of the total number of small somatic mutations (coding and noncoding single nucleotide and indel variants) identified per megabase for exome and WGS sequence data were analyzed separately. The group of samples with high mutation load, at the top of each distribution, were defined as the upper distribution outliers for mutations per megabase, that is, ≥75th centile + (1.5× interquartile range). The threshold for detecting outliers in the exome and WGS groups was 3.4 and 4.2 mutations/Mb, respectively. From within the highly mutated set of tumors, hypermutated samples were identified as those with a mutation rate exceeding the thresholds for extreme distribution outliers (≥75th centile + [5× interquartile range]) of 7.4 and 8.1 mutations/Mb for exome and WGS sequencing, respectively. MSIsensor was used to detect microsatellite instability by directly comparing microsatellite repeat lengths between paired normal and tumor sequencing data.10Niu B. et al.Bioinformatics. 2014; 30: 1015-1016Crossref PubMed Scopus (378) Google Scholar A MSIsensor score of >3.5% of somatic microsatellites with repeat length shifts was the detection threshold used to indicate microsatellite instability as published for endometrial cancer.10Niu B. et al.Bioinformatics. 2014; 30: 1015-1016Crossref PubMed Scopus (378) Google Scholar This correlated well with the 5 and 7 microsatellite panels recommended in the Bethesda guidelines.10Niu B. et al.Bioinformatics. 2014; 30: 1015-1016Crossref PubMed Scopus (378) Google Scholar, 11Umar A. et al.J Natl Cancer Inst. 2004; 96: 261-268Crossref PubMed Scopus (2461) Google Scholar Tissue microarrays were constructed using at least three 1-mm formalin-fixed, paraffin-embedded tumor cores. Immunohistochemistry for MSH6 and PMS2 proteins was performed on tissue microarray sections as a screen for MMR deficiency due to MMR proteins forming heterodimers with concordant mismatch repair loss (ie, loss of MLH1 and PMS2 or loss of MSH2 and MSH6).12Hall G. et al.Pathology. 2010; 42: 409-413Abstract Full Text PDF PubMed Scopus (98) Google Scholar Immunohistochemistry on full tumor sections for MSH2, MLH1, MSH6, and PMS2 was performed in those with abnormal staining in core sections. The immunohistochemistry was performed as described previously12Hall G. et al.Pathology. 2010; 42: 409-413Abstract Full Text PDF PubMed Scopus (98) Google Scholar and scored by a senior pathologist. Somatic mutational signatures were extracted from the whole genome sequenced samples using the framework described previously.13Alexandrov L.B. et al.Cell Rep. 2013; 3: 246-259Abstract Full Text Full Text PDF PubMed Scopus (734) Google Scholar High confidence somatic substitutions were classified by the substitution change and sequence context, that is, the type of immediately neighboring bases to the variant. The framework processes the counts of somatic mutations at each context within each sample using non-negative factorization to produce the different signature profiles that are present in the data. The profiles identified were matched against reported signatures from the Cancer of Somatic Mutations in Cancer (http://cancer.sanger.ac.uk/cosmic/signatures). The major contributory signatures, defined as the mutational signature with the highest number of contributing somatic substitution variants, is reported for highly mutated whole genome samples. Bisulfite-converted whole-genome amplified DNA was hybridized to Infinium Human Methylation 450K Beadchips according to the manufacturers protocol (Illumina). Methylation arrays were performed on DNA from 174 pancreatic ductal adenocarcinoma samples, which were compared to DNA from 29 adjacent nonmalignant pancreata. A subset of the methylation data has been published previously.14Nones K. et al.Int J Cancer. 2014; 135: 1110-1118Crossref PubMed Scopus (156) Google Scholar We examined the data for evidence of tumor-specific hypermethylation of the promoter region of MLH1 and MSH2 genes. The methylation array data have been deposited into the International Cancer Genome Consortium data portal (dcc.icgc.org, project PACA-AU). Download .xlsx (.08 MB) Help with xlsx files Supplementary Tables 1 and 2

The Global Alliance for Genomics and Health (GA4GH) aims to accelerate biomedical advances by enabling the responsible sharing of clinical and genomic data through both harmonized data aggregation and federated approaches. The decreasing cost of genomic sequencing (along with other genome-wide molecular assays) and increasing evidence of its clinical utility will soon drive the generation of sequence data from tens of millions of humans, with increasing levels of diversity. In this perspective, we present the GA4GH strategies for addressing the major challenges of this data revolution. We describe the GA4GH organization, which is fueled by the development efforts of eight Work Streams and informed by the needs of 24 Driver Projects and other key stakeholders. We present the GA4GH suite of secure, interoperable technical standards and policy frameworks and review the current status of standards, their relevance to key domains of research and clinical care, and future plans of GA4GH. Broad international participation in building, adopting, and deploying GA4GH standards and frameworks will catalyze an unprecedented effort in data sharing that will be critical to advancing genomic medicine and ensuring that all populations can access its benefits.

Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarker-driven therapeutic strategy targeting DDR and replication stress in PC.We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids.Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P < .001) and PARP inhibitor therapy (P < .001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P < .018) and WEE1 inhibitor (P < .029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < .001) but was not associated with DDR deficiency.Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy.

Abstract Cancer immunoediting 1 is a hallmark of cancer 2 that predicts that lymphocytes kill more immunogenic cancer cells to cause less immunogenic clones to dominate a population. Although proven in mice 1,3 , whether immunoediting occurs naturally in human cancers remains unclear. Here, to address this, we investigate how 70 human pancreatic cancers evolved over 10 years. We find that, despite having more time to accumulate mutations, rare long-term survivors of pancreatic cancer who have stronger T cell activity in primary tumours develop genetically less heterogeneous recurrent tumours with fewer immunogenic mutations (neoantigens). To quantify whether immunoediting underlies these observations, we infer that a neoantigen is immunogenic (high-quality) by two features—‘non-selfness’ based on neoantigen similarity to known antigens 4,5 , and ‘selfness’ based on the antigenic distance required for a neoantigen to differentially bind to the MHC or activate a T cell compared with its wild-type peptide. Using these features, we estimate cancer clone fitness as the aggregate cost of T cells recognizing high-quality neoantigens offset by gains from oncogenic mutations. With this model, we predict the clonal evolution of tumours to reveal that long-term survivors of pancreatic cancer develop recurrent tumours with fewer high-quality neoantigens. Thus, we submit evidence that that the human immune system naturally edits neoantigens. Furthermore, we present a model to predict how immune pressure induces cancer cell populations to evolve over time. More broadly, our results argue that the immune system fundamentally surveils host genetic changes to suppress cancer.

Purpose Individuals with adenocarcinoma of the ampulla of Vater demonstrate a broad range of outcomes, presumably because these cancers may arise from any one of the three epithelia that converge at that location. This variability poses challenges for clinical decision making and the development of novel therapeutic strategies. Patients and Methods We assessed the potential clinical utility of histomolecular phenotypes defined using a combination of histopathology and protein expression (CDX2 and MUC1) in 208 patients from three independent cohorts who underwent surgical resection for adenocarcinoma of the ampulla of Vater. Results Histologic subtype and CDX2 and MUC1 expression were significant prognostic variables. Patients with a histomolecular pancreaticobiliary phenotype (CDX2 negative, MUC1 positive) segregated into a poor prognostic group in the training (hazard ratio [HR], 3.34; 95% CI, 1.69 to 6.62; P &lt; .001) and both validation cohorts (HR, 5.65; 95% CI, 2.77 to 11.5; P &lt; .001 and HR, 2.78; 95% CI, 1.25 to 7.17; P = .0119) compared with histomolecular nonpancreaticobiliary carcinomas. Further stratification by lymph node (LN) status defined three clinically relevant subgroups: one, patients with histomolecular nonpancreaticobiliary (intestinal) carcinoma without LN metastases who had an excellent prognosis; two, those with histomolecular pancreaticobiliary carcinoma with LN metastases who had a poor outcome; and three, the remainder of patients (nonpancreaticobiliary, LN positive or pancreaticobiliary, LN negative) who had an intermediate outcome. Conclusion Histopathologic and molecular criteria combine to define clinically relevant histomolecular phenotypes of adenocarcinoma of the ampulla of Vater and potentially represent distinct diseases with significant implications for current therapeutic strategies, the ability to interpret past clinical trials, and future trial design.

The ampulla of Vater is a complex cellular environment from which adenocarcinomas arise to form a group of histopathologically heterogenous tumors. To evaluate the molecular features of these tumors, 98 ampullary adenocarcinomas were evaluated and compared to 44 distal bile duct and 18 duodenal adenocarcinomas. Genomic analyses revealed mutations in the WNT signaling pathway among half of the patients and in all three adenocarcinomas irrespective of their origin and histological morphology. These tumors were characterized by a high frequency of inactivating mutations of ELF3, a high rate of microsatellite instability, and common focal deletions and amplifications, suggesting common attributes in the molecular pathogenesis are at play in these tumors. The high frequency of WNT pathway activating mutation, coupled with small-molecule inhibitors of β-catenin in clinical trials, suggests future treatment decisions for these patients may be guided by genomic analysis.

Pancreatic cancer is one of the most lethal and molecularly diverse malignancies. Repurposing of therapeutics that target specific molecular mechanisms in different disease types offers potential for rapid improvements in outcome. Although HER2 amplification occurs in pancreatic cancer, it is inadequately characterized to exploit the potential of anti-HER2 therapies.HER2 amplification was detected and further analyzed using multiple genomic sequencing approaches. Standardized reference laboratory assays defined HER2 amplification in a large cohort of patients (n = 469) with pancreatic ductal adenocarcinoma (PDAC).An amplified inversion event (1 MB) was identified at the HER2 locus in a patient with PDAC. Using standardized laboratory assays, we established diagnostic criteria for HER2 amplification in PDAC, and observed a prevalence of 2%. Clinically, HER2- amplified PDAC was characterized by a lack of liver metastases, and a preponderance of lung and brain metastases. Excluding breast and gastric cancer, the incidence of HER2-amplified cancers in the USA is >22,000 per annum.HER2 amplification occurs in 2% of PDAC, and has distinct features with implications for clinical practice. The molecular heterogeneity of PDAC implies that even an incidence of 2% represents an attractive target for anti-HER2 therapies, as options for PDAC are limited. Recruiting patients based on HER2 amplification, rather than organ of origin, could make trials of anti-HER2 therapies feasible in less common cancer types.

Extensive molecular heterogeneity of pancreatic ductal adenocarcinoma (PDA), few effective therapies and high mortality make this disease a prime model for advancing development of tailored therapies. The p16-cyclin D-cyclin-dependent kinase 4/6-retinoblastoma (RB) protein (CDK4) pathway, regulator of cell proliferation, is deregulated in PDA. Our aim was to develop a novel personalised treatment strategy for PDA based on targeting CDK4.Sensitivity to potent CDK4/6 inhibitor PD-0332991 (palbociclib) was correlated to protein and genomic data in 19 primary patient-derived PDA lines to identify biomarkers of response. In vivo efficacy of PD-0332991 and combination therapies was determined in subcutaneous, intrasplenic and orthotopic tumour models derived from genome-sequenced patient specimens and genetically engineered model. Mechanistically, monotherapy and combination therapy were investigated in the context of tumour cell and extracellular matrix (ECM) signalling. Prognostic relevance of companion biomarker, RB protein, was evaluated and validated in independent PDA patient cohorts (>500 specimens).Subtype-specific in vivo efficacy of PD-0332991-based therapy was for the first time observed at multiple stages of PDA progression: primary tumour growth, recurrence (second-line therapy) and metastatic setting and may potentially be guided by a simple biomarker (RB protein). PD-0332991 significantly disrupted surrounding ECM organisation, leading to increased quiescence, apoptosis, improved chemosensitivity, decreased invasion, metastatic spread and PDA progression in vivo. RB protein is prevalent in primary operable and metastatic PDA and may present a promising predictive biomarker to guide this therapeutic approach.This study demonstrates the promise of CDK4 inhibition in PDA over standard therapy when applied in a molecular subtype-specific context.

Tumour cellularity, the relative proportion of tumour and normal cells in a sample, affects the sensitivity of mutation detection, copy number analysis, cancer gene expression and methylation profiling. Tumour cellularity is traditionally estimated by pathological review of sectioned specimens; however this method is both subjective and prone to error due to heterogeneity within lesions and cellularity differences between the sample viewed during pathological review and tissue used for research purposes. In this paper we describe a statistical model to estimate tumour cellularity from SNP array profiles of paired tumour and normal samples using shifts in SNP allele frequency at regions of loss of heterozygosity (LOH) in the tumour. We also provide qpure, a software implementation of the method. Our experiments showed that there is a medium correlation 0.42 ([Formula: see text]-value=0.0001) between tumor cellularity estimated by qpure and pathology review. Interestingly there is a high correlation 0.87 ([Formula: see text]-value [Formula: see text] 2.2e-16) between cellularity estimates by qpure and deep Ion Torrent sequencing of known somatic KRAS mutations; and a weaker correlation 0.32 ([Formula: see text]-value=0.004) between IonTorrent sequencing and pathology review. This suggests that qpure may be a more accurate predictor of tumour cellularity than pathology review. qpure can be downloaded from https://sourceforge.net/projects/qpure/.

Cancer-associated fibroblasts (CAF) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression through protumor signaling and the generation of fibrosis, the latter of which creates a physical barrier to drugs. CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11 is a cystine transporter that has been identified as a potential therapeutic target in PDAC cells. However, no prior study has evaluated the role of SLC7A11 in PDAC tumor stroma and its prognostic significance. Here we show that high expression of SLC7A11 in human PDAC tumor stroma, but not tumor cells, is independently prognostic of poorer overall survival. Orthogonal approaches showed that PDAC-derived CAFs are highly dependent on SLC7A11 for cystine uptake and glutathione synthesis and that SLC7A11 inhibition significantly decreases CAF proliferation, reduces their resistance to oxidative stress, and inhibits their ability to remodel collagen and support PDAC cell growth. Importantly, specific ablation of SLC7A11 from the tumor compartment of transgenic mouse PDAC tumors did not affect tumor growth, suggesting the stroma can substantially influence PDAC tumor response to SLC7A11 inhibition. In a mouse orthotopic PDAC model utilizing human PDAC cells and CAFs, stable knockdown of SLC7A11 was required in both cell types to reduce tumor growth, metastatic spread, and intratumoral fibrosis, demonstrating the importance of targeting SLC7A11 in both compartments. Finally, treatment with a nanoparticle gene-silencing drug against SLC7A11, developed by our laboratory, reduced PDAC tumor growth, incidence of metastases, CAF activation, and fibrosis in orthotopic PDAC tumors. Overall, these findings identify an important role of SLC7A11 in PDAC-derived CAFs in supporting tumor growth. SIGNIFICANCE: This study demonstrates that SLC7A11 in PDAC stromal cells is important for the tumor-promoting activity of CAFs and validates a clinically translatable nanomedicine for therapeutic SLC7A11 inhibition in PDAC.

Background: The long-term outcomes following surgical resection for pancreatic ductal adenocarcinoma (PDAC) remains poor, with only 20% of patients surviving 5 years after pancreatectomy. Patient selection for surgery remains suboptimal largely due to the absence of consideration of aggressive tumor biology. Objective: The aim of this study was to evaluate traditional staging criteria for PDAC in the setting of molecular subtypes. Methods: Clinicopathological data were obtained for 5 independent cohorts of consecutive unselected patients, totaling n = 1298, including n = 442 that underwent molecular subtyping. The main outcome measure was disease-specific survival following surgical resection for PDAC stratified according to the American Joint Commission for Cancer (TNM) staging criteria, margin status, and molecular subtype. Results: TNM staging criteria and margin status confers prognostic value only in tumors with classical pancreatic subtype. Patients with tumors that are of squamous subtype, have a poor outcome irrespective of favorable traditional pathological staging [hazard ratio (HR) 1.54, 95% confidence interval (CI) 1.04–2.28, P = 0.032]. Margin status has no impact on survival in the squamous subtype (16.0 vs 12.1 months, P = 0.374). There were no differences in molecular subtype or gene expression of tumors with positive resection margin status. Conclusions: Aggressive tumor biology as measured by molecular subtype predicts poor outcome following pancreatectomy for PDAC and should be utilized to inform patient selection for surgery.

Aim We examined whether introduction of a standardised pancreatic cancer minimum data set improved the reporting of key pathological features across multiple institutions. Methods From seven different pathology departments that are members of the New South Wales Pancreatic Cancer Network, 109 free text reports and 68 synoptic reports were compared. Results AJCC stage could not be inferred from 44% of free text reports, whereas stage was reported in all 68 synoptic reports. In the free text reports 28 different names were used to designate margins. All margins were reported in only 12 (11%) of the free text reports compared with 64 (94%) of the synoptic reports (p = 0.0011). The presence or absence of lymphovascular or perineural invasion was reported in 72 (66%) and 92 (84%) of free text reports, respectively. In contrast, lymphovascular space and perineural invasion were reported in all synoptic reports (p = 0.0011 and p = 0.0058). Conclusion: We conclude that synoptic reporting of pancreatic resections without any other intervention increases the information contained within histopathology reports. Therefore, the introduction of minimal data set synoptic reports is a simple and feasible mechanism to immediately improve reporting for pancreatectomy specimens.

Somatic mutation calling from next-generation sequencing data remains a challenge due to the difficulties of distinguishing true somatic events from artifacts arising from PCR, sequencing errors or mis-mapping. Tumor cellularity or purity, sub-clonality and copy number changes also confound the identification of true somatic events against a background of germline variants. We have developed a heuristic strategy and software (http://www.qcmg.org/bioinformatics/qsnp/) for somatic mutation calling in samples with low tumor content and we show the superior sensitivity and precision of our approach using a previously sequenced cell line, a series of tumor/normal admixtures, and 3,253 putative somatic SNVs verified on an orthogonal platform.

We examined the expression of cyclin D1 in conjunction with β-catenin and the phosphorylated inactive form of glycogen synthase kinase 3β (GSK-3β) in benign, nonneoplastic thyroid tissue as well as papillary thyroid carcinoma primary tumors and nodal metastases. We aim to unravel the regulation of cyclin D1 and determine if this cell cycle protein is a useful biomarker for metastatic disease. It is clear that expression of cyclin D1 (P < .0001), β-catenin (P < .0001), and inactive form of GSK-3β (P < .0001) are significantly higher in papillary thyroid carcinoma primary tumors than in corresponding benign, nonneoplastic tissue thyroid specimens. Interestingly, β-catenin and cyclin D1 expressions in papillary thyroid carcinoma are correlated (P = .025), implying that β-catenin is a factor driving higher levels of cyclin D1 consistent with previous cell models linking Wnt/β-catenin signaling and cyclin D1 expression. Conversely, inactive form of GSK-3β expression does not correlate with cyclin D1 (P = .52) or β-catenin expression (P = .54). We also did not observe any relationship between tumor size and marker expression. Comparing papillary thyroid carcinoma primary tumors with or without nodal metastases, we did not see any differences in expression of inactive form of GSK-3β (P = .95), β-catenin (P = .14), or cyclin D1 (P = .46). However, in papillary thyroid carcinoma lymph node specimens, the up-regulation of cyclin D1 (P = .0083) was highly significant compared with primary tumors. pGSK-3β and β-catenin expression did not vary between primary tumors and nodal specimens. In conclusion, we have demonstrated that expression of cyclin D1 is linked to nodal metastases and that cyclin D1 levels are regulated by Wnt/β-catenin signaling. GSK pathway–mediated regulation of β-catenin or cyclin D1 expression does not appear operative in papillary thyroid carcinoma.

Adjuvant chemotherapy improves survival for patients with resected pancreatic cancer. Elderly patients are under-represented in Phase III clinical trials, and as a consequence the efficacy of adjuvant therapy in older patients with pancreatic cancer is not clear. We aimed to assess the use and efficacy of adjuvant chemotherapy in older patients with pancreatic cancer.We assessed a community cohort of 439 patients with a diagnosis of pancreatic ductal adenocarcinoma who underwent operative resection in centres associated with the Australian Pancreatic Cancer Genome Initiative.The median age of the cohort was 67 years. Overall only 47% of all patients received adjuvant therapy. Patients who received adjuvant chemotherapy were predominantly younger, had later stage disease, more lymph node involvement and more evidence of perineural invasion than the group that did not receive adjuvant treatment. Overall, adjuvant chemotherapy was associated with prolonged survival (median 22.1 vs 15.8 months; P<0.0001). Older patients (aged ≥70) were less likely to receive adjuvant chemotherapy (51.5% vs 29.8%; P<0.0001). Older patients had a particularly poor outcome when adjuvant therapy was not delivered (median survival=13.1 months; HR 1.89, 95% CI: 1.27-2.78, P=0.002).Patients aged ≥70 are less likely to receive adjuvant therapy although it is associated with improved outcome. Increased use of adjuvant therapy in older individuals is encouraged as they constitute a large proportion of patients with pancreatic cancer.

Pancreatic cancer is a leading cause of cancer-related deaths in Western societies.This poor prognosis is due to chemotherapeutic drug resistance and metastatic spread.Evidence suggests that microtubule proteins namely, β-tubulins are dysregulated in tumor cells and are involved in regulating chemosensitivity.However, the role of β-tubulins in pancreatic cancer are unknown.We measured the expression of different β-tubulin isotypes in pancreatic adenocarcinoma tissue and pancreatic cancer cells.Next, we used RNAi to silence βIII-tubulin expression in pancreatic cancer cells, and measured cell growth in the absence and presence of chemotherapeutic drugs.Finally, we assessed the role of βIII-tubulin in regulating tumor growth and metastases using an orthotopic pancreatic cancer mouse model.We found that βIII-tubulin is highly expressed in pancreatic adenocarcinoma tissue and pancreatic cancer cells.Further, we demonstrated that silencing βIII-tubulin expression reduced pancreatic cancer cell growth and tumorigenic potential in the absence and presence of chemotherapeutic drugs.Finally, we demonstrated that suppression of βIII-tubulin reduced tumor growth and metastases in vivo.Our novel data demonstrate that βIII-tubulin is a key player in promoting pancreatic cancer growth and survival, and silencing its expression may be a potential therapeutic strategy to increase the long-term survival of pancreatic cancer patients.

BACKGROUND Inherited predisposition to pancreatic cancer contributes significantly to its incidence and presents an opportunity for the development of early detection strategies. The genetic basis of predisposition remains unexplained in a high proportion of patients with familial PC (FPC). METHODS Clinicopathologic features were assessed in a cohort of 766 patients who had been diagnosed with pancreatic ductal adenocarcinoma (PC). Patients were classified with FPC if they had ≥1 affected first‐degree relatives; otherwise, they were classified with sporadic PC (SPC). RESULTS The prevalence of FPC in this cohort was 8.9%. In FPC families with an affected parent‐child pair, 71% in the subsequent generation were 12.3 years younger at diagnosis. Patients with FPC had more first‐degree relatives who had an extrapancreatic malignancy (EPM) (42.6% vs 21.2; P &lt;.0001), particularly melanoma and endometrial cancer, but not a personal history of EPM. Patients with SPC were more likely to be active smokers, have higher cumulative tobacco exposure, and have fewer multifocal precursor lesions, but these were not associated with differences in survival. Long‐standing diabetes mellitus (&gt;2 years) was associated with poor survival in both groups. CONCLUSIONS FPC represents 9% of PC, and the risk of malignancy in kindred does not appear to be confined to the pancreas. Patients with FPC have more precursor lesions and include fewer active smokers, but other clinicopathologic factors and outcome are similar to those in patients with SPC. Furthermore, some FPC kindreds may exhibit anticipation. A better understanding of the clinical features of PC will facilitate efforts to uncover novel susceptibility genes and the development of early detection strategies. Cancer 2014;120:3669–3675. © 2014 American Cancer Society .

Despite the plethora of empirical studies conducted to date, debate continues about whether and to what extent results should be returned to participants of genomic research. We aimed to systematically review the empirical literature exploring stakeholders' perspectives on return of individual research results (IRR) from genomic research. We examined preferences for receiving or willingness to return IRR, and experiences with either receiving or returning them. The systematic searches were conducted across five major databases in August 2018 and repeated in April 2020, and included studies reporting findings from primary research regardless of method (quantitative, qualitative, mixed). Articles that related to the clinical setting were excluded. Our search identified 221 articles that met our search criteria. This included 118 quantitative, 69 qualitative and 34 mixed methods studies. These articles included a total number of 118,874 stakeholders with research participants (85,270/72%) and members of the general public (40,967/35%) being the largest groups represented. The articles spanned at least 22 different countries with most (144/65%) being from the USA. Most (76%) discussed clinical research projects, rather than biobanks. More than half (58%) gauged views that were hypothetical. We found overwhelming evidence of high interest in return of IRR from potential and actual genomic research participants. There is also a general willingness to provide such results by researchers and health professionals, although they tend to adopt a more cautious stance. While all results are desired to some degree, those that have the potential to change clinical management are generally prioritized by all stakeholders. Professional stakeholders appear more willing to return results that are reliable and clinically relevant than those that are less reliable and lack clinical relevance. The lack of evidence for significant enduring psychological harm and the clear benefits to some research participants suggest that researchers should be returning actionable IRRs to participants.

Abstract Here we report the DNA methylation profile of 84 sporadic pancreatic neuroendocrine tumors (PanNETs) with associated clinical and genomic information. We identified three subgroups of PanNETs, termed T1, T2 and T3, with distinct patterns of methylation. The T1 subgroup was enriched for functional tumors and ATRX , DAXX and MEN1 wild-type genotypes. The T2 subgroup contained tumors with mutations in ATRX , DAXX and MEN1 and recurrent patterns of chromosomal losses in half of the genome with no association between regions with recurrent loss and methylation levels. T2 tumors were larger and had lower methylation in the MGMT gene body, which showed positive correlation with gene expression. The T3 subgroup harboured mutations in MEN1 with recurrent loss of chromosome 11, was enriched for grade G1 tumors and showed histological parameters associated with better prognosis. Our results suggest a role for methylation in both driving tumorigenesis and potentially stratifying prognosis in PanNETs.

Radical new possibilities of improved treatment of cancer are on offer from an advanced medical technology already demonstrating its significance: next-generation sequencing (NGS). This refined testing provides unprecedentedly precise diagnoses and permits the use of focused and highly personalized treatments. However, across regions globally, many cancer patients will continue to be denied the benefits of NGS as long as some of the yawning gaps in its implementation remain unattended. The challenges at the regional and national levels are linked because putting the solutions into effect is highly dependent on cooperation between regional- and national-level cooperation, which could be hindered by shortfalls in interpretation or understanding. The aim of the paper was to define and explore the necessary conditions for NGS and make recommendations for effective implementation based on extensive exchanges with policy makers and stakeholders. As a result, the European Alliance for Personalised Medicine (EAPM) developed a maturity framework structured around demand-side and supply-side issues to enable interested stakeholders in different countries to self-evaluate according to a common matrix. A questionnaire was designed to identify the current status of NGS implementation, and it was submitted to different experts in different institutions globally. This revealed significant variability in the different aspects of NGS uptake. Within different regions globally, to ensure those conditions are right, this can be improved by linking efforts made at the national level, where patients have needs and where care is delivered, and at the global level, where major policy initiatives in the health field are underway or in preparation, many of which offer direct or indirect pathways for building those conditions. In addition, in a period when consensus is still incomplete and catching up is needed at a political level to ensure rational allocation of resources-even within individual countries-to enable the best ways to make the necessary provisions for NGS, a key recommendation is to examine where closer links between national and regional actions could complement, support, and mutually reinforce efforts to improve the situation for patients.

Abstract Background and Aim: Clinicopathological data regarding pancreatic solid pseudopapillary tumors (SPT) in a multiethnic country are limited. The aim of the present study was to characterize pancreatic SPT in Australia. Methods: Clinicopathological features, treatment, immunohistochemical findings and outcome data of 34 patients (79% Caucasian, 12% Asian, 6% South Pacific Islander and 3% African) with pancreatic SPT were reviewed. Results: The most presenting complaint was abdominal pain. Median diameter of tumors was 60 mm (range: 20–220); predominantly located in the pancreatic tail (tail : body : head = 23:3:8). All tumors were resected and patients underwent surgery, including a liver resection for metastasis, all patients were alive after a median follow up of 70 months (IQR: 48–178). Two patients underwent repeated surgery for local recurrences with liver metastases after 8 and 18 months, which were successfully managed by surgical resection. Completeness of excision, perineural spread, vascular space invasion, mitotic rate and cellular atypia did not predict recurrence. In all cases, there was aberrant nuclear staining of beta‐catenin and a loss of membranous expression of E‐cadherin with aberrant nuclear localization of the cytoplasmic domain. Most pancreatic SPT were also strongly positive for CD10 (96%), progesterone receptor (79%), cytokeratin (28%), synapthophysin (26%) and chromogranin (15%). Conclusions: Pancreatic SPT occur in all races and are uniformly indolent. Given complete resection of a pancreatic SPT is usually curative and recurrences can be treated with re‐operation, correct diagnosis is important.

We aimed to define preoperative clinical and molecular characteristics that would allow better patient selection for operative resection.Although we use molecular selection methods for systemic targeted therapies, these principles are not applied to surgical oncology. Improving patient selection is of vital importance for the operative treatment of pancreatic cancer (pancreatic ductal adenocarcinoma). Although surgery is the only chance of long-term survival, 80% still succumb to the disease and approximately 30% die within 1 year, often sooner than those that have unresected local disease.In 3 independent pancreatic ductal adenocarcinoma cohorts (total participants = 1184) the relationship between aberrant expression of prometastatic proteins S100A2 and S100A4 and survival was assessed. A preoperative nomogram based on clinical variables available before surgery and expression of these proteins was constructed and compared to traditional measures, and a postoperative nomogram.High expression of either S100A2 or S100A4 was independent poor prognostic factors in a training cohort of 518 participants. These results were validated in 2 independent patient cohorts (Glasgow, n = 198; Germany, n = 468). Aberrant biomarker expression stratified the cohorts into 3 distinct prognostic groups. A preoperative nomogram incorporating S100A2 and S100A4 expression predicted survival and nomograms derived using postoperative clinicopathological variables.Of those patients with a poor preoperative nomogram score, approximately 50% of patients died within a year of resection. Nomograms have the potential to improve selection for surgery and neoadjuvant therapy, avoiding surgery in aggressive disease, and justifying more extensive resections in biologically favorable disease.

Intravital imaging guides a personalized medicine approach to target mechanoreciprocity in pancreatic cancer.

The lysyl oxidase family represents a promising target in stromal targeting of solid tumors due to the importance of this family in crosslinking and stabilizing fibrillar collagens and its known role in tumor desmoplasia. Using small-molecule drug-design approaches, we generated and validated PXS-5505, a first-in-class highly selective and potent pan-lysyl oxidase inhibitor. We demonstrate in vitro and in vivo that pan-lysyl oxidase inhibition decreases chemotherapy-induced pancreatic tumor desmoplasia and stiffness, reduces cancer cell invasion and metastasis, improves tumor perfusion and enhances the efficacy of chemotherapy in the autochthonous genetically engineered KPC model, while also demonstrating antifibrotic effects in human patient-derived xenograft models of pancreatic cancer. PXS-5505 is orally bioavailable, safe and effective at inhibiting lysyl oxidase activity in tissues. Our findings present the rationale for progression of a pan-lysyl oxidase inhibitor aimed at eliciting a reduction in stromal matrix to potentiate chemotherapy in pancreatic ductal adenocarcinoma.

Abstract Pancreatic cancer is the fourth leading cause of cancer death in our society, with a mortality that virtually parallels its incidence, a median survival of &lt;12 months even with maximal therapy, and a 5‐year survival rate of &lt;5 %. The diversity of clinical outcomes and the molecular heterogeneity of histopathologically similar cancer types, incomplete knowledge of the genomic aberrations that drive carcinogenesis and the lack of therapeutics that specifically target most known genomic aberrations necessitates large‐scale detailed analysis of cancer genomes to identify novel potential therapeutic strategies. As part of the International Cancer Genome Consortium (ICGC), the Australian Pancreatic Cancer Genome Initiative (APGI) used exomic sequencing and copy number analysis to define genomic aberrations that characterize a large, clinically focused, prospectively accrued cohort of patients with pancreatic cancer. The cohort consisted of early (clinical stages I and II) non‐pre‐treated patients with pancreatic ductal adenocarcinoma who underwent operative resection with curative intent. We devised approaches to adjust for low epithelial content in primary tumours and to define the genomic landscape of pancreatic cancer to identify novel candidate driver genes and mechanisms. We aim to develop stratified, molecular phenotype‐guided therapeutic strategies using existing therapeutics that are either rescued, repurposed, in development, or are known to be effective in an undefined subgroup of PC patients. These are then tested in primary patient‐derived xenografts and cell lines from the above deeply characterized cohort. In addition, we return information to treating clinicians that influences patient care and are launching a clinical trial called IMPaCT (Individualized Molecular Pancreatic Cancer Therapy). This umbrella design trial randomizes patients with metastatic disease to either standard first‐line therapy with gemcitabine, or a molecular phenotype‐guided approach using next‐generation sequencing strategies to screen for actionable mutations defined through the ICGC effort.

The return of research results (RoR) remains a complex and well-debated issue. Despite the debate, actual data related to the experience of giving individual results back, and the impact these results may have on clinical care and health outcomes, is sorely lacking. Through the work of the Australian Pancreatic Cancer Genome Initiative (APGI) we: (1) delineate the pathway back to the patient where actionable research data were identified; and (2) report the clinical utilisation of individual results returned. Using this experience, we discuss barriers and opportunities associated with a comprehensive process of RoR in large-scale genomic research that may be useful for others developing their own policies. We performed whole-genome (n = 184) and exome (n = 208) sequencing of matched tumour-normal DNA pairs from 392 patients with sporadic pancreatic cancer (PC) as part of the APGI. We identified pathogenic germline mutations in candidate genes (n = 130) with established predisposition to PC or medium–high penetrance genes with well-defined cancer associated syndromes or phenotypes. Variants from candidate genes were annotated and classified according to international guidelines. Variants were considered actionable if clinical utility was established, with regard to prevention, diagnosis, prognostication and/or therapy. A total of 48,904 germline variants were identified, with 2356 unique variants undergoing annotation and in silico classification. Twenty cases were deemed actionable and were returned via previously described RoR framework, representing an actionable finding rate of 5.1%. Overall, 1.78% of our cohort experienced clinical benefit from RoR. Returning research results within the context of large-scale genomics research is a labour-intensive, highly variable, complex operation. Results that warrant action are not infrequent, but the prevalence of those who experience a clinical difference as a result of returning individual results is currently low.

We have made substantial progress in developing cancer treatments that target specific molecular vulnerabilities in an increasing number of cancer types.1 Interrogation of complex genomic, transcriptomic, and metabolomic (referred to as omics) profiles, integrated with comprehensive clinical data, represents the essential next step to inform the development and application of selective approaches to cancer prevention, diagnosis, and treatment.2 However, the majority of patients with cancer around the world, and particularly those living in resource-limited settings, cannot access molecular testing and targeted therapies due to regulatory, financial, logistical, educational, and clinical barriers.

The treatment of advanced pancreatic cancer has not moved much beyond single agent gemcitabine until recently when protocols such as FOLFIRINOX (fluorouracil, leucovorin, irinotecan and oxaliplatin) and nab-paclitaxel-gemcitabine have demonstrated some improved outcomes. Advances in technology especially in massively parallel genome sequencing has progressed our understanding of the biology of pancreatic cancer especially the candidate signalling pathways that are involved in tumourogenesis and disease course. This has allowed identification of potentially actionable mutations that may be targeted by new biological agents. The heterogeneity of pancreatic cancer makes tumour tissue collection important with the aim of being able to personalise therapies for the individual as opposed to a one size fits all approach to treatment of the condition. This paper reviews the developments in this area of translational research and the ongoing clinical studies that will attempt to move this into the everyday oncology practice.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies. It is phenotypically heterogeneous with a highly unstable genome and provides few common therapeutic targets. We found that MCL1, Cofilin1 (CFL1) and SRC mRNA were highly expressed by a wide range of these cancers, suggesting that a strategy of dual MCL-1 and SRC inhibition might be efficacious for many patients. Immunohistochemistry revealed that MCL-1 protein was present at high levels in 94.7% of patients in a cohort of PDACs from Australian Pancreatic Genome Initiative (APGI). High MCL1 and Cofilin1 mRNA expression was also strongly predictive of poor outcome in the TCGA dataset and in the APGI cohort. In culture, MCL-1 antagonism reduced the level of the cytoskeletal remodeling protein Cofilin1 and phosphorylated SRC on the active Y416 residue, suggestive of reduced invasive capacity. The MCL-1 antagonist S63845 synergized with the SRC kinase inhibitor dasatinib to reduce cell viability and invasiveness through 3D-organotypic matrices. In preclinical murine models, this combination reduced primary tumor growth and liver metastasis of pancreatic cancer xenografts. These data suggest that MCL-1 antagonism, while reducing cell viability, may have an additional benefit in increasing the antimetastatic efficacy of dasatinib for the treatment of PDAC.

Next generation sequencing has revolutionised genomic studies of cancer, having facilitated the development of precision oncology treatments based on a tumour's molecular profile. We aimed to develop a targeted gene sequencing panel for application to disparate cancer types with particular focus on tumours of the head and neck, plus test for utility in liquid biopsy. The final panel designed through Roche/Nimblegen combined 451 cancer-associated genes (2.01 Mb target region). 136 patient DNA samples were collected for performance and application testing. Panel sensitivity and precision were measured using well-characterised DNA controls (n = 47), and specificity by Sanger sequencing of the Aryl Hydrocarbon Receptor Interacting Protein (AIP) gene in 89 patients. Assessment of liquid biopsy application employed a pool of synthetic circulating tumour DNA (ctDNA). Library preparation and sequencing were conducted on Illumina-based platforms prior to analysis with our accredited (ISO15189) bioinformatics pipeline. We achieved a mean coverage of 395x, with sensitivity and specificity of >99% and precision of >97%. Liquid biopsy revealed detection to 1.25% variant allele frequency. Application to head and neck tumours/cancers resulted in detection of mutations aligned to published databases. In conclusion, we have developed an analytically-validated panel for application to cancers of disparate types with utility in liquid biopsy.

Papillary thyroid cancer (PTC) has an impressive propensity for lymphatic spread. Signal transducers and activators of transcription 3 (STAT3), constitutively activated in many different cancers, may play a role in PTC lymphatic metastases. We examined 49 patients with PTC, 22 with and 27 without lymphatic metastases. All patients had a total thyroidectomy with lymph node dissection to document true node negative cases. The level of STAT3 expression in benign, non-neoplastic thyroid tissue is barely detectable by immunohistochemistry. Only 11 of the 35 (31%) specimens exhibited weak immunostainingfor STAT3 and pSTAT3 was found weakly positive in 3 of 35 (9%) benign specimens. Expression of STAT3 in all PTC primary tumors was 98% (40/41) and thus significantly higher than corresponding benign thyroid tissue (p=0.0001). pSTAT3 was found in 37% of primary tumors (15/41) and this was significantly higher than pSTAT3 expression in benign tissue (p=0.006). Comparing node-positive and node-negative primary tumors, there was no difference in staining intensity for STAT3 where strong (2+) staining was seen 12/19 node-positive tumors and 13/22 node-negative tumors (p=1). Regarding pSTAT3 expression in primary PTC tumors, node negative cases (n=22) exhibited significantly less staining compared to node positive cases (n=19). Only 4 of 22 (18%) cases in the node-negative group were weakly (1+) positive for pSTAT3 while 12 of 19 (58%) cases in the node-positive group were positive (p=0.011) with 45% of these specimens exhibiting strong (2+) staining. Lymphatic metastases were highly positive (>93%) for both STAT3 and pSTAT3. The STAT3 pathway is ubiquitous in PTC and activated pSTAT3 is significantly upregulated in PTC tumors with metastatic disease. This study is the first to suggest a potential role for activated pSTAT3 in lymphatic metastases in thyroid cancer.

Disclosure of individual results to participants in genomic research is a complex and contentious issue. There are many existing commentaries and opinion pieces on the topic, but little empirical data concerning actual cases describing how individual results have been returned. Thus, the real life risks and benefits of disclosing individual research results to participants are rarely if ever presented as part of this debate.The Australian Pancreatic Cancer Genome Initiative (APGI) is an Australian contribution to the International Cancer Genome Consortium (ICGC), that involves prospective sequencing of tumor and normal genomes of study participants with pancreatic cancer in Australia. We present three examples that illustrate different facets of how research results may arise, and how they may be returned to individuals within an ethically defensible and clinically practical framework. This framework includes the necessary elements identified by others including consent, determination of the significance of results and which to return, delineation of the responsibility for communication and the clinical pathway for managing the consequences of returning results.Of 285 recruited patients, we returned results to a total of 25 with no adverse events to date. These included four that were classified as medically actionable, nine as clinically significant and eight that were returned at the request of the treating clinician. Case studies presented depict instances where research results impacted on cancer susceptibility, current treatment and diagnosis, and illustrate key practical challenges of developing an effective framework.We suggest that return of individual results is both feasible and ethically defensible but only within the context of a robust framework that involves a close relationship between researchers and clinicians.

We previously reported two patients with metastatic colorectal adenocarcinoma who, after referral to the precision medicine tumour board, had tissue and longitudinal liquid biopsies and responded to targeted therapy based on their molecular profile. These patients are from the SCRUM-MONSTAR programme, a nationwide genome-screening project in Japan that is part of the International Cancer Genome Consortium–Accelerating Research in Genomic Oncology. Here, we summarise the clinical history of the two patients and report an update on clinical course and longitudinal molecular assessment using circulating-tumour DNA (ctDNA) sequencing. The first patient was diagnosed with RAS–BRAF wild-type and microsatellite-stable (MSS) metastatic adenocarcinoma of the sigmoid colon and was treated with multiple lines of standard systemic treatments from January, 2018, to November, 2020. At progression after sixth-line regorafenib, an emerging BRAF Val600Glu mutation in ctDNA sequencing (Guardant360, Guardant Health, Redwood City, CA, USA) was identified and confirmed on biopsy of a liver metastasis. Based on these findings, the molecular tumour board recommended triplet targeted therapy with the BRAF inhibitor encorafenib, MEK inhibitor binimetinib, and the anti-EGFR antibody cetuximab, leading to partial response for 11 weeks. At the time of disease progression, the BRAF V600E mutation was no longer detected on ctDNA sequencing and an EML4–ALK fusion emerged (appendix pp 2–3). These findings were confirmed at endoscopic, ultrasound-guided, fine needle aspiration of abdominal lymph node metastasis (FoundationOne CDx, Foundation Medicine, Cambridge, MA, USA), which identified an EML4–ALK v5a/b variant (appendix pp 3–4). The molecular tumour board recommended the ALK inhibitor alectinib (600 mg twice per day) for EML4–ALK fusion colorectal adenocarcinoma. The patient died after 3 months of treatment due to tumour progression. The variant allelic frequency of EML4–ALK fusions in ctDNA sequencing was stable before and after alectinib (appendix pp 2–3). The clinical course of subsequent therapy in the first patient for emerging EML4–ALK fusion in ctDNA sequencing is summarised in the appendix (pp 5–6). The second patient was diagnosed with metastatic colorectal cancer in December, 2020, and was initially treated with first-line FOLFOX. Second-line pembrolizumab monotherapy was started at progression based on the results of ctDNA sequencing (Guardant360 assay) showing RAS wild-type, a BRAF V600E mutation, and high microsatellite instability (MSI-h). Molecular analysis of an archival biopsy sample also showed RAS wild-type, the BRAF V600E mutation, and an MSS tumour. A mixed response to pembrolizumab was observed and, because of discrepancy in microsatellite status between ctDNA and tumour biopsy, the board considered heterogeneous tumour microsatellite status. Meanwhile, consistent results in BRAF V600E mutation between biopsy sample and ctDNA sequencing led the molecular tumour board to recommend subsequent combination of encorafenib (300 mg once per day) and cetuximab (400 mg/m2 initial dose followed by 250 mg/m2 once per week) as third-line therapy, started in April, 2021. Notably, a BRAF Val600Glu mutation was detected in ctDNA sequencing both before and after pembrolizumab (before: Guardant360; after: FoundationOne Liquid CDx, Foundation Medicine), whereas MSI-h was not observed in ctDNA sequencing after pembrolizumab (appendix pp 7–8). A durable response according to Response Evaluation Criteria in Solid Tumors version 1.1 to combination therapy with encorafenib and cetuximab was confirmed at last follow-up (August, 2022), after more than 60 weeks of treatment. The patient is currently continuing the combination therapy without serious adverse events. The first patient lived for approximately 8 months after becoming refractory to all standard treatments for metastatic colorectal adenocarcinoma, and for 42 months from initiation of systemic chemotherapy, with massive liver and lymph node metastases. Targeted therapy, especially triplet therapy with encorafenib, binimetinib, and cetuximab based on the emerging BRAF Val600Glu mutation in ctDNA sequencing, might have contributed to extension of survival in this patient. However, treatment with ALK tyrosine kinase inhibitor alectinib was ineffective, despite the identification of an EML4–ALK fusion, which is a recognised actionable alteration in other tumour types, such as non-small cell lung cancer (NSCLC). A potential explanation for the primary resistance to this agent in the first patient is the concomitance of TP53 mutation, detected both in ctDNA sequencing and tissue-based next generation sequencing, which might be associated with lack of response to alectinib. In patients with ALK-rearranged NSCLC, concomitant TP53 mutations reduce the efficacy of ALK inhibitors in preclinical and clinical studies. Furthermore, the most common variants of EML4–ALK in NSCLC are variant 1 and variant 3, accounting for 75–80% of EML4–ALK variants. The tumour of the first patient had variant 5a/b of EML4–ALK, which is reported in only 2% of patients with EML4–ALK-positive NSCLC. Although impaired efficacy for ALK inhibitors in patients with NSCLC with EML4-ALK variant 3 has been suggested compared with patients with variant 1, little is known about the efficacy of ALK inhibitors for EML4–ALK variant 5a/b due to its low incidence, even in patients with NSCLC. With regard to structural motifs, variant 5a/b belongs to the category of short variants, which include variant 3, and this could be another reason why alectinib was not effective in this case. The second patient highlights the challenges in interpretation of molecular findings when there is a difference in assay results, in this case between Guardant360 and FoundationOne Liquid CDx. MSI-h was not detected in ctDNA sequencing after pembrolizumab treatment; these results suggest that microsatellite instability status was heterogeneous between lesions and MSS tumours and had become dominant after pembrolizumab. Meanwhile, the BRAF Val600Glu mutation was consistent in ctDNA sequencing before and after pembrolizumab, with durable response to combination treatment with encorafenib and cetuximab. Although ctDNA sequencing at baseline is able to evaluate tumour heterogeneity, longitudinal assessment is important to understand clonality and changes in therapeutic targets during the clinical course for treatment decisions in patients with metastatic colorectal cancer. DK reports honoraria from Ono Pharmaceutical, Merck Biopharma, and MSD; and research funding from Ono Pharmaceutical and MSD. YN reports research funding from Guardant Health. TY reports research funding from Ono Pharmaceutical, Sanofi, Daiichi Sankyo, PAREXEL international, Pfizer Japan, Taiho Pharmaceutical, MSD, Amgen, Genomedia, SysmexCorporation, Chugai Pharmaceutical, and Nippon Boehringer Ingelheim; and honoraria from Taiho Pharmaceutical, Chugai Pharmaceutical, Eli Lilly Japan, Merck Biopharma, Bayer Yakuhin, Ono Pharmaceutical, and MSD. All other authors declare no competing interests. Download .pdf (.52 MB) Help with pdf files Supplementary appendix ICGC-ARGO precision medicine: targeted therapy according to longitudinal assessment of tumour heterogeneity in colorectal cancerColorectal cancer is characterised by high molecular heterogeneity and genomic alterations in common cancer drivers, including RAS, BRAF, and mismatch repair genes, which are routinely assessed to inform precision treatments. However, constant clonal evolution is common and leads to therapeutic resistance. Longitudinal molecular analysis of integrated tissue and liquid biopsies is essential to monitor the molecular evolution of colorectal cancer during the continuum of care and inform sequential adaptive therapies based on real-time genomic changes. Full-Text PDF

Pancreatic cancer (PC) is a lethal disease which is characterized by chemoresistance. Components of the cell cytoskeleton are therapeutic targets in cancer. βIV-tubulin is one such component that has two isotypes-βIVa and βIVb. βIVa and βIVb isotypes only differ in two amino acids at their C-terminus. Studies have implicated βIVa-tubulin or βIVb-tubulin expression with chemoresistance in prostate, breast, ovarian and lung cancer. However, no studies have examined the role of βIV-tubulin in PC or attempted to identify isotype specific roles in regulating cancer cell growth and chemosensitivity. We aimed to determine the role of βIVa- or βIVb-tubulin on PC growth and chemosensitivity. PC cells (MiaPaCa-2, HPAF-II, AsPC1) were treated with siRNA (control, βIVa-tubulin or βIVb-tubulin). The ability of PC cells to form colonies in the presence or absence of chemotherapy was measured by clonogenic assays. Inhibition of βIVa-tubulin in PC cells had no effect chemosensitivity. In contrast, inhibition of βIVb-tubulin in PC cells sensitized to vinca alkaloids (Vincristine, Vinorelbine and Vinblastine), which was accompanied by increased apoptosis and enhanced cell cycle arrest. We show for the first time that βIVb-tubulin, but not βIVa-tubulin, plays a role in regulating vinca alkaloid chemosensitivity in PC cells. The results from this study suggest βIVb-tubulin may be a novel therapeutic target and predictor of vinca alkaloid sensitivity for PC and warrants further investigation.

Patients with pancreatic ductal adenocarcinoma (PC) have a poor prognosis due to metastases and chemoresistance. PC is characterized by extensive fibrosis, which creates a hypoxic microenvironment, and leads to increased chemoresistance and intracellular oxidative stress. Thus, proteins that protect against oxidative stress are potential therapeutic targets for PC. A key protein that maintains genomic integrity against oxidative damage is MutY-Homolog (MYH). No prior studies have investigated the function of MYH in PC cells. Using siRNA, we showed that knockdown of MYH in PC cells 1) reduced PC cell proliferation and increased apoptosis; 2) further decreased PC cell growth in the presence of oxidative stress and chemotherapy agents (gemcitabine, paclitaxel and vincristine); 3) reduced PC cell metastatic potential; and 4) decreased PC tumor growth in a subcutaneous mouse model in vivo. The results from this study suggest MYH may be a novel therapeutic target for PC that could potentially improve patient outcome by reducing PC cell survival, increasing the efficacy of existing drugs and reducing metastatic spread.

The International Cancer Genome Consortium–Accelerating Research in Genomic Oncology (ICGC-ARGO)1 will analyse the tumours of more than 100 000 patients with cancer during the next 10 years in a standardised way, using high-quality multiomic and clinical data (in particular outcome and treatment information) to address outstanding questions that are vital to our quest to defeat cancer (ie, the delineation of markers of prognosis, therapeutic response, and resistance). ICGC-ARGO aims to deliver a million patient-years of precision oncology knowledge, by making data available to the research community in a rapid and responsible way, to accelerate research into the causes, and control, of cancer. Here, we present the first report of a series of precision medicine-informed cases discussed by tumour boards within ICGC-ARGO programmes: two sisters, diagnosed with pancreatic ductal adenocarcinoma 7 years apart, whose disease was characterised by an uncommon clinical course; the somatic and germline profiling of these two sisters informed treatment decisions and risk management for a third sister who was not diagnosed with pancreatic ductal adenocarcinoma, but with thyroid cancer and meningioma.

<ns3:p>Genomic science is increasingly central to the provision of health care. Producing and applying robust genomics knowledge is a complex endeavour in which no single individual, profession, discipline or community holds all the answers. Engagement and involvement of diverse stakeholders can support alignment of societal and scientific interests, understandings and perspectives and promises better science and fairer outcomes. In this context we argue for F.A.I.R.E.R. data and data use that is Findable, Accessible, Interoperable, Reproducible, <ns3:italic>Equitable</ns3:italic> and <ns3:italic>Responsible. </ns3:italic>Yet there is a paucity of international guidance on how to engage publics, patients and participants in genomics. To support meaningful and effective engagement and involvement we developed an <ns3:italic>Engagement Framework for</ns3:italic><ns3:italic> involving and engaging participants, patients and publics in genomics research and health</ns3:italic><ns3:italic> implementation</ns3:italic>.</ns3:p><ns3:p> The <ns3:italic>Engagement Framework </ns3:italic>is intended to support all those working in genomics research, medicine, and healthcare to deliberatively consider approaches to participant, patient and public engagement and involvement in their work. Through a series of questions, the <ns3:italic>Engagement Framework</ns3:italic> prompts new ways of thinking about<ns3:italic> </ns3:italic>the aims and purposes of engagement, and support reflection on the strengths, limitations, likely outcomes and impacts of choosing different approaches to engagement. To guide genomics activities, we describe four themes and associated questions for deliberative reflection: (i) fairness; (ii) context; (iii) heterogeneity, and (iv) recognising tensions and conflict.</ns3:p><ns3:p> The four key components in the <ns3:italic>Engagement </ns3:italic>provide a framework to assist those involved in genomics to reflect on decisions they make for their initiatives, including the strategies selected, the participant, patient and public stakeholders engaged, and the anticipated goals. <ns3:italic>The Engagement Framework</ns3:italic> is one step in an actively evolving process of building genomics research and implementation cultures which foster responsible leadership and are attentive to objectives which increase equality, diversity and inclusion in participation and outcomes.</ns3:p>

This corrects the article DOI: 10.1038/nature21063.

Whereas biological materials were once transferred freely, there has been a marked shift in the formalisation of exchanges involving these materials, primarily through the use of Material Transfer Agreements (MTAs). This paper considers how risk aversion dominates MTA negotiations and the impact it may have on scientific progress. Risk aversion is often based on unwarranted fears of incurring liability through the use of a material or loss of control or missing out on commercialisation opportunities. Evidence to date has suggested that complexity tends to permeate even straightforward transactions despite extensive efforts to implement simple, standard MTAs. We argue that in most cases, MTAs need do little more than establish provenance, and any attempt to extend MTAs beyond this simple function constitutes stifling behaviour. Drawing on available examples of favourable practice, we point to a number of strategies that may usefully be employed to reduce risk-averse tendencies, including the promotion of simplicity, education of those engaged in the MTA process, and achieving a cultural shift in the way in which technology transfer office (TTO) success is measured in institutions employing MTAs.

Pancreatic cancer (PC) is an aggressive disease with a dismal 5-year survival rate. Surveillance of high-risk individuals is hoped to improve survival outcomes by detection of precursor lesions or early-stage malignancy.Since 2011, a national high-risk cohort recruited through St Vincent's Hospital, Sydney, has undergone prospective PC screening incorporating annual endoscopic ultrasound, formal genetic counselling and mutation analysis as appropriate. PancPRO, a Bayesian PC risk assessment model, was used to estimate 5-year and lifetime PC risks for familial pancreatic cancer (FPC) participants and this was compared to their perceived chance of pancreatic and other cancers. Genetic counselling guidelines were developed to improve consistency. Follow-up questionnaires were used to assess the role of genetic counselling and testing.We describe the Australian PC screening program design and recruitment strategy and the results of the first 102 individuals who have completed at least one-year of follow-up. Seventy-nine participants met the FPC criteria (≥ two first-degree relatives affected), 22 individuals had both a BRCA2 pathogenic variant and a close relative with PC and one had a clinical diagnosis of Peutz-Jeghers syndrome. Participants reported a high perceived chance of developing PC regardless of their genetic testing status. PancPRO reported FPC participants' mean 5-year and lifetime PC risks as 1.81% (range 0.2-3.2%) and 10.17% (range 2.4-14.4%), respectively. Participants' perceived PC chance did not correlate with their PancPRO 5-year (r = - 0.17, p = 0.128) and lifetime PC risks (r = 0.19, p = 0.091). Two-thirds felt that current genetic testing would help them, and 91% of tested participants were glad to have undergone genetic testing. Overall, 79% of participants found genetic counselling to be helpful, and 88% reported they would recommend counselling to their relatives.Participants reported multiple benefits of genetic counselling and testing but continue to seek greater clarification about their individual PC risk. Extension of PancPRO is required to enable personalised PC risk assessment for all high-risk sub-groups. More detailed discussion of PC risk for BRCA2 pathogenic variant carriers, providing a written summary in all cases and a plan for genetics review were identified as areas for improvement.

Colorectal cancer is characterised by high molecular heterogeneity and genomic alterations in common cancer drivers, including RAS, BRAF, and mismatch repair genes, which are routinely assessed to inform precision treatments. However, constant clonal evolution is common and leads to therapeutic resistance. Longitudinal molecular analysis of integrated tissue and liquid biopsies is essential to monitor the molecular evolution of colorectal cancer during the continuum of care and inform sequential adaptive therapies based on real-time genomic changes.

We previously described a case of two sisters (M and A) diagnosed with pancreatic ductal adenocarcinoma. M and A had somatic and germline tumour molecular profiling as part of the International Cancer Genome Consortium-Accelerating Research in Genomic Oncology (ICGC ARGO) programme, the results of which informed treatment for M and A and risk management for a third sister (Ma), who did not have pancreatic ductal adenocarcinoma but was diagnosed with thyroid cancer at age 36 years and meningioma at age 50 years. Here, we report an update on the treatment history and the clinical course of M and A.

Tackling cancer is a major challenge right on the global level. Europe is only the tip of an iceberg of cancer around the world. Prosperous developed countries share the same problems besetting Europe–and the countries and regions with fewer resources and less propitious conditions are in many cases struggling often heroically against a growing tide of disease. This paper offers a view on these geographically wider, but essentially similar, challenges, and on the prospects for and barriers to better results in this ceaseless battle. A series of panels have been organized by the European Alliance for Personalised Medicine (EAPM) to identify different aspects of cancer care around the globe. There is significant diversity in key issues such as NGS, RWE, molecular diagnostics, and reimbursement in different regions. In all, it leads to disparities in access and diagnostics, patients’ engagement, and efforts for a better understanding of cancer.

The Australian Pancreatic Cancer Screening Program (APCSP) offers endoscopic ultrasound surveillance for individuals at increased risk of pancreatic ductal adenocarcinoma (PDAC) with all participants requiring assessment by a Familial Cancer Service before or after study enrolment.Individuals aged 40-80 years (or 10 years younger than the earliest PDAC diagnosis) were eligible for APCSP study entry if they had 1) ≥ two blood relatives with PDAC (at least one of first-degree association); 2) a clinical or genetic diagnosis of Hereditary Pancreatitis or Peutz-Jeghers syndrome irrespective of PDAC family history; or 3) a known PDAC predisposition germline pathogenic variant (BRCA2, PALB2, CDKN2A, or Lynch syndrome) with ≥one PDAC-affected first- or second-degree relative. Retrospective medical record review was conducted for APCSP participants enrolled at the participating Australian hospitals from January 2011 to December 2019. We audited the genetic investigations offered by multiple Familial Cancer Services who assessed APCSP participants according to national guidelines, local clinical protocol and/or the availability of external research-funded testing, and the subsequent findings. Descriptive statistical analysis was performed using Microsoft Excel.Of 189 kindreds (285 participants), 50 kindreds (71 participants) had a known germline pathogenic variant at enrolment (BRCA2 n = 35, PALB2 n = 6, CDKN2A n = 3, STK11 n = 3, PRSS1 n = 2, MLH1 n = 1). Forty-eight of 136 (35%) kindreds with no known germline pathogenic variant were offered mutation analysis; 89% was clinic-funded, with increasing self-funded testing since 2016. The relatively low rates of genetic testing performed reflects initial strict criteria for clinic-funded genetic testing. New germline pathogenic variants were detected in five kindreds (10.4%) after study enrolment (BRCA2 n = 3 kindreds, PALB2 n = 1, CDKN2A n = 1). Of note, only eight kindreds were reassessed by a Familial Cancer Service since enrolment, with a further 21 kindreds identified as being suitable for reassessment.Germline pathogenic variants associated with PDAC were seen in 29.1% of our high-risk cohort (55/189 kindreds; 82/285 participants). Importantly, 10.4% of kindreds offered genetic testing were newly identified as having germline pathogenic variants, with majority being BRCA2. As genetic testing standards evolve rapidly in PDAC, 5-yearly reassessment of high-risk individuals by Familial Cancer Services is warranted.

&lt;div&gt;Abstract&lt;p&gt;Cancer-associated fibroblasts (CAF) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression through protumor signaling and the generation of fibrosis, the latter of which creates a physical barrier to drugs. CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11 is a cystine transporter that has been identified as a potential therapeutic target in PDAC cells. However, no prior study has evaluated the role of SLC7A11 in PDAC tumor stroma and its prognostic significance. Here we show that high expression of SLC7A11 in human PDAC tumor stroma, but not tumor cells, is independently prognostic of poorer overall survival. Orthogonal approaches showed that PDAC-derived CAFs are highly dependent on SLC7A11 for cystine uptake and glutathione synthesis and that SLC7A11 inhibition significantly decreases CAF proliferation, reduces their resistance to oxidative stress, and inhibits their ability to remodel collagen and support PDAC cell growth. Importantly, specific ablation of SLC7A11 from the tumor compartment of transgenic mouse PDAC tumors did not affect tumor growth, suggesting the stroma can substantially influence PDAC tumor response to SLC7A11 inhibition. In a mouse orthotopic PDAC model utilizing human PDAC cells and CAFs, stable knockdown of SLC7A11 was required in both cell types to reduce tumor growth, metastatic spread, and intratumoral fibrosis, demonstrating the importance of targeting SLC7A11 in both compartments. Finally, treatment with a nanoparticle gene-silencing drug against SLC7A11, developed by our laboratory, reduced PDAC tumor growth, incidence of metastases, CAF activation, and fibrosis in orthotopic PDAC tumors. Overall, these findings identify an important role of SLC7A11 in PDAC-derived CAFs in supporting tumor growth.&lt;/p&gt;Significance:&lt;p&gt;This study demonstrates that SLC7A11 in PDAC stromal cells is important for the tumor-promoting activity of CAFs and validates a clinically translatable nanomedicine for therapeutic SLC7A11 inhibition in PDAC.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;SLC7A11 knockdown in PDAC CAFs does not affect glutamate secretion and is maintained in the presence of oxidative stress.&lt;/p&gt;

&lt;p&gt;Confirmation of SLC7A11 knockdown in KPC tumour cells and CAFs and collagen fibril analysis in tumour sections at therapeutic model endpoint.&lt;/p&gt;

&lt;p&gt;Validation of SLC7A11 antibodies and SLC7A11 knockdown in CAFs and PDAC cells in vitro.&lt;/p&gt;

&lt;p&gt;Anti-proliferative effect of SLC7A11 knockdown in CAFs and the effect of SLC7A11 inhibition in MiaPaCa-2 PDAC cells and normal human pancreatic ductal epithelial (HPDE) cells.&lt;/p&gt;

&lt;p&gt;Australian Pancreatic Cancer Genome Initiative International Cancer Genome Cohort patient cohort characteristics.&lt;/p&gt;

&lt;p&gt;List of antibodies&lt;/p&gt;

&lt;p&gt;SLC7A11 expression in iCAFs, myCAFs and quiescent PSCs.&lt;/p&gt;

&lt;p&gt;SLC7A11 multivariate survival analysis parameters.&lt;/p&gt;

&lt;p&gt;SLC7A11 multivariate survival analysis parameters.&lt;/p&gt;

&lt;p&gt;SLC7A11 expression in iCAFs, myCAFs and quiescent PSCs.&lt;/p&gt;

&lt;p&gt;Confirmation of SLC7A11 knockdown in KPC tumour cells and CAFs and collagen fibril analysis in tumour sections at therapeutic model endpoint.&lt;/p&gt;

&lt;p&gt;Anti-proliferative effect of SLC7A11 knockdown in CAFs and the effect of SLC7A11 inhibition in MiaPaCa-2 PDAC cells and normal human pancreatic ductal epithelial (HPDE) cells.&lt;/p&gt;

&lt;p&gt;List of antibodies&lt;/p&gt;

&lt;p&gt;SLC7A11 knockdown in PDAC CAFs does not affect glutamate secretion and is maintained in the presence of oxidative stress.&lt;/p&gt;

&lt;p&gt;Validation of SLC7A11 antibodies and SLC7A11 knockdown in CAFs and PDAC cells in vitro.&lt;/p&gt;

&lt;p&gt;Australian Pancreatic Cancer Genome Initiative International Cancer Genome Cohort patient cohort characteristics.&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;Cancer-associated fibroblasts (CAF) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression through protumor signaling and the generation of fibrosis, the latter of which creates a physical barrier to drugs. CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11 is a cystine transporter that has been identified as a potential therapeutic target in PDAC cells. However, no prior study has evaluated the role of SLC7A11 in PDAC tumor stroma and its prognostic significance. Here we show that high expression of SLC7A11 in human PDAC tumor stroma, but not tumor cells, is independently prognostic of poorer overall survival. Orthogonal approaches showed that PDAC-derived CAFs are highly dependent on SLC7A11 for cystine uptake and glutathione synthesis and that SLC7A11 inhibition significantly decreases CAF proliferation, reduces their resistance to oxidative stress, and inhibits their ability to remodel collagen and support PDAC cell growth. Importantly, specific ablation of SLC7A11 from the tumor compartment of transgenic mouse PDAC tumors did not affect tumor growth, suggesting the stroma can substantially influence PDAC tumor response to SLC7A11 inhibition. In a mouse orthotopic PDAC model utilizing human PDAC cells and CAFs, stable knockdown of SLC7A11 was required in both cell types to reduce tumor growth, metastatic spread, and intratumoral fibrosis, demonstrating the importance of targeting SLC7A11 in both compartments. Finally, treatment with a nanoparticle gene-silencing drug against SLC7A11, developed by our laboratory, reduced PDAC tumor growth, incidence of metastases, CAF activation, and fibrosis in orthotopic PDAC tumors. Overall, these findings identify an important role of SLC7A11 in PDAC-derived CAFs in supporting tumor growth.&lt;/p&gt;Significance:&lt;p&gt;This study demonstrates that SLC7A11 in PDAC stromal cells is important for the tumor-promoting activity of CAFs and validates a clinically translatable nanomedicine for therapeutic SLC7A11 inhibition in PDAC.&lt;/p&gt;&lt;/div&gt;

Abstract Background: The Individualized Molecular Pancreatic Cancer Therapy (IMPaCT) trial is designed to exploit results from whole genome sequencing of pancreatic cancer collected under the auspices of the ICGC in Australia. Results showed that small subsets of patients had actionable changes in their tumor genome that could be druggable with currently available therapies. Only 7% of cases were found to be KRAS wildtype, and this phenotype may enrich for susceptibility to EGFR inhibition. Her2 positivity occurs in 2% and may confer sensitivity to Her inhibition. Tumors displaying defects in the DNA damage repair pathway (∼5%) respond to DNA damaging chemotherapy. Trial Design: The IMPaCT trial has recently been amended to a single arm pilot study of first line molecularly guided therapy for advanced pancreas cancer. Patients are permitted to begin their first cycle of chemotherapy with gemcitabine with or without nab-paclitaxel while awaiting molecular results. We screen potential patients for the three molecular targets: Her2 amplification: trastuzumab + gemcitabine; KRAS wildtype: erlotinib + gemcitabine; and DNA damage: platinum-based chemotherapy. In our initial cohort of patients who underwent resection with curative intent, 70% recurred. Recurrence occurred 16m after initial surgery. Collection of tissue commenced in 2009. The first site to open was in April 2013 by which time, only 8 patients for whom we had complete sequence data and actionable mutations were still alive, so we changed the trial to screen de novo metastatic patients. Using the WGS data, we constructed a custom sequencing panel to use DNA extracted from FFPE core biopsies to screen in real time for mutations in KRAS, BRCA2, BRCA1, PALB2 and ATM. Her2 screening is undertaken with IHC and FISH. We have screened 89 cases in 18m, 8 have relevant molecular targets. The average time from biopsy to delivery of results is 21d. 2 of the 8 eligible cases have commenced precision therapy on trial. Citation Format: Lorraine Chantrill, Skye Simpson, Amber Johns, Mona Martyn-Smith, Angela Chou, Clare Watson, Adnan Nagrial, Venessa Chin, Lucille Sebastian, Sonia Yip, John Simes, Nick Pavlakis, Peter Grimison, Ray Asghari, Sandra Harvey, Andrew Biankin. Precision medicine for advanced pancreas cancer: the individualized molecular pancreatic cancer therapy (IMPaCT) trial. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT210. doi:10.1158/1538-7445.AM2015-CT210

Here we describe two cases of metastatic squamous cell carcinoma of the head and neck, discussed at the molecular tumour board of the Profiling Orphan Neoplasms for Treatment Election (PONTE) project, part of the International Cancer Genome Consortium Accelerating Research in Genomic Oncology (ICGC ARGO). These patients had variability in response to immunotherapy due to biological heterogeneity of head and neck squamous cell carcinoma, highlighting the importance of complete molecular characterisation—including PD-L1 expression, tumour mutational burden, and microsatellite instability—to accurately predict treatment response in patients with these tumours.

The effectiveness of anti-PD-1/PD-L1 therapies in Stage III/IV (advanced) non-small cell lung cancer (NSCLC) is correlated with tumor percentage score (TPS) of PD-L1, such that patients with TPS ≥ 50% may benefit from single agent checkpoint blockade without the need for augmentation with chemotherapy. Preclinical evidence demonstrates that mRNA vaccines stimulate interferon-mediated increases in PD-L1 expression at the tumor site. We therefore hypothesized that COVID-mRNA vaccines would similarly increase PD-L1 expression in biopsy samples from patients with advanced NSCLC.

595 Background: Neoadjuvant (Neo) chemotherapy (CT) with trastuzumab (H) improves pathologic complete response (pCR) rate for HER2+ breast cancer. Dose-dense regimens improve outcome in the adjuvant setting but have not been fully evaluated as preoperative therapy. We designed this regimen to utilize full doses of active agents including docetaxel (T) and H in a novel biweekly schedule to explore efficacy and safety. Methods: Patients (pts) with biopsy proven, clinical stage IIA-IIIC, noninflammatory breast cancer were eligible. HER2+ by FISH was determined locally. CT consisted of epirubicin (E) 100 mg/m2 and cyclophosphamide (C) 600 mg/m2 Q 14 days x 4 followed by T 75 mg/m2 and H 6 mg/kg loading dose, then 4 mg/kg Q 14 days x 4, all with pegfilgrastim support. Surgery was scheduled 20–24 weeks from start after a fifth cycle of H 4mg/kg. EF was measured prior to CT, after EC, after TH and at 6, 12 and 24 months after surgery. Additional adjuvant H to complete 1 year of therapy by conventional schedule was recommended after surgery. The primary endpoint was pCR for invasive cancer in breast and lymph nodes. Results: 30 pts were enrolled at 5 centers: median age was 50.1 (range, 31–72); ethnicity African-American 14, Caucasian 14, other 2; clinical stage IIA, 14, IIB, 4, IIIA, 7, IIIB/C, 5; ER+ 18, PR+ 14; grade 3, 21 and grade 2, 8. Twenty eight pts were evaluable for pathologic response- 2 withdrew before completing treatment, 1 for toxicity. Dose delivery on schedule was &gt;95% for all drugs. Clinical response prior to surgery was cCR 20; cPR 5; and stable 2 pts. Pathologic response: pCR 16 (57%) including 4 with residual DCIS only; 9 pPR, and 2 stable. Mean EF was 63.1 (range, 51–81) before treatment, 62.4 (49–75) after EC and 58.3 (35–74) after TH. Two pts had EF &lt;50% during Neo, one with clinical CHF and 1 additional pt developed CHF during adjuvant single agent H. Both pts had symptomatic improvement with cessation of H. Adverse events were generally mild with 14 grade 3 AEs including 3 episodes of dyspnea and no grade 3 skin toxicity or any grade 4 toxicity noted. Conclusions: Sequential Neo dose-dense Q 14 day EC followed by Q 14 day TH yields a high pCR rate in HER2+ breast cancer with acceptable toxicity profile and no new safety signals noted. No significant financial relationships to disclose.

Abstract Aim: To determine the prevalence of pathogenic germline mutations in known pancreatic cancer (PC) predisposition genes in an unselected PC cohort; identify clinico-pathological characteristics associated with gene carrier status; and analyse somatic genomic data for evidence of biallelic inactivation. Methods: Whole-genome (n = 184) and exome (n = 208) sequencing was performed on matched tumor-normal DNA pairs from 392 predominantly sporadic cases of PC. Pathogenic mutations were assessed in 13 cancer predisposition genes associated with PC risk (APC, ATM, BRCA1, BRCA2, CDKN2A, MLH1, MSH2, MSH6, PALB2, PMS2, PRSS1, STK11, TP53. Results: A total of 377 unique high confidence germline variants were observed in the 13 PC predisposition genes. 22 were classified as pathogenic and identified in 23/392 (5.9%) PC patients. The mutations occurred in BRCA2 (n=9), ATM (n=4), BRCA1 (n=3), PALB2 (n=3), CDKN2A (n=2) and one each in PMS2 and STK11. Truncating BRCA2 and PALB2 mutations were detected in 2 cases classified as familial PC. There was no significant difference in average age at diagnosis (67.9 vs 66.6, P = 0.5468), post-resection survival (20.3 vs 20.5 months, P = 0.9788), family history of malignancy (55.6% vs 40.5%, P = 0.2249) or personal history of malignancy (31.8% vs 15.0%, P = 0.0642) between those with and those without a pathogenic germline PC risk mutation. Patients harboring BRCA1, BRCA2 or PALB2 mutations were associated with an increased family history of breast or ovarian cancer (27.3% vs 6.3%, P = 0.0348). The second-hit mechanism could be assessed in tumors with &amp;gt;30% cellularity (n=16), of which 75% (7 BRCA2, 4 ATM, 1 BRCA1) showed evidence of biallelic inactivation. Conclusion: 5.9% of PC patients with predominantly sporadic disease have a pathogenic germline mutation in cancer predisposition genes associated with PC risk. However, carrier status did not significantly affect age at diagnosis, survival, personal or family history of malignancy in this cohort. Second hit mechanisms were identified in 12 (52.2%) cases, supporting a potential role in pancreatic tumorgenesis. An expanded analysis of the germline data is required to further understand the genetic basis of both sporadic and familial PC. Citation Format: Skye McKay, Jeremy Humphris, Amber Johns, Mark Pinese, Ann-Marie Patch, Katia Nones, Australian Pancreatic Cancer Genome Initiative (APGI), Sean Grimmond, Andrew Biankin, Nicola Waddell.{Authors}. Assessment of germline cancer predisposition genes in 392 unselected pancreatic cancer patients. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A02.

ABSTRACT Cancer-Associated Fibroblasts (CAFs) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression, through pro-tumour cross-talk and the generation of fibrosis (physical barrier to drugs). CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11 is a cystine transporter that has been identified as a potential therapeutic target in PDAC cells. However, no prior study has evaluated the role of SLC7A11 in PDAC tumour stroma and its prognostic significance. Herein we show that high expression of SLC7A11 in PDAC tumour stroma (but not tumour cells) is independently prognostic of poorer overall survival. We demonstrate using orthogonal approaches that PDAC-derived CAFs are highly dependent on SLC7A11 for cystine uptake and glutathione synthesis, and that SLC7A11 inhibition significantly decreases their proliferation, reduces their resistance to oxidative stress and inhibits their ability to remodel collagen and support PDAC cell growth. Importantly, our paradigm-shifting work demonstrates the need to inhibit SLC7A11 in the PDAC stroma, as genetic ablation of SLC7A11 in PDAC cells alone is not enough to reduce tumour growth. Finally, our work validates that a nano-based gene-silencing drug against SLC7A11, developed by our group, reduces PDAC tumour growth, CAF activation and fibrosis in a mouse model of PDAC.

Purpose: The long-term outcomes following surgical resection for Pancreatic Ductal Adenocarcinoma (PDAC) remains poor, with only 20% of patients surviving 5 years after pancreatectomy. Patient selection for surgery remains sub-optimal largely due to the absence of consideration of aggressive tumour biology. Molecular subtyping has demonstrated 2 broad subtypes in PDAC, called Classical Pancreatic and Squamous.

e11087 Background: The standard approach to HR+ MBC is sequential single endocrine agents, but in the current era of adjuvant AI therapy, alternatives to AIs for first line MBC endocrine treatment are desirable. We designed this trial to assess the feasibility, safety and efficacy of a combined chemoendocrine approach with continuous low dose oral chemotherapy (metronomic) along with a potentially non-cross resistant estrogen receptor down regulator. Methods: Eligible patients (pts) in this single arm, multicenter trial had HR +, HER2- biopsy proven MBC, were post-menopausal, had no prior chemotherapy for metastatic disease, had evaluable or measurable disease by RECIST criteria, and no prior exposure to C or F. C was given at a fixed divided dose of 1500-2000 mg, depending on weight, continuously daily without interruption. F was given IM at standard 250 mg after loading doses. Cycles were 28 days, with assessment for response Q 2 cycles. Primary endpoints were progression free survival (PFS) and time to progression (TTP). Results: 42 pts were accrued, 41 are evaluable. Mean age 64.5 years (range 37-85), 37% African American, ECOG 0/1/2: 17/20/4; ER+ 40, PR+ 27, prior endocrine therapy 21, prior adjuvant chemo 16. The median (range) cycles delivered were 8 (2-27+), with 12 pts receiving 12 or more months of therapy. As of 1/1/11, 17 pts remain on study. Discontinuation occurred due to disease progression in 18, toxicity in 2, investigator discretion in 3 and 1 withdrew consent. The only grade 3/4 toxicity occurring in >5% of pts was grade 3 hand/foot syndrome in 4 (9.8%). Dose reduction of C occurred in 12 (29.3%) pts, typically during cycles 2-8. F was given in full dose in 383/391 (97.7%) total cycles delivered. Overall response rate was 22%, with 2 (4.9%) achieving CR and 29 (70.7%) pts with stable disease. The clinical benefit rate (CR+ PR+ stable > 6 months) was 53.7 %. The PFS and TTP were both [median (95% CI)] 10.87 (7.26- NR) months. Conclusions: Metronomic continuous capecitabine and fulvestrant monthly following loading dose is feasible, well tolerated even with prolonged periods of administration in HR+ MBC, and is effective in maintaining PFS and TTP.

196 Background: Individuals with adenocarcinoma of the ampulla of Vater demonstrate a broad range of outcomes, as they may arise from any of the three epithelia that converge at that location. This variation in outcomes poses specific challenges in clinical decision making concerning the aggressiveness of treatment, and the appropriateness and type of adjuvant therapy. Methods: We assessed the relationship between molecular pathologic phenotypes defined using a combination of histopathology and protein expression (CDX2 [caudal-type homeodomain transcription factor 2] and MUC1), and outcome in 72 patients who underwent operative resection for adenocarcinoma of the ampulla of Vater. Results: Patients with a pancreaticobiliary phenotype (CDX2 negative, MUC1 positive) compared to an intestinal phenotype carcinoma segregated into an independently poor prognostic group (HR = 3.40, 95% CI: 1.71 – 6.76, p = 0.0005). Stratification using lymph node (LN) status (the only other independent poor prognostic factor) (HR = 3.19, 95% CI: 1.54 – 6.58, p = 0.0017) defined three clinically relevant phenotypes: 1) those with a non-pancreaticobiliary (intestinal) phenotype without LN metastases who had an excellent outcome (5-year survival 88.4%, median survival 172.8 months); 2) those that had a pancreaticobiliary phenotype and lymph node metastases that had a poor outcome (5-year survival 20.0%, median survival 7.4 months); and 3) the remainder (intestinal/LN positive or pancreaticobiliary/LN negative) who had an intermediate outcome (5-year survival 46.9%, median survival 57.0 months). Conclusions: A combination of histopathologic and molecular criteria identified three distinct clinically relevant phenotypes of adenocarcinoma of the ampulla of Vater that have significantly different outcomes. This distinction, which requires validation, may be used to refine current therapeutic strategies by guiding aggressiveness of surgery and selection for adjuvant therapy, which would improve overall outcomes. No significant financial relationships to disclose.

Abstract The ampulla of Vater is a region where the pancreatic duct, biliary duct, and intestinal duodenum converge creating a complex epithelial cellular environment from which the ampullary adenocarcinomas (AMPAC) arise to form a group of histopathologically heterogenous tumors. The confluence of three organs at the ampulla of Vater gives rise to complexity in diagnosis, treatment and outcomes. Previous studies have focused mainly on marker identification to differentiate between the AMPAC intestinal and pancreatobiliary subtypes. It is established by immunohistochemistry and expression analysis that the intestinal subtype correlates with better prognosis. However, no large scale genomic profiling has been untaken. To better understand the diverse make-up of the AMPAC subtypes and to potentially help guide treatment, we analyzed 94 AMPAC of intestinal, pancreatobiliary, and mixed subtypes; 33 cholangioadenocarcinomas (CAC); and 11 duodenal adenocarcinomas (DUOAC) and their normal matched samples by whole exome sequencing, SNP array and RNA seq. Whole exome revealed that AMPAC, DUOAC, and CAC shared in common with pancreatic ductal adenocarcinoma (PDAC) high mutation rate in KRAS and TP53, loss and mutation in CDKN2A and SMAD4. ATM, a key component of the DNA damage repair pathway significantly mutated in PDAC, was also mutated in AMPAC, but not in DUOA or CAC. In contrast, loss of function mutation in NF1, an important component of the KRAS pathway, rarely mutated in PDAC, was significantly mutated (12%) and exhibited allelic loss uniquely in AMPAC. Mutations in major gene components of the PIK and wnt signaling, observed in intestinal cancers were detected in DUOAC and AMPAC but not in CAC. The AMPAC, mutations in these genes were found in both intestinal and pancreatobiliary subtypes. We also observed microsatellite instability (MSI), a common feature of gastrointestinal cancers, in 5% AMPAC, 36% of DUOAC , and 3% CAC tumors. The MSI tumors showed mutation frequencies of 17 to 113 mutations per megabase compared to 0.1 to 9.3 mutations per megabase in non-MSI tumors. In our previous study of 94 PDAC, none were MSI. Copy number analysis by SNP array revealed frequent arm-level loss in chromosome 9p, 17p, and 18q in common with all cancers. The patters of copy number change differed most clearly between CAC and DUOAC; AMPAC shared characteristics of both. Gene expression by RNAseq revealed a strong intestinal signature, including high expression of the colon cancer biomarker KRT20, in the intestinal AMPAC but not in pancreatobiliary subtype. The theme that emerges from our study is that the AMPAC shares more similarity in molecular characteristics with gastrointestinal than pancreatic ductal cancers. Interestingly, the histological subtypes of intestinal (30), pancreatobilliary (43) or mixed (10) tumors recognized by pathologists is not strongly reflected in the molecular properties of the AMPAC tumors. Pancreatobiliary subtype shows mutations associated with intestinal tumors but not PDAC, for example 32% of pancreatobilary tumors harbor PIK3CA, PIK3R1, PTEN or wnt pathway members such as CTNNB1, and APC. Patient outcomes for these various mutation profiles are under investigation. From a clinical and therapeutic perspective, targets potentially include ERB-family, ALK and BRCA1/2, mutated tumors, which collectively represent 20% of the patients. An additional 5% of patients are MSI. MSI is a favorable prognostic marker in colorectal and endometrial cancers, and has therapeutic implications. In conclusion, future diagnosis and treatment decision may gain insight by including screening for specific mutations in the characterization of AMPAC. This abstract is also presented as Poster A1. Citation Format: Marie-Claude Gingras, Amber Johns, Anthony Gill, Michael Overman, Christian Pilarsky, Sean Grimmond, Andrew Biankin, David Wheeler, Richard Gibbs. The ampullary adenocarcinoma, its molecular characterization and differentiation from the pancreatic ductal adenocarcinoma, duodenal adenocarcinoma, and cholangiocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr PR06.

There is an increasing emphasis on community and consumer engagement in cancer research, from identifying priorities to reviewing grants from a consumer perspective. It is clear that there is great interest from the community and consumers to be more actively involved in research, and many organisations and research institutions have responded by convening consumer advisory panels, including consumers on boards and committees, and including consumers and the community in forums and research seminars. While the opportunities available for consumers to participate in research are welcome, current mechanisms to engage with consumers often appear to be tokenistic and bureaucratic. Bedside to Bench, a research, community engagement and health education organisation, conducted an online, consumer engagement in research survey over four weeks. The aim of the survey was to determine when and how cancer patients and their families how they would like to be involved in research. The survey was developed following feedback from consumers at the Australian Pancreatic Genome Initiative's annual research symposium, that suggested current opportunities for consumers to engage in research were limited. Eighty two cancer patients and carers responded to the survey. The majority of respondents (82%) stated that they were interested in being involved in the decision-making process in relation to cancer research. The greatest area of interest was in having access to the results of research projects (23%) and providing feedback to researchers once the projects are developed (23%). Other areas of interest were the development of research projects with researchers (17%), identification of research priorities (17%), with the lowest area of interest being grant reviews (13%). The results of this study suggest that the majority of consumers want to be involved in research in some way, however, given the option, there is potentially only a subset of consumers interested in the review of research grants. What is clear is that, whatever the mechanisms for consumer engagement, strategies, policies and resources need to be available in order to support all stakeholders improve the practice of research involving consumers. The results of this study will be useful to guide future research and policy decisions in relation to consumer engagement in research.

ABSTRACT Background: The Individualized Molecular Pancreatic Cancer Therapy (IMPaCT) trial was designed to exploit results from whole genome sequencing of pancreatic cancer collected under the auspices of the ICGC in Australia. Results showed that small subsets of patients had actionable changes in their tumor genome that could be druggable with currently available therapies. 93% of cases analyzed harbored a KRAS mutation, KRAS wildtype phenotype may enrich for susceptibility to EGFR inhibition. Her2 positivity occurs in 2% and may confer sensitivity to Her inhibition. Tumors displaying defects in the DNA damage repair pathway (∼5%) respond to DNA damaging chemotherapy. Trial design: The IMPaCT trial is a randomized phase 2 study of first line molecularly guided therapy against standard therapy with gemcitabine in advanced pancreas cancer. We screen potential patients for the three molecular targets: Her2 amplification: trastuzumab+gemcitabine; KRAS wildtype: erlotinib+gemcitabine; and DNA damage: mitomycin C+5-fluorouracil chemotherapy. If a patient's tumour has one of these signals, they consent to be randomized to precision treatment tailored for them or standard therapy with gemcitabine. For analysis, the precision treatment arm will be considered as a whole and compared to the standard therapy arm. In our initial cohort of patients who underwent resection with curative intent, 70% recurred. Recurrence occurred on average 16m after initial surgery. Collection of tissue commenced in 2009. The first site to open was in June 2013 by which time, only 6 patients for whom we had complete sequence data and actionable mutations were still alive, so we changed the trial to screen de novo metastatic patients. Using the WGS data, we constructed a custom sequencing panel for DNA extracted from FFPE core biopsies to screen for mutations in KRAS, BRCA2, BRCA1, PALB2 and ATM. Her2 screening is undertaken with IHC and FISH. We have screened 49 cases, and found 10 with relevant molecular targets, 5 of whom were eligible. The average time from biopsy to delivery of results is 23d, an amendment has allowed patients to commence standard therapy whilst waiting for the results of molecular analysis. The IMPaCT trial is breaking new ground in the treatment of pancreas cancer. Disclosure: All authors have declared no conflicts of interest.

305 Background: The most important modifiable risk factor for the development of pancreas cancer is smoking, which accounts for up to 25% of pancreatic ductal adenocarcinomas (PDAC; Maisonneuve P, Lowenfels AB: Epidemiology of pancreatic cancer: an update. Digestive Diseases 28:645-656, 2010), and the incidence of PDAC correlates with smoking prevalence (Weiss W, Benarde MA: The temporal relation between cigarette smoking and pancreatic cancer. American journal of public health 73:1403-1404, 1983). A recently published Ingenuity Pathway Analysis of GWAS genotype and risk factor data from the Pancreatic Cancer Case Control Consortium demonstrated that axon guidance signalling genes were significantly overrepresented in smokers (Tang H, Wei P, Duell EJ, et al: Axonal guidance signaling pathway interacting with smoking in modifying the risk of pancreatic cancer: A gene and pathway-based interaction analysis of GWAS data. Carcinogenesis:bgu010, 2014) and we aimed to investigate this link in our cohort. Methods: Tissue from resected PDAC were obtained from 200 patients via the Australian Pancreatic Cancer Genome Initiative with patient consent and ethical approval. Immunohistochemistry was performed on TMAs with the anti-Robo1 antibody (ab7279). The sections were scored on a 4-point scale of 0, 1, 2 and 3 intensity. All sections were scored blindly by two independent reviewers. Results: Robo1 protein expression is found in the normal pancreas, predominantly in acinar cells. In PDAC, Robo1 expression is predominantly in epithelial ductal cells. Most PDACs have Robo1 expression with an overall mean score of 1.65±0.05. 20 out of 200 patients were current smokers at the time of their pancreatectomy, 97 were never smokers, the remainder were ex-smokers. The mean score for smokers was 2.1± 0.1 and for never smokers 1.6±0.1 (p = 0.0003). Genomic analysis did not demonstrate any mutations in the Robo1 gene. There were 7 cases of loss of heterozygosity for Robo1; none of these were current smokers and they had an average score of 1.25±0.17. Conclusions: In addition to our genomic (Biankin AV, Waddell N, Kassahn KS, et al: Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature 491:399-405, 2012) and methylation data (Nones K, Waddell N, Song S, et al: Genome‐wide DNA methylation patterns in pancreatic ductal adenocarcinoma reveal epigenetic deregulation of SLIT‐ROBO, ITGA2 and MET signaling. International Journal of Cancer, 2014), we provide the results of protein expression of Robo1 in a clinically annotated cohort of 200 cases of PDAC. We demonstrate that patients who are currently smoking have enhanced Robo1 expression. Preliminary results indicate that this may confer a poorer prognosis when coupled with high SLIT2 expression.

312 Background: Pancreatic cancer (PC) is a highly lethal and genetically heterogenous disease. Genomic sequence data from the Australian Pancreatic Cancer Genome Initiative (APGI) has identified a subset of patients with ROCK-1 amplification. ROCK-1 is a downstream target of Rho, a small GTPase that plays an important role in regulating proliferation, invasion and metastasis of cancer cells. Our aim was to analyse the effects of inhibiting ROCK-1 using specific small molecule inhibitors (Fasudil and Y-27632) in well annotated and robust pre-clinical model systems generated as part of our APGI efforts. Methods: Patient derived cell lines (PDCL) and xenografts (PDX) were used to test the effectiveness of ROCK-1 inhibitors (RI). Colony formation and 3-D organotypic assays tested cellular proliferation and invasion. In vivo, pre-clinical trials assessed the effect gemcitabine (G) +/- RI on a range of PDXs with varying tumour ROCK expression, including one G resistant model. A PDCL shown to form metastases when injected orthotopically into mice has been labeled with firefly luciferase. The effect of RI on metastasis formation will be assessed in vivo using real time imaging. Results: ROCK inhibition has a differential effect on colony formation on PDCLs in vitro, and inhibits cellular invasion. A statistically significant increase in median survival in the G + RI group compared with G alone, was seen in 3 PDXs, including the G resistant tumour. 1 PDX showed a decrease in tumour size at 200 days in the G + RI group. Conclusions: ROCK inhibition has a differential effect in vitro, but an anti-tumour effect in vivo, including overcoming resistance to G. This suggests that effects on the tumour micro-environment are an important mechanism of action. RI have the potential to be an effective therapy in PC.

Abstract Purpose: Defining clinically and biologically relevant phenotypes to inform treatment decisions in other cancers have led to substantial improvements in overall outcomes. About 80% of patients with pancreatic cancer (PC) succumb to the disease despite curative resection, many of whom recur within 6 months of surgery. These early recurrences demonstrate that current staging is inadequate and that there is a clear need to better define prognostic phenotypes prior to significant intervention. This study aimed to evaluate the potential clinical utility of biologically relevant molecules as prognostic factors in resected PC to define strategies that may improve current preoperative staging accuracy and inform treatment decisions. Method: We assessed the relationship of aberrant expression of two pro-metastatic calcium-binding proteins, S100A2 and S100A4 with disease-specific survival in four independent cohorts of patients (total n = 547) who underwent operative resection for PC. A preoperative nomogram using pre-operatively assessable variables including biomarkers was derived and was compared to traditional prognostic variables and the postoperative nomogram. Results: High S100A2 and S100A4 expressions were an independent poor prognostic factor in both the training (n = 76; HR = 2.00, 95% CI = 1.15 - 3.49, P = 0.0216 and HR = 3.34, 95% CI = 1.69 - 6.62, P = 0.0005 respectively) and validation (n = 316; HR = 1.73, 95% CI = 1.20 - 2.48, P = 0.0030 and HR = 1.65, 95% CI = 1.23 - 2.22, P = 0.0009 respectively) cohorts. These results were further validated in a prospective cohort (ICGC-APGI, n = 100) and another retrospective population based cohort (NCI SEER, n = 55). Aberrant expression of S100A2/A4 protein again stratified the cohorts into 3 distinct prognostic groups. A preoperative nomogram using only variables that could be measured preoperatively (age, tumour size, tumour location and molecular biomarkers), predicted survival better than nomograms derived using clinico-pathological variables, which are only determinable after surgery. A proof-of-principle study demonstrated that biomarker expression can be reliably assessed in preoperative EUS-FNA cell block samples. Conclusion: S100A4 and S100A2 are one of the very few biomarkers that have been independently validated in multiple patient cohorts. Their aberrant expression stratifies PC into distinct prognostic groups and potentially enables more accurate preoperative prognostication through a nomogram. Biomarker assessment potentially enables accurate preoperative prognostication, which is more accurate than current staging methods. The development and application of such nomograms has the potential to improve patient selection for surgery and assist clinicians in making “tie-breaker” decisions that would ultimately improve the overall survival and quality of life for patients with PC. Citation Format: David K. Chang, Mark Pinese, Christopher J. Scarlett, Marina Pajic, Emily K. Colvin, Amber L. Johns, Jianmin Wu, Mark J. Cowley, Jeremy L. Humphris, Angela Chou, Nam Q. Nguyen, Adnan M. Nagrial, Lorraine Chantrill, Venessa T. Chin, Elizabeth A. Musgrove, Sean Altekruse, Anthony J. Gill, James G. Kench, Andrew V. Biankin. A molecular pre-operative prognostic nomogram for resectable pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1156. doi:10.1158/1538-7445.AM2013-1156

Unprecedented advances in biomolecular technology have greatly increased the power and precision of analytical tools used in cancer research and have accelerated the drive toward personalised medicine. Human specimens that are analysed using these new and developing technology platforms have emerged as a critical resource for basic and translational research in cancer because they are a direct source of molecular data from which targets for therapy, detection, and prevention are identified. As a result this has stimulated a growing demand for carefully collected, appropriately stored and well-annotated tumour specimens worldwide. This requirement has put pathologists in the centre of the personalised medicine world as providers of information identifying tissues and also as decision makers on what materials should be biobanked, on the preservation conditions, and on the timeline of events that precede preservation and storage. This critical position in the research process places extraordinary demands on all aspects of pathology practice, in particular as pathology laboratories are ultimately key players in delivering new medicines through molecular diagnostics. We present a national biobank network infrastructure that promotes multi-disciplinary research and has pathology underpinning tumour banking in order to facilitate multi-institutional, high throughput genomic studies. This new era of medicine requires a strategic view of ways forward for better integration of pathology, not only as ‘tissue providers’ but also as essential participants in the design, performance, interpretation, and implementation of research and clinical trials. Unprecedented advances in biomolecular technology have greatly increased the power and precision of analytical tools used in cancer research and have accelerated the drive toward personalised medicine. Human specimens that are analysed using these new and developing technology platforms have emerged as a critical resource for basic and translational research in cancer because they are a direct source of molecular data from which targets for therapy, detection, and prevention are identified. As a result this has stimulated a growing demand for carefully collected, appropriately stored and well-annotated tumour specimens worldwide. This requirement has put pathologists in the centre of the personalised medicine world as providers of information identifying tissues and also as decision makers on what materials should be biobanked, on the preservation conditions, and on the timeline of events that precede preservation and storage. This critical position in the research process places extraordinary demands on all aspects of pathology practice, in particular as pathology laboratories are ultimately key players in delivering new medicines through molecular diagnostics. We present a national biobank network infrastructure that promotes multi-disciplinary research and has pathology underpinning tumour banking in order to facilitate multi-institutional, high throughput genomic studies. This new era of medicine requires a strategic view of ways forward for better integration of pathology, not only as ‘tissue providers’ but also as essential participants in the design, performance, interpretation, and implementation of research and clinical trials.

Abstract Background: Less than 5% of patients with metastatic pancreatic cancer survive to 5 years and there have been no major improvements in outcomes over the last 20 years. The use of treatments targeted according to the molecular phenotype of individual tumours may result in improved response and survival compared to standard therapy. Methods: The IMPaCT trial is a multidisciplinary collaboration between the AGITG, NHMRC Clinical Trials Centre, Sydney Catalyst, and the Kinghorn Cancer Centre at Garvan Institute of Medical Research, which houses the Australian Pancreatic Cancer Genome Initiative (APGI). Patients who have available sequence data will be screened for actionable molecular phenotypes and randomized 1:1 to receive standard therapy (gemcitabine) or personalized treatment. Recruitment to the IMPaCT trial is based on the following defined molecular phenotypes: HER2/neu overexpression: personalized treatment with gemcitabine + trastuzumab; BRCA1, BRCA2, and PALB2 mutations: personalized treatment with 5-FU and mitomycin C; Kras wildtype: personalized treatment with gemcitabine + erlotinib. The study will be conducted in two parts: an initial 20 patient pilot trial across 4 Australian sites assessing feasibility, followed by an additional 70 patients to assess progression (90 patients in total). The pilot study is now open and active. Results: The novel trial design involves personalized treatment, where therapies are assigned based on a defined molecular phenotype, in a standard care setting. Stratifying randomization for individual molecular signatures will provide evidence, albeit in small numbers, for confirmation in a larger Phase III trial and broader clinical applicability. Additionally, the study offers the opportunity to explore a number of unique tertiary/correlative objectives, including the planned examination of circulating DNA as a surrogate of survival. Conclusion: The IMPaCT trial exemplifies a strong collaboration between basic scientists, clinicians and clinical trial investigators to illustrate the promises and challenges facing the development and successful testing of personalized therapeutic strategies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A75. Citation Format: Lorraine Chantrill, Amber Johns, Adnan Nagrial, Venessa Chin, Angela Chou, Mark Pinese, Scott Mead, Val Gebski, Katrin Sjoquist, Chee Lee, Sonia Yip, Danielle Miller, Lucille Sebastian, Ray Asghari, Sandra Harvey, Nick Pavlakis, Sanjay Mukhedkar, Peter Grimison, David Miller, John Pearson, Nicola Waddell, Sean Grimmond, John Simes, Andrew Biankin. The IMPaCT trial: Individualised Molecular Pancreatic Cancer Therapy. A pilot, randomized, open label Phase II trial assessing first line treatment with gemcitabine or personalized treatment based on tumour molecular signature in patients with metastatic pancreatic cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A75.

e15029 Background: Pancreatic cancer (PC) is a highly lethal disease with few effective treatment options for metastatic disease. PC is a genetically highly heterogeneous disease that lends itself to a personalised approach to translational research (Biankin et al. Nature. 2012 Nov 15;491(7424):399-405.). Genomic sequence data from the Australian Pancreatic Genome Initiative (APGI) has identified a subset of patients with ROCK-1 amplification. ROCK-1 is a downstream target of Rho, a small GTPase, which is involved in cell adhesion and motility and has been shown to play a role in cancer cell growth and invasiveness (S. Boeck, P. Stieber, et al. Oncology 2006; 70:255., Lane J, Martin T et al. Int J Onc 2008; 33:585-593.). Our aim is to identify biologically and clinically relevant, targetable phenotypes for PC by examining therapeutic efficacy of two ROCK-1 inhibitors, Y-27632 and fasudil in model systems with aberrant Rho/ROCK signaling. Methods: Using extensively characterised model systems of PC, including unique primary patient derived xenografts (PDX) and patient derived cell lines (PDCL) established in the laboratory, we are examining the efficacy of individualized rationally designed combination therapies based on targeting Rho/ROCK signaling. Using innovative techniques, including organotypic assays, we are examining the effect of the targeted approach on cell proliferation, migration, invasion and metastasis in relevant models of PC. Results: Here we demonstrate significant differential sensitivity of pancreatic PDXs and PDCLs with aberrant Rho/ROCK signaling to ROCK combination therapy when compared with standard therapy, gemcitabine. Finally, these compounds impair invasiveness of human pancreatic tumour cells, suggesting that ROCK-1 is a targetable phenotype for the treatment of PC. Conclusions: By carefully defining the key cancer subtypes and expanding our knowledge about the interplay of the key regulators and activators of the Rho/ROCK pathway, the promise of ROCK inhibitors for PC therapy may be realized. As fasudil is already in clinical use for the treatment of cerebral vasospasm, this work may be quickly translated to the clinic.

314 Background: The IMPaCT trial screened patients with advanced pancreas cancer for molecular targets and aimed to test molecularly guided therapy in a pilot randomized study, but closed in December 2015 having delivered personalized treatment to only 1 patient. A follow up analysis of the screened cohort provides insight into the clinical utilisation of targetable molecular results in pancreas cancer. Methods: IMPaCT screened for 3 genetic phenotypes matched to precision treatment: Her2 amplification (trastuzamab + gemcitabine), KRAS wildtype (erlotinib + gemcitabine) and DNA damage repair pathway defects (platinum-based). Previously recruited primary resected APGI participants known to be alive were also screened for these targets. The initial pilot protocol included randomisation, but after amendment in early 2015, became a single-arm study allowing one cycle of gemcitabine + nab-paclitaxel during screening. Results: A total of 101 potential patients were screened. This was enriched for patients with known targets where sequence data was available from primary pancreatectomy. 23 had one of the 3 molecular targets in this enriched population (4 Her2 amplified, 16 KRAS wt, 2 BRCA2, 1 ATM). The prospectively recruited de novo metastatic cohort (n = 63) had an actionable mutation detection rate of 14.3%. Of the 23 patients identified, 3 received personalised treatment off study, 1 on study, 3 are currently alive without recurrence, 1 is monitored for suspected recurrence, 2 developed second primary malignancies, 10 died or progressed prior to result and 3 decided not to use personalised treatment. The identification of a molecular target did not significantly affect overall survival. Conclusions: Although actionable molecular targets were detected, recruitment rates on the IMPaCT trial were low (1%). Follow up illustrates that these molecular results were incorporated into clinical care off trial or still have the potential to be utilised in another 6.9% of the cohort. Further follow up is planned for long term outcomes. Clinical trial information: ACTRN12612000777897.

Abstract The microtubule protein, βIII-tubulin, has been implicated as a prognostic, pro-survival, and chemoresistance factor in some of the most lethal malignancies including pancreatic ductal adenocarcinoma (PDAC). However, precise survival mechanisms controlled by βIII-tubulin in cancer cells are unknown. Here, we report an unexpected role of βIII-tubulin as a brake on extrinsic caspase 8-dependent apoptosis in PDAC. We show that βIII-tubulin knockdown frees death-receptor DR5 to increase its membrane diffusion, clustering, and activation of cell-death. We demonstrate that βIII-ubulin silencing increases sensitivity of PDAC cells to chemotherapeutic and microenvironment-derived extrinsic cell-death signals including TRAIL, TNFα, and FasL. Finally, nanoparticle delivery of βIII-tubulin siRNA to mouse orthotopic PDAC tumours in vivo and human patient-derived PDAC tumour explants ex vivo increases extrinsic apoptosis and reduces tumour progression. Thus, silencing of βIII-tubulin represents an innovative strategy to unleash a suicide signal in PDAC cells and render them sensitive to microenvironment and chemotherapy-derived death signals.

The introduction of Personalised Medicine (PM) into healthcare systems could benefit from a clearer understanding of the distinct national and regional frameworks around the world. Recent engagement by international regulators on maximising the use of real-world evidence (RWE) has highlighted the scope for improving the exploitation of the treasure-trove of health data that is currently largely neglected in many countries. The European Alliance for Personalised Medicine (EAPM) led an international study aimed at identifying the current status of conditions.A literature review examined how far such frameworks exist, with a view to identifying conducive factors - and crucial gaps. This extensive review of key factors across 22 countries and 5 regions revealed a wide variety of attitudes, approaches, provisions and conditions, and permitted the construction of a comprehensive overview of the current status of PM. Based on seven key pillars identified from the literature review and expert panels, the data was quantified, and on the basis of further analysis, an index was developed to allow comparison country by country and region by region.The results show that United States of America is leading according to overall outcome whereas Kenya scored the least in the overall outcome.Still, common approaches exist that could help accelerate take-up of opportunities even in the less prosperous parts of the world.

Gene List, which includes the list of candidate genes selected for the RoR process. (XLSX 60 kb)