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Background Human papilloma virus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) continues to increase in incidence. Patients are younger, non-smokers and most commonly present with a neck mass often with no other symptoms. This altered presentation compared with non-HPV OPSCC may not be recognized by medical practitioners, leading to delayed diagnosis. Methods Patients with histopathological confirmation of OPSCC and known HPV and/or P16 status who presented to our institution between 2012-2017 inclusive were included in the study. Demographic data, tumour characteristics and presenting symptoms were retrospectivxely obtained from both electronic- and paper-based records. Descriptive statistics were used to report demographic data and the two sample t-test and Fisher's exact test were used to compare groups based on HPV status. Time to diagnosis was also reported. Results A total of 184 patients were included in the study. The majority of patients were male (85.4%) and HPV + (85.3%). The tonsillar complex (53.8%) and tongue base (42.4%) were the most common primary sites. HPV+ patients were less likely to smoke (17.8%) and they commonly presented with a neck mass (39.5% alone or with other symptoms 61.2%). Time to diagnosis in the HPV+ group was longer (15 weeks). Conclusion Our review has highlighted the altered presentation of OPSCC due to the increased incidence of HPV infection. We showed a delayed time to diagnosis in HPV+ OPSCC compared with non-HPV disease. This confirms the importance of focusing our efforts on educating medical practitioners and creating further awareness to facilitate early detection and treatment.

Pre-clinical and early clinical data suggests the microbiome plays an important role in oncogenesis and influences response to immune checkpoint blockade (ICB). The objective of this systematic review and meta-analysis was to determine whether antibiotics affect overall survival (OS) and progression free survival (PFS) in patients with solid malignancies treated with ICB. A systematic search of EMBASE, MEDLINE and conference proceedings was conducted for observational studies examining the effect of antibiotics on ICB. A random effects study-level meta-analysis was performed with pooling of the hazards ratio (HR) for OS and PFS. Meta-regression was used to determine the impact of the timing of antibiotic exposure on OS. 766 studies were identified, and 18 studies met the inclusion criteria. Of the 2889 patients included, 826 (28.6%) were exposed to antibiotics. The most common malignancies were lung (59%), renal cell carcinoma (RCC) or urothelial carcinoma (16.3%) and melanoma (18.7%). OS was prolonged in those without antibiotic exposure (pooled HR 1.92, 95% CI 1.37–2.68, p

This study analysed nutritional parameters (baseline body mass index (BMI), weight changes and enteral nutrition (EN) use, and their association with hospital admissions during radiotherapy in patients with head and neck cancer (HNC)). A retrospective review of patients diagnosed with HNC and treated with radiotherapy between October 2012 and April 2014 was conducted. Data on each subject’s diagnosis, age, sex, chemotherapy, previous surgery, EN use, weight changes, and BMI were examined for their association with hospital admissions during treatment. Eighty-three patients were included, mean age (±standard deviation) = 61 (± 11 years). Thirty-four percent had self-reported weight loss at diagnosis, and mean BMI was 26.2 ± 5.3 kg/m2. Mean weight change during treatment was − 5.1 ± 6.2%. Ten patients used EN, with mean weight stabilisation during EN use (0.3 ± 5.1%). Higher presenting BMI, younger age, and definitive radiotherapy ± chemotherapy predicted greater weight loss (p

Objective We aimed to define preoperative clinical and molecular characteristics that would allow better patient selection for operative resection. Background 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. Method 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. Results 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. Conclusions 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.

Background & Aims 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. Methods 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. Results 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. Conclusions Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy., Graphical abstract

We previously used a pulse-based in vitro assay to unveil targetable signalling pathways associated with innate cisplatin resistance in lung adenocarcinoma (Hastings et al., 2020). Here, we advanced this model system and identified a non-genetic mechanism of resistance that drives recovery and regrowth in a subset of cells. Using RNAseq and a suite of biosensors to track single-cell fates both in vitro and in vivo, we identified that early S phase cells have a greater ability to maintain proliferative capacity, which correlated with reduced DNA damage over multiple generations. In contrast, cells in G1, late S or those treated with PARP/RAD51 inhibitors, maintained higher levels of DNA damage and underwent prolonged S/G2 phase arrest and senescence. Combined with our previous work, these data indicate that there is a non-genetic mechanism of resistance in human lung adenocarcinoma that is dependent on the cell cycle stage at the time of cisplatin exposure.

The identification of clinically viable strategies for overcoming resistance to platinum chemotherapy in lung adenocarcinoma has previously been hampered by inappropriately tailored in vitro assays of drug response. Therefore, using a pulse model that closely mimics the in vivo pharmacokinetics of platinum therapy, we profiled cisplatin-induced signalling, DNA-damage and apoptotic responses across a panel of human lung adenocarcinoma cell lines. By coupling this data to real-time, single-cell imaging of cell cycle and apoptosis we provide a fine-grained stratification of response, where a P70S6K-mediated signalling axis promotes resistance on a TP53 wildtype or null background, but not a mutant TP53 background. This finding highlights the value of in vitro models that match the physiological pharmacokinetics of drug exposure. Furthermore, it also demonstrates the importance of a mechanistic understanding of the interplay between somatic mutations and the signalling networks that govern drug response for the implementation of any consistently effective, patient-specific therapy., eLife digest Lung adenocarcinoma is the most common type of lung cancer, and it emerges because of a variety of harmful genetic changes, or mutations. Two lung cancer patients – or indeed, two different sets of cancerous cells within a patient – may therefore carry different damaging mutations. A group of drugs called platinum-based chemotherapies are currently the most effective way to treat lung adenocarcinoma. Yet, only 30% of patients actually respond to the therapy. Many studies conducted in laboratory settings have tried to understand why most cases are resistant to treatment, with limited success. Here, Hastings, Gonzalez-Rajal et al. propose that previous research has been inconclusive because studies done in the laboratory do not reflect how the treatment is actually administered. In patients, platinum-based drugs are cleared from the body within a few hours, but during experiments, the treatment is continually administered to cells growing in a dish. Hastings, Gonzalez-Rajal et al. therefore developed a laboratory method that mimics the way cells are exposed to platinum-based chemotherapy in the body. These experiments showed that the lung adenocarcinoma cells which resisted treatment also carried high levels of a protein known as P70S6K. Pairing platinum-based chemotherapy with a drug that blocks the activity of P70S6K killed these resistant cells. This combination also treated human lung adenocarcinoma tumours growing under the skin of mice. However, it was ineffective on cancerous cells that carry a mutation in a protein called p53, which is often defective in cancers. Overall, this work demonstrates the need to refine how drugs are tested in the laboratory to better reflect real-life conditions. It also underlines the importance of personalizing drug combinations to the genetic background of each tumour, a concept that will be vital to consider in future clinical trials.

Pancreatic ductal adenocarcinoma (PDAC) can be divided into transcriptomic subtypes with two broad lineages referred to as classical (pancreatic) and squamous. We find that these two subtypes are driven by distinct metabolic phenotypes. Loss of genes that drive endodermal lineage specification, HNF4A and GATA6, switch metabolic profiles from classical (pancreatic) to predominantly squamous, with glycogen synthase kinase 3 beta (GSK3β) a key regulator of glycolysis. Pharmacological inhibition of GSK3β results in selective sensitivity in the squamous subtype; however, a subset of these squamous patient-derived cell lines (PDCLs) acquires rapid drug tolerance. Using chromatin accessibility maps, we demonstrate that the squamous subtype can be further classified using chromatin accessibility to predict responsiveness and tolerance to GSK3β inhibitors. Our findings demonstrate that distinct patterns of chromatin accessibility can be used to identify patient subgroups that are indistinguishable by gene expression profiles, highlighting the utility of chromatin-based biomarkers for patient selection in the treatment of PDAC.

Our understanding of genomic heterogeneity in lung cancer is largely based on the analysis of early-stage surgical specimens. Here we used endoscopic sampling of paired primary and intrathoracic metastatic tumors from 11 lung cancer patients to map genomic heterogeneity inoperable lung cancer with deep whole-genome sequencing. Intra-patient heterogeneity in driver or targetable mutations was predominantly in the form of copy number gain. Private mutation signatures, including patterns consistent with defects in homologous recombination, were highly variable both within and between patients. Irrespective of histotype, we observed a smaller than expected number of private mutations, suggesting that ancestral clones accumulated large mutation burdens immediately prior to metastasis. Single-region whole-genome sequencing of from 20 patients showed that tumors in ever-smokers with the strongest tobacco signatures were associated with germline variants in genes implicated in the repair of cigarette-induced DNA damage. Our results suggest that lung cancer precursors in ever-smokers accumulate large numbers of mutations prior to the formation of frank malignancy followed by rapid metastatic spread. In advanced lung cancer, germline variants in DNA repair genes may interact with the airway environment to influence the pattern of founder mutations, whereas similar interactions with the tumor microenvironment may play a role in the acquisition of mutations following metastasis.

Summary Durvalumab is a programmed cell death ligand 1 inhibitor, which is now approved in Australia for use in non-small-cell lung and urothelial cancers. Autoimmune diabetes is a rare immune-related adverse effect associated with the use of immune checkpoint inhibitor therapy. It is now being increasingly described reflecting the wider use of immune checkpoint inhibitor therapy. We report the case of a 49-year-old female who presented with polyuria, polydipsia and weight loss, 3 months following the commencement of durvalumab. On admission, she was in severe diabetic ketoacidosis with venous glucose: 20.1 mmol/L, pH: 7.14, bicarbonate 11.2 mmol/L and serum beta hydroxybutyrate: >8.0 mmol/L. She had no personal or family history of diabetes or autoimmune disease. Her HbA1c was 7.8% and her glutamic acid decarboxylase (GAD) antibodies were mildly elevated at 2.2 mU/L (reference range: Learning points: Durvalumab use is rarely associated with fulminant autoimmune diabetes, presenting with severe DKA. Multiple endocrinopathies can co-exist with the use of a single immune checkpoint inhibitors; thus, patients should be regularly monitored. Regular blood glucose levels should be performed on routine pathology on all patients on immune checkpoint inhibitor. Clinician awareness of immunotherapy-related diabetes needs to increase in an attempt to detect hyperglycaemia early and prevent DKA.

Metastatic pancreatic ductal adenocarcinoma (mPDAC) is a lethal disease with a poor 5-year survival. Systemic treatments can be used to control symptoms and prolong life. Cytotoxic chemotherapies are commonly administered, with combination treatments, such as fluorouracil, folinic acid, irinotecan and oxaliplatin (FOLFIRINOX) or nab-paclitaxel and gemcitabine showing the largest clinical benefits. Newer genomic classifications of PDAC may provide a rationale for targeted therapies or immunotherapies, although at present these remain largely experimental. This review discusses the evidence behind the currently used regimens, while introducing the potential future of pancreatic cancer care.

MASTL kinase is essential for correct progression through mitosis, with loss of MASTL causing chromosome segregation errors, mitotic collapse and failure of cytokinesis. However, in cancer MASTL is most commonly amplified and overexpressed. This correlates with increased chromosome instability in breast cancer and poor patient survival in breast, ovarian and lung cancer. Global phosphoproteomic analysis of immortalised breast MCF10A cells engineered to overexpressed MASTL revealed disruption to desmosomes, actin cytoskeleton, PI3K/AKT/mTOR and p38 stress kinase signalling pathways. Notably, these pathways were also disrupted in patient samples that overexpress MASTL. In MCF10A cells, these alterations corresponded with a loss of contact inhibition and partial epithelial-mesenchymal transition, which disrupted migration and allowed cells to proliferate uncontrollably in 3D culture. Furthermore, MASTL overexpression increased aberrant mitotic divisions resulting in increased micronuclei formation. Mathematical modelling indicated that this delay was due to continued inhibition of PP2A-B55, which delayed timely mitotic exit. This corresponded with an increase in DNA damage and delayed transit through interphase. There were no significant alterations to replication kinetics upon MASTL overexpression, however, inhibition of p38 kinase rescued the interphase delay, suggesting the delay was a G2 DNA damage checkpoint response. Importantly, knockdown of MASTL, reduced cell proliferation, prevented invasion and metastasis of MDA-MB-231 breast cancer cells both in vitro and in vivo, indicating the potential of future therapies that target MASTL. Taken together, these results suggest that MASTL overexpression contributes to chromosome instability and metastasis, thereby decreasing breast cancer patient survival.

Identification of clinically viable strategies for overcoming resistance to platinum chemotherapy in lung adenocarcinoma has been hampered by inappropriately tailoredin vitroassays of drug response. Therefore, using a pulse model that closely recapitulates thein vivopharmacokinetics of platinum therapy, we profiled cisplatin-induced signalling, DNA damage and apoptotic responses across a panel of lung adenocarcinoma cell lines. By coupling this data with real-time, single cell imaging of cell cycle and apoptosis, we show thatTP53mutation status influenced the mode of cisplatin induced cell cycle arrest, but could not predict cisplatin sensitivity. In contrast, P70S6K-mediated signalling promoted resistance by increasing p53/p63 and p21 expression, reducing double-stranded DNA breaks and apoptosis. Targeting P70S6K sensitised bothTP53wildtype and null lines to cisplatin, but notTP53mutant lines. In summary, usingin vitroassays that mimicin vivopharmacokinetics identified P70S6K as a robust mediator of cisplatin resistance and highlighted the importance of considering somatic mutation status when designing patient-specific combination therapies.

ObjectiveExtensive 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.DesignSensitivity 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).ResultsSubtype-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.ConclusionThis study demonstrates the promise of CDK4 inhibition in PDA over standard therapy when applied in a molecular subtype-specific context.

e21737 Background: Lung cancer is the leading cause of cancer death in Australia with 13,000 new cases per year. Although targeted therapy and immunotherapy have drastically changed the treatment landscape, the majority of patients will receive platinum-based chemotherapy for which the response rate is approximately 30% (Reck et al, 2016). An immunohistochemistry-based, predictive biomarker would be beneficial for patients and help avoid toxicity for patients unlikely to respond. Marini et al (2018) identified 3 biomarkers associated with in-vitro platinum resistance – activin A, growth differentiation factor-11 and transforming growth factor-b – which were investigated in a real-world retrospective cohort to determine their relation to objective radiological response and overall survival. Methods: We identified 101 patients with advanced non-small cell lung cancer who received platinum chemotherapy at 2 cancer centres between 2014-2015. Archival formalin-fixed paraffin embedded tissue samples were stained with activin A. Slides were manually scored by 2 independent clinicians using the multiplicative quickscore method (Detre et al, 1995). Kaplan Meier analysis for overall survival, a Cox-proportional hazards model for confounding variables and a chi-square analysis was performed to analyse the relationship between high immunohistochemistry scores (greater or less than 6) and radiological response. Results: We performed statistical analysis around the median cytoplasmic score (6). The overall median survival was 15.3 months. No significant difference in survival was detected between the two populations (p value = 0.97). The immunohistochemistry score was also not associated with rates of partial response (p value = 0.98) or progressive disease (p value 0.22). Conclusions: Despite an association with lower progression-free survival in a retrospective cohort in a previous study, high expression of activin does not appear to be a useful biomarker for platinum response in the setting of non-small cell lung cancer. Further research into associated antibodies including GDF-11 and TGF-b is in progress.

Background HGSOC and BLBC are aggressive cancers that share aetiology, co-occur frequently and share molecular signatures including cyclin E1 deregulation. In HGSOC, we have described two cyclin E1 high groups, with and without associated gene amplification. We aimed to determine which subsets of these disease have the greatest similarity in terms of cyclin E1 as a biomarkers of response with the ultimate aim of groping similar subsets in the future for therapeutic purposes. Methods Using automated tissue based assays, we assessed the expression and the amplification status of cyclin E1 in a cohort enriched for familial cancer mutations against each of the expression of cyclin E1 degradation associated proteins, i.e., FBXW7 and USP28, BRCA status and clinical outcome and in comparison with cyclin E1 high subsets of HGSOC. Results For BLBC cyclin E1 overexpression but not CCNE1 amplification was found to be prognostic for overall survival. By comparison, both cyclin E1 expression and CCNE1 gene amplification are prognostic for HGSOC, which we establish with a meta-analysis of existing studies. We examined the nature of CCNE1 amplification to find that HGSOC has an enhanced degree of amplification of CCNE1 with 5.6x copies compared to 3.9x CCNE1 copies in BLBC, and this was associated with widely different expression of cyclin E1 protein. We find that BLBC cancers, with or without CCNE1 amplification, have similarities in dysregulation of regulators of cyclin E1 protein stability and those shared more features with the non-amplified cyclin E1 high HGSOC subset. Since cyclin E1 is only expressed at high levels in HGSOC and not BLBC, we examined the consequence of very high cyclin E1 expression in a BLBC cell line, MDA-MB-468. Moderate overexpression of cyclin E1 showed increased survival upon treatment with Paclitaxel, however very high expression of cyclin E1 gave less survival benefit. Conclusions We conclude that cyclin E1 high BLBC shared features with the non-amplified cyclin E1 high subset, high cyclin E1 expression associated with CCNE1 amplification gives rise to a unique phenotype in HGSOC, but otherwise the high expression of cyclin E1 protein may be a similar prognostic marker for BLBC and HGSOC. Legal entity responsible for the study The authors. Funding Ventana Medical System. D. Aziz: Research grant / Funding (self), provided reagents and technical support: Ventana Medical System. P.M. Waring: Research grant / Funding (institution), Full / Part-time employment, provided reagents and technical support, worked for about 10 months for ventana during the project: Ventana Medical System. All other authors have declared no conflicts of interest.

Background: \ud Pancreatic cancer (PC) is a highly lethal disease with few effective treatment options. Over the past few decades, many anti-cancer therapies have been tested in the locally advanced and metastatic setting, with mixed results. This review attempts to synthesise all the randomised data available to help better inform patient and clinician decision-making when dealing with this difficult disease.\ud \ud Objectives: \ud To assess the effect of chemotherapy, radiotherapy or both for first-line treatment of advanced pancreatic cancer. Our primary outcome was overall survival, while secondary outcomes include progression-free survival, grade 3/4 adverse events, therapy response and quality of life.\ud \ud Search methods: \ud We searched for published and unpublished studies in CENTRAL (searched 14 June 2017), Embase (1980 to 14 June 2017), MEDLINE (1946 to 14 June 2017) and CANCERLIT (1999 to 2002) databases. We also handsearched all relevant conference abstracts published up until 14 June 2017.\ud \ud Selection criteria: \ud All randomised studies assessing overall survival outcomes in patients with advanced pancreatic ductal adenocarcinoma. Chemotherapy and radiotherapy, alone or in combination, were the eligible treatments.\ud \ud Data collection and analysis: \ud Two review authors independently analysed studies, and a third settled any disputes. We extracted data on overall survival (OS), progression-free survival (PFS), response rates, adverse events (AEs) and quality of life (QoL), and we assessed risk of bias for each study.\ud \ud Main results: \ud We included 42 studies addressing chemotherapy in 9463 patients with advanced pancreatic cancer. We did not identify any eligible studies on radiotherapy.\ud \ud We did not find any benefit for chemotherapy over best supportive care. However, two identified studies did not have sufficient data to be included in the analysis, and many of the chemotherapy regimens studied were outdated.\ud \ud Compared to gemcitabine alone, participants receiving 5FU had worse OS (HR 1.69, 95% CI 1.26 to 2.27, moderate-quality evidence), PFS (HR 1.47, 95% CI 1.12 to 1.92) and QoL. On the other hand, two studies showed FOLFIRINOX was better than gemcitabine for OS (HR 0.51 95% CI 0.43 to 0.60, moderate-quality evidence), PFS (HR 0.46, 95% CI 0.38 to 0.57) and response rates (RR 3.38, 95% CI 2.01 to 5.65), but it increased the rate of side effects. The studies evaluating CO-101, ZD9331 and exatecan did not show benefit or harm when compared with gemcitabine alone.\ud \ud Giving gemcitabine at a fixed dose rate improved OS (HR 0.79, 95% CI 0.66 to 0.94, high-quality evidence) but increased the rate of side effects when compared with bolus dosing.\ud \ud When comparing gemcitabine combinations to gemcitabine alone, gemcitabine plus platinum improved PFS (HR 0.80, 95% CI 0.68 to 0.95) and response rates (RR 1.48, 95% CI 1.11 to 1.98) but not OS (HR 0.94, 95% CI 0.81 to 1.08, low-quality evidence). The rate of side effects increased. Gemcitabine plus fluoropyrimidine improved OS (HR 0.88, 95% CI 0.81 to 0.95), PFS (HR 0.79, 95% CI 0.72 to 0.87) and response rates (RR 1.78, 95% CI 1.29 to 2.47, high-quality evidence), but it also increased side effects. Gemcitabine plus topoisomerase inhibitor did not improve survival outcomes but did increase toxicity. One study demonstrated that gemcitabine plus nab-paclitaxel improved OS (HR 0.72, 95% CI 0.62 to 0.84, high-quality evidence), PFS (HR 0.69, 95% CI 0.58 to 0.82) and response rates (RR 3.29, 95% CI 2.24 to 4.84) but increased side effects. Gemcitabine-containing multi-drug combinations (GEMOXEL or cisplatin/epirubicin/5FU/gemcitabine) improved OS (HR 0.55, 95% CI 0.39 to 0.79, low-quality evidence), PFS (HR 0.43, 95% CI 0.30 to 0.62) and QOL.\ud \ud We did not find any survival advantages when comparing 5FU combinations to 5FU alone.\ud \ud Authors' conclusions: \ud Combination chemotherapy has recently overtaken the long-standing gemcitabine as the standard of care. FOLFIRINOX and gemcitabine plus nab-paclitaxel are highly efficacious, but our analysis shows that other combination regimens also offer a benefit. Selection of the most appropriate chemotherapy for individual patients still remains difficult, with clinicopathological stratification remaining elusive. Biomarker development is essential to help rationalise treatment selection for patients.

Metastatic pancreatic ductal adenocarcinoma (mPDAC) is a lethal disease with a poor 5-year survival. Systemic treatments can be used to control symptoms and prolong life. Cytotoxic chemotherapies are commonly administered, with combination treatments, such as fluorouracil, folinic acid, irinotecan and oxaliplatin (FOLFIRINOX) or nab-paclitaxel and gemcitabine showing the largest clinical benefits. Newer genomic classifications of PDAC may provide a rationale for targeted therapies or immunotherapies, although at present these remain largely experimental. This review discusses the evidence behind the currently used regimens, while introducing the potential future of pancreatic cancer care.

Our recent publication demonstrates that transient inhibition of Rho-associated kinase signaling within stroma, significantly decreased in vivo primary tumor growth, metastasis and improved response to standard-of-care therapy in pancreatic cancer. Automated analysis of collagen organization in patient tumors may present a promising tool to predict response to our proposed treatment.

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 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 survivors, 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.

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).

e20563 Background: Lung cancer (LC) is common with a dismal prognosis. Although treatment with immunotherapy (IO) has improved survival outcomes, these therapies remain expensive. Even using biomarker selection, response rates still fall short of 50%. The majority of immune cell profiling done previously uses samples taken from early stage patients focusing on a single immune cell subtype. Here we use single cell RNA sequencing (scRNA-seq) and Cellular Indexing of Transcriptome and Epitopes by sequencing (CITE-seq) to characterise the innate and adaptive immune cell activation state and assess the response to exposure to IO in vitro from patients with advanced LC. Methods: Patients with locally advanced or metastatic LC having an Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration (EBUS-TBNA) have biopsies collected for analysis. Cells are grown in culture +/- nivolumab for 48 hours. scRNA-seq and CITE-seq is conducted using established protocols. Transcriptomic and proteomic data on the innate and adaptive immune cell subsets assess markers of immune activation and/or suppression and the changes after nivolumab are quantified. Results are correlated with clinical outcomes. Results: In a locally advanced/metastatic population, EBUS-TBNA biopsies yield highly cellular samples with a heterogeneous population of immune cells able to be cultured +/- IO using novel, inclusive techniques. Transcriptomic clustering reveals distinct sub-populations within the T-cell, B-cell and innate immune cell compartments. Within these clusters, CITE-seq shows protein expression on individual cells can determine states of exhaustion, cytolytic ability, migratory potential and innate immune activation. Conclusions: Single cell genomics is feasible and informative in patients with advanced LC. This work will form the basis of a functional, real-time assay to assess individualised responses to IO therapy that has the potential to predict IO response.

background: 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.\ud methods: 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.\ud results: 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\ud conclusion: Patients aged greater than or equal to70 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.

The emerging standard of care for patients with inoperable pancreatic cancer is a combination of cytotoxic drugs gemcitabine and Abraxane, but patient response remains moderate. Pancreatic cancer development and metastasis occur in complex settings, with reciprocal feedback from microenvironmental cues influencing both disease progression and drug response. Little is known about how sequential dual targeting of tumor tissue tension and vasculature before chemotherapy can affect tumor response. We used intravital imaging to assess how transient manipulation of the tumor tissue, or "priming," using the pharmaceutical Rho kinase inhibitor Fasudil affects response to chemotherapy. Intravital Forster resonance energy transfer imaging of a cyclin-dependent kinase 1 biosensor to monitor the efficacy of cytotoxic drugs revealed that priming improves pancreatic cancer response to gemcitabine/Abraxane at both primary and secondary sites. Transient priming also sensitized cells to shear stress and impaired colonization efficiency and fibrotic niche remodeling within the liver, three important features of cancer spread. Last, we demonstrate a graded response to priming in stratified patient-derived tumors, indicating that fine-tuned tissue manipulation before chemotherapy may offer opportunities in both primary and metastatic targeting of pancreatic cancer. Refereed/Peer-reviewed

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.

Background: Current 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.\ud \ud Methods: We 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.\ud \ud Results By 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%.\ud \ud Conclusions: Perioperative serum CA19.9 measurements are informative in patients with detectable CA19.9 (defined by serum levels of 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.

Purpose: 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. Experimental Design: 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). Results: 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. Conclusions: 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. Clin Cancer Res; 21(9); 2029–37. ©2015 AACR.

The Rho/ROCK pathway is involved in numerous pivotal cellular processes that have made it an area of intense study in cancer medicine, however, Rho-associated coiled-coil containing protein kinase (ROCK) inhibitors are yet to make an appearance in the clinical cancer setting. Their performance as an anti-cancer therapy has been varied in pre-clinical studies, however, they have been shown to be effective vasodilators in the treatment of hypertension and post-ischaemic stroke vasospasm. This review addresses the various roles the Rho/ROCK pathway plays in angiogenesis, tumour vascular tone and reciprocal feedback from the tumour microenvironment and explores the potential utility of ROCK inhibitors as effective vascular normalising agents. ROCK inhibitors may potentially enhance the delivery and efficacy of chemotherapy agents and improve the effectiveness of radiotherapy. As such, repurposing of these agents as adjuncts to standard treatments may significantly improve outcomes for patients with cancer. A deeper understanding of the controlled and dynamic regulation of the key components of the Rho pathway may lead to effective use of the Rho/ROCK inhibitors in the clinical management of cancer.

Nature 543, 65–71 (2017); doi:10.1038/nature21063 It has been brought to our attention that in Fig. 2d of this Article, an incorrect Sanger trace was used to represent the breakpoint of the EWSR1 and FLI1 type 2 fusion. This was due to an error during manuscript preparation, when we inadvertently inserted the electrophoretic trace referring to EWSR1 splicing variants.

e15759 Background: PC is a highly lethal disease which metastasises early. The sequencing efforts of the Australian Pancreatic Cancer Genome Initiative (APGI) identified ROCK-1 as a target of interest. The Rho/ROCK pathway is essential in cellular movement, metastasis, vasculogenesis and stromal signaling. Fasudil (F) is a ROCK inhibitor which has been shown to be safe for human use. Using unique, patient derived models, we have previously shown that F plus gemcitabine (G) does not reduce cellular proliferation in vitro but reduces tumour size and improves overall survival (OS) in subcutaneous patient-derived xenografts (PDX). However, PDXs are critisised for not fully recapitulating the human condition. Here we aimed to generate a patient-derived orthotopic xenograft (O-PDX) which could be used to assess the effects of F + G on metastasis formation and OS. Methods: One patient-derived cell line (PDCL) was labled with firefly luciferase. This was injected into the pancreas of NOD/Shi- scid/IL-2Rγnull (NSG) mice to generate the O-PDX model. The O-PDX was imaged weekly with the IVIS imaging system. The O-PDXs were treated with saline control (S) (n = 8), gemcitabine (G) (n = 10) fasudil (F) (n = 6) or gemcitabine + fasudil (GF) (n = 10). Mice were culled once radiographic or clinical evidence of ascites was detected. Metastasis free (MFS) and OS were recorded. Vessel quantification was performed via CD-31 staining. Results: Metastases seen (spleen, liver, lung, ovary) on imaging were confirmed using GFP stains on immunohistochemistry. F prolonged MFS but not OS when compared with S. GF significantly prolonged MFS (33.5 versus 43 days; p = 0.0339) and OS (51.5 versus 64 days; p = 0.0035). CD-31 staining showed that treatment with F resulted in increased vascularity in the liver metastases and normal liver tissue but not in the pancreas tumour. Conclusions: The addition of Fasudil to gemcitabine improves MFS and OS in an orthotopic model of PC and is a promising new therapeutic option. The increase in vascularity of the metastases and normal liver tissue suggest that changes in vascularity are important and may result in improved delivery of gemcitabine.

6596 Background: Knowing which factors compromise quality of life (QoL) in patients undergoing cancer treatments can help oncologists provide more effective care. To identify these factors, we conducted a single-centered cross-sectional study examining the relationships between patient-reported QoL, adverse events (AE), and treatment characteristics. Methods: Consecutive patients attending an outpatient chemotherapy unit completed two questionnaires (EORTC QLQ-C30 and National Cancer Institute PRO-CTCAE) per visit to identify factors contributing to the lowest global QoL score [QLQ-C30 QL2, range 0 (worst)–100 (best)] over a 6-week period. QL2 was correlated to each PRO-CTCAE item and treatment characteristic (tumor type, drug class, number of cycles, and treatment intent) using multiple regression, adjusted for age, sex, and use of concurrent radiotherapy. To determine whether QoL can be reliably modeled by machine learning, ten algorithms were compared for performance in classifying patients into dichotomized QL2 subgroups. Results: One hundred and fifteen of 130 patients (157/244 visits) completed up to 6 sets of questionnaires (median QL2: 67, IQR: 50–83). No difference was found between QL2 and treatment characteristics (at α Bonferroni=5×10-4). However, QL2 was significantly associated with AE in gastrointestinal, respiratory, attention, pain, sleep/wake, and mood categories. Using AE as covariates, support vector machine with radial basis kernel was the best at classifying patients into QoL groups (mean bootstrapped area under ROC curve 0.812, 95% CI 0.700–0.925). Conclusions: Patient-reported QoL is associated with multiple AE, but not with characteristics of systemic therapy. Machine learning analysis suggests that a combined AE analysis may reliably characterize a patient’s QoL. [Table: see text]

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.

There remains uncertainty regarding the optimal second-line chemotherapy in advanced pancreatic ductal adenocarcinoma (PDAC). The current recommendation of 5-fluorouracil and oxaliplatin may not be relevant in current practice, as FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan and oxaliplatin) has become a more popular first line therapy in fit patients. The majority of studies in this setting are single-arm Phase II trials with significant heterogeneity of patient populations, treatments and outcomes. In this review, we sought to systematically review and synthesise all prospective data available for the second-line treatment of advanced PDAC.

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).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).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.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 (2 years) was associated with poor survival in both groups.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.

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 < .001) and both validation cohorts (HR, 5.65; 95% CI, 2.77 to 11.5; P < .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.

Background:\ud 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.\ud \ud Methods:\ud 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).\ud \ud Results:\ud 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.\ud \ud Conclusions:\ud 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.

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

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.

348 Background: The role of combined modality therapy in the management of locally advanced pancreatic cancer (LAPC) is uncertain. Current guidelines recommend combined modality therapy for select patients. We sought to review the evidence for combined modality versus single modality therapy in LAPC. We performed a systematic review and meta-analysis of randomised controlled trials (RCT) comparing chemoradiation versus chemotherapy alone as well as chemoradiation versus radiation alone in patients with LAPC. Methods: We searched MEDLINE, EMBASE and CENTRAL from inception to Oct 2013 for RCTs comparing chemotherapy alone versus chemotherapy plus radiation and radiation alone versus chemotherapy plus radiation. We also searched abstracts of major cancer meetings from 1990 to 2013. The primary outcome was overall survival (OS). Secondary outcomes included progression-free survival (PFS), response rate and adverse events. Hazard ratios (HR), confidence intervals (CI) and p-values (p) were estimated with a random-effects model due to the heterogeneity of included studies using Revman 5.3. Results: We included 7 eligible trials including 753 patients. Induction chemotherapy was delivered prior to randomisation in one study. Chemoradiation versus chemotherapy (n= 5 studies) did not improve PFS (HR 1.02, 95% CI 0.87 - 1.20, p = 0.80) or OS (HR 0.91, 95% CI 0.64 - 1.31, p = 0.61). However, chemoradiation versus radiation (n=2 studies) improved PFS (HR 0.63, 95% CI 0.41 - 0.96, p = 0.03) and OS (HR 0.65, 95% CI 0.43 – 0.96, p = 0.03). There was significant statistical heterogeneity in the included studies and subsequently wide confidence intervals in the pooled results. This is most likely due to the small participant numbers in each study. Adverse events were more frequent in the chemoradiation arms. Response rates were assessed in only two studies. Conclusions: Chemoradiation is not superior to chemotherapy alone in LAPC, but may be superior to radiation alone. The studies were small with significant heterogeneity. Combined modality therapy results in increased adverse events. Our results do not provide evidence for a universal standard of care, thus more studies of combined modality therapy in LAPC are needed.

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.