Nigel B. Jamieson

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.

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.

CXCR2 has been suggested to have both tumor-promoting and tumor-suppressive properties. Here we show that CXCR2 signaling is upregulated in human pancreatic cancer, predominantly in neutrophil/myeloid-derived suppressor cells, but rarely in tumor cells. Genetic ablation or inhibition of CXCR2 abrogated metastasis, but only inhibition slowed tumorigenesis. Depletion of neutrophils/myeloid-derived suppressor cells also suppressed metastasis suggesting a key role for CXCR2 in establishing and maintaining the metastatic niche. Importantly, loss or inhibition of CXCR2 improved T cell entry, and combined inhibition of CXCR2 and PD1 in mice with established disease significantly extended survival. We show that CXCR2 signaling in the myeloid compartment can promote pancreatic tumorigenesis and is required for pancreatic cancer metastasis, making it an excellent therapeutic target.

TP53 mutation occurs in 50-75% of human pancreatic ductal adenocarcinomas (PDAC) following an initiating activating mutation in the KRAS gene. These p53 mutations frequently result in expression of a stable protein, p53(R175H), rather than complete loss of protein expression. In this study we elucidate the functions of mutant p53 (Trp53(R172H)), compared to knockout p53 (Trp53(fl)), in a mouse model of PDAC. First we find that although Kras(G12D) is one of the major oncogenic drivers of PDAC, most Kras(G12D)-expressing pancreatic cells are selectively lost from the tissue, and those that remain form premalignant lesions. Loss, or mutation, of Trp53 allows retention of the Kras(G12D)-expressing cells and drives rapid progression of these premalignant lesions to PDAC. This progression is consistent with failed growth arrest and/or senescence of premalignant lesions, since a mutant of p53, p53(R172P), which can still induce p21 and cell cycle arrest, is resistant to PDAC formation. Second, we find that despite similar kinetics of primary tumor formation, mutant p53(R172H), as compared with genetic loss of p53, specifically promotes metastasis. Moreover, only mutant p53(R172H)-expressing tumor cells exhibit invasive activity in an in vitro assay. Importantly, in human PDAC, p53 accumulation significantly correlates with lymph node metastasis. In summary, by using 'knock-in' mutations of Trp53 we have identified two critical acquired functions of a stably expressed mutant form of p53 that drive PDAC; first, an escape from Kras(G12D)-induced senescence/growth arrest and second, the promotion of metastasis.

Here we show that Rab25 permits the sorting of ligand-occupied, active-conformation α5β1 integrin to late endosomes/lysosomes. Photoactivation and biochemical approaches show that lysosomally targeted integrins are not degraded but are retrogradely transported and recycled to the plasma membrane at the back of invading cells. This requires CLIC3, a protein upregulated in Rab25-expressing cells and tumors, which colocalizes with active α5β1 in late endosomes/lysosomes. CLIC3 is necessary for release of the cell rear during migration on 3D matrices and is required for invasion and maintenance of active Src signaling in organotypic microenvironments. CLIC3 expression predicts lymph node metastasis and poor prognosis in operable cases of pancreatic ductal adenocarcinoma (PDAC). The identification of CLIC3 as a regulator of a recycling pathway and as an independent prognostic indicator in PDAC highlights the importance of active integrin trafficking as a potential drive to cancer progression in vivo.

FOLFIRINOX is a standard treatment for metastatic pancreatic cancer patients. The effectiveness of neoadjuvant FOLFIRINOX in patients with borderline resectable pancreatic cancer (BRPC) remains debated.We performed a systematic review and patient-level meta-analysis on neoadjuvant FOLFIRINOX in patients with BRPC. Studies with BRPC patients who received FOLFIRINOX as first-line neoadjuvant treatment were included. The primary endpoint was overall survival (OS), and secondary endpoints were progression-free survival, resection rate, R0 resection rate, and grade III-IV adverse events. Patient-level survival outcomes were obtained from authors of the included studies and analyzed using the Kaplan-Meier method.We included 24 studies (8 prospective, 16 retrospective), comprising 313 (38.1%) BRPC patients treated with FOLFIRINOX. Most studies (n = 20) presented intention-to-treat results. The median number of administered neoadjuvant FOLFIRINOX cycles ranged from 4 to 9. The resection rate was 67.8% (95% confidence interval [CI] = 60.1% to 74.6%), and the R0-resection rate was 83.9% (95% CI = 76.8% to 89.1%). The median OS varied from 11.0 to 34.2 months across studies. Patient-level survival data were obtained for 20 studies representing 283 BRPC patients. The patient-level median OS was 22.2 months (95% CI = 18.8 to 25.6 months), and patient-level median progression-free survival was 18.0 months (95% CI = 14.5 to 21.5 months). Pooled event rates for grade III-IV adverse events were highest for neutropenia (17.5 per 100 patients, 95% CI = 10.3% to 28.3%), diarrhea (11.1 per 100 patients, 95% CI = 8.6 to 14.3), and fatigue (10.8 per 100 patients, 95% CI = 8.1 to 14.2). No deaths were attributed to FOLFIRINOX.This patient-level meta-analysis of BRPC patients treated with neoadjuvant FOLFIRINOX showed a favorable median OS, resection rate, and R0-resection rate. These results need to be assessed in a randomized trial.

Research Article15 June 2015Open Access Source Data Targeting the LOX/hypoxia axis reverses many of the features that make pancreatic cancer deadly: inhibition of LOX abrogates metastasis and enhances drug efficacy Bryan W Miller Bryan W Miller Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Jennifer P Morton Jennifer P Morton Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Mark Pinese Mark Pinese The Garvan Institute of Medical Research, Sydney, NSW, Australia Search for more papers by this author Grazia Saturno Grazia Saturno Cancer Research UK Manchester Institute, Withington, Manchester, UK Search for more papers by this author Nigel B Jamieson Nigel B Jamieson West of Scotland Pancreatic Unit, Glasgow Royal Infirmary, Glasgow, UK Search for more papers by this author Ewan McGhee Ewan McGhee Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Paul Timpson Paul Timpson The Garvan Institute of Medical Research, Sydney, NSW, Australia Search for more papers by this author Joshua Leach Joshua Leach Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Lynn McGarry Lynn McGarry Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Emma Shanks Emma Shanks Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Peter Bailey Peter Bailey Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Search for more papers by this author David Chang David Chang Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Search for more papers by this author Karin Oien Karin Oien Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Search for more papers by this author Saadia Karim Saadia Karim Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Amy Au Amy Au Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Colin Steele Colin Steele Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Christopher Ross Carter Christopher Ross Carter West of Scotland Pancreatic Unit, Glasgow Royal Infirmary, Glasgow, UK Search for more papers by this author Colin McKay Colin McKay West of Scotland Pancreatic Unit, Glasgow Royal Infirmary, Glasgow, UK Search for more papers by this author Kurt Anderson Kurt Anderson Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Thomas R Jeffry Evans Thomas R Jeffry Evans Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Search for more papers by this author Richard Marais Richard Marais Cancer Research UK Manchester Institute, Withington, Manchester, UK Search for more papers by this author Caroline Springer Caroline Springer Institute of Cancer Research, London, UK Search for more papers by this author Andrew Biankin Andrew Biankin Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Search for more papers by this author Janine T Erler Corresponding Author Janine T Erler Biotech Research & Innovation Centre (BRIC), University of Copenhagen, Copenhagen (UCPH), Denmark Search for more papers by this author Owen J Sansom Corresponding Author Owen J Sansom Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Bryan W Miller Bryan W Miller Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Jennifer P Morton Jennifer P Morton Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Mark Pinese Mark Pinese The Garvan Institute of Medical Research, Sydney, NSW, Australia Search for more papers by this author Grazia Saturno Grazia Saturno Cancer Research UK Manchester Institute, Withington, Manchester, UK Search for more papers by this author Nigel B Jamieson Nigel B Jamieson West of Scotland Pancreatic Unit, Glasgow Royal Infirmary, Glasgow, UK Search for more papers by this author Ewan McGhee Ewan McGhee Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Paul Timpson Paul Timpson The Garvan Institute of Medical Research, Sydney, NSW, Australia Search for more papers by this author Joshua Leach Joshua Leach Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Lynn McGarry Lynn McGarry Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Emma Shanks Emma Shanks Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Peter Bailey Peter Bailey Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Search for more papers by this author David Chang David Chang Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Search for more papers by this author Karin Oien Karin Oien Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Search for more papers by this author Saadia Karim Saadia Karim Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Amy Au Amy Au Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Colin Steele Colin Steele Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Christopher Ross Carter Christopher Ross Carter West of Scotland Pancreatic Unit, Glasgow Royal Infirmary, Glasgow, UK Search for more papers by this author Colin McKay Colin McKay West of Scotland Pancreatic Unit, Glasgow Royal Infirmary, Glasgow, UK Search for more papers by this author Kurt Anderson Kurt Anderson Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Thomas R Jeffry Evans Thomas R Jeffry Evans Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Search for more papers by this author Richard Marais Richard Marais Cancer Research UK Manchester Institute, Withington, Manchester, UK Search for more papers by this author Caroline Springer Caroline Springer Institute of Cancer Research, London, UK Search for more papers by this author Andrew Biankin Andrew Biankin Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Search for more papers by this author Janine T Erler Corresponding Author Janine T Erler Biotech Research & Innovation Centre (BRIC), University of Copenhagen, Copenhagen (UCPH), Denmark Search for more papers by this author Owen J Sansom Corresponding Author Owen J Sansom Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK Search for more papers by this author Author Information Bryan W Miller1,‡, Jennifer P Morton1,‡, Mark Pinese2,‡, Grazia Saturno3,‡, Nigel B Jamieson4, Ewan McGhee1, Paul Timpson2, Joshua Leach1, Lynn McGarry1, Emma Shanks1, Peter Bailey5, David Chang5, Karin Oien5, Saadia Karim1, Amy Au1, Colin Steele1, Christopher Ross Carter4, Colin McKay4, Kurt Anderson1, Thomas R Jeffry Evans1,5, Richard Marais3, Caroline Springer6,‡, Andrew Biankin5,‡, Janine T Erler 7,‡ and Owen J Sansom 1,‡ 1Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK 2The Garvan Institute of Medical Research, Sydney, NSW, Australia 3Cancer Research UK Manchester Institute, Withington, Manchester, UK 4West of Scotland Pancreatic Unit, Glasgow Royal Infirmary, Glasgow, UK 5Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK 6Institute of Cancer Research, London, UK 7Biotech Research & Innovation Centre (BRIC), University of Copenhagen, Copenhagen (UCPH), Denmark ‡These authors contributed equally to this work *Corresponding author. Tel: +45 3532 5666; Fax: +45 3532 5669; E-mail: [email protected] *Corresponding author. Tel: +44 141 330 3656; Fax: +44 141 942 6521; E-mail: [email protected] EMBO Mol Med (2015)7:1063-1076https://doi.org/10.15252/emmm.201404827 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related mortality. Despite significant advances made in the treatment of other cancers, current chemotherapies offer little survival benefit in this disease. Pancreaticoduodenectomy offers patients the possibility of a cure, but most will die of recurrent or metastatic disease. Hence, preventing metastatic disease in these patients would be of significant benefit. Using principal component analysis (PCA), we identified a LOX/hypoxia signature associated with poor patient survival in resectable patients. We found that LOX expression is upregulated in metastatic tumors from Pdx1-Cre KrasG12D/+ Trp53R172H/+ (KPC) mice and that inhibition of LOX in these mice suppressed metastasis. Mechanistically, LOX inhibition suppressed both migration and invasion of KPC cells. LOX inhibition also synergized with gemcitabine to kill tumors and significantly prolonged tumor-free survival in KPC mice with early-stage tumors. This was associated with stromal alterations, including increased vasculature and decreased fibrillar collagen, and increased infiltration of macrophages and neutrophils into tumors. Therefore, LOX inhibition is able to reverse many of the features that make PDAC inherently refractory to conventional therapies and targeting LOX could improve outcome in surgically resectable disease. Synopsis Lysyl oxidase (LOX) is identified as a therapeutic target in pancreatic ductal adenocarcinoma (PDAC). Inhibition of LOX resulted in increased drug efficacy and stromal changes and reduction in metastasis. A signature of hazardous and protective genes in PDAC was defined. High expression of hypoxia-associated genes, including LOX, was associated with poor patient prognosis. Using transgenic mouse models of PDAC, LOX was found to be overexpressed in metastatic disease and its expression was required for PDAC cell invasion. Inhibition of LOX in transgenic mice inhibited metastasis, while combination therapy with LOX inhibition and gemcitabine induced stromal alterations, immune cell infiltration and tumor necrosis and improved survival. Introduction Pancreatic cancer is one of the leading causes of cancer-related death in the UK with around 7,000 cases being diagnosed every year (Mukherjee et al, 2008). Pancreatic ductal adenocarcinoma (PDAC) is almost universally lethal. Aggressive invasion and early metastases are characteristic of the disease, such that 80–90% of patients have surgically unresectable disease at the time of diagnosis (Giovinazzo et al, 2012). The majority of patients who are selected to undergo potentially curative resection for small, localized lesions almost inevitably develop recurrent or metastatic disease (Yeo et al, 2002), presumably due to the presence of undetected micro-metastases at initial diagnosis. Adjuvant (post-operative) chemotherapy can improve outcome despite the modest anti-tumor efficacy of these agents (Neoptolemos et al, 2004; Stocken et al, 2005). Nevertheless, overall survival remains disappointing with most patients developing local recurrence or extra-pancreatic metastasis within 2 years (Giovinazzo et al, 2012). Hence, there is a requirement for biomarkers to predict/prognosticate those patients that do not succumb to early recurrence post-resection and identify potentially targetable pathways that may be associated with poor prognosis. This has been made even more important by recent sequencing studies of pancreatic cancer, which have found PDAC to be very complex and contain multiple low-frequency mutations of unknown functional significance. Moreover, none of the major mutations (KRAS, TP53, SMAD4 and INK4A) currently confer treatment opportunities. Importantly, these surgically resectable patients who die of recurrent metastatic disease represent a set of patients where suppression of metastasis (either prevention or suppression of growth of micro-metastasis) may be a realistic therapeutic option and trials are underway to test whether SRC inhibitors may be beneficial in this patient set. A characteristic feature of PDAC is the highly desmoplastic stromal microenvironment. This stroma consists of immune cells, collagen and fibronectin laid down by fibroblasts and stellate cells (Chu et al, 2007). In addition to contributing to disease progression, and promoting tumor growth and invasion, recent studies suggest that the stroma also limits drug delivery to tumor cells, partly explaining the profound resistance of pancreatic tumors to systemic chemotherapy agents (Olive et al, 2009). This situation is made worse by a very poor tumor vasculature which may also limit drug access to tumors (Olive et al, 2009). Consistent with this, a number of studies have shown that potential stromal markers such as S100A2 and A4 have prognostic value in patients with resected PDAC (Jamieson et al, 2011). More recently, there has been great interest in the role of the enzyme lysyl oxidase (LOX) in driving metastasis in epithelial cancers such as breast and colorectal cancer (Payne et al, 2005; Baker et al, 2011; Cox & Erler, 2013). LOX is a copper-dependent enzyme which cross-links collagen and elastins to drive tissue stiffness (Barker et al, 2012). It has previously been shown to be induced by hypoxia-inducible factor 1α (HIF1α) and can function upstream of the SRC/FAK tyrosine kinases (Payne et al, 2005; Erler et al, 2006; Baker et al, 2011, 2013). These studies suggest that LOX inhibition will have the potential to suppress metastasis rather than causing regression of established tumors (Cox et al, 2013; Cox & Erler, 2014), consistent with observations using SRC inhibitors in cancer cell lines and mouse models (Morton et al, 2010a). Additionally, LOX has been reported to have direct effects on cancer cells themselves through regulation of senescence (Wiel et al, 2013). There is a plethora of evidence that inflammation is tumor promoting in pancreatic cancer. Pancreatitis leads to increased risk of PDAC, while cerulein promotes acinar-to-ductal metaplasia and disease progression (Guerra et al, 2007; Rhim et al, 2012). However, it is also clear that the presence of leukocytes such as neutrophils within tumors is often associated with a good prognosis in many different cancer types including pancreatic cancer (Caruso et al, 2002; Schaider et al, 2003; Jamieson et al, 2012). Thus far, there are no mechanistic data to explain this phenomenon, nor is it known how the desmoplastic "stiff" stroma would affect the ability of leukocytes to penetrate tumor tissue. Given the complex interplay between the tumor and the stroma, modeling therapies in these systems require immunocompetent mice that develop tumors that closely recapitulate human disease. Genetically engineered mice carrying the common mutations that occur in PDAC rapidly generate invasive and metastatic disease (Morton et al, 2010b). Therefore, these can serve as excellent models in which to test therapies aimed at targeting the stroma and assess the impact of the mutations that occur in pancreatic cancer upon the stroma, invasion and metastasis. Our and others' previous studies have shown that the accumulating mutant p53 (p53R172H) has gain-of-function properties over loss-of-function mutations (Olive et al, 2004; Jackson et al, 2005; Adorno et al, 2009; Morton et al, 2010b). Consistent with this, we and others have previously found that mice expressing p53R172H display a far higher frequency of liver metastasis than mice with loss of p53 function (Morton et al, 2010b; Weissmueller et al, 2014). However, it has been reported that mice carrying loss of function of p53 and INK4 loss alongside KrasG12D targeted to the pancreas develop adenocarcinoma that metastasizes to the liver (Bardeesy et al, 2006). As yet, most work has focused on how mutant p53 facilitates cell autonomous migration and invasion and has not examined whether it affects tumor–stromal interactions. While the majority of studies had suggested that targeting the stroma may be a promising therapeutic option in this disease, recent studies have shown dramatically different results. Here, targeted sonic hedgehog depletion or myofibroblast disruption has reported decreased survival in mouse models (Lee et al, 2014; Ozdemir et al, 2014; Rhim et al, 2014).These studies suggest that thinking of the tumor stroma simply as a tumor-promoting entity needs to be re-evaluated and it will be very important to understand which elements of the stroma drive the chemoresistance and early progression of pancreatic cancer, and which act to constrain the tumor. It should be noted that these studies also showed that subsequent co-targeting with either anti-angiogenesis or immunotherapy could then lead to significant tumor regression. In this study, we identify LOX, driven by mutant p53, as an important therapeutic target in pancreatic cancer, inhibition of which causes tumor necrosis in combination with gemcitabine. Importantly, the LOX–hypoxia axis defines the poor prognosis of surgically resectable cancers and can explain many of the features that contribute to making PDAC inherently refractory to conventional cytotoxic chemotherapy. Most importantly, inhibition of LOX can reverse most of these features, suggesting that this may be a promising therapeutic approach. Results LOX/hypoxia marks poor prognosis in patients with resected PDAC To elucidate the key components that contribute to poor prognosis in patients with PDAC, a gene signature analysis was performed on the transcriptome of 73 PDACs (Biankin et al, 2012). From this, a single signature was identified (called PC-1) that following cross-validation could predict survival in the discovery cohort (Fig 1A). PC-1 could be split into two gene sets: a hazardous set of 321 transcripts, for which increased expression was associated with poor prognosis, and a protective set of 238 transcripts, for which increased expression was beneficial. To identify potential biological processes underlying the survival signature, we tested the hazardous and protective sets of PC-1 for overlap against the MSigDB gene set database (Subramanian et al, 2005). We observed that the hazardous subset of PC-1 showed significant overlap with numerous hypoxia signatures, and the protective subset was linked to lymphocyte and antigen presentation signatures (Supplementary Table S1). We confirmed that high expression of a hypoxia signature correlated with poor prognosis (Fig 1B). From this hypoxia signature, we selected LOX for further analysis given we wished to investigate factors that could modulate the stroma that are targetable, its previous association with the mesenchymal subtype and reports of its involvement in metastasis in breast and colon cancer (Payne et al, 2005; Baker et al, 2011). We examined overall survival in terms of the expression of LOX using an expanded APGI cohort of 266 patients (Chou et al, 2013). Expression of LOX correlated with patient survival (Fig 1C), and we confirmed this correlation using multivariate analysis (Supplementary Table S2). We next looked in our Glasgow cohort of patients where 47 patients have been profiled and once again high LOX expression correlated with a poor prognosis (Fig 1D). Moreover, LOX was 1 out of only 5 probe sets that overlapped between the Glasgow (Jamieson et al, 2011) cohort and the Collisson (Collisson et al, 2011) signatures of poor prognosis in pancreatic cancer (the others being S100A2, TWIST, NT5E and PAPPA) (Supplementary Fig S1). Additionally, high expression of further LOX family members (LOXL1, 2, 3 and 4) were found to be associated with poor prognosis in the Glasgow data set (Supplementary Fig S1B–E). Taken together, and given the reproducibility across multiple patient cohorts, these findings argued for a significant role for LOX expression as a determinant of poor survival in pancreatic cancer. Figure 1. LOX/hypoxia marks poor prognosis in resectable PDAC Kaplan–Meier analysis showing cases from the Australian cohort (n = 73) delineated on the basis of expression of a set of hazardous genes, termed PC-1. Patients with the highest expression had a significantly poorer prognosis (red line, median survival: 11.6 months) compared with those patients with the lowest expression (blue line, median survival: 34.4 months, P = 0.01). The black line shows those with medium expression. Kaplan–Meier analysis showing that cases in the Australian cohort that fell in the highest quartile of a hypoxia signature (red line, median survival: 11.4 months) have significantly decreased survival compared with those in the lowest quartile (blue line, median survival: 25.2 months; P = 0.03). Kaplan–Meier analysis showing that cases in the APGI cohort (n = 266) with high LOX expression above the 3rd quantile (red line) have significantly decreased survival compared to those with low expression below the 1st quantile (blue line). Kaplan–Meier analysis showing that cases in the Glasgow cohort with high LOX expression (red line, n = 24) have significantly decreased survival compared with those with low expression (blue line, n = 23; P = 0.005). Kaplan–Meier analysis showing that fibrillar collagen is significantly associated with reduced survival in human PDAC (20 months vs. 28.3 months, P = 0.033). Mean decay distance of the second harmonic generation (SHG) signal emitted by human PDAC-associated collagen. Mean decay distance is represented by boxplots showing the second and third quartile of the data with the whiskers indicating the maximum and minimum data points. Outliers are indicated by individual markers. Stage T3 tumors have a higher collagen mean decay distance score compared with Stage T2 tumors (P = 0.02). Lymph node-positive tumors have a higher collagen mean decay distance score vs. lymph node-negative tumors (P = 0.047). Tumors showing vascular invasion have a higher collagen mean decay distance score than non-vascular invasive tumors (P = 0.037). Download figure Download PowerPoint As LOX is associated with regulation of collagen cross-linking, we assessed the status of fibrillar collagen in human PDAC. Using multiphoton microscopy, we analyzed the second harmonic resonance signal of collagen in a human pancreatic tissue microarray (TMA) consisting of 80 patients. Second harmonic generation (SHG) imaging of collagen has been used previously to identify collagen "signatures" in mammary tumors, which may impact on the ability of cancer cell migration (Provenzano et al, 2006, 2009). In our study, we found that fibrillar collagen was significantly associated with reduced survival (20 months vs. 28.3 months, P = 0.033) (Fig 1E). Further, we showed that fibrillar collagen was significantly associated with increased tumor stage, lymph node spread and vascular invasion (Fig 1F). This suggested that the generation of fibrillar collagen may be important in PDAC disease progression and that the inhibition of cross-linking needed to generate collagen fibers via LOX inhibition may be important therapeutically. In order to further investigate this, we used immunocompetent, autochthonous genetically engineered mouse models of PDAC, which are initiated by targeting Kras mutation to the murine pancreas using the Pdx1-Cre transgene. Without the addition of cooperating mutations, only one-third of Pdx1-Cre KrasG12D/+ mice develop PDAC by 500 days (Hingorani et al, 2003). However, when p53 is additionally targeted by deletion or mutation, mice rapidly develop invasive adenocarcinoma with a median latency of between 120 and 180 days (Hingorani et al, 2005) and we have previously found that p53 mutation, but not loss, could drive metastasis in this model (Morton et al, 2010b). In order to characterize the gene expression changes that underlie metastatic disease, we carried out microarray analysis of tumors driven by Kras mutation and mutation of one copy of p53 or deletion of p53. To address whether transcriptional changes observed in pancreatic cancer patients were reflected in our mouse models, we applied loading values obtained from the PC-1 human tumor signature to our mouse microarray data sets (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE67358). This allowed us to derive risk scores for each individual mouse transcriptome. While the majority of mice that had non-metastatic tumors had negative risk scores, 100% of KPC mice that developed metastatic disease had positive risk scores (Fig 2A, top). Of the gene expression changes observed between metastatic and non-metastatic tumors, multiple members of the LOX family were overexpressed in metastatic disease (Fig 2A, bottom). Figure 2. LOX expression is required for invasion in a mutant p53-driven model of PDAC PC-1 signature is predictive of metastatic disease in mouse models of pancreatic cancer. Log-transformed expressions of signature transcripts from mouse tumor microarrays were mean-centered across samples and scaled to unit variance. These values were then multiplied by the matching loading values from the PC-1 signature and summarized for each sample across all transcripts to yield the risk score for that sample. Inverted invasion assays were performed with PDAC tumor cell lines from KPC and KPflC mice. Tumor cell lines bearing mutant p53R172H (KPC) invade significantly further than tumor cells with deletion of 1 copy of p53 (KPflC) (P ≤ 0.01). Data are shown as the average of four wells + SEM. Introduction of shRNA targeting Lox into KPC tumor cells significantly inhibits invasion (P ≤ 0.01 by unpaired Student's t-test). Data are shown as the average of four wells + SEM. Introduction of exogenous LOX into KPflC tumor cells significantly promotes invasion (left panel, P ≤ 0.01 by unpaired Student's t-test). LOX expression was assessed by immunoblotting (right panel). Columns indicate the mean of four well and error bars indicate SEM. Integration of heterogeneous data sets identifies LOX as a therapeutic target in pancreatic cancer. Components of the hazardous PC-1 signature are overlaid with genes found to be overexpressed in a microarray and hits that cause a reduction in viable cell number in an RNAi functional screen. Overlap is shown as a proportional Venn diagram. Source data are available online for this figure. Source Data for Figure 2 [emmm201404827-sup-0012-SDataFig2.pdf] Download figure Download PowerPoint LOX is required for mutant p53-driven invasion Given that our data suggest a role for LOX in mutant p53-driven metastatic PDAC, we decided to address the consequences for cell migration and invasion, of manipulating LOX expression in PDAC cell models. For this, we used both KPC cell lines and cells derived from Pdx1-Cre KrasG12D/+ p53flox/+ (KPflC) tumors. Initially, to assess whether LOX expression was required for invasion, we used siRNA to knockdown LOX expression and measured migration of KPC cells through a Matrigel matrix (Supplementary Fig S2A). Loss of LOX expression significantly impaired KPC cell migration, and this could be rescued by recombinant LOX protein. Inhibition of LOX in KPC cells led to a reduction in SRC phosphorylation (Supplementary Fig S2B), and we confirmed that low doses of the SRC inhibitor dasatinib resulted in a slowing of wound healing (Supplementary Fig S2C). Given our previous data showing a requirement for SRC in KPC cell invasion (Morton et al, 2010a), this suggests that the pro-migratory effects of LOX could be mediated at least in part by SRC activation. We confirmed that LOX knockdown in human Panc-1 cells had similar effects on migration (Supplementary Fig S2D). We have previously shown that the ability of mutant p53 cells (and not p53-deleted cells) to invade in vitro correlates well with the ability of these cells to metastasize in vivo (Fig 2B). Analysis of the expression of LOX family members in these cell lines showed a clear increase in the expression of LOX and LOX family members in cell lines carrying mutant but not loss-of-function p53, consistent with our microarray analyses (Supplementary Fig S2E). Thus, we carried out stable knockdown of LOX in KPC cells using shRNA and found this significantly reduced invasion of mutant p53 cells (Fig 2C; Supplementary Fig S2F). Conversely, overexpression of LOX in p53 loss-of-function tumor cells promoted invasion of these cells (Fig 2D). In order to determine the effects of the modulation of LOX expression in these cell models in vivo, we carried out a series of allograft experiments. LOX knockdown slowed allograft growth of KPC cells (Supplementary Fig S2G), while LOX overexpression resulted in faster growth of KPflC cells (Supplementary Fig S2H). In parallel, we also performed a functional screen on primary cells from Pdx1-Cre KrasG12D p53R172H (KPC) metastatic tumors. Overall, we found that 920 siRNAs reduced cell viability using a Z-score threshold of -3 (Supplementary Table S3). A number of these screen hits were extracellular matrix components, including LOX, fi

Abstract Purpose: MicroRNAs (miRNA) have potential as diagnostic and prognostic biomarkers and as therapeutic targets in cancer. We sought to establish the relationship between miRNA expression and clinicopathologic parameters, including prognosis, in pancreatic ductal adenocarcinoma (PDAC). Experimental Design: Global miRNA microarray expression profiling of prospectively collected fresh-frozen PDAC tissue was done on an initial test cohort of 48 patients, who had undergone pancreaticoduodenectomy between 2003 and 2008 at a single institution. We evaluated association with tumor stage, lymph node status, and site of recurrence, in addition to overall survival, using Cox regression multivariate analysis. Validation of selected potentially prognostic miRNAs was done in a separate cohort of 24 patients. Results: miRNA profiling identified expression signatures associated with PDAC, lymph node involvement, high tumor grade, and 20 miRNAs were associated with overall survival. In the initial cohort of 48 PDAC patients, high expression of miR-21 (HR = 3.22, 95% CI: 1.21–8.58) and reduced expression of miR-34a (HR = 0.15, 95% CI: 0.06–0.37) and miR-30d (HR = 0.30, 95% CI: 0.12–0.79) were associated with poor overall survival following resection independent of clinical covariates. In a further validation set of 24 patients, miR-21 and miR-34a expression again significantly correlated with overall survival (P = 0.031 and P = 0.001). Conclusion: Expression patterns of miRNAs are significantly altered in PDAC. Aberrant expression of a number of miRNAs was independently associated with reduced survival, including overexpression of miR-21 and underexpression of miR-34a. Summary: miRNA expression profiles for resected PDAC were examined to identify potentially prognostic miRNAs. miRNA microarray analysis identified statistically unique profiles, which could discriminate PDAC from paired nonmalignant pancreatic tissues as well as molecular signatures that differ according to pathologic features. miRNA expression profiles correlated with overall survival of PDAC following resection, indicating that miRNAs provide prognostic utility. Clin Cancer Res; 18(2); 534–45. ©2011 AACR.

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

Objective: To identify a clinical fistula risk score following distal pancreatectomy. Background: Clinically relevant pancreatic fistula (CR-POPF) following distal pancreatectomy (DP) is a dominant contributor to procedural morbidity, yet risk factors attributable to CR-POPF and effective practices to reduce its occurrence remain elusive. Methods: This multinational, retrospective study of 2026 DPs involved 52 surgeons at 10 institutions (2001–2016). CR-POPFs were defined by 2016 International Study Group criteria, and risk models generated using stepwise logistic regression analysis were evaluated by c-statistic. Mitigation strategies were assessed by regression modeling while controlling for identified risk factors and treating institution. Results: CR-POPF occurred following 306 (15.1%) DPs. Risk factors independently associated with CR-POPF included: age (&lt;60 yrs: OR 1.42, 95% CI 1.05–1.82), obesity (OR 1.54, 95% CI 1.19–2.12), hypoalbuminenia (OR 1.63, 95% CI 1.06–2.51), the absence of epidural anesthesia (OR 1.59, 95% CI 1.17–2.16), neuroendocrine or nonmalignant pathology (OR 1.56, 95% CI 1.18–2.06), concomitant splenectomy (OR 1.99, 95% CI 1.25–3.17), and vascular resection (OR 2.29, 95% CI 1.25–3.17). After adjusting for inherent risk between cases by multivariable regression, the following were not independently associated with CR-POPF: method of transection, suture ligation of the pancreatic duct, staple size, the use of staple line reinforcement, tissue patches, biologic sealants, or prophylactic octreotide. Intraoperative drainage was associated with a greater fistula rate (OR 2.09, 95% CI 1.51–3.78) but reduced fistula severity ( P &lt; 0.001). Conclusions: From this large analysis of pancreatic fistula following DP, CR-POPF occurrence cannot be reliably predicted. Opportunities for developing a risk score model are limited for performing risk-adjusted analyses of mitigation strategies and surgeon performance.

To determine the prognostic influence of residual tumor at or within 1 mm of the mobilization margins (R1Mobilization) compared with transection margins (R1Transection) following pancreaticoduodenectomy for pancreatic ductal adenocarcinoma (PDAC).The prognostic strength of R1 status increases with frequency of margin positivity and is enhanced by protocol driven pathology reporting. Currently, margins are treated uniformly with tumor at or close to any margin considered of equal prognostic significance. The resection involves a mobilization phase freeing the posterior margin and anterior surface then a transection phase requiring lympho-vascular division forming the medial resection and pancreatic transection margin. The comparative assessment of the relative importance of tumor involvement of these different margins has not previously been investigated.Retrospective analysis of 148 consecutive resections for PDAC from 1996-2007 was performed. The individual (pancreatic transection, medial, posterior, and anterior surface) margins were separately identified and analyzed by a senior pathologist. An R1 resection was defined as microscopic evidence of tumor < or = 1 mm from a resection margin. R1Mobilization tumor extension included both R1Anterior and R1Posterior cases; while R1Transection included pancreatic neck/body transection, R1Medial and adjacent transection margins.R1 status was confirmed in 109 patients (74%). The medial (46%) and posterior (44%) margins were most commonly involved. R1 status was found to an independent predictor of poor outcome (P < 0.001). R1Mobilization involvement only (n = 48) was associated with a significantly longer median survival of 18.9 months (95% CI, 13.7-24.8) versus 11.1 months (95% CI, 7.1-15.0) for those with R1Transection tumor involvement (n = 61) (P < 0.001). There was no significant difference in the survival of the R1Mobilization compared with R0 group (P = 0.52).Following pancreaticoduodenectomy for PDAC, involvement of the transection margins in contrast to mobilization margins defines a group whose outcome is significantly worse. This may impact upon the allocation of adjuvant therapy within the setting of randomized controlled trials.

Mutations in both RAS and the PTEN/PIK3CA/AKT signaling module are found in the same human tumors. PIK3CA and AKT are downstream effectors of RAS, and the selective advantage conferred by mutation of two genes in the same pathway is unclear. Based on a comparative molecular analysis, we show that activated PIK3CA/AKT is a weaker inducer of senescence than is activated RAS. Moreover, concurrent activation of RAS and PIK3CA/AKT impairs RAS-induced senescence. In vivo, bypass of RAS-induced senescence by activated PIK3CA/AKT correlates with accelerated tumorigenesis. Thus, not all oncogenes are equally potent inducers of senescence, and, paradoxically, a weak inducer of senescence (PIK3CA/AKT) can be dominant over a strong inducer of senescence (RAS). For tumor growth, one selective advantage of concurrent mutation of RAS and PTEN/PIK3CA/AKT is suppression of RAS-induced senescence. Evidence is presented that this new understanding can be exploited in rational development and targeted application of prosenescence cancer therapies.

Patients with pancreatic cancer have one of the poorest survival rates and selection of patients for active treatment remains problematical. The present study assesses the value of an inflammation-based score (Glasgow Prognostic Score, GPS) in patients with inoperable pancreatic cancer.The GPS was constructed as follows: patients with both an elevated C-reactive protein (>10 mg/l) and hypoalbuminaemia (<35 g/l) were allocated a score of 2. Patients in whom only 1 or none of these biochemical abnormalities was present were allocated a score of 1 or 0, respectively.One hundred and eighty-seven patients were studied and 49 (26%) underwent an operative palliative bypass procedure. At the end of follow-up, 181 (97%) patients died, 17% of patients were alive at 12 months. On univariate analysis, age (p < 0.01), TNM stage (p < 0.001) and the GPS (p < 0.001) were significant predictors of survival. On multivariate survival analysis, stratified for bypass procedure, age (hazard ratio 1.53, 95%CI 1.12-2.10, p = 0.008), TNM stage (hazard ratio 1.70, 95%CI 1.33-2.18, p < 0.001) and the GPS (hazard ratio 1.72, 95%CI 1.40-2.11, p < 0.001) remained independent significant predictors of survival.At diagnosis, the presence of a systemic inflammatory response (as measured by the GPS) appears to be a useful indicator of poor outcome, independent of TNM stage, in patients with inoperable pancreatic cancer.

525 different drugs that can, as an adverse reaction, induce acute pancreatitis are listed in a WHO database. Compared to other causes drugs represent a relatively rare cause of pancreatitis. They should be considered as a triggering event in patients with no other identifiable cause of the disease, who takes medications that have been shown to induce pancreatitis. The prevalence of drug-induced pancreatitis is still unclear because most incidences have been documented only as isolated case reports. The overall incidence probably ranges from between 0.1 and 2% of pancreatitis cases. For only very few substances evidence from controlled trials has been obtained. Epidemiologic data suggest the risk of pancreatitis is highest for mesalazine (HR 3.5,) azathioprine (HR 2,5) and simvastatine (HR 1,8). Even when a definite association has been demonstrated it is often impossible to determine whether the drug, or the underlying condition for which the drug was taken has conferred the risk of pancreatitis (e.g. Azathioprine and Crohns disease or Pentamidine and HIV). Knowledge about the underlying pathophysiologic mechanisms as well as evidence for a direct causality often remains sparse. For only 31 drugs a definite causality has been established. The most frequently reported are mesalazine (nine cases in total, three cases with re-exposure), azathioprine (five cases in total, two cases with re-exposure) and simvastatin (one case in total, this one with re-exposure). As cause–effect relationship is generally accepted when symptoms re-occur upon re-challenge. Available data from case control studies suggest that even drugs with solid evidence for an association with pancreatitis only rarely cause the disease. Even when pancreatitis is induced as an adverse drug event the disease course is usually mild or even subclinical.

Adiponectin is a recently identified, insulin-sensitizing and anti-inflammatory protein released by adipocytes, which is paradoxically reduced in obesity. It suppresses endothelial activation. Physiological insulin resistance occurs in normal pregnancy and is exaggerated in women with preeclampsia (PE), together with enhanced inflammatory and endothelial activation. Women with increased body mass index (BMI) and insulin resistance are predisposed to PE. We hypothesized that adiponectin concentrations are reduced in normal pregnancy compared with postpartum values and further reduced in women with PE. Fifteen women with PE and 30 control subjects with similar first trimester BMI had adiponectin concentrations measured in the third trimester; postpartum measurements were repeated in 16 control subjects. Adiponectin concentration in healthy pregnant women correlated inversely with early pregnancy BMI (r=-0.47, P=0.01) and fasting insulin concentrations (r=-0.58, P=0.001). However, adiponectin concentrations did not differ significantly in pregnancy and postpartum samples (mean change, -0.15 microg/mL; 95% CI, -2.28 to 1.98, P=0.88). Plasma adiponectin concentrations were markedly elevated (P=0.01) in women with PE (mean, 21.6; SD, 8.18 microg/mL) compared with control subjects (mean, 14.7; SD, 7.06 microg/mL). Moreover, in PE, adiponectin concentrations did not correlate with first trimester BMI or insulin or with serum urate or creatinine concentrations or urinary protein levels. We conclude that plasma adiponectin concentrations are not elevated in normal human pregnancy and paradoxically elevated (by 47%) in women with PE. This may be secondary to exaggerated nonspecific adipocyte lipolysis or as a physiological response to enhance fat utilization and attenuate endothelial damage. Future studies should determine whether adiponectin concentrations help improve prediction of PE.

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.

<h3>Background & Aims</h3> Patients carrying germline mutations of <i>LKB1</i> have an increased risk of pancreatic cancer; however, it is unclear whether down-regulation of <i>LKB1</i> is an important event in sporadic pancreatic cancer. In this study, we aimed to investigate the impact of LKB1 down-regulation for pancreatic cancer in mouse and human and to elucidate the mechanism by which Lkb1 deregulation contributes to this disease. <h3>Methods</h3> We first investigated the consequences of Lkb1 deficiency in a genetically modified mouse model of pancreatic cancer, both in terms of disease progression and at the molecular level. To test the relevance of our findings to human pancreatic cancer, we investigated levels of LKB1 and its potential targets in human pancreatic cancer. <h3>Results</h3> We definitively show that <i>Lkb1</i> haploinsufficiency can cooperate with oncogenic <i>Kras</i>^G12D to cause pancreatic ductal adenocarcinoma (PDAC) in the mouse. Mechanistically, this was associated with decreased p53/p21-dependent growth arrest. Haploinsufficiency for p21 (<i>Cdkn1a)</i> also synergizes with <i>Kras</i>^G12D to drive PDAC in the mouse. We also found that levels of LKB1 expression were decreased in around 20% of human PDAC and significantly correlated with low levels of p21 and a poor prognosis. Remarkably, all tumors that had low levels of LKB1 had low levels of p21, and these tumors did not express mutant p53. <h3>Conclusions</h3> We have identified a novel LKB1-p21 axis that suppresses PDAC following <i>Kras</i> mutation in vivo. Down-regulation of LKB1 may therefore serve as an alternative to p53 mutation to drive pancreatic cancer in vivo.

<h3>Background & Aims</h3> Pancreatic ductal adenocarcinoma (PDAC) is a highly invasive and metastatic disease for which conventional treatments are of limited efficacy. A number of agents in development are potential anti-invasive and antimetastatic agents, including the Src kinase inhibitor dasatinib. The aim of this study was to assess the importance of Src in human PDAC and to use a genetically engineered mouse model of PDAC to determine the effects of dasatinib on PDAC progression. <h3>Methods</h3> Src expression and activity was measured by immunohistochemistry in 114 human PDACs. Targeting expression of Trp53^R172H and Kras^G12D to the mouse pancreas results in the formation of invasive and metastatic PDAC. These mice were treated with dasatinib, and disease progression monitored. Cell lines were derived from mouse PDACs, and in vitro effects of dasatinib assessed. <h3>Results</h3> Src expression and activity were up-regulated in human PDAC and this correlated with reduced survival. Dasatinib inhibited the migration and invasion of PDAC cell lines, although no effects on proliferation were seen at concentrations that inhibited Src kinase activity. In addition, dasatinib significantly inhibited the development of metastases in <i>Pdx1-Cre</i>, <i>Z/EGFP</i>, <i>LSL-Kras</i>^G12D/+, <i>LSL-Trp53</i>^R172H/+ mice. However, there was no survival advantage in the dasatinib-treated animals owing to continued growth of the primary tumor. <h3>Conclusions</h3> This study confirms the importance of Src in human PDAC and shows the usefulness of a genetically engineered mouse model of PDAC for assessing the activity of potential antimetastatic agents and suggests that dasatinib should be evaluated further as monotherapy after resection of localized invasive PDAC.

Objective: The aim of this study was to identify the optimal fistula mitigation strategy following pancreaticoduodenectomy. Background: The utility of technical strategies to prevent clinically relevant postoperative pancreatic fistula (CR-POPF) following pancreatoduodenectomy (PD) may vary by the circumstances of the anastomosis. The Fistula Risk Score (FRS) identifies a distinct high-risk cohort (FRS 7 to 10) that demonstrates substantially worse clinical outcomes. The value of various fistula mitigation strategies in these particular high-stakes cases has not been previously explored. Methods: This multinational study included 5323 PDs performed by 62 surgeons at 17 institutions. Mitigation strategies, including both technique related (ie, pancreatogastrostomy reconstruction; dunking; tissue patches) and the use of adjuvant strategies (ie, intraperitoneal drains; anastomotic stents; prophylactic octreotide; tissue sealants), were evaluated using multivariable regression analysis and propensity score matching. Results: A total of 522 (9.8%) PDs met high-risk FRS criteria, with an observed CR-POPF rate of 29.1%. Pancreatogastrostomy, prophylactic octreotide, and omission of externalized stents were each associated with an increased rate of CR-POPF (all P < 0.001). In a multivariable model accounting for patient, surgeon, and institutional characteristics, the use of external stents [odds ratio (OR) 0.45, 95% confidence interval (95% CI) 0.25–0.81] and the omission of prophylactic octreotide (OR 0.49, 95% CI 0.30–0.78) were independently associated with decreased CR-POPF occurrence. In the propensity score matched cohort, an “optimal” mitigation strategy (ie, externalized stent and no prophylactic octreotide) was associated with a reduced rate of CR-POPF (13.2% vs 33.5%, P < 0.001). Conclusions: The scenarios identified by the high-risk FRS zone represent challenging anastomoses associated with markedly elevated rates of fistula. Externalized stents and omission of prophylactic octreotide, in the setting of intraperitoneal drainage and pancreaticojejunostomy reconstruction, provides optimal outcomes.

Abstract Purpose: The management of pancreatic ductal adenocarcinoma (PDAC) continues to present a great challenge particularly with regard to prediction of outcome following pancreaticoduodenectomy. Molecular markers have been extensively investigated by numerous groups with the aim of enhancing prognostication; however, despite hundreds of studies that have sought to assess the potential prognostic value of molecular markers in predicting the clinical course following resection of PDAC, at this time, no molecular marker assay forms part of recommended clinical practice. Experimental Design: We conducted a systematic review and meta-analysis of the published literature for immunohistochemistry-based biomarkers of PDAC outcome. A dual search strategy was applied to the PubMed database on January 6, 2010, to identify cohort studies that reported associations between immunohistochemical biomarker expression and survival outcomes in PDAC, and conformed to the REMARK (REporting recommendations for tumor MARKer prognostic studies) criteria. Results: A total of 103 distinct proteins met all inclusion criteria. Promising markers that emerged for the prediction of overall survival included BAX (HR = 0.31, 95% CI: 0.71–0.56), Bcl-2 (HR = 0.41, 95% CI: 0.27–0.63), survivin (HR = 0.46, 95% CI: 0.29–0.73), Ki-67: (HR = 2.42, 95% CI: 1.87–3.14), COX-2 (HR = 1.39, 95% CI: 1.13–1.71), E-cadherin (HR = 1.80, 95% CI: 1.33–2.42), and S100 calcium-binding proteins, in particular S100A2 (HR = 3.23, 95% CI: 1.58–6.62). Conclusions: We noted that that there was incomplete adherence to the REMARK guidelines with inadequate methodology reporting as well as failure to perform multivariate analysis. Addressing the persistent incomplete adoption of these criteria may eventually result in the incorporation of molecular marker assessment within PDAC management algorithms. Clin Cancer Res; 17(10); 3316–31. ©2011 AACR.

<h3>Objective</h3> Pancreatic cancer is a leading cause of cancer-related death in the Western world. Current chemotherapy regimens have modest survival benefit. Thus, novel, effective therapies are required for treatment of this disease. <h3>Design</h3> Activating <i>KRAS</i> mutation almost always drives pancreatic tumour initiation, however, deregulation of other potentially druggable pathways promotes tumour progression. PTEN loss leads to acceleration of <i>Kras^G12D</i>-driven pancreatic ductal adenocarcinoma (PDAC) in mice and these tumours have high levels of mammalian target of rapamycin (mTOR) signalling. To test whether these KRAS PTEN pancreatic tumours show mTOR dependence, we compared response to mTOR inhibition in this model, to the response in another established model of pancreatic cancer, KRAS P53. We also assessed whether there was a subset of pancreatic cancer patients who may respond to mTOR inhibition. <h3>Results</h3> We found that tumours in KRAS PTEN mice exhibit a remarkable dependence on mTOR signalling. In these tumours, mTOR inhibition leads to proliferative arrest and even tumour regression. Further, we could measure response using clinically applicable positron emission tomography imaging. Importantly, pancreatic tumours driven by activated KRAS and mutant p53 did not respond to treatment. In human tumours, approximately 20% of cases demonstrated low PTEN expression and a gene expression signature that overlaps with murine KRAS PTEN tumours. <h3>Conclusions</h3> KRAS PTEN tumours are uniquely responsive to mTOR inhibition. Targeted anti-mTOR therapies may offer clinical benefit in subsets of human PDAC selected based on genotype, that are dependent on mTOR signalling. Thus, the genetic signatures of human tumours could be used to direct pancreatic cancer treatment in the future.

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.

<h2>Summary</h2> Integrin trafficking is key to cell migration, but little is known about the spatiotemporal organization of integrin endocytosis. Here, we show that α5β1 integrin undergoes tensin-dependent centripetal movement from the cell periphery to populate adhesions located under the nucleus. From here, ligand-engaged α5β1 integrins are internalized under control of the Arf subfamily GTPase, Arf4, and are trafficked to nearby late endosomes/lysosomes. Suppression of centripetal movement or Arf4-dependent endocytosis disrupts flow of ligand-bound integrins to late endosomes/lysosomes and their degradation within this compartment. Arf4-dependent integrin internalization is required for proper lysosome positioning and for recruitment and activation of mTOR at this cellular subcompartment. Furthermore, nutrient depletion promotes subnuclear accumulation and endocytosis of ligand-engaged α5β1 integrins via inhibition of mTORC1. This two-way regulatory interaction between mTORC1 and integrin trafficking in combination with data describing a role for tensin in invasive cell migration indicate interesting links between nutrient signaling and metastasis.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant desmoplastic reaction driven by pancreatic stellate cells (PSCs) that contributes to tumor progression.Here we sought to characterize the interactions between pancreatic cancer cells (PCCs) and PSCs that affect the inflammatory and immune response in pancreatic tumors.Conditioned media from mono-and cocultures of PSCs and PCCs were assayed for expression of cytokines and growth factors.IP-10/ CXCL10 was the most highly induced chemokine in coculture of PSCs and PCCs.Its expression was induced in the PSCs by PCCs.IP-10 was elevated in human PDAC specimens, and positively correlated with high stroma content.Furthermore, gene expression of IP-10 and its receptor CXCR3 were significantly associated with the intratumoral presence of regulatory T cells (Tregs).In an independent cohort of 48 patients with resectable pancreatic ductal adenocarcinoma, high IP-10 expression levels correlated with decreased median overall survival.Finally, IP-10 stimulated the ex vivo recruitment of CXCR3 + effector T cells as well as CXCR3 + Tregs derived from patients with PDAC.Our findings suggest that, in pancreatic cancer, CXCR3 + Tregs can be recruited by IP-10 expressed by PSCs in the tumor stroma, leading to immunosuppressive and tumor-promoting effects.

Background & AimsPancreatic ductal adenocarcinoma (PDAC) is often lethal because it is highly invasive and metastasizes rapidly. The actin-bundling protein fascin has been identified as a biomarker of invasive and advanced PDAC and regulates cell migration and invasion in vitro. We investigated fascin expression and its role in PDAC progression in mice.MethodsWe used KRasG12D p53R172H Pdx1-Cre (KPC) mice to investigate the effects of fascin deficiency on development of pancreatic intraepithelial neoplasia (PanIn), PDAC, and metastasis. We measured levels of fascin in PDAC cell lines and 122 human resected PDAC samples, along with normal ductal and acinar tissues; we associated levels with patient outcomes.ResultsPancreatic ducts and acini from control mice and early-stage PanINs from KPC mice were negative for fascin, but approximately 6% of PanIN3 and 100% of PDAC expressed fascin. Fascin-deficient KRasG12D p53R172H Pdx1-Cre mice had longer survival times, delayed onset of PDAC, and a lower PDAC tumor burdens than KPC mice; loss of fascin did not affect invasion of PDAC into bowel or peritoneum in mice. Levels of slug and fascin correlated in PDAC cells; slug was found to regulate transcription of Fascin along with the epithelial−mesenchymal transition. In PDAC cell lines and cells from mice, fascin concentrated in filopodia and was required for their assembly and turnover. Fascin promoted intercalation of filopodia into mesothelial cell layers and cell invasion. Nearly all human PDAC samples expressed fascin, and higher fascin histoscores correlated with poor outcomes, vascular invasion, and time to recurrence.ConclusionsThe actin-bundling protein fascin is regulated by slug and involved in late-stage PanIN and PDAC formation in mice. Fascin appears to promote formation of filopodia and invasive activities of PDAC cells. Its levels in human PDAC correlate with outcomes and time to recurrence, indicating it might be a marker or therapeutic target for pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) is often lethal because it is highly invasive and metastasizes rapidly. The actin-bundling protein fascin has been identified as a biomarker of invasive and advanced PDAC and regulates cell migration and invasion in vitro. We investigated fascin expression and its role in PDAC progression in mice. We used KRasG12D p53R172H Pdx1-Cre (KPC) mice to investigate the effects of fascin deficiency on development of pancreatic intraepithelial neoplasia (PanIn), PDAC, and metastasis. We measured levels of fascin in PDAC cell lines and 122 human resected PDAC samples, along with normal ductal and acinar tissues; we associated levels with patient outcomes. Pancreatic ducts and acini from control mice and early-stage PanINs from KPC mice were negative for fascin, but approximately 6% of PanIN3 and 100% of PDAC expressed fascin. Fascin-deficient KRasG12D p53R172H Pdx1-Cre mice had longer survival times, delayed onset of PDAC, and a lower PDAC tumor burdens than KPC mice; loss of fascin did not affect invasion of PDAC into bowel or peritoneum in mice. Levels of slug and fascin correlated in PDAC cells; slug was found to regulate transcription of Fascin along with the epithelial−mesenchymal transition. In PDAC cell lines and cells from mice, fascin concentrated in filopodia and was required for their assembly and turnover. Fascin promoted intercalation of filopodia into mesothelial cell layers and cell invasion. Nearly all human PDAC samples expressed fascin, and higher fascin histoscores correlated with poor outcomes, vascular invasion, and time to recurrence. The actin-bundling protein fascin is regulated by slug and involved in late-stage PanIN and PDAC formation in mice. Fascin appears to promote formation of filopodia and invasive activities of PDAC cells. Its levels in human PDAC correlate with outcomes and time to recurrence, indicating it might be a marker or therapeutic target for pancreatic cancer.

Background Reports have described the prognostic relevance of microRNAs (miRNAs) in patients treated for pancreatic ductal adenocarcinoma (PDAC). However, many of these include small numbers of patients. To increase statistical power and improve translation, we performed a systematic review and meta-analysis to determine a pooled conclusion. We examined the impact of miRNAs on overall survival (OS) and disease-free survival (DFS) in PDAC. Methods Eligible studies were identified and quality assessed using multiple search strategies (last search December 2014). Data were collected from studies correlating clinical outcomes with dysregulated tumoural or blood miRNAs. Studies were pooled, and combined hazard ratios (HRs) with 95% confidence intervals (CIs) were used to estimate strength of the associations. Results Twenty studies involving 1525 patients treated for PDAC were included. After correcting for publication bias, OS was significantly shortened in patients with high tumoural miR-21 (adjusted HR = 2.48; 1.96–3.14). This result persisted when only studies adjusting for adjuvant chemotherapy were combined (adjusted HR = 2.72; 1.91–3.89). High miR-21 also predicted reduced DFS (adjusted HR = 3.08; 1.78–5.33). Similarly, we found significant adjusted HRs for poor OS for high miR-155, high miR-203, and low miR-34a; and unadjusted HRs for high miR-222 and high miR-10b. The small number of studies, limited number of miRNAs and paucity of multivariate analyses are the limitations of our study. Conclusions This is the first rigorous pooled analysis assessing miRNAs as prognostic biomarkers in PDAC. Tumoural miR-21 overexpression emerged as an important predictor of poor prognosis after PDAC resection independent of other clinicopathologic factors, including adjuvant chemotherapy use.

Objective: This multicenter study sought to evaluate the accuracy of the American College of Surgeons National Surgical Quality Improvement Program's (ACS-NSQIP) surgical risk calculator for predicting outcomes after pancreatoduodenectomy (PD) and to determine whether incorporating other factors improves its predictive capacity. Background: The ACS-NSQIP surgical risk calculator has been proposed as a decision-support tool to predict complication risk after various operations. Although it considers 21 preoperative factors, it does not include procedure-specific variables, which have demonstrated a strong predictive capacity for the most common and morbid complication after PD – clinically relevant pancreatic fistula (CR-POPF). The validated Fistula Risk Score (FRS) intraoperatively predicts the occurrence of CR-POPF and serious complications after PD. Methods: This study of 1480 PDs involved 47 surgeons at 17 high-volume institutions. Patient complication risk was calculated using both the universal calculator and a procedure-specific model that incorporated the FRS and surgeon/institutional factors. The performance of each model was compared using the c-statistic and Brier score. Results: The FRS was significantly associated with 30-day mortality, 90-day mortality, serious complications, and reoperation (all P < 0.0001). The procedure-specific model outperformed the universal calculator for 30-day mortality (c-statistic: 0.79 vs 0.68; Brier score: 0.020 vs 0.021), 90-day mortality, serious complications, and reoperation. Neither surgeon experience nor institutional volume significantly predicted mortality; however, surgeons with a career PD volume >450 were less likely to have serious complications (P < 0.001) or perform reoperations (P < 0.001). Conclusions: Procedure-specific complication risk influences outcomes after pancreatoduodenectomy; therefore, risk adjustment for performance assessment and comparative research should consider these preoperative and intraoperative factors along with conventional ACS-NSQIP preoperative variables.

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) remains a dismal disease, with very little improvement in survival over the past 50 years. Recent large-scale genomic studies have improved understanding of the genomic and transcriptomic landscape of the disease, yet very little is known about molecular heterogeneity according to tumour location in the pancreas; body and tail PDACs especially tend to have a significantly worse prognosis. The aim was to investigate the molecular differences between PDAC of the head and those of the body and tail of the pancreas. Methods Detailed correlative analysis of clinicopathological variables, including tumour location, genomic and transcriptomic data, was performed using the Australian Pancreatic Cancer Genome Initiative (APGI) cohort, part of the International Cancer Genome Consortium study. Results Clinicopathological data were available for 518 patients recruited to the APGI, of whom 421 underwent genomic analyses; 179 of these patients underwent whole-genome and 96 RNA sequencing. Patients with tumours of the body and tail had significantly worse survival than those with pancreatic head tumours (12·1 versus 22·0 months; P = 0·001). Location in the body and tail was associated with the squamous subtype of PDAC. Body and tail PDACs enriched for gene programmes involved in tumour invasion and epithelial-to-mesenchymal transition, as well as features of poor antitumour immune response. Whether this is due to a molecular predisposition from the outset, or reflects a later time point on the tumour molecular clock, requires further investigation using well designed prospective studies in pancreatic cancer. Conclusion PDACs of the body and tail demonstrate aggressive tumour biology that may explain worse clinical outcomes.

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a 5‐year survival rate of less than 4% and desperately needs novel effective therapeutics. Integrin αvβ6 has been linked with poor prognosis in cancer but its potential as a target in PDAC remains unclear. We report that transcriptional expression analysis revealed that high levels of β6 mRNA correlated strongly with significantly poorer survival ( n = 491 cases, p = 3.17 × 10 −8 ). In two separate cohorts, we showed that over 80% of PDACs expressed αvβ6 protein and that paired metastases retained αvβ6 expression. In vitro, integrin αvβ6 promoted PDAC cell growth, survival, migration, and invasion. Treatment of both αvβ6‐positive human PDAC xenografts and transgenic mice bearing αvβ6‐positive PDAC with the αvβ6 blocking antibody 264RAD, combined with gemcitabine, significantly reduced tumour growth ( p &lt; 0.0001) and increased survival (log‐rank test, p &lt; 0.05). Antibody therapy was associated with suppression of tumour cell activity (suppression of pErk growth signals, increased apoptosis seen as activated caspase‐3) and suppression of the pro‐tumourigenic microenvironment (suppression of TGFβ signalling, fewer αSMA‐positive myofibroblasts, decreased blood vessel density). These data show that αvβ6 promotes PDAC growth through both tumour cell and tumour microenvironment mechanisms and represents a valuable target for PDAC therapy. © 2019 The Authors. The Journal of Pathology published by John Wiley &amp; Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Recent clinical successes with immunotherapy have resulted in expanding indications for cancer therapy. To enhance antitumor immune responses, and to better choose specific strategies matched to patient and tumor characteristics, genomic-driven precision immunotherapy will be necessary. Herein, we explore the role that tumor gene-expression profiling (GEP) may have in the prediction of an immunotherapeutic response. Genetic markers associated with response to immunotherapy are addressed as they pertain to the tumor genomic landscape, the extent of DNA damage, tumor mutational load and tumor-specific neoantigens. Furthermore, genetic markers associated with resistance to checkpoint blockade and relapse are reviewed. Finally, the utility of GEP to identify new tumor types for immunotherapy and implications for combinatorial strategies are summarized.

The aim of the present study was to examine the relationship between the clinicopathological status, the pre- and postoperative systemic inflammatory response and survival in patients undergoing potentially curative resection for ductal adenocarcinoma of the head of the pancreas. Patients (n = 65) who underwent resection of ductal adenocarcinoma of the head of pancreas between 1993 and 2001, and had pre- and postoperative measurements of C-reactive protein, were included in the study. The majority of patients had stage III disease (International Union Against Cancer Criteria, IUCC), positive circumferential margin involvement (R1), tumour size greater than 25 mm with perineural and lymph node invasion and died within the follow-up period. On multivariate analysis, tumour size (hazard ratio (HR) 2.10, 95% confidence interval (CI) 1.20-3.68, P = 0.009), vascular invasion (HR 2.58, 95% CI 1.48-4.50, P < 0.001) and postoperative C-reactive protein (HR 2.00, 95% CI 1.14-3.52, P = 0.015) retained independent significance. Those patients with a postoperative C-reactive protein < or = 10 mg l(-1) had a median survival of 21.5 months compared with 8.4 months in those patients with a C-reactive protein >10 mg l(-1) (P < 0.001). The results of the present study indicate that, in patients who have undergone potentially curative resection for ductal adenocarcinoma of the head of pancreas, the presence of a systemic inflammatory response predicts poor outcome.

Multiple mechanisms of senescence induction exist including telomere attrition, oxidative stress, oncogene expression and DNA damage signalling. The regulation of the cellular changes required to respond to these stimuli and create the complex senescent cell phenotype has many different mechanisms. MiRNAs present one mechanism by which genes with diverse functions on multiple pathways can be simultaneously regulated. In this study we investigated 12 miRNAs previously identified as senescence regulators. Using pathway analysis of their target genes we tested the relevance of miRNA regulation in the induction of senescence. Our analysis highlighted the potential of these senescence-associated miRNAs (SA-miRNAs) to regulate the cell cycle, cytoskeletal remodelling and proliferation signalling logically required to create a senescent cell. The reanalysis of publicly available gene expression data from studies exploring different senescence stimuli also revealed their potential to regulate core senescence processes, regardless of stimuli. We also identified stimulus specific apoptosis survival pathways theoretically regulated by the SA-miRNAs. Furthermore the observation that miR-499 and miR-34c had the potential to regulate all 4 of the senescence induction types we studied highlights their future potential as novel drug targets for senescence induction.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy associated with <5% 5-year survival, in which standard chemotherapeutics have limited benefit. The disease is associated with significant intra- and peritumoral inflammation and failure of protective immunosurveillance. Indeed, inflammatory signals are implicated in both tumour initiation and tumour progression. The major pathways regulating PDAC-associated inflammation are now being explored. Activation of leukocytes, and upregulation of cytokine and chemokine signalling pathways, both have been shown to modulate PDAC progression. Therefore, targeting inflammatory pathways may be of benefit as part of a multi-target approach to PDAC therapy. This review explores the pathways known to modulate inflammation at different stages of tumour development, drawing conclusions on their potential as therapeutic targets in PDAC.

ObjectivesDrainage after pancreaticoduodenectomy (PD) remains controversial because the risk for uncontrolled postoperative pancreatic fistula (POPF) must be balanced against the potential morbidity associated with prolonged and possibly unnecessary drainage. This study investigated the utility of the level of serum amylase on the night of surgery [postoperative day (PoD) 0 serum amylase] to predict POPF.MethodsA total of 185 patients who underwent PD were studied. Occurrences of POPF were graded using the International Study Group on Pancreatic Fistula (ISGPF) classification. Receiver operating characteristic (ROC) analysis identified a threshold value of PoD0 serum amylase associated with clinically significant POPF (ISGPF Grades B and C) in a test cohort (n = 45). The accuracy of this threshold value was then tested in a validation cohort (n = 140).ResultsOverall, 43 (23.2%) patients developed clinically significant POPF. The threshold value of PoD0 serum amylase for the identification of clinically significant POPF was ≥130IU/l (P = 0.003). Serum amylase of <130IU/l had a negative predictive value of 88.8% for clinically significant POPF (P < 0.001). Serum amylase of ≥130IU/l on PoD0 and a soft pancreatic parenchyma were independent risk factors for clinically significant POPF.ConclusionsPostoperative day 0 serum amylase of <130IU/l allows for the early and accurate categorization of patients at least risk for clinically significant POPF and may identify patients suitable for early drain removal.

Maker, Ajay V. MD, FACSa, *; Carrara, Silvia MDb; Jamieson, Nigel B. MD, PhDc; Pelaez-Luna, Mario MDd; Lennon, Anne Marie MD, PhDe; Dal Molin, Marco MDf; Scarpa, Aldo MD, PhDg; Frulloni, Luca MDh; Brugge, William R. MDi Author Information

Pancreas-specific complications (PSCs), comprising postoperative pancreatic fistula, haemorrhage and intra-abdominal collections, are drivers of morbidity and mortality after pancreaticoduodenectomy (PD). A serum amylase concentration of 130 units/l or more on postoperative day (POD) 0 has been shown to be an objective surrogate of pancreatic texture, a determinant of PSCs. This study evaluated serial measurements of C-reactive protein (CRP) to refine PSC risk stratification.Consecutive patients undergoing PD between 2008 and 2014, with vascular resection if required and without preoperative chemoradiotherapy, had serum investigations from the day before operation until discharge. Receiver operating characteristic (ROC) curve analysis was used to identify a threshold value of serum CRP with clinically relevant PSCs for up to 30 days after discharge as outcome measure.Of 230 patients, 95 (41·3 per cent) experienced a clinically relevant PSC. A serum CRP level of 180 mg/l or higher on POD 2 was associated with PSCs, prolonged critical care stay and relaparotomy (all P < 0·050). Patients with a serum amylase concentration of 130 units/l or more on POD 0 who developed a serum CRP level of at least 180 mg/l on POD 2 had a higher incidence of morbidity. Patients were stratified into high-, intermediate- and low-risk groups using these markers. The low-risk category was associated with a negative predictive value of 86·5 per cent for development of clinically relevant PSCs. There were no deaths among 52 patients in the low-risk group, but seven deaths among 79 (9 per cent) in the high-risk group.A serum amylase level below 130 units/l on POD 0 combined with a serum CRP level under 180 mg/l on POD 2 constitutes a low-risk profile following PD, and may help identify patients suitable for early discharge.

The sirtuin gene family has been linked with tumourigenesis, in both a tumour promoter and suppressor capacity. Information regarding the function of sirtuins in pancreatic cancer is sparse and equivocal. We undertook a novel study investigating SIRT1-7 protein expression in a cohort of pancreatic tumours. The aim of this study was to establish a protein expression profile for SIRT1-7 in pancreatic ductal adenocarcinomas (PDAC) and to determine if there were associations between SIRT1-7 expression, clinico-pathological parameters and patient outcome.Immunohistochemical analysis of SIRT1-7 protein levels was undertaken in a tissue micro-array comprising 77 resected PDACs. Statistical analyses determined if SIRT1-7 protein expression was associated with clinical parameters or outcome.Two sirtuin family members demonstrated significant associations with clinico-pathological parameters and patient outcome. Low level SIRT3 expression in the tumour cytoplasm correlated with more aggressive tumours, and a shorter time to relapse and death, in the absence of chemotherapeutic intervention. Low levels of nuclear SIRT7 expression were also associated with an aggressive tumour phenotype and poorer outcome, as measured by disease-free and disease-specific survival time, 12 months post-diagnosis.Our data suggests that SIRT3 and SIRT7 possess tumour suppressor properties in the context of pancreatic cancer. SIRT3 may also represent a novel predictive biomarker to determine which patients may or may not respond to chemotherapy. This study opens up an interesting avenue of investigation to potentially identify predictive biomarkers and novel therapeutic targets for pancreatic cancer, a disease that has seen no significant improvement in survival over the past 40 years.

Objective: The aim of this study was to elucidate the impact of intraoperative blood loss on outcomes following pancreatoduodenectomy (PD). Background: The negative impact of intraoperative blood loss on outcomes in PD has long been suspected but not well characterized, particularly those factors that may be within surgeons’ control. Methods: From 2001 to 2015, 5323 PDs were performed by 62 surgeons from 17 institutions. Estimated blood loss (EBL) was discretized (0 to 300, 301 to 750, 751 to 1300, and &gt;1300 mL) using optimal scaling methodology. Multivariable regression, adjusted for patient, surgeon, and institutional variables, was used to identify associations between EBL and perioperative outcomes. Factors associated with both increased and decreased EBL were elucidated. The relative impact of surgeon-modifiable contributors was estimated through beta coefficient standardization. Results: The median EBL of the series was 400 mL [interquartile range (IQR) 250 to 600]. Intra-, post-, and perioperative transfusion rates were 15.8%, 24.8%, and 37.2%, respectively. Progressive EBL zones correlated with intra- but not postoperative transfusion in a dose-dependent fashion ( P &lt; 0.001), with a key threshold of 750 mL EBL (8.14% vs 40.9%; P &lt; 0.001). Increasing blood loss significantly correlated with poor perioperative outcomes. Factors associated with increased EBL were trans-anastomotic stent placement, neoadjuvant chemotherapy, pancreaticogastrostomy reconstruction, multiorgan or vascular resection, and elevated operative time, of which 38.7% of the relative impact was “potentially modifiable” by the surgeon. Conversely, female sex, small duct, soft gland, minimally invasive approach, pylorus-preservation, biological sealant use, and institutional volume (≥67/year) were associated with decreased EBL, of which 13.6% was potentially under the surgeon's influence. Conclusion: Minimizing blood loss contributes to fewer intraoperative transfusions and better perioperative outcomes for PD. Improvements might be achieved by targeting modifiable factors that influence EBL.

Objective: The aim of this study was to investigate the relationship between drain fluid amylase value on the first postoperative day (DFA1) and clinically relevant fistula (CR-POPF) after distal pancreatectomy (DP), and to identify the cut-off of DFA1 that optimizes CR-POPF prediction. Background: DFA1 is a well-recognized predictor of CR-POPF after pancreatoduodenectomy, but its role in DP is largely unexplored. Methods: DFA1 levels were correlated with CR-POPF in 2 independent multi-institutional sets of DP patients: developmental (n = 338; years 2012 to 2017) and validation cohort (n = 166; years 2006 to 2016). Cut-off choice was based on Youden index calculation, and its ability to predict CR-POPF occurrence was tested in a multivariable regression model adjusted for clinical, demographic, operative, and pathological variables. Results: In the developmental set, median DFA1 was 1745 U/L and the CR-POPF rate was 21.9%. DFA1 correlated with CR-POPF with an area under the curve of 0.737 ( P &lt; 0.001). A DFA1 of 2000 U/L had the highest Youden index, with 74.3% sensitivity and 62.1% specificity. Patients in the validation cohort displayed different demographic and operative characteristics, lower values of DFA1 (784.5 U/L, P &lt; 0.001), and reduced CR-POPF rate (10.2%, P &lt; 0.001). However, a DFA1 of 2000 U/L had the highest Youden index in this cohort as well, with 64.7% sensitivity and 75.8% specificity. At multivariable analysis, DFA1 ≥2000 U/L was the only factor significantly associated with CR-POPF in both cohorts. Conclusion: A DFA1 of 2000 U/L optimizes CR-POPF prediction after DP. These results provide the substrate to define best practices and improve outcomes for patients receiving DP.

Colorectal cancer (CRC) is a heterogenous malignancy underpinned by dysregulation of cellular signaling pathways. Previous literature has implicated aberrant JAK/STAT3 signal transduction in the development and progression of solid tumors. In this study we investigate the effectiveness of inhibiting JAK/STAT3 in diverse CRC models, establish in which contexts high pathway expression is prognostic and perform in depth analysis underlying phenotypes. In this study we investigated the use of JAK inhibitors for anti-cancer activity in CRC cell lines, mouse model organoids and patient-derived organoids. Immunohistochemical staining of the TransSCOT clinical trial cohort, and 2 independent large retrospective CRC patient cohorts was performed to assess the prognostic value of JAK/STAT3 expression. We performed mutational profiling, bulk RNASeq and NanoString GeoMx® spatial transcriptomics to unravel the underlying biology of aberrant signaling. Inhibition of signal transduction with JAK1/2 but not JAK2/3 inhibitors reduced cell viability in CRC cell lines, mouse, and patient derived organoids (PDOs). In PDOs, reduced Ki67 expression was observed post-treatment. A highly significant association between high JAK/STAT3 expression within tumor cells and reduced cancer-specific survival in patients with high stromal invasion (TSPhigh) was identified across 3 independent CRC patient cohorts, including the TrasnSCOT clinical trial cohort. Patients with high phosphorylated STAT3 (pSTAT3) within the TSPhigh group had higher influx of CD66b + cells and higher tumoral expression of PDL1. Bulk RNAseq of full section tumors showed enrichment of NFκB signaling and hypoxia in these cases. Spatial deconvolution through GeoMx® demonstrated higher expression of checkpoint and hypoxia-associated genes in the tumor (pan-cytokeratin positive) regions, and reduced lymphocyte receptor signaling in the TME (pan-cytokeratin- and αSMA-) and αSMA (pan-cytokeratin- and αSMA +) areas. Non-classical fibroblast signatures were detected across αSMA + regions in cases with high pSTAT3. Therefore, in this study we have shown that inhibition of JAK/STAT3 represents a promising therapeutic strategy for patients with stromal-rich CRC tumors. High expression of JAK/STAT3 proteins within both tumor and stromal cells predicts poor outcomes in CRC, and aberrant signaling is associated with distinct spatially-dependant differential gene expression.

As more therapeutic options for pancreatic cancer are becoming available, there is a need to improve outcome prediction to support shared decision-making. A systematic evaluation of prediction models in resectable pancreatic cancer is lacking.This systematic review followed the CHARMS and PRISMA guidelines. PubMed, Embase and Cochrane Library databases were searched up to 11 October 2017. Studies reporting development or validation of models predicting survival in resectable pancreatic cancer were included. Models without performance measures, reviews, abstracts or more than 10 per cent of patients not undergoing resection in postoperative models were excluded. Studies were appraised critically.After screening 4403 studies, 22 (44 319 patients) were included. There were 19 model development/update studies and three validation studies, altogether concerning 21 individual models. Two studies were deemed at low risk of bias. Eight models were developed for the preoperative setting and 13 for the postoperative setting. Most frequently included parameters were differentiation grade (11 of 21 models), nodal status (8 of 21) and serum albumin (7 of 21). Treatment-related variables were included in three models. The C-statistic/area under the curve values ranged from 0·57 to 0·90. Based on study design, validation methods and the availability of web-based calculators, two models were identified as the most promising.Although a large number of prediction models for resectable pancreatic cancer have been reported, most are at high risk of bias and have not been validated externally. This overview of prognostic factors provided practical recommendations that could help in designing easily applicable prediction models to support shared decision-making.

To develop a nomogram estimating the probability of recurrence free at 5 years after resection for localized grade 1 (G1)/ grade 2 (G2) pancreatic neuroendocrine tumors (PanNETs).Among patients undergoing resection of PanNETs, approximately 17% experience recurrence. It is not established which patients are at risk, with no consensus on optimal follow-up.A multi-institutional database of patients with G1/G2 PanNETs treated at 2 institutions was used to develop a nomogram estimating the rate of freedom from recurrence at 5 years after curative resection. A second cohort of patients from 3 additional institutions was used to validate the nomogram. Prognostic factors were assessed by univariate analysis using Cox regression model. The nomogram was internally validated using bootstrap resampling method and on the external cohort. Performance was assessed by concordance index (c-index) and a calibration curve.The nomogram was constructed using a cohort of 632 patients. Overall, 68% of PanNETs were G1, the median follow-up was 51 months, and we observed 74 recurrences. Variables included in the nomogram were the number of positive nodes, tumor diameter, Ki-67, and vascular/perineural invasion. The model bias-corrected c-index from the internal validation was 0.85, which was higher than European Neuroendocrine Tumors Society/ American Joint Committee on Cancer 8th staging scheme (c-index 0.76, P = <0.001). On the external cohort of 328 patients, the nomogram c-index was 0.84 (95% confidence interval 0.79-0.88).Our externally validated nomogram predicts the probability of recurrence-free survival at 5 years after PanNETs curative resection, with improved accuracy over current staging systems. Estimating individual recurrence risk will guide the development of personalized surveillance programs after surgery.

Abstract Purpose: Precise mechanism-based gene expression signatures (GES) have been developed in appropriate in vitro and in vivo model systems, to identify important cancer-related signaling processes. However, some GESs originally developed to represent specific disease processes, primarily with an epithelial cell focus, are being applied to heterogeneous tumor samples where the expression of the genes in the signature may no longer be epithelial-specific. Therefore, unknowingly, even small changes in tumor stroma percentage can directly influence GESs, undermining the intended mechanistic signaling. Experimental Design: Using colorectal cancer as an exemplar, we deployed numerous orthogonal profiling methodologies, including laser capture microdissection, flow cytometry, bulk and multiregional biopsy clinical samples, single-cell RNA sequencing and finally spatial transcriptomics, to perform a comprehensive assessment of the potential for the most widely used GESs to be influenced, or confounded, by stromal content in tumor tissue. To complement this work, we generated a freely-available resource, ConfoundR; https://confoundr.qub.ac.uk/, that enables users to test the extent of stromal influence on an unlimited number of the genes/signatures simultaneously across colorectal, breast, pancreatic, ovarian and prostate cancer datasets. Results: Findings presented here demonstrate the clear potential for misinterpretation of the meaning of GESs, due to widespread stromal influences, which in-turn can undermine faithful alignment between clinical samples and preclinical data/models, particularly cell lines and organoids, or tumor models not fully recapitulating the stromal and immune microenvironment. Conclusions: Efforts to faithfully align preclinical models of disease using phenotypically-designed GESs must ensure that the signatures themselves remain representative of the same biology when applied to clinical samples.

Objectives: Aggressive invasion and early metastases are characteristic features of pancreatic ductal adenocarcinoma (PDAC). More than 90% of patients have surgically nonresectable disease at presentation. Despite increasing knowledge of the genetics of this complex disease, systemic therapies, particularly gemcitabine, have modest clinical benefit and marginal survival advantage. MicroRNAs have been shown to have a role in oncogenesis, invasion, and metastases via epigenetic posttranscriptional gene regulation. Our objective was to discuss the clinical impact of microRNAs within PDAC. Methods: This review details the understanding of microRNAs to date and explores the clinical utility of microRNAs in PDAC. Results: Recent studies have focused on the impact of microRNA expression in PDAC, many of which have shown the diagnostic, predictive, and prognostic utility of microRNA profiling in PDAC identifying numerous potential targets including miR-21, miR-196a, and miR-217. Conclusions: MicroRNA stability in body fluid and tissue samples makes this area one of the most promising for earlier detection of PDAC. Indeed, microRNAs may in the future serve as a long-awaited screening tool for PDAC. Furthermore, microRNA expression profiling in PDAC may be incorporated into modern treatment algorithms to enhance therapeutic management. Equally as exciting is the potential for novel therapeutics directed against these important disease mediators.

Pancreatico-biliary adenocarcinomas (PBA) have a poor prognosis. Diagnosis is usually achieved by imaging and/or endoscopy with confirmatory cytology. Cytological interpretation can be difficult especially in the setting of chronic pancreatitis/cholangitis. Immunohistochemistry (IHC) biomarkers could act as an adjunct to cytology to improve the diagnosis. Thus, we performed a meta-analysis and selected KOC, S100P, mesothelin and MUC1 for further validation in PBA resection specimens. Tissue microarrays containing tumour and normal cores in a ratio of 3:2, from 99 surgically resected PBA patients, were used for IHC. IHC was performed on an automated platform using antibodies against KOC, S100P, mesothelin and MUC1. Tissue cores were scored for staining intensity and proportion of tissue stained using a Histoscore method (range, 0–300). Sensitivity and specificity for individual biomarkers, as well as biomarker panels, were determined with different cut-offs for positivity and compared by summary receiver operating characteristic (ROC) curve. The expression of all four biomarkers was high in PBA versus normal ducts, with a mean Histoscore of 150 vs. 0.4 for KOC, 165 vs. 0.3 for S100P, 115 vs. 0.5 for mesothelin and 200 vs. 14 for MUC1 (p < .0001 for all comparisons). Five cut-offs were carefully chosen for sensitivity/specificity analysis. Four of these cut-offs, namely 5%, 10% or 20% positive cells and Histoscore 20 were identified using ROC curve analysis and the fifth cut-off was moderate-strong staining intensity. Using 20% positive cells as a cut-off achieved higher sensitivity/specificity values: KOC 84%/100%; S100P 83%/100%; mesothelin 88%/92%; and MUC1 89%/63%. Analysis of a panel of KOC, S100P and mesothelin achieved 100% sensitivity and 99% specificity if at least 2 biomarkers were positive for 10% cut-off; and 100% sensitivity and specificity for 20% cut-off. A biomarker panel of KOC, S100P and mesothelin with at least 2 biomarkers positive was found to be an optimum panel with both 10% and 20% cut-offs in resection specimens from patients with PBA.

Next-generation sequencing is enabling molecularly guided therapy for many cancer types, yet failure rates remain relatively high in pancreatic cancer (PC).The aim of this study is to investigate the feasibility of genomic profiling using endoscopic ultrasound (EUS) biopsy samples to facilitate personalised therapy for PC.Ninty-five patients underwent additional research biopsies at the time of diagnostic EUS.Diagnostic formalin-fixed (FFPE) and fresh frozen EUS samples underwent DNA extraction, quantification and targeted gene sequencing.Whole genome (WGS) and RNA sequencing was performed as proof of concept.Only 2 patients (2%) with a diagnosis of PC had insufficient material for targeted sequencing in both FFPE and frozen specimens.Targeted panel sequencing (n=54) revealed mutations in PC genes (KRAS, GNAS, TP53, CDKN2A, SMAD4) in patients with histological evidence of PC, including potentially actionable mutations (BRCA1, BRCA2, ATM, BRAF).WGS (n=5) of EUS samples revealed mutational signatures that are potential biomarkers of therapeutic responsiveness.RNA sequencing (n=35) segregated patients into clinically relevant molecular subtypes based on transcriptome.Integrated multi-omic analysis of PC using standard EUS guided biopsies offers clinical utility to guide personalized therapy and study the molecular pathology in all patients with PC.

In post-coronavirus disease-19 (post-COVID-19) conditions (long COVID), systemic vascular dysfunction is implicated, but the mechanisms are uncertain, and the treatment is imprecise.Patients convalescing after hospitalization for COVID-19 and risk factor matched controls underwent multisystem phenotyping using blood biomarkers, cardiorenal and pulmonary imaging, and gluteal subcutaneous biopsy (NCT04403607). Small resistance arteries were isolated and examined using wire myography, histopathology, immunohistochemistry, and spatial transcriptomics. Endothelium-independent (sodium nitroprusside) and -dependent (acetylcholine) vasorelaxation and vasoconstriction to the thromboxane A2 receptor agonist, U46619, and endothelin-1 (ET-1) in the presence or absence of a RhoA/Rho-kinase inhibitor (fasudil), were investigated. Thirty-seven patients, including 27 (mean age 57 years, 48% women, 41% cardiovascular disease) 3 months post-COVID-19 and 10 controls (mean age 57 years, 20% women, 30% cardiovascular disease), were included. Compared with control responses, U46619-induced constriction was increased (P = 0.002) and endothelium-independent vasorelaxation was reduced in arteries from COVID-19 patients (P < 0.001). This difference was abolished by fasudil. Histopathology revealed greater collagen abundance in COVID-19 arteries {Masson's trichrome (MT) 69.7% [95% confidence interval (CI): 67.8-71.7]; picrosirius red 68.6% [95% CI: 64.4-72.8]} vs. controls [MT 64.9% (95% CI: 59.4-70.3) (P = 0.028); picrosirius red 60.1% (95% CI: 55.4-64.8), (P = 0.029)]. Greater phosphorylated myosin light chain antibody-positive staining in vascular smooth muscle cells was observed in COVID-19 arteries (40.1%; 95% CI: 30.9-49.3) vs. controls (10.0%; 95% CI: 4.4-15.6) (P < 0.001). In proof-of-concept studies, gene pathways associated with extracellular matrix alteration, proteoglycan synthesis, and viral mRNA replication appeared to be upregulated.Patients with post-COVID-19 conditions have enhanced vascular fibrosis and myosin light change phosphorylation. Rho-kinase activation represents a novel therapeutic target for clinical trials.

Nucleophosmin (NPM) is known to regulate ARF subcellular localization and MDM2 activity in response to oncogenic stress, though the precise mechanism has remained elusive.Here we describe how NPM and ARF associate in the nucleoplasm to form a MDM2 inhibitory complex.We find that oligomerization of NPM drives nucleolar accumulation of ARF.Moreover, the formation of NPM and ARF oligomers antagonizes MDM2 association with the inhibitory complex, leading to activation of MDM2 E3-ligase activity and targeting of p53.We find that AKT phosphorylation of NPM-Ser48 prevents oligomerization that results in nucleoplasmic localization of ARF, constitutive MDM2 inhibition and stabilization of p53.We also show that ARF promotes p53 mutant stability in tumors and suppresses p73 mediated p21 expression and senescence.We demonstrate that AKT and PI3K inhibitors may be effective in treatment of therapeutically resistant tumors with elevated AKT and carrying gain of function mutations in p53.Our results show that the clinical candidate AKT inhibitor MK-2206 promotes ARF nucleolar localization, reduced p53 mut stability and increased sensitivity to ionizing radiation in a xenograft model of pancreatic cancer.Analysis of human tumors indicates that phospho-S48-NPM may be a useful biomarker for monitoring AKT activity and in vivo efficacy of AKT inhibitor treatment.Critically, we propose that combination therapy involving PI3K-AKT inhibitors would benefit from a patient stratification rationale based on ARF and p53 mut status.

Evaluation of: Ma MZ, Kong X, Weng MZ et al. Candidate microRNA biomarkers of pancreatic ductal adenocarcinoma: meta-analysis, experimental validation and clinical significance. J. Exp. Clin. Cancer Res. 32(1), 71 (2013).Due to its aggressive and late presentation, there is an urgent need for novel and reliable biomarkers for the diagnosis and prognostication of pancreatic ductal adenocarcinoma (PDAC). MiRNAs have been extensively profiled in PDAC tissues, biopsies, blood samples and other biofluids and their expression levels compared to normal and chronic pancreatitis (CP) specimens in order to identify the most relevant candidates. Consolidation of these activities has not been attempted until now. The evaluated meta-review by Ma et al. helps to define the use of miRNAs as biomarkers for detecting this tumor-type and predicting survival outcomes in PDAC. Based on frequency and consistency between microarray studies, they identified a miRNA meta-signature for recognising PDAC: upregulation of miR-21, 23a, 31, 100, 143, 155, and 221; with downregulation of miR-148a, 217 and 375. Furthermore, they validated high miR-21, high miR-31 and low miR-375 tumoural expression as independently prognostic for poor overall-survival (OS; n = 70).

Abstract Strong immune responses in primary colorectal cancer correspond with better patient survival following surgery compared with tumors with predominantly stromal microenvironments. However, biomarkers to identify patients with colorectal cancer liver metastases (CRLM) with good prognosis following surgery for oligometastatic disease remain elusive. The aim of this study was to determine the practical application of a simple histological assessment of immune cell infiltration and stromal content in predicting outcome following synchronous resection of primary colorectal cancer and CRLM and to interrogate the underlying functional biology that drives disease progression. Samples from patients undergoing synchronous resection of primary colorectal cancer and CRLM were evaluated in detail through histological assessment, panel genomic and bulk transcriptomic assessment, IHC, and GeoMx spatial transcriptomics (ST) analysis. High immune infiltration of metastases was associated with improved cancer-specific survival. Bulk transcriptomic analysis was confounded by stromal content, but ST demonstrated that the invasive edge of the metastases of long-term survivors was characterized by adaptive immune cell populations enriched for type II IFN signaling and MHC-class II antigen presentation. In contrast, patients with poor prognosis demonstrated increased abundance of regulatory T cells and neutrophils with enrichment of Notch and TGFβ signaling pathways at the metastatic tumor center. In summary, histological assessment can stratify outcomes in patients undergoing synchronous resection of CRLM, suggesting that it has potential as a prognostic biomarker. Furthermore, ST analysis has revealed significant intratumoral and interlesional heterogeneity and identified the underlying transcriptomic programs driving each phenotype. Significance: Spatial transcriptomics uncovers heterogeneity between patients, between matched lesions in the same patient, and within individual lesions and identifies drivers of metastatic progression in colorectal cancer with reactive and suppressed immune microenvironments.

Abstract Tumor budding (TB) is a strong biomarker of poor prognosis in colorectal cancer and other solid cancers. TB is defined as isolated single cancer cells or clusters of up to four cancer cells at the invasive tumor front. In areas with a large inflammatory response at the invasive front, single cells and cell clusters surrounding fragmented glands are observed appearing like TB. Occurrence of these small groups is referred to as pseudobudding (PsB), which arises due to external influences such as inflammation and glandular disruption. Using a combination of orthogonal approaches, we show that there are clear biological differences between TB and PsB. TB is representative of active invasion by presenting features of epithelial‐mesenchymal transition and exhibiting increased deposition of extracellular matrix within the surrounding tumor microenvironment (TME), whereas PsB represents a reactive response to heavy inflammation where increased levels of granulocytes within the surrounding TME are observed. Our study provides evidence that areas with a strong inflammatory reaction should be avoided in the routine diagnostic assessment of TB. © 2023 The Authors. The Journal of Pathology published by John Wiley &amp; Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract Background Intraductal Papillary Mucinous Neoplasms (IPMN) remain the largest subtype of cystic pancreatic cancer precursors. Cancer risk has been observed to vary between histological grades and histological subtypes. Spatial characterisation has revealed variation in immune cell distribution disease subtypes. Methods A cohort of 14 surgically resected IPMN tumors across a range of histological grades (Low-grade LG, high-grade HG and invasive IPMN cancers IPMN-PDAC) and subtypes (gastric, intestinal, pancreaticobiliary) underwent multimodal spatial interrogation. After expert histopathological annotation; tissue sections underwent regional whole transcriptome analysis with TempO-seq and 10x Visium. Extensive region analysis with NanoString GeoMx employing segmentation using staining for epithelium (PanCK) fibroblast stroma (aSMA) was performed. A tissue microarray was then constructed, and representative cores underwent single-cell and subcellular spatial transcriptomic analysis with NanoString CosMx. Raw count data and digital images were exported and analysed using Seurat, Giotto, SPATA2 and custom R pipelines. Region deconvolution, Harmony integration, Leiden Clustering, Gene Set Enrichment Analysis, Trajectory Mapping, and Moran’s I Analysis were performed. Results TempO-seq revealed up-regulation of canonical oncogene expression in all IPMN-PDAC cases as compared to LG and HG IPMN. There were no statistically significant differences in expression comparing LG and HG IPMN lesions. Visium differential gene expression and clustering models identified cancer epithelium, stroma and lymphatic components that correlated with histopathological annotations. Immune cell rich spots were identified via gene ontology of top 50 marker genes. The spatial trajectory of B-cell expression signatures in IPMN-PDAC showed greater concentration and less variability than HG tumors, Moran’s I -0.34 vs 0.12 (P = 0.021). B-cells in HG IPMN were showed greater concentration and less variability than LG IPMN, Moran’s I -0.15 vs 0.02 (P=0.05). Much of this concentration was in identified lymphoid aggregates or tertiary lymphoid structures. The spatial trajectory of T-cell expression. The spatial trajectory of macrophage expression showed greater concentration and less variability than in LG and HG tumors. GeoMx analysis of stromal regions identified up regulation of B cell signatures in IPMN PDAC and HG IPMN when compared to LG IPMN. Markers associated with TLS formation were found to be significantly upregulated including CXCL13, CXCR5 and LAMP3. Pancreaticobiliary subtypes were found to have the least concentrated B-cell distribution across subtypes. Conclusions Immune cell composition was found to vary across histological grades and subtypes of IPMN. A paradoxical relationship was observed between T-cell infiltrates and B-cell and macrophage populations. The greater proportion of tertiary lymphoid structures, and high levels of TLS specific gene expression, may indicate a propensity for b-cell activation in IPMN tumors which progress to malignancy. Citation Format: Andrew J. Cameron, Assya Legrini, Colin S. Wood, Craig Nourse, Yoana Doncheva, Claire Kennedy Dietrich, Colin Nixon, Jennifer Hay, Fraser Duthie, Pawel Herzyk, Jennifer Morton, Nigel B. Jamieson. Multiomic modelling of pancreatic IPMN stroma reveals distinct tertiary lymphoid structure distribution: Mapping the transcriptomic landscape via regional bulk, single-cell and subcellular approaches [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A028.

Abstract Aim: Patients who undergo intended curative resection of colorectal cancer (CRC) have a 20-25% chance of metachronous recurrence, the site and timing of which are unpredictable and resistant to current treatment. The biological basis for such heterogeneous disease behavior remains to be elucidated. Bulk transcriptomic profiling and subsequently single-cell RNAseq have provided insight in the epithelial subtypes and immune microenvironment. Recently spatially resolved transcriptomic assessment allow molecular profiling of tissue while preserving tissue architecture. Utilizing ST technology we sought to perform deep characterization of a large cohort of patients with primary CRC with the intention to decipher the biological determinants of spatio-temporal patterns of recurrence. Methods: 750 patients who underwent resection of CRC with mature follow up were studied. Bulk transcriptomic, genomic and multiplex immune characterization was performed using a TMA format. Of these, 28 patients were assessed using single cell spatial transcriptomics (Nanostring CosMx Spatial Molecular Imager (SMI, 1000plex gene panel)), 120 tumors underwent regional whole transcriptome profiling of epithelium and TME (Nanostring GeoMx Digital Spatial Profiler using FFPE tissue). We used image analysis and bioinformatics to integrate these complex datasets in over 120000 single-cells in the context of their spatial tissue architecture and clinicopatholgical outcome and recurrence data. Results: Using the CosMx SMI we characterized cells with complete topographic detail and defined 2 unique epithelial cell states defined. Each epithelial state had distinct spatial properties including cell size and morphology, average distance to nearest lymphocyte, neighboring cell types and stromal neighborhood. This epithelial signature was integrated in GeoMx samples and were found to predict recurrence (p&amp;lt;0.05). The Epithelial, Fibroblast and Immune GeoMx transcriptomic compartments were expanded and grouped using unsupervised clustering. Each compartment demonstrated groups of patients where specific spatially derived signatures could predict site and time of recurrence (p&amp;lt;0.05). Conclusions: By maintaining the tissue structure, we have directly measured cellular interactions and captured cells commonly missed during dissociative studies whilst defining novel molecular subtypes of CRC with clinical relevance. Novel platforms such as CosMx allow deeper characterization of unique cell types and cellular interactions that may pave the way for novel therapeutics and precision medicine for patients with CRC. Citation Format: Colin Stuart Wood, Joao Da Silva Filho, Andrew Cameron, Assya Legrini, Holly Leslie, Tengyu Zhang, Yoana Doncheva, Claire Kennedy-Dietrich, Matthias Marti, Joanne Edwards, Paul Horgan, Campbell Roxburgh, Colin Steele, Nigel Jamieson. Multi-omic, multi-scale characterisation of colorectal cancer defines spatiotemporal patterns of recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5072.

The aim of the present study was to examine the relationship between adiponectin and the systemic inflammatory response in weight-losing patients with non-small cell lung cancer (NSCLC). Measurement of anthropometry, acute phase proteins, interleukin-6, leptin (total and free) and adiponectin were carried out on healthy subjects (n=13) and non-small cell lung cancer patients with weight loss (n=20). The groups were age and sex matched. Compared with the controls the cancer group had a lower BMI (p<0.01), mid-upper arm circumference (p<0.001), triceps skinfold thickness (p<0.05) and circulating concentrations of albumin (p<0.001), haemoglobin (p<0.05), free and total leptin (p<0.05) and adiponectin (p<0.01). In contrast, the cancer group had elevated circulating concentrations of interleukin-6 and C-reactive protein concentrations (p<0.001). In the cancer group circulating adiponectin concentrations were significantly inversely correlated with both free (rs=−0.675, p=0.001) and total leptin concentrations (rs=−0.690, p=0.001). However, neither weight loss, interleukin-6 or C-reactive protein concentrations were correlated with either adiponectin, free or total leptin concentrations in the cancer group. These results suggest that adipokine production is normal and is unlikely to play a major role in the abnormal fat metabolism in weight-losing cancer patients.

Background Up to 60% of patients who undergo curative-intent pancreatic ductal adenocarcinoma (PDAC) resection experience disease recurrence within six months. We recently published a systematic review of prognostic immunohistochemical biomarkers in PDAC and shortlisted a panel of those reported with the highest level of evidence, including p53, p16, Ca-125, S100A4, FOXC1, EGFR, mesothelin, CD24 and UPAR. This study aims to discover and validate the prognostic significance of a combinatorial panel of tumor biomarkers in patients with resected PDAC. Methods Patients who underwent PDAC resection were included from a single institution discovery cohort and a multi-institutional validation cohort. Tumors in the discovery cohort were stained immunohistochemically for all nine shortlisted biomarkers. Biomarkers significantly associated with overall survival (OS) were reevaluated as a combinatorial panel in both discovery and validation cohorts for its prognostic significance. Results 224 and 191 patients were included in the discovery and validation cohorts, respectively. In both cohorts, S100A4, Ca-125 and mesothelin expression were associated with shorter OS. In both cohorts, the number of these biomarkers expressed was significantly associated with OS (discovery cohort 36.8 vs. 26.4 vs 16.3 vs 12.8 months, P < 0.001; validation cohort 25.2 vs 18.3 vs 13.6 vs 11.9 months, P = 0.008 for expression of zero, one, two and three biomarkers, respectively). On multivariable analysis, expression of at least one of three biomarkers was independently associated with shorter OS. Conclusion Combinations of S100A4, Ca-125 and mesothelin expression stratify survival after resection of localized PDAC. Co-expression of all three biomarkers is associated with the poorest prognostic outcome.

Background: Adiponectin, an adipocyte-derived hormone found in lower concentration with greater adiposity, is suggested to reduce the risk of insulin resistance, atherosclerosis, and cardiovascular disease. We tested this hypothesis in a healthy, nonobese population.

Pancreatic cancer is the third leading cause of cancer mortality in the West, after recently overtaking breast cancer [[1]Siegel R.L. Miller K.D. Jemal A. Cancer statistics, 2018.CA Cancer J Clin. 2018; 68: 7-30Crossref PubMed Scopus (6441) Google Scholar]. There has been little improvement in the overall outcomes for pancreatic cancer in the last 50 years. Recently there have been small incremental improvements in overall survival in the metastatic setting based on the PRODIGE-4/ACCORD-11 and MPACT clinical trials [2Von Hoff D.D. Ervin T. Arena F.P. Chiorean E.G. Infante J. Moore M. et al.Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine.N Engl J Med. 2013; 369: 1691-1703Crossref PubMed Scopus (4141) Google Scholar, 3Conroy T. Desseigne F. Ychou M. Bouche O. Guimbaud R. Becouarn Y. et al.FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer.N Engl J Med. 2011; 364: 1817-1825Crossref PubMed Scopus (5137) Google Scholar] and in the adjuvant setting with gemcitabine-capecitabine and FOLFIRINOX combinations [4Neoptolemos J.P. Palmer D.H. Ghaneh P. Psarelli E.E. Valle J.W. Halloran C.M. et al.Comparison of adjuvant gemcitabine and capecitabine with gemcitabine monotherapy in patients with resected pancreatic cancer (ESPAC-4): a multicentre, open-label, randomised, phase 3 trial.Lancet. 2017; 389: 1011-1024Abstract Full Text Full Text PDF PubMed Scopus (1136) Google Scholar, 5Conroy T. Hammel P. Hebbar M. Abdelghani M.B. Wei AC-C Raoul J.-L. et al.Unicancer GI PRODIGE 24/CCTG PA.6 trial: A multicenter international randomized phase III trial of adjuvant mFOLFIRINOX versus gemcitabine (gem) in patients with resected pancreatic ductal adenocarcinomas.J Clin Oncol. 2018; 36: LBA4001Crossref Google Scholar]. Neoadjuvant therapy is gaining popularity in the borderline resectable and locally advanced settings, with some exceptional responses leading to long-term survival in undefined subgroups [6Murphy J.E. Wo J.Y. Ryan D.P. Jiang W. Yeap B.Y. Drapek L.C. et al.Total neoadjuvant therapy with FOLFIRINOX followed by individualized chemoradiotherapy for borderline resectable pancreatic adenocarcinoma: a phase 2 clinical trial.JAMA Oncol. 2018; 4: 963-969Crossref PubMed Scopus (306) Google Scholar, 7He J. Blair A.B. Groot V.P. Javed A.A. Burkhart R.A. Gemenetzis G. et al.Is a pathological complete response following neoadjuvant chemoradiation associated with prolonged survival in patients with pancreatic cancer?.Ann Surg. 2018 Jul; 268: 1-8Crossref Scopus (101) Google Scholar, 8Gemenetzis G. Groot V.P. Blair A.B. Laheru D.A. Zheng L. Narang A.K. et al.Survival in locally advanced pancreatic cancer after neoadjuvant therapy and surgical resection.Ann Surg. 2019 Aug; 270: 340-347Crossref Scopus (194) Google Scholar, 9Unno M. Motoi F. Matsuyama Y. Satoi S. Matsumoto I. Aosasa S. et al.Randomized phase II/III trial of neoadjuvant chemotherapy with gemcitabine and S-1 versus upfront surgery for resectable pancreatic cancer (Prep-02/JSAP-05).J Clin Oncol. 2019; 37: 189Crossref Google Scholar]. The incremental improvements are probably due to the effects of these small undefined subgroups of responders, with most patients gaining minimal or no benefit from systemic chemotherapy such as gemcitabine combinations and the FOLFIRINOX regimen [10Dreyer S.B. Chang D.K. Bailey P. Biankin A.V. Pancreatic cancer genomes: implications for clinical management and therapeutic development.Clin Cancer Res. 2017; 23: 1638-1646Crossref PubMed Scopus (112) Google Scholar, 11Chang D.K. Grimmond S.M. Evans T.R. Biankin A.V. Mining the genomes of exceptional responders.Nat Rev Cancer. 2014; 14: 291-292Crossref PubMed Scopus (35) Google Scholar]. Therefore, a selection biomarker-driven precision oncology approach is urgently needed in pancreatic cancer. Here we summarise some of the attempts to translate clinically relevant genome discoveries into the real world and highlight a few candidate therapeutic segments in development. Recently, there has been a vast expansion in the understanding of the molecular pathology of many cancer types, including pancreatic cancer. The ‘omics’ revolution is rapidly generating large-scale datasets including whole genome and whole transcriptome sequencing from a large number of tumours. The Australian Pancreatic Cancer Genome Initiative (APGI; http://www.pancreaticcancer.net.au) led the contribution of about 450 pancreatic cancer cases to the International Cancer Genome Consortium (ICGC; https://icgc.org) [12Waddell N. Pajic M. Patch A.M. Chang D.K. Kassahn K.S. Bailey P. et al.Whole genomes redefine the mutational landscape of pancreatic cancer.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1687) Google Scholar, 13Biankin A.V. Waddell N. Kassahn K.S. Gingras M.C. Muthuswamy L.B. Johns A.L. et al.Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1513) Google Scholar, 14Bailey P. Chang D.K. Nones K. Johns A.L. Patch A.M. Gingras M.C. et al.Genomic analyses identify molecular subtypes of pancreatic cancer.Nature. 2016; 531: 47-52Crossref PubMed Scopus (1974) Google Scholar], making significant headway in the understanding of the mutational landscape of the disease, as well as clinically relevant molecular subtypes. Whole exome sequencing and copy number alteration analyses of 142 resected pancreatic cancers reaffirmed the mutational profile originally described in a landmark study describing 12 core signalling pathways and gained further insights into the molecular pathology of pancreatic cancer [13Biankin A.V. Waddell N. Kassahn K.S. Gingras M.C. Muthuswamy L.B. Johns A.L. et al.Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1513) Google Scholar, 15Jones S. Zhang X. Parsons D.W. Lin J.C. Leary R.J. Angenendt P. et al.Core signaling pathways in human pancreatic cancers revealed by global genomic analyses.Science. 2008; 321: 1801-1806Crossref PubMed Scopus (3100) Google Scholar]. The most frequent mutations in pancreatic cancer include KRAS, TP53, SMAD4 and CDKN2A, all of which do not confer effective therapeutic options at present [10Dreyer S.B. Chang D.K. Bailey P. Biankin A.V. Pancreatic cancer genomes: implications for clinical management and therapeutic development.Clin Cancer Res. 2017; 23: 1638-1646Crossref PubMed Scopus (112) Google Scholar, 13Biankin A.V. Waddell N. Kassahn K.S. Gingras M.C. Muthuswamy L.B. Johns A.L. et al.Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1513) Google Scholar]. Yet, this study from the APGI revealed therapeutic vulnerabilities in the disease, including mutations in genes such as BRCA1, BRCA2, PALB2, ARID1A and ATM [[13]Biankin A.V. Waddell N. Kassahn K.S. Gingras M.C. Muthuswamy L.B. Johns A.L. et al.Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1513) Google Scholar]. Similar findings have also been described by large-scale studies from Canada and The Cancer Genome Atlas initiative, resulting in about 1000 published genomes of pancreatic cancer to date [16Cancer Genome Atlas Research NetworkIntegrated genomic characterization of pancreatic ductal adenocarcinoma.Cancer Cell. 2017; 32: 185-203 e13Abstract Full Text Full Text PDF PubMed Scopus (967) Google Scholar, 17Connor A.A. Denroche R.E. Jang G.H. Lemire M. Zhang A. Chan-Seng-Yue M. et al.Integration of genomic and transcriptional features in pancreatic cancer reveals increased cell cycle progression in metastases.Cancer Cell. 2019; 35: 267-282Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar]. However, the frequency of actionable mutations in pancreatic cancer remains low and highlights the need to identify alternative biomarkers of therapeutic response beyond point mutation. Waddell et al. [[12]Waddell N. Pajic M. Patch A.M. Chang D.K. Kassahn K.S. Bailey P. et al.Whole genomes redefine the mutational landscape of pancreatic cancer.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1687) Google Scholar] carried out an analysis of the first 100 whole genomes sequenced from the APGI and revealed mutational signatures and structural variation patterns associated with defects in the DNA damage response (DDR). Four patterns of structural variation were identified and can be associated with underlying mutational processes. The unstable pattern (over 200 structural variations evenly distributed throughout the genome) overlaps with the high COSMIC BRCA point mutational signature and mutations in genes involved in DNA maintenance, such as BRCA1, BRCA2 and PALB2 [[12]Waddell N. Pajic M. Patch A.M. Chang D.K. Kassahn K.S. Bailey P. et al.Whole genomes redefine the mutational landscape of pancreatic cancer.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1687) Google Scholar]. Up to 24% of patients with pancreatic cancer exhibit one or more of these putative biomarkers of DDR deficiency, demonstrating that the potential responsive subgroup to DNA damaging agents (e.g. platinum) may be higher than previously thought [[12]Waddell N. Pajic M. Patch A.M. Chang D.K. Kassahn K.S. Bailey P. et al.Whole genomes redefine the mutational landscape of pancreatic cancer.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1687) Google Scholar]. There has been growing interest in molecular ‘subtyping’ beyond the genome to gain insights into the molecular processes driving tumour progression and therapeutic response. The APGI carried out the first large-scale multi-omic analyses in pancreatic cancer, using transcriptomes, methylome, mutational data and histopathology from 451 patients and identified clinically relevant differential gene programs that cluster tumours into unique molecular subtypes [[14]Bailey P. Chang D.K. Nones K. Johns A.L. Patch A.M. Gingras M.C. et al.Genomic analyses identify molecular subtypes of pancreatic cancer.Nature. 2016; 531: 47-52Crossref PubMed Scopus (1974) Google Scholar]. The study identified four potentially clinically relevant molecular subtypes of pancreatic cancer, termed squamous, pancreatic progenitor, aberrantly differentiated endocrine exocrine (ADEX) and immunogenic. The immunogenic and ADEX subtypes were found to be subclasses of the pancreatic subtype. The squamous subtype was so-called as it is enriched for gene programs that have previously been described in squamous cancers of the head and neck, breast, bladder and lung [[18]Hoadley K.A. Yau C. Wolf D.M. Cherniack A.D. Tamborero D. Ng S. et al.Multiplatform analysis of 12 cancer types reveals molecular classification within and across tissues of origin.Cell. 2014; 158: 929-944Abstract Full Text Full Text PDF PubMed Scopus (999) Google Scholar]. It is characterised by epigenetic changes such as hypermethylation leading to loss of expression of key genes in endodermal differentiation such as HNF1B, GATA6, PDX1 and MNX1 [[14]Bailey P. Chang D.K. Nones K. Johns A.L. Patch A.M. Gingras M.C. et al.Genomic analyses identify molecular subtypes of pancreatic cancer.Nature. 2016; 531: 47-52Crossref PubMed Scopus (1974) Google Scholar]. This subtype is associated with inflammatory pathways, immune evasion and epithelial-to-mesenchymal transition [[14]Bailey P. Chang D.K. Nones K. Johns A.L. Patch A.M. Gingras M.C. et al.Genomic analyses identify molecular subtypes of pancreatic cancer.Nature. 2016; 531: 47-52Crossref PubMed Scopus (1974) Google Scholar]. These molecular features of aggressive disease, in turn, are associated with poor survival [[14]Bailey P. Chang D.K. Nones K. Johns A.L. Patch A.M. Gingras M.C. et al.Genomic analyses identify molecular subtypes of pancreatic cancer.Nature. 2016; 531: 47-52Crossref PubMed Scopus (1974) Google Scholar]. By contrast, the pancreatic progenitor subtype described by Bailey et al. [[14]Bailey P. Chang D.K. Nones K. Johns A.L. Patch A.M. Gingras M.C. et al.Genomic analyses identify molecular subtypes of pancreatic cancer.Nature. 2016; 531: 47-52Crossref PubMed Scopus (1974) Google Scholar] is enriched for genes involved in pancreatic differentiation. The immunogenic subtype is enriched for immune cell infiltrates including cytotoxic CD8+ T cells, regulatory T cells and B cells. However, this also includes increased expression of CTLA-4 and PD-1 immune checkpoints, demonstrating immune modulation that can potentially be targeted by immune checkpoint inhibition [[14]Bailey P. Chang D.K. Nones K. Johns A.L. Patch A.M. Gingras M.C. et al.Genomic analyses identify molecular subtypes of pancreatic cancer.Nature. 2016; 531: 47-52Crossref PubMed Scopus (1974) Google Scholar]. Our increasing understanding of the molecular pathology of pancreatic cancer has revealed therapeutic vulnerabilities that extend beyond simple somatic mutations. As discussed above, up to a quarter of patients with pancreatic cancer may harbour genomic evidence of DDR deficiency. This extends beyond germline mutations in BRCA1 and BRCA2, which is currently the selection biomarker for the vast majority of DDR targeted treatment strategies and clinical trials. Using surrogate readouts of DDR deficiency, such as mutational signatures and structural variation patterns, would probably benefit more patients than just point mutations alone. Platinum-based chemotherapy regimens have shown a benefit in all stages of pancreatic cancer [3Conroy T. Desseigne F. Ychou M. Bouche O. Guimbaud R. Becouarn Y. et al.FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer.N Engl J Med. 2011; 364: 1817-1825Crossref PubMed Scopus (5137) Google Scholar, 19Hackert T. Sachsenmaier M. Hinz U. Schneider L. Michalski C.W. Springfeld C. et al.Locally advanced pancreatic cancer: neoadjuvant therapy with folfirinox results in resectability in 60% of the patients.Ann Surg. 2016; 264: 457-463Crossref PubMed Scopus (224) Google Scholar, 20Rombouts S.J. Mungroop T.H. Heilmann M.N. van Laarhoven H.W. Busch O.R. Molenaar I.Q. et al.FOLFIRINOX in locally advanced and metastatic pancreatic cancer: a single centre cohort study.J Cancer. 2016; 7: 1861-1866Crossref PubMed Scopus (28) Google Scholar] and the response is probably related to homologous recombination deficiency (HRD), a dominant subset of DDR deficiency, which occurs in 10–20% of patients with pancreatic cancer. Significant responses to platinum-based chemotherapy occur in up to a fifth of patients and improvements in overall outcomes are probably due to this subgroup of responders to therapy [10Dreyer S.B. Chang D.K. Bailey P. Biankin A.V. Pancreatic cancer genomes: implications for clinical management and therapeutic development.Clin Cancer Res. 2017; 23: 1638-1646Crossref PubMed Scopus (112) Google Scholar, 19Hackert T. Sachsenmaier M. Hinz U. Schneider L. Michalski C.W. Springfeld C. et al.Locally advanced pancreatic cancer: neoadjuvant therapy with folfirinox results in resectability in 60% of the patients.Ann Surg. 2016; 264: 457-463Crossref PubMed Scopus (224) Google Scholar]. However, platinum-based regimens can be associated with significant adverse effects and even mortality. Thus, it is crucial to identify responsive patient subgroups to prevent unnecessary morbidity in patients who do not benefit from platinum. Surrogate readouts of HRD can be used as a selection biomarker to identify platinum responders. This is one of the key research objectives of PRECISION-Panc, a therapeutic development platform that aims to rapidly translate key molecular discoveries into a precision oncology approach for pancreatic cancer (see accompanying Editorial [[21]Dreyer S.B. Jamieson N.B. Cooke S.L. Valle J.W. McKay C.J. Biankin A.V. et al.PRECISION-Panc: the next generation therapeutic development platform for pancreatic cancer.Clin Oncol. 2019; 32: 1-4Abstract Full Text Full Text PDF Scopus (11) Google Scholar]). To date, single-agent immunotherapy trials in pancreatic cancer have been disappointing. Immune checkpoint (PD-1 and CTLA4) inhibitors have failed to show significant benefit in pancreatic cancer. The microenvironment of pancreatic cancer varies greatly between tumours and this is probably due to differential immune signalling between subtypes of tumour epithelium and stroma. The squamous subtype of pancreatic cancer is enriched for immunosuppressive myeloid cell infiltration, with an abundance of neutrophil and macrophage signalling resulting in sparse T and B cell infiltration leading to immune invasion. Recent genetically engineered mouse model studies showed significant benefits in pharmacologically induced myeloid depletion in pancreatic cancer. In an attempt to enhance immune checkpoint inhibition, several preclinical studies have targeted the immunosuppressive myeloid cell infiltration seen in pancreatic cancer. Combining CXCR2 inhibitors with a PD-1 checkpoint inhibitor improves survival in mouse models of pancreatic cancer [[22]Steele C.W. Karim S.A. Leach J.D. Bailey P. Upstill-Goddard R. Rishi L. et al.CXCR2 inhibition profoundly suppresses metastases and augments immunotherapy in pancreatic ductal adenocarcinoma.Cancer Cell. 2016; 29: 832-845Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar]. In another study, CSF1R inhibitors reduced immunosuppressive macrophage infiltration and resulted in an active T cell response [[23]Candido J.B. Morton J.P. Bailey P. Campbell A.D. Karim S.A. Jamieson T. et al.CSF1R(+) macrophages sustain pancreatic tumor growth through T cell suppression and maintenance of key gene programs that define the squamous subtype.Cell Rep. 2018; 23: 1448-1460Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar]. Whether these preclinical findings translate into patient benefit or if these are subtype-specific phenomena remains to be determined in the clinical setting. The immunogenic subtype, on the other hand, is enriched for signalling from CD4+ and CD8+ T and B cells [[14]Bailey P. Chang D.K. Nones K. Johns A.L. Patch A.M. Gingras M.C. et al.Genomic analyses identify molecular subtypes of pancreatic cancer.Nature. 2016; 531: 47-52Crossref PubMed Scopus (1974) Google Scholar]. This is coupled with upregulation of PD-1 and CTLA-4 expression. Checkpoint inhibition may benefit this subgroup of patients, whereas it is unlikely to show any response in the squamous group, as a single agent. This indicates that unselected immunotherapy trials will result in dilution of any therapeutic signals leading to clinical trial failures. In an effort to accelerate therapeutic development, a UK-wide PRECISION-Panc consortium was established in 2016 [[21]Dreyer S.B. Jamieson N.B. Cooke S.L. Valle J.W. McKay C.J. Biankin A.V. et al.PRECISION-Panc: the next generation therapeutic development platform for pancreatic cancer.Clin Oncol. 2019; 32: 1-4Abstract Full Text Full Text PDF Scopus (11) Google Scholar]. Within the clinical development pillar is the PRIMUS (Pancreatic Cancer Individualised Multi-arm Umbrella Study) clinical trial platform (Figure 1). It was designed to rapidly translate preclinical advances into clinical trials for patients with all stages of pancreatic cancer. Clinical trials are traditionally dominated by exclusion criteria, resulting in only around 5% of pancreatic cancer patients currently entering clinical trials. The molecular heterogeneity of pancreatic cancer and often the prolonged period between translating preclinical advances into clinical trials have severely hampered progress in improving outcomes for pancreatic cancer. A PRECISION-Panc Therapeutic Testing Board has been established to regularly review preclinical concepts and assess the readiness of the concepts to be developed into clinical trials. The Therapeutic Testing Board also meets with the National Cancer Research Institute clinical studies group pancreatic cancer workstream twice a year to ensure complementing and aligning therapeutic development. The trials on the PRIMUS platform are designed specifically by the scientific advances described above. PRIMUS-001 and PRIMUS-002 aim to investigate the efficacy of the platinum-containing regimen FOLFOX-abraxane compared with gemcitabine-abraxane in all and biomarker-enriched patients in the first-line metastatic and neoadjuvant settings, respectively. The initial biomarker being tested is a candidate HRD signature, derived from the specific pattern of genomic structural rearrangements seen in known HRD cancers from published and unpublished data sets. This is hypothesised to be a biomarker of response to platinum-based therapy. The adaptive design of these studies allows the discovery and development of the predictive signatures and to inform a future molecularly stratified phase III trial design if needed. This strategy is an overarching goal of PRECISION-Panc by integrating discovery, with preclinical development and clinical trial testing to allow rapid forward and backward translation, thereby accelerating scientific advances into the clinic [[21]Dreyer S.B. Jamieson N.B. Cooke S.L. Valle J.W. McKay C.J. Biankin A.V. et al.PRECISION-Panc: the next generation therapeutic development platform for pancreatic cancer.Clin Oncol. 2019; 32: 1-4Abstract Full Text Full Text PDF Scopus (11) Google Scholar]. The concept of PRECISION-Panc is further demonstrated by the PRIMUS-003 trial (NCT02583477), which has recently completed recruitment. Based on the preclinical data that CXCR2 inhibition blockade significantly abrogated metastasis, and in combination with PD-1 extended survival in a genetically engineered mouse model [[22]Steele C.W. Karim S.A. Leach J.D. Bailey P. Upstill-Goddard R. Rishi L. et al.CXCR2 inhibition profoundly suppresses metastases and augments immunotherapy in pancreatic ductal adenocarcinoma.Cancer Cell. 2016; 29: 832-845Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar], a clinical trial was rationally designed to assess clinical efficacy in a phase Ib signal seeking manner. The PRIMUS-004 umbrella was designed to offer patients a portfolio of second-line options, with an early phase, signal seeking trial design with clear biomarker-based hypotheses using well-tolerated targeted agents. It aims to offer patients a range of options targeting various disease mechanisms and potentially provide therapies for those less fit for further cytotoxic chemotherapy. The first Appendix is investigating if the combination of an ATR inhibitor (AZD6738) with the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib is able to overcome acquired resistance to first-line platinum treatment. Patients who had stable disease or a better response to platinum-containing regimens are stratified by mutations in key genes in the homologous recombination repair (HRR) pathway to allow comparison between these groups (HRR mutant versus wild-type). With the growing knowledge base of the molecular pathology of pancreatic cancer we are revealing novel therapeutic opportunities in this recalcitrant disease. Furthermore, it highlights that underlying inter-patient molecular heterogeneity may have contributed to the significant failure rate associated with unselected clinical trials in pancreatic cancer. The generation and translation of clinically relevant and robust preclinical platforms of evidence into well-designed clinical trials with parallel molecular profiling of patients' tumours will ideally lead to improvements in overall survival for this devastating disease.

Triple-negative breast cancer (TNBC) patients have the poorest clinical outcomes compared to other molecular subtypes of breast cancer. IL6/JAK/STAT3 signalling is upregulated in breast cancer; however, there is limited evidence for its role in TNBC. This study aimed to assess the expression of IL6/JAK/STAT3 in TNBC as a prognostic biomarker.Tissue microarrays consisting of breast cancer specimens from a retrospective cohort (n = 850) were stained for IL6R, JAK1, JAK2 and STAT3 via immunohistochemistry. Staining intensity was assessed by weighted histoscore and analysed for association with survival/clinical characteristics. In a subset of patients (n = 14) bulk transcriptional profiling was performed using TempO-Seq. Nanostring GeoMx® digital spatial profiling was utilised to establish the differential spatial gene expression in high STAT3 tumours.In TNBC patients, high expression of stromal STAT3 was associated with reduced cancer-specific survival (HR = 2.202, 95% CI: 1.148-4.224, log rank p = 0.018). TNBC patients with high stromal STAT3 had reduced CD4+ T-cell infiltrates within the tumour (p = 0.001) and higher tumour budding (p = 0.003). Gene set enrichment analysis (GSEA) of bulk RNA sequencing showed high stromal STAT3 tumours were characterised by enrichment of IFNγ, upregulation of KRAS signalling and inflammatory signalling Hallmark pathways. GeoMx™ spatial profiling showed high stromal STAT3 samples. Pan cytokeratin (panCK)-negative regions were enriched for CD27 (p < 0.001), CD3 (p < 0.05) and CD8 (p < 0.001). In panCK-positive regions, high stromal STAT3 regions had higher expression of VEGFA (p < 0.05).High expression of IL6/JAK/STAT3 proteins was associated with poor prognosis and characterised by distinct underlying biology in TNBC.

Pancreatic Ductal Adenocarcinoma (PDAC) patients exhibit varied responses to multimodal therapy. RNA gene sequencing has unravelled distinct tumour biology subtypes, forming the focus of this review exploring its impact on survival outcomes.A systematic search across PubMed, Medline, Embase, and CINAHL databases targeted studies assessing long-term overall and disease-free survival in PDAC patients with molecular subtyping.Fifteen studies including 2731 patients were identified. Molecular subtyping was performed by RNA sequencing and Immunohistochemistry in 14 studies and by Mass Spectrometry in 1 study. Two main tumour subtypes were identified (classical and basal-like or squamous) with basal like associated with poorer outcomes. Further subtypes were identified in individual studies. Superior survival was seen with classical subtype in all other analyses that compared the classical and basal subtypes. High risk stromal subtypes were identified on further analysis of the stroma and were associated with a worse survival independent of the tumour subtype.Molecular subtyping of PDAC specimens can identify patients with high-risk tumour biology and poor survival outcomes. Routine subtyping is limited by the cost of RNA sequencing and the volume of raw data generated which has made its translation into routine clinical practice difficult.

Background: The incidence of pancreatic ductal adenocarcinoma (PC) is rising in the western world. There is a particularly concerning trend in the rapid increase in incidence of early onset PC (EOPC). The reason for this, and the clinical implications of EOPC is not well understood. The aim of this study was to investigate the clinical outcomes of patients with EOPC and the molecular heterogeneity between EOPC and late onset disease.

Introduction: The prognostic value of CT-derived liver volume in terms of cancer outcomes is not clear. The aim of the present study was to examine the relationship between liver area on a single axial CT-slice and the total liver volume in patients with colonic cancer. Furthermore, we examine the relationship between liver volume, determined using this novel method, clinicopathological variables and survival. Methods: Consecutive patients who underwent potentially curative surgery for colonic cancer were identified from a prospectively maintained database. Maximal liver area on axial CT-slice (cm2) and total volume (cm3), were obtained by the manual segmentation of pre-operative CT-images in a PACS viewer. The maximal liver area was normalized for body height2 to create the liver index (LI) and values, categorized into tertiles. The primary outcome of interest was overall survival (OS). Relationships between LI and clinico-pathological variables were examined using chi-square analysis and binary logistic regression. The relationship between LI and OS was examined using cox proportional hazard regression. Results: A total of 359 patients were included. A total of 51% (n = 182) of patients were male and 73% (n = 261) were aged 65 years or older. 81% (n = 305) of patients were alive 3-years post-operatively. The median maximal liver area on the axial CT slice was 178.7 (163.7–198.4) cm2. The median total liver volume was 1509.13 (857.8–3337.1) cm3. Maximal liver area strongly correlated with total liver volume (R2 = 0.749). The median LI was 66.8 (62.0–71.6) cm2/m2. On multivariate analysis, age (p &lt; 0.001), sex (p &lt; 0.05), BMI (p &lt; 0.001) and T2DM (p &lt; 0.05) remained significantly associated with LI. On univariate analysis, neither LI (continuous) or LI (tertiles) were significantly associated with OS (p = 0.582 and p = 0.290, respectively). Conclusions: The simple, reliable method proposed in this study for quantifying liver volume using CT-imaging was found to have an excellent correlation between observers and provided results consistent with the contemporary literature. This method may facilitate the further examination of liver volume in future cancer studies.

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The aim of the present study was to compare long-term post-resection oncological outcomes between A-IPMN and PDAC.Knowledge of long term oncological outcomes (e.g recurrence and survival data) comparing between adenocarcinoma arising from intraductal papillary mucinous neoplasms (A-IPMN) and pancreatic ductal adenocarcinoma (PDAC) is scarce.Patients undergoing pancreatic resection (2010-2020) for A-IPMN were identified retrospectively from 18 academic pancreatic centres and compared with PDAC patients from the same time-period. Propensity-score matching (PSM) was performed and survival and recurrence were compared between A-IPMN and PDAC.459 A-IPMN patients (median age,70; M:F,250:209) were compared with 476 PDAC patients (median age,69; M:F,262:214). A-IPMN patients had lower T-stage, lymphovascular invasion (51.4%vs. 75.6%), perineural invasion (55.8%vs. 71.2%), lymph node positivity (47.3vs. 72.3%) and R1 resection (38.6%vs. 56.3%) compared to PDAC(P<0.001). The median survival and time-to-recurrence for A-IPMN versus PDAC were 39.0 versus19.5months (P<0.001) and 33.1 versus 14.8months (P<0.001), respectively (median follow-up,78 vs.73 months). Ten-year overall survival for A-IPMN was 34.6%(27/78) and PDAC was 9%(6/67). A-IPMN had higher rates of peritoneal (23.0 vs. 9.1%, P<0.001) and lung recurrence (27.8% vs. 15.6%, P<0.001) but lower rates of locoregional recurrence (39.7% vs. 57.8%; P<0.001). Matched analysis demonstrated inferior overall survival (P=0.005), inferior disease-free survival (P=0.003) and higher locoregional recurrence (P<0.001) in PDAC compared to A-IPMN but no significant difference in systemic recurrence rates (P=0.695).PDACs have inferior survival and higher recurrence rates compared to A-IPMN in matched cohorts. Locoregional recurrence is higher in PDAC but systemic recurrence rates are comparable and constituted by their own distinctive site-specific recurrence patterns.

The clinical impact of adjuvant chemotherapy after resection for adenocarcinoma arising from intraductal papillary mucinous neoplasia is unclear. The aim of this study was to identify factors related to receipt of adjuvant chemotherapy and its impact on recurrence and survival.This was a multicentre retrospective study of patients undergoing pancreatic resection for adenocarcinoma arising from intraductal papillary mucinous neoplasia between January 2010 and December 2020 at 18 centres. Recurrence and survival outcomes for patients who did and did not receive adjuvant chemotherapy were compared using propensity score matching.Of 459 patients who underwent pancreatic resection, 275 (59.9%) received adjuvant chemotherapy (gemcitabine 51.3%, gemcitabine-capecitabine 21.8%, FOLFIRINOX 8.0%, other 18.9%). Median follow-up was 78 months. The overall recurrence rate was 45.5% and the median time to recurrence was 33 months. In univariable analysis in the matched cohort, adjuvant chemotherapy was not associated with reduced overall (P = 0.713), locoregional (P = 0.283) or systemic (P = 0.592) recurrence, disease-free survival (P = 0.284) or overall survival (P = 0.455). Adjuvant chemotherapy was not associated with reduced site-specific recurrence. In multivariable analysis, there was no association between adjuvant chemotherapy and overall recurrence (HR 0.89, 95% c.i. 0.57 to 1.40), disease-free survival (HR 0.86, 0.59 to 1.30) or overall survival (HR 0.77, 0.50 to 1.20). Adjuvant chemotherapy was not associated with reduced recurrence in any high-risk subgroup (for example, lymph node-positive, higher AJCC stage, poor differentiation). No particular chemotherapy regimen resulted in superior outcomes.Chemotherapy following resection of adenocarcinoma arising from intraductal papillary mucinous neoplasia does not appear to influence recurrence rates, recurrence patterns or survival.

Cellular senescence is a major barrier to tumour progression, though its role in pathogenesis of cancer and other diseases is poorly understood in vivo. Improved understanding of the degree to which latent senescence signalling persists in tumours might identify intervention strategies to provoke "accelerated senescence" responses as a therapeutic outcome. Senescence involves convergence of multiple pathways and requires ongoing dynamic signalling throughout its establishment and maintenance. Recent discovery of several new markers allows for an expression profiling approach to study specific senescence phenotypes in relevant tissue samples. We adopted a "senescence scoring" methodology based on expression profiles of multiple senescence markers to examine the degree to which signals of damage-associated or secretory senescence persist in various human tumours.We first show that scoring captures differential induction of damage or inflammatory pathways in a series of public datasets involving radiotherapy of colon adenocarcinoma, chemotherapy of breast cancer cells, replicative senescence of mesenchymal stem cells, and progression of melanoma. We extended these results to investigate correlations between senescence score and growth inhibition in response to ~1500 compounds in the NCI60 panel. Scoring of our own mesenchymal tumour dataset highlighted differential expression of secretory signalling pathways between distinct subgroups of MPNST, liposarcomas and peritoneal mesothelioma. Furthermore, a pro-inflammatory signature yielded by hierarchical clustering of secretory markers showed prognostic significance in mesothelioma.We find that "senescence scoring" accurately reports senescence signalling in a variety of situations where senescence would be expected to occur and highlights differential expression of damage associated and secretory senescence pathways in a context-dependent manner.

Cytotoxic agents are the cornerstone of treatment for patients with advanced intrahepatic cholangiocarcinoma (iCCA), despite heterogeneous benefit. We hypothesised that the pretreatment molecular profiles of diagnostic biopsies can predict patient benefit from chemotherapy and define molecular bases of innate chemoresistance.We identified a cohort of advanced iCCA patients with comparable baseline characteristics who diverged as extreme outliers on chemotherapy (survival <6 m in rapid progressors, RP; survival >23 m in long survivors, LS). Diagnostic biopsies were characterised by digital pathology, then subjected to whole-transcriptome profiling of bulk and geospatially macrodissected tissue regions. Spatial transcriptomics of tumour-infiltrating myeloid cells was performed using targeted digital spatial profiling (GeoMx). Transcriptome signatures were evaluated in multiple cohorts of resected cancers. Signatures were also characterised using in vitro cell lines, in vivo mouse models and single cell RNA-sequencing data.Pretreatment transcriptome profiles differentiated patients who would become RPs or LSs on chemotherapy. Biologically, this signature originated from altered tumour-myeloid dynamics, implicating tumour-induced immune tolerogenicity with poor response to chemotherapy. The central role of the liver microenviroment was confrmed by the association of the RPLS transcriptome signature with clinical outcome in iCCA but not extrahepatic CCA, and in liver metastasis from colorectal cancer, but not in the matched primary bowel tumours.The RPLS signature could be a novel metric of chemotherapy outcome in iCCA. Further development and validation of this transcriptomic signature is warranted to develop precision chemotherapy strategies in these settings.

Both lymph node metastases (LNMs) and tumour deposits (TDs) are included in colorectal cancer (CRC) staging, although knowledge regarding their biological background is lacking. This study aimed to compare the biology of these prognostic features, which is essential for a better understanding of their role in CRC spread. Spatially resolved transcriptomic analysis using digital spatial profiling was performed on TDs and LNMs from 10 CRC patients using 1,388 RNA targets, for the tumour cells and tumour microenvironment. Shotgun proteomics identified 5,578 proteins in 12 different patients. Differences in RNA and protein expression were analysed, and spatial deconvolution was performed. Image-based consensus molecular subtype (imCMS) analysis was performed on all TDs and LNMs included in the study. Transcriptome and proteome profiles identified distinct clusters for TDs and LNMs in both the tumour and tumour microenvironment segment, with upregulation of matrix remodelling, cell adhesion/motility, and epithelial-mesenchymal transition (EMT) in TDs (all p < 0.05). Spatial deconvolution showed a significantly increased abundance of fibroblasts, macrophages, and regulatory T-cells (p < 0.05) in TDs. Consistent with a higher fibroblast and EMT component, imCMS classified 62% of TDs as poor prognosis subtype CMS4 compared to 36% of LNMs (p < 0.05). Compared to LNMs, TDs have a more invasive state involving a distinct tumour microenvironment and upregulation of EMT, which are reflected in a more frequent histological classification of TDs as CMS4. These results emphasise the heterogeneity of locoregional spread and the fact that TDs should merit more attention both in future research and during staging. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The incidence of pancreatic ductal adenocarcinoma ( PDAC ) is steadily increasing and the annual death‐to‐incidence ratio approaches one. This is a figure that has not changed for several decades. Surgery remains the only chance of cure; however, only less than 20% of patients are amenable to operative resection. Despite successful surgical resection, the majority of the patients still succumb to recurrent metastatic disease. Therefore, there is an urgent need to develop novel therapeutic strategies and to better select patients for current therapies. In this review, we will discuss current management by highlighting the landmark clinical trials that have shaped current care. We will then discuss the challenges of therapeutic development using the current randomized‐controlled trial paradigm when confronted with the molecular heterogeneity of PDAC . Finally, we will discuss strategies that may help to shape the management of PDAC in the near future.

Background: Despite being relatively rare pancreatic cancer is one of the highest causes of death. Even within the potentially resectable group outcomes are poor. We present our initial experiences utilising a neoadjuvant approach to localised pancreatic cancer, evaluating survival, response rates and tolerability.

The value of routine surveillance after resection of pancreatic ductal adenocarcinoma (PDAC) is unclear, and expert guidelines offer conflicting recommendations. This study is a systematic review of evidence for surveillance programs.A systematic review of studies evaluating different surveillance methods was undertaken. A meta-analysis was performed for those studies reporting rates of asymptomatic recurrence, treatment of recurrence and overall survival, according to different surveillance methods.Ten studies were included in the literature review, with five studies appropriate for meta-analysis (1596 patients). Patients within active surveillance programs were more likely to have recurrence detected at an asymptomatic stage (Pooled Rate: 49.3% vs. 19.1%, p = 0.043). Within studies reporting these outcomes, patients with asymptomatic recurrence were more likely to receive treatment for recurrence (Odds Ratio 3.49; 95% CI: 1.73-7.07; p < 0.001) and had longer overall survival (Mean Difference: 9.5 months; 95% CI: 4.1-14.8; p < 0.001) than those with symptoms at time of recurrence.Routine surveillance after surgery for PDAC appears to detect more patients at an asymptomatic stage. Data from these non-randomised trials also suggest that treatment rates and survival may be superior in patients were recurrence is detected when asymptomatic. As such, these data suggest that routine surveillance may improve patient outcomes, although an appropriately conducted trial would be required to address concerns that various sources of bias may be affecting these results.

Abstract High vagal nerve activity, reliability measured by HRV, is considered protective in cancer, reducing oxidative stress, inflammation and opposing sympathetic nerve activity. The present monocentric study examines the relationship between HRV, TNM stage, co-morbidity, systemic inflammation and survival in patients who underwent potentially curative resections for colorectal cancer (CRC). Time-domain HRV measures, Standard Deviation of NN-intervals (SDNN) and Root Mean Square of Successive Differences (RMSSD), were examined as categorical (median) and continuous variables. Systemic inflammation was determined using systemic inflammatory grade (SIG) and co-morbidity using ASA. The primary end point was overall survival (OS) and was analysed using Cox regression. There were 439 patients included in the study and the median follow-up was 78 months. Forty-nine percent (n = 217) and 48% (n = 213) of patients were categorised as having low SDNN (&lt; 24 ms) and RMSSD (&lt; 29.8 ms), respectively. On univariate analysis, SDNN was not significantly associated with TNM stage (p = 0.830), ASA (p = 0.598) or SIG (p = 0.898). RMSSD was not significantly associated with TNM stage (p = 0.267), ASA (p = 0.294) or SIG (p = 0.951). Neither SDNN or RMSSD, categorical or continuous, were significantly associated with OS. In conclusion, neither SDNN or RMSSD were associated with TNM stage, ASA, SIG or survival in patients undergoing potentially curative surgery for CRC.

The rising incidence and the accumulating prevalence of neuroendocrine neoplasia (NEN) in the population makes this a common, prevalent and a clinically relevant disease group. Surgical resection represents the only potentially curative treatment for digestive NENs. Thus, resection should in principle be considered for all patients with NEN, although taking the patients age, relevant comorbidity, and performance status into account for operability. Patients with insulinomas, NEN of the appendix and rectal NENs are usually cured by surgery alone. However, less than a third of patients are amendable to curative surgery alone at time of diagnosis. Furthermore, recurrence is common and may occur years after primary surgery, hence the long follow-up time recommended in most NENs (>10 years). As many patients with NENs present with locoregional or metastatic disease, there is considerable debate regarding the role of debulking surgery in these settings. However, good long-term survival can be achieved in a considerable proportion of patients, with 50-70% alive up to 10 years after surgery. Location and grade are the main determinants of long-term survival. Here we present considerations to surgery for primary neuroendocrine tumors in the digestive tract.

Objective: This international multicentre cohort study aims to identify recurrence patterns and treatment of first and second recurrence in a large cohort of patients after pancreatic resection for adenocarcinoma arising from IPMN. Summary Background Data: Recurrence patterns and treatment of recurrence post resection of adenocarcinoma arising from IPMN are poorly explored. Method: Patients undergoing pancreatic resection for adenocarcinoma from IPMN between January 2010 to December 2020 at 18 pancreatic centres were identified. Survival analysis was performed by the Kaplan-Meier log rank test and multivariable logistic regression by Cox-Proportional Hazards modelling. Endpoints were recurrence (time-to, location, and pattern of recurrence) and survival (overall survival and adjusted for treatment provided). Results: Four hundred and fifty-nine patients were included (median, 70 y; IQR, 64-76; male, 54 percent) with a median follow-up of 26.3 months (IQR, 13.0-48.1 mo). Recurrence occurred in 209 patients (45.5 percent; median time to recurrence, 32.8 months, early recurrence [within 1 y], 23.2 percent). Eighty-three (18.1 percent) patients experienced a local regional recurrence and 164 (35.7 percent) patients experienced distant recurrence. Adjuvant chemotherapy was not associated with reduction in recurrence (HR 1.09; P =0.669) One hundred and twenty patients with recurrence received further treatment. The median survival with and without additional treatment was 27.0 and 14.6 months ( P &lt;0.001), with no significant difference between treatment modalities. There was no significant difference in survival between location of recurrence ( P =0.401). Conclusion: Recurrence after pancreatic resection for adenocarcinoma arising from IPMN is frequent with a quarter of patients recurring within 12 months. Treatment of recurrence is associated with improved overall survival and should be considered.

Pancreatic ductal adenocarcinoma remains an unyielding adversary, with a 5-year survival of 5%, a figure unchanged in 50 years. Characterised by marked genetic heterogeneity, recent genomic sequencing efforts demonstrate that with the exclusion of a few known mutations, most mutations occur at a prevalence of <5%. Current systemic chemotherapeutics, when used in an all-comer approach, are at best modestly effective, yet are associated with responses in small groups of undefined patients. Defining these subgroups and targeting them with the appropriate therapy in a personalized or stratified approach holds the promise of improved outcomes for this disease.

There is now compelling evidence that the molecular heterogeneity of cancer is associated with disparate phenotypes with variable outcomes and therapeutic responsiveness to therapy in histologically indistinguishable cancers. This diversity may explain why conventional clinical trial designs have mostly failed to show efficacy when patients are enrolled in an unselected fashion. Knowledge of the molecular phenotype has the potential to improve therapeutic selection and hence the early delivery of the optimal therapeutic regimen. Resolution of the challenges associated with a more stratified approach to health care will ensure more precise diagnostics and enhance therapeutic selection, which will improve overall outcomes.

Background: Clinical translation of immunohistochemistry (IHC) biomarkers requires reliable and reproducible cutoffs or thresholds for interpretation of immunostaining. Most IHC biomarker research focuses on the clinical relevance (diagnostic, prognostic, or predictive utility) of cutoffs, with less emphasis on observer agreement using these cutoffs. From the literature, we identified 3 commonly used cutoffs of 10% positive epithelial cells, 20% positive epithelial cells, and moderate to strong staining intensity (+2/+3 hereafter) to use for investigating observer agreement. Materials and Methods: A series of 36 images of microarray cores stained for 4 different IHC biomarkers, with variable staining intensity and percentage of positive cells, was used for investigating interobserver and intraobserver agreement. Seven pathologists scored the immunostaining in each image using the 3 cutoffs for positive and negative staining. Kappa (κ) statistic was used to assess the strength of agreement for each cutoff. Results: The interobserver agreement between all 7 pathologists using the 3 cutoffs was reasonably good, with mean κ scores of 0.64, 0.59, and 0.62, respectively, for 10%, 20%, and +2/+3 cutoffs. A good agreement was observed for experienced pathologists using the 10% cutoff, and their agreement was statistically higher than for junior pathologists ( P =0.02). In addition, the mean intraobserver agreement for all 7 pathologists using the 3 cutoffs was reasonably good, with mean κ scores of 0.71, 0.60, and 0.73, respectively, for 10%, 20%, and +2/+3 cutoffs. For all 3 cutoffs, a positive correlation was observed with perceived ease of interpretation ( P &lt;0.003). Finally, cytoplasmic-only staining achieved higher agreement using all 3 cutoffs than mixed staining patterns. Conclusions: All 3 cutoffs investigated achieve reasonable strength of agreement, modestly decreasing interobserver and intraobserver variability in IHC interpretation. These cutoffs have previously been used in cancer pathology, and this study provides evidence that these cutoffs can be reproducible between practicing pathologists.

Given the importance placed on awareness and participation in research by Speciality and Training organisations, we sought to survey Scottish trainee attitudes to exposure to research practice during training and research in or out of programme.An online survey was distributed to core and specialist trainees in general surgery in Scotland.Over a 4-month period, 108 trainees (75 ST/SPRs and 33 CTs) completed the survey. In their current post, most were aware of ongoing research projects (77%) and 55% were aware of trial recruitment. Only 47% attend regular journal clubs. Most believe that they are expected to present (89%) and publish (82%) during training. Most (59%) thought that participation in research is well supported. 57% were advised to undertake time out of programme research, mostly by consultants (48%) and training committee (36%). Of the 57 with time out of programme research experience, most did so in early training (37%) or between ST3-5 (47%). 28 out of the 36 (78%) without a national training number secured one after starting research. Most undertook research in a local academic unit (80%) funded by small grants (47%) or internally (33%). Most research (69%) was clinically orientated (13/55 clinical, 25/55 translational). 56% of those completing time out of programme research obtained an MD or PhD. About 91% thought that research was relevant to a surgical career.Most trainees believe that research is an important part of training. Generally, most trainees are exposed to research practices including trial recruitment. However, <50% attend regular journal clubs, a pertinent point, given the current 'exit exam' includes the assessment of critical appraisal skills.

Supplementary Table from Biological Misinterpretation of Transcriptional Signatures in Tumor Samples Can Unknowingly Undermine Mechanistic Understanding and Faithful Alignment with Preclinical Data

Supplementary Figure from Biological Misinterpretation of Transcriptional Signatures in Tumor Samples Can Unknowingly Undermine Mechanistic Understanding and Faithful Alignment with Preclinical Data

Supplementary Data from Biological Misinterpretation of Transcriptional Signatures in Tumor Samples Can Unknowingly Undermine Mechanistic Understanding and Faithful Alignment with Preclinical Data

Supplementary Figure from Biological Misinterpretation of Transcriptional Signatures in Tumor Samples Can Unknowingly Undermine Mechanistic Understanding and Faithful Alignment with Preclinical Data

Supplementary Figure from Biological Misinterpretation of Transcriptional Signatures in Tumor Samples Can Unknowingly Undermine Mechanistic Understanding and Faithful Alignment with Preclinical Data

Supplementary Figure from Biological Misinterpretation of Transcriptional Signatures in Tumor Samples Can Unknowingly Undermine Mechanistic Understanding and Faithful Alignment with Preclinical Data

&lt;div&gt;AbstractPurpose:&lt;p&gt;Precise mechanism-based gene expression signatures (GES) have been developed in appropriate &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt; model systems, to identify important cancer-related signaling processes. However, some GESs originally developed to represent specific disease processes, primarily with an epithelial cell focus, are being applied to heterogeneous tumor samples where the expression of the genes in the signature may no longer be epithelial-specific. Therefore, unknowingly, even small changes in tumor stroma percentage can directly influence GESs, undermining the intended mechanistic signaling.&lt;/p&gt;Experimental Design:&lt;p&gt;Using colorectal cancer as an exemplar, we deployed numerous orthogonal profiling methodologies, including laser capture microdissection, flow cytometry, bulk and multiregional biopsy clinical samples, single-cell RNA sequencing and finally spatial transcriptomics, to perform a comprehensive assessment of the potential for the most widely used GESs to be influenced, or confounded, by stromal content in tumor tissue. To complement this work, we generated a freely-available resource, ConfoundR; &lt;a href="https://confoundr.qub.ac.uk/" target="_blank"&gt;https://confoundr.qub.ac.uk/&lt;/a&gt;, that enables users to test the extent of stromal influence on an unlimited number of the genes/signatures simultaneously across colorectal, breast, pancreatic, ovarian and prostate cancer datasets.&lt;/p&gt;Results:&lt;p&gt;Findings presented here demonstrate the clear potential for misinterpretation of the meaning of GESs, due to widespread stromal influences, which in-turn can undermine faithful alignment between clinical samples and preclinical data/models, particularly cell lines and organoids, or tumor models not fully recapitulating the stromal and immune microenvironment.&lt;/p&gt;Conclusions:&lt;p&gt;Efforts to faithfully align preclinical models of disease using phenotypically-designed GESs must ensure that the signatures themselves remain representative of the same biology when applied to clinical samples.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;Precise mechanism-based gene expression signatures (GES) have been developed in appropriate &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt; model systems, to identify important cancer-related signaling processes. However, some GESs originally developed to represent specific disease processes, primarily with an epithelial cell focus, are being applied to heterogeneous tumor samples where the expression of the genes in the signature may no longer be epithelial-specific. Therefore, unknowingly, even small changes in tumor stroma percentage can directly influence GESs, undermining the intended mechanistic signaling.&lt;/p&gt;Experimental Design:&lt;p&gt;Using colorectal cancer as an exemplar, we deployed numerous orthogonal profiling methodologies, including laser capture microdissection, flow cytometry, bulk and multiregional biopsy clinical samples, single-cell RNA sequencing and finally spatial transcriptomics, to perform a comprehensive assessment of the potential for the most widely used GESs to be influenced, or confounded, by stromal content in tumor tissue. To complement this work, we generated a freely-available resource, ConfoundR; &lt;a href="https://confoundr.qub.ac.uk/" target="_blank"&gt;https://confoundr.qub.ac.uk/&lt;/a&gt;, that enables users to test the extent of stromal influence on an unlimited number of the genes/signatures simultaneously across colorectal, breast, pancreatic, ovarian and prostate cancer datasets.&lt;/p&gt;Results:&lt;p&gt;Findings presented here demonstrate the clear potential for misinterpretation of the meaning of GESs, due to widespread stromal influences, which in-turn can undermine faithful alignment between clinical samples and preclinical data/models, particularly cell lines and organoids, or tumor models not fully recapitulating the stromal and immune microenvironment.&lt;/p&gt;Conclusions:&lt;p&gt;Efforts to faithfully align preclinical models of disease using phenotypically-designed GESs must ensure that the signatures themselves remain representative of the same biology when applied to clinical samples.&lt;/p&gt;&lt;/div&gt;

Supplementary Table from Biological Misinterpretation of Transcriptional Signatures in Tumor Samples Can Unknowingly Undermine Mechanistic Understanding and Faithful Alignment with Preclinical Data

Supplementary Figure from Biological Misinterpretation of Transcriptional Signatures in Tumor Samples Can Unknowingly Undermine Mechanistic Understanding and Faithful Alignment with Preclinical Data

Supplementary Figure from Biological Misinterpretation of Transcriptional Signatures in Tumor Samples Can Unknowingly Undermine Mechanistic Understanding and Faithful Alignment with Preclinical Data

Supplementary Figure from Biological Misinterpretation of Transcriptional Signatures in Tumor Samples Can Unknowingly Undermine Mechanistic Understanding and Faithful Alignment with Preclinical Data

Supplementary Data from Biological Misinterpretation of Transcriptional Signatures in Tumor Samples Can Unknowingly Undermine Mechanistic Understanding and Faithful Alignment with Preclinical Data

Supplementary Figure from Biological Misinterpretation of Transcriptional Signatures in Tumor Samples Can Unknowingly Undermine Mechanistic Understanding and Faithful Alignment with Preclinical Data