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Several prognostic nomograms designed to predict survival after curative resection for colorectal cancer (CRC) have been proposed. Recently, routine pathological assessment has evolved with subtle changes to the AJCC staging system, and routine screening for mismatch repair deficiency (MMRd). Therefore we sought to develop and validate a new prognostic nomogram. All cause survival data from 4517 consecutive patients with primary CRC were used as independent training and validation cohorts to develop a final model including only: age, sex, tumour stage, nodal status, number of lymph nodes resected, apical node status, distant metastases, thin-walled vascular invasion, and MMR status. Patients were stratified into four risk groups to assess model discrimination and calibration. To assess discrimination, the area-under-the-curve (AUC) of a receiver-operator-curve (ROC), concordance-index (C-index), and D-index were calculated. The model was compared to the Memorial Sloan Kettering Cancer Center (MSKCC) CRC nomogram and the AJCC TNM staging. Based on the 5-year ROC analysis, the AUC for our model was 0.81 (0.79 and 0.74 for MSKCC and AJCC, respectively). Moreover, our model demonstrated a concordance index of 0.77 (95% CI 0.70-0.82) compared to 0.75 (95% CI 0.68-0.81) for MSKCC and 0.73 (95% CI 0.65-0.79) for AJCC. In conclusion, our new prognostic nomogram incorporates a larger number of clinically relevant prognostic markers, including MMR status, and therefore demonstrates improved predictive capability. As these factors are routinely assessed, it is hoped that this model will inform prognostication and difficult management decisions, such as patient selection for adjuvant therapy.

Aims Myxoid liposarcoma (MLPS) is characterised by DNA damage-inducible transcript 3 (DDIT3) gene rearrangements, confirmation of which is commonly used diagnostically. Recently, DDIT3 immunohistochemistry (IHC) has been reported to be highly sensitive and, when strict criteria are employed, specific for the diagnosis of MLPS. The aim of this study was to independently investigate DDIT3 IHC as a diagnostic marker for MLPS. Methods and results DDIT3 IHC was performed on 52 MLPS and on 152 mimics on whole sections, and on 515 non-MLPS sarcomas in tissue microarray format. Only one MLPS (which had undergone acid-based decalcification) was completely negative. With inclusion of this case if any nuclear expression is considered to indicate positivity, the overall sensitivity of DDIT3 is 98% (51 of 52 cases) and the specificity is 94% (633 of 667 non-MLPS cases are negative). If a cut-off of >10% of neoplastic cells is required for positivity, then the sensitivity remains 98% (51/52) and the specificity is 98.5% (657 of 667 non-MLPS cases are negative). If a cut-off of >50% of cells is required for positivity, then the sensitivity is 96% (50 of 52 cases) but the specificity improves to 100%. Conclusions Diffuse nuclear DDIT3 expression occurs in the overwhelming majority of MLPSs, and can be used to confirm the diagnosis in most cases without the need for molecular testing. A complete absence of expression argues strongly against MLPS, and almost completely excludes this diagnosis, particularly if there is consideration of technical factors such as decalcification. The significance of focal DDIT3 expression should be interpreted in the morphological and clinical context, although most tumours showing only focal expression are not MLPS.

Currently, there is no consensus on the optimal tumor response score (TRS) system to assess regression in pancreatic cancers resected after neoadjuvant therapy. We developed a novel TRS (Royal North Shore [RNS] system) based on estimating the percentage of tumor bed occupied by viable cancer and categorized into 3 tiers: grade 1 (≤10%), grade 2 (11% to 75%), and grade 3 (>75%). We assessed 147 resected carcinomas with this and other TRS systems (College of American Pathologists [CAP], MD Anderson Cancer Center [MDACC], and Evans). The 3-tiered RNS system predicted median survival after surgery for grades 1, 2, and 3 of 54, 23, and 9 months, respectively (P

Aims Synovial sarcoma is defined by recurrent t(X;18)(p11;q11) translocations creating SS18-SSX1, SS18-SSX2 or SS18-SSX4 fusions. Recently, a novel rabbit monoclonal antibody designed to identify these fusions (SS18-SSX, clone E9X9V) was proposed to be highly specific (100%), but not completely sensitive (95%) for this diagnosis. Another antibody designed to identify the C-terminal end of SSX (SSX_CT, clone E5A2C) was proposed to be highly sensitive (100%), but not completely specific (96%). We sought to validate these antibodies in an independent cohort. Methods and results We performed immunohistochemistry for SS18-SSX and SSX_CT on 39 synovial sarcoma samples from 25 patients with confirmed gene rearrangements. Thirty-four (87%) and 36 (92%) were positive for SS18-SSX and SSX_CT, respectively. False-negative staining was associated with suboptimally handled small biopsies and decalcified specimens, even when staining was diffuse and strong in subsequent optimally processed excisions and non-decalcified areas. None of 580 non-synovial sarcoma tumours (76 whole sections, 504 TMA samples) were positive for SS18-SSX (100% specificity), whereas 39 (93% specificity) were positive for SSX_CT. Conclusions SS18-SSX fusion-specific IHC is 87-95% sensitive for the diagnosis of synovial sarcoma and highly (perhaps perfectly) specific. Therefore, positive SS18-SSX staining definitively confirms the diagnosis of synovial sarcoma. SSX_CT is less specific (93-96%) but highly sensitive (92%, but approaching 100% when suboptimally processed biopsies and decalcified specimens are excluded). Negative SSX_CT staining may therefore have an ancillary role as a rule-out test for synovial sarcoma. We caution that both antibodies are prone to false-negative staining in decalcified specimens.

There is now evidence that gene fusions activating the MAPK pathway are relatively common in pancreatic acinar cell carcinoma with potentially actionable BRAF or RET fusions being found in ~30%. We sought to investigate the incidence of RAF1 fusions in pancreatic malignancies with acinar cell differentiation. FISH testing for RAF1 was undertaken on 30 tumors comprising 25 'pure' acinar cell carcinomas, 2 mixed pancreatic acinar-neuroendocrine carcinomas, 1 mixed acinar cell-low grade neuroendocrine tumor and 2 pancreatoblastomas. RAF1 rearrangements were identified in 5 cases and confirmed by DNA and RNA sequencing to represent oncogenic fusions (GATM-RAF1, GOLGA4-RAF1, PDZRN3-RAF1, HERPUD1-RAF1 and TRIM33-RAF1) and to be mutually exclusive with BRAF and RET fusions, as well as KRAS mutations. Large genome-wide copy number changes were common and included 1q gain and/or 1p loss in all five RAF1 FISH-positive acinar cell carcinomas. RAF1 expression by immunohistochemistry was found in 3 of 5 (60%) of fusion-positive cases and no FISH-negative cases. Phospho-ERK1/2 expression was found in 4 of 5 RAF1-fusion-positive cases. Expression of both RAF1 and phospho-ERK1/2 was heterogeneous and often only detected at the tumor-stroma interface, thus limiting their clinical utility. We conclude that RAF1 gene rearrangements are relatively common in pancreatic acinar cell carcinomas (14.3% to 18.5% of cases) and can be effectively identified by FISH with follow up molecular testing. The combined results of several studies now indicate that BRAF, RET or RAF1 fusions occur in between one third and one-half of these tumors but are extremely rare in other pancreatic malignancies. As these fusions are potentially actionable with currently available therapies, a strong argument can be made to perform FISH or molecular testing on all pancreatic acinar cell carcinomas.

The presence of increased tumour infiltrating lymphocytes (TILs) is established as a positive prognostic factor in triple-negative breast cancer (TNBC). The majority of studies have examined the role of TILs in predicting response to chemotherapy, but their role as a general prognostic marker in TNBC is unclear. Moreover, there is a lack of consensus in the literature regarding a suitable cut-off point by which to stratify patients into prognostic groups. Therefore, we sought to confirm the prognostic value of TILs in an independent cohort of unselected TNBCs, and to determine an appropriate cut-off point by which to stratify TIL scores into prognostically significant categories. We used the International TILs Working Group (ITWG) methodology to assess the density of stromal TILs in our cohort of 139 TNBC patients undergoing curative resection at our institution. The percentage TILs scores were categorised first into three groups: low (0-10%), intermediate (15-50%), and high (55-100%). A second binary variable was also created by separating cases into low TILs (≤50%) and high TILs (>50%) groups. Using the three-tiered system, mean disease-free survival was 156, 99 and 94 months for the high, intermediate and low TILs groups, respectively (p=0.030). However, no statistically significant improvement was observed for overall survival. Using the two-tiered system, statistically significant improvements in both overall survival (p=0.030) and disease-free survival (p=0.010) were observed. This survival benefit remained statistically significant in multivariate analyses (p=0.010, p=0.014). We conclude that TILs scored using the ITWG system and dichotomised at a cut-off score of 50%, are a powerful predictor of all-cause and disease-free survival in TNBC regardless of chemotherapy treatment.

NTRK gene rearrangements are important to identify as predictors of response to targeted therapy in many malignancies. Only 0.16–0.3% of colorectal carcinomas (CRCs) harbor these fusions making universal screening difficult. We therefore investigated whether pan-Trk immunohistochemistry (IHC), mismatch repair deficiency (MMRd), and BRAFV600E mutation status could be used to triage molecular testing for NTRK gene rearrangements in CRC. CRCs from 4569 unselected patients underwent IHC in TMA format with two different anti-pan-Trk rabbit monoclonal antibodies. All positive cases were confirmed on whole sections and underwent RNA-sequencing. Pan-Trk IHC was positive in 0.2% of CRCs (9/4569). Both antibodies demonstrated similar staining characteristics with diffuse positive staining in all neoplastic cells. Of note 8/9 (89%) IHC positive cases were both MMRd (all showing MLH1/PMS2 loss) and lacked BRAFV600E mutation. That is, IHC was positive in 5.3% (8/152) MLH1/PMS2/BRAFV600E triple negative CRCs, but only 0.02% (1/4417) not showing this phenotype. All nine IHC positive CRCs demonstrated gene rearrangements (LMNA-NTRK1 in 5 CRCs, TPR-NTRK1, STRM-NTRK1, MUC2-NTRK2, and NTRK1 with an unknown partner in one each), suggesting close to 100% specificity for IHC in this sub-population. NTRK fusions were associated with right sided (p = 0.02), larger tumors (p = 0.029) with infiltrative growth (p = 0.021). As a part of universal Lynch syndrome screening many institutions routinely test all CRCs for MMRd, and then proceed to reflex BRAFV600E mutation testing in MLH1/PMS2 negative CRCs. We conclude that performing pan-Trk IHC on this preselected subgroup of MLH1/PMS2/BRAFV600E triple negative CRCs (only 3.3% of all CRC patients) is a resource effective approach to identify the overwhelming majority of CRC patients with NTRK gene fusions.

Background Drug resistance is a major obstacle in cancer therapy. To elucidate the genetic factors that regulate sensitivity to anti-cancer drugs, we performed CRISPR-Cas9 knockout screens for resistance to a spectrum of drugs. Results In addition to known drug targets and resistance mechanisms, this study revealed novel insights into drug mechanisms of action, including cellular transporters, drug target effectors, and genes involved in target-relevant pathways. Importantly, we identified ten multi-drug resistance genes, including an uncharacterized gene C1orf115, which we named Required for Drug-induced Death 1 (RDD1). Loss of RDD1 resulted in resistance to five anti-cancer drugs. Finally, targeting RDD1 leads to chemotherapy resistance in mice and low RDD1 expression is associated with poor prognosis in multiple cancers. Conclusions Together, we provide a functional landscape of resistance mechanisms to a broad range of chemotherapeutic drugs and highlight RDD1 as a new factor controlling multi-drug resistance. This information can guide personalized therapies or instruct rational drug combinations to minimize acquisition of resistance.

The tumor immune microenvironment is a main contributor to cancer progression and a promising therapeutic target for oncology. However, immune microenvironments vary profoundly between patients, and biomarkers for prognosis and treatment response lack precision. A comprehensive compendium of tumor immune cells is required to pinpoint predictive cellular states and their spatial localization. We generated a single-cell tumor immune atlas, jointly analyzing published data sets of >500,000 cells from 217 patients and 13 cancer types, providing the basis for a patient stratification based on immune cell compositions. Projecting immune cells from external tumors onto the atlas facilitated an automated cell annotation system. To enable in situ mapping of immune populations for digital pathology, we applied SPOTlight, combining single-cell and spatial transcriptomics data and identifying colocalization patterns of immune, stromal, and cancer cells in tumor sections. We expect the tumor immune cell atlas, together with our versatile toolbox for precision oncology, to advance currently applied stratification approaches for prognosis and immunotherapy. This publication is part of a project (BCLLATLAS) that has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 810287). This work has received funding from the Ministerio de Ciencia, Innovación y Universidades (SAF2017-89109-P; AEI/FEDER, UE) and the Fundació La Marató de TV3 (201903-30-31-32).We further acknowledge funding from the St. Vincent’s Clinic Foundation (V.T.C.) and the National Health and Medical Research Council Investigator Grant (APP1175781, J.E.P.), the Fundación Asociación Española contra el Cáncer (AECC), FERO (EDM), Ramón Areces Foundation, Cellex Foundation, BBVA (CAIMI), the ISCIII, FIS (PI16/01278), Juan de la Cierva formación fellowship (C.R.-P.) and Sara Borrell fellowship (E.P.-R.). Core funding is from the ISCIII and the Generalitat de Catalunya. We acknowledge support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the Centro de Excelencia Severo Ochoa, the CERCA Programme / Generalitat de Catalunya, the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) through the nstituto de Salud Carlos III and the Generalitat de Catalunya through Departament de Salut and Departament d’Empresa i Coneixement. We also acknowledge the cofinancing by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) with funds from the European Regional Development Fund (ERDF) corresponding to the 2014–2020 Smart Growth Operating Program

The presence of increased tumor-infiltrating lymphocytes (TILs) is established as a positive prognostic factor in many malignancies including colorectal carcinoma (CRC). However, multiple different approaches have been used to assess TILs. In 2014, the International TILs Working Group (ITWG) proposed a standardized methodology for evaluating TILs, initially in the context of breast cancer, but subsequently expanded to other malignancies. To date, the efficacy of the ITWG system has not been investigated in a large cohort of all-stage CRC. We, therefore, sought to validate this system in CRC. We used the ITWG system to assess the density of stromal TILs in an unselected cohort of 1034 CRC patients undergoing primary tumor resection at our institution. The percentage TILs' score was categorized into 3 groups: low (0% to 10%), intermediate (15% to 50%), and high (55% to 100%). The mean survival was 53, 67, and 75 months, respectively (P=0.0001). This survival benefit remained statistically significant in multivariate analyses (P=0.0001) and subgroup analyses of mismatch repair-proficient CRCs (P=0.0001), mismatch repair-deficient CRCs (P=0.031), BRAFV600E-mutant CRCs (P=0.0001), and BRAF wild-type CRCs (P=0.001). The predictive value of TILs assessed using the ITWG system was superior to the assessment of intraepithelial lymphocyte performed prospectively using a standard system requiring ≥5 lymphocytes per high-powered field in direct contact with tumor cells or between tumor clusters. We conclude that the ITWG system for assessing TILs is a powerful predictor of all-cause survival in CRC independent of many prognostic factors and superior to the assessment of intraepithelial lymphocytes using a traditional system.

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

© 2018 The Author(s). Published by Wolters Kluwer Health, Inc. The gene CDC73 (previously known as HRPT2) encodes the protein parafibromin. Biallelic mutation of CDC73 is strongly associated with malignancy in parathyroid tumors. Heterozygous germline mutations cause hyperparathyroidism jaw tumor syndrome,which is associated with a high life-time risk of parathyroid carcinoma. Therefore loss of parafibromin expression by immunohistochemistry may triage genetic testing for hyperparathyroidism jaw tumor syndrome and be associated with malignant behavior in atypical parathyroid tumors. We share our experience that parafibromin-negative parathyroid tumors show distinctive morphology. We searched our institutional database for parathyroid tumors demonstrating complete loss of nuclear expression of parafibromin with internal positive controls. Forty-three parafibromin-negative tumors from 40 (5.1%) of 789 patients undergoing immunohistochemistry were identified. Thirty-three (77%) were external consultation cases; the estimated incidence in unselected tumors was 0.19%. Sixteen (37.2%) fulfilled World Health Organization 2017 criteria for parathyroid carcinoma and 63% had serum calcium greater than 3mmol/L. One of 27 (3.7%) noninvasive but parafibromin-negative tumors subsequently metastasized. Parafibromin-negative patients were younger (mean, 36 vs. 63 y; P

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

Item does not contain fulltext Pancreatic neuroendocrine tumors (PanNETs) are rare neoplasms accounting for 1% to 2% of all pancreatic tumors. The biological behavior of PanNETs is heterogeneous and unpredictable, adding to the difficulties of clinical management. The DAXX (death domain associated protein) and ATRX (alpha-thalassemia/mental retardation syndrome X-linked) genes encode proteins involved in SWI/SNF-like chromatin remodeling. Somatic inactivating mutations in DAXX and ATRX are frequent in PanNETs, mutually exclusive, and associated with telomere dysfunction, resulting in genomic instability and alternate lengthening of telomeres. We sought to assess the clinical significance of the loss of the ATRX and DAXX proteins as determined by immunohistochemistry (IHC) in patients with PanNET. From an unselected cohort of 105 patients, we found ATRX loss in 10 tumors (9.5%) and DAXX loss in 16 (15.2%). DAXX and ATRX losses were confirmed mutually exclusive and associated with other adverse clinicopathological variables and poor survival in univariate analysis. In addition, ATRX loss was also associated with higher AJCC stage and infiltrative tumor borders. However, only ATRX loss, lymphovascular invasion, and perineural spread were independent predictors of poor overall survival in multivariate analysis. In conclusion, loss of expression of ATRX as determined by IHC is a useful independent predictor of poor overall survival in PanNETs. Given its relative availability, ATRX loss as determined by IHC may have a role in routine clinical practice to refine prognostication in patients with PanNET.

Cancer-associated fibroblasts (CAF) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression through protumor signaling and the generation of fibrosis, the latter of which creates a physical barrier to drugs. CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11 is a cystine transporter that has been identified as a potential therapeutic target in PDAC cells. However, no prior study has evaluated the role of SLC7A11 in PDAC tumor stroma and its prognostic significance. Here we show that high expression of SLC7A11 in human PDAC tumor stroma, but not tumor cells, is independently prognostic of poorer overall survival. Orthogonal approaches showed that PDAC-derived CAFs are highly dependent on SLC7A11 for cystine uptake and glutathione synthesis and that SLC7A11 inhibition significantly decreases CAF proliferation, reduces their resistance to oxidative stress, and inhibits their ability to remodel collagen and support PDAC cell growth. Importantly, specific ablation of SLC7A11 from the tumor compartment of transgenic mouse PDAC tumors did not affect tumor growth, suggesting the stroma can substantially influence PDAC tumor response to SLC7A11 inhibition. In a mouse orthotopic PDAC model utilizing human PDAC cells and CAFs, stable knockdown of SLC7A11 was required in both cell types to reduce tumor growth, metastatic spread, and intratumoral fibrosis, demonstrating the importance of targeting SLC7A11 in both compartments. Finally, treatment with a nanoparticle genesilencing drug against SLC7A11, developed by our laboratory, reduced PDAC tumor growth, incidence of metastases, CAF activation, and fibrosis in orthotopic PDAC tumors. Overall, these findings identify an important role of SLC7A11 in PDAC-derived CAFs in supporting tumor growth. Significance: this study demonstrates that SLC7A11 in PDAC stromal cells is important for the tumor-promoting activity of CAFs and validates a clinically translatable nanomedicine for therapeutic SLC7A11 inhibition in PDAC., NHMRC Project Grant; Avner Innovation Grant; NHMRC CDF-I; NHMRC Ideas Grant ; Cancer-Institute NSW ECF/CDFs ; Cancer Institute NSW Innovation Grant; Cancer Institute NSW The Professor Rob Sutherland AO Make a Difference Award; Cancer Australia/Cancer Council; Cancer Australia/Kids Cancer Project; Cure Cancer Australia; Tour de Cure PhD Support Scholarship; Tour de Cure Established Research Grant; Tour de Cure Pioneering Research Grant; UNSW Interlude Grant Scheme; Cancer Research UK Core Funding and Grand Challenge Grants; NHMRC CDF-II; NHMRC Senior Research Fellowship; Suttons, Cancer Council NSW; Avner Grant from PanKind; Australian Pancreatic Cancer Foundation; Translational Cancer Research Network and Australian Postgraduate Award Scholarships; Australian Government Research Training Program Scholarship; UNSW Sydney Scientia PhD Scholarship; Pancreatic Cancer UK Future Leaders Academy; Len Ainsworth Pancreatic Cancer Fellowship

Description, Intravital imaging guides a personalized medicine approach to target mechanoreciprocity in pancreatic cancer., Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic, chemoresistant malignancy and is characterized by a dense, desmoplastic stroma that modulates PDAC progression. Here, we visualized transient manipulation of focal adhesion kinase (FAK), which integrates bidirectional cell-environment signaling, using intravital fluorescence lifetime imaging microscopy of the FAK-based Förster resonance energy transfer biosensor in mouse and patient-derived PDAC models. Parallel real-time quantification of the FUCCI cell cycle reporter guided us to improve PDAC response to standard-of-care chemotherapy at primary and secondary sites. Critically, micropatterned pillar plates and stiffness-tunable matrices were used to pinpoint the contribution of environmental cues to chemosensitization, while fluid flow–induced shear stress assessment, patient-derived matrices, and personalized in vivo models allowed us to deconstruct how FAK inhibition can reduce PDAC spread. Last, stratification of PDAC patient samples via Merlin status revealed a patient subset with poor prognosis that are likely to respond to FAK priming before chemotherapy.

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

Calcineurin inhibitors, such as Cyclosporin (CsA), are the mainstay of anti-rejection therapy in solid organ transplants but can paradoxically induce progressive nephropathy characterised by renal dysfunction and interstitial fibrosis. Lysyl oxidases (LOXs), a group of enzymes that catalyse extracellular matrix (ECM) crosslinking, were shown to implicate in tissue scarring. It is hypothesized that inhibition of these enzymes may render therapeutic effects against CsA-induced nephropathy. In this study, 6-to-8 weeks old C57BL/6 J mice were administered saline or CsA (30 mg/kg/day s.c) for 16 weeks. At 8 weeks, CsA-treated animals were divided into 5 groups respectively treated with: (1) vehicle, (2) PXS-5505 (Pan-LOX inhibitor), (3) PXS-5382 (LOX-like 2 inhibitor), (4) PXS-5505 for 4 weeks then PXS-5382 for 4 weeks (sequential therapy), and (5) Telmisartan (standard therapy). Our results indicate that CsA administration significantly increased the levels of blood urea nitrogen, glomerular and tubular injury, tubulointerstitial fibrosis, inflammation and oxidative stress in mouse kidney. These changes were associated with upregulated mRNA expression of LOX and LOXL2. Administration of Pan-LOX or LOXL2 inhibitors or the sequential therapy suppressed the expression of ECM proteins (α-SMA, FN and COL1A), matrix metalloproteases (MMP)2 and 9, inflammatory markers (TNFα and MCP-1) and TGF-β1-Smad3 signalling. Among all regimens including telmisartan, only Pan-LOX inhibitor PXS-5505 was able to attenuate uraemia. Collectively, our study suggests that Pan-LOX and LOXL2 inhibition can attenuate progressive nephropathy due to CsA administration.

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

The recently published 5th edition 2019 World Health Organization (WHO) Classification of Tumours of the Digestive System brings significant changes from the 2010 4th edition. An emphasis on uniformity in nomenclature and grading for tumours across all organ systems is a particular feature of the 5th edition blue book series that is reflected in the gastrointestinal tract (GIT) classification. For example, simplified two tiered grading is reinforced for preinvasive lesions throughout the GIT, with dysplasia at all sites now being considered either low or high grade. Similarly, a uniform approach to classification and grading of GIT neuroendocrine neoplasms has been consolidated, with an emphasis on distinguishing grade 3 neuroendocrine tumours from neuroendocrine carcinomas. In this review, we discuss and critically assess the important and sometimes controversial changes made to the classification of tumours of the lower GIT, comprising the colorectum, vermiform appendix and anal canal. The particularly controversial decision to endorse the term 'sessile serrated lesion' for lesions previously termed 'sessile serrated polyp/adenoma' is explored. The morphological, molecular, and clinical insights behind the substitution of the term 'goblet cell adenocarcinoma' for 'goblet cell carcinoid' are assessed. The evolution of the classification of appendiceal mucinous neoplasms is considered. Inflammatory bowel disease related dysplasia and its evolving subtypes, with major implications for pathologists in routine practice, is explained.

We investigated the prognostic value of a range of histologic parameters in medullary thyroid carcinoma (MTC) to design a grading system to predict overall survival. We assessed 76 patients with MTCs undergoing primary tumor resection for age, sex, tumor size, vascular space invasion, lymph node metastasis, multiple endocrine neoplasia type 2 (MEN2) status, mitotic count, Ki-67 proliferative index, spindled morphology, sheet-like growth pattern, coagulative necrosis, incipient necrosis, nuclear grade, multinucleation, prominent nucleoli, fibrosis, and amyloid deposition. In addition to the clinical features of age and the diagnosis of MEN2, the only histologic features that significantly predicted reduced overall survival were Ki-67 proliferative index, mitotic count, and the presence of coagulative necrosis. Using a combination of these 3 variables, we propose a 3-tiered grading system based solely on proliferative activity (Ki-67 proliferative index and mitotic count) and necrosis. There were 62 (82%) low-grade MTCs (low proliferative activity, no necrosis), 9 (12%) intermediate grade (low proliferative activity and necrosis present, or intermediate proliferative activity and no necrosis), and 5 (7%) high grade (intermediate proliferative activity and necrosis present, or high proliferative activity with or without necrosis). The mean overall survival was 193, 146, and 45 months, respectively (P=0.0001) for the 3 grades. The grading system remained prognostic when controlled for other factors associated with survival including age and known MEN2 syndrome. We conclude that this proposed grading system, which uses only a combination of proliferative activity (Ki-67 index, mitotic count) and coagulative necrosis, is a strong predictor of overall survival in MTC.

Rationale: Crystalglobulinemia is a rare complication of monoclonal gammopathy wherein crystallized immunoglobulins deposit in various organs causing occlusive vasculopathy, endothelial damage, and thrombosis. It should be differentiated from light chain cast nephropathy without crystalline nephropathy through timely diagnosis with a kidney biopsy. Presenting concerns of the patient: We report a case of a 74-year-old female with polyarthralgia, chest pain, petechial rash, and acute kidney injury. Diagnoses: Kidney biopsy revealed eosinophilic casts in the tubular lumen and similar occlusive crystalline deposits within the glomerular vasculature and interlobular arteries. Bone marrow biopsy and serum electrophoresis confirmed immunoglobulin G (IgG) κ multiple myeloma. Interventions: Dialysis was initiated for severe oligoanuric acute kidney injury. The patient was treated with 5 sessions of plasmapheresis and 11 cycles of clone reduction chemotherapy with CyBorD (cyclophosphamide, bortezomib, and dexamethasone). Outcomes: This patient achieved excellent kidney recovery and is no longer dialysis dependent. Teaching points: Crystalglobulinemia should be suspected in patients with rapidly progressive acute kidney injury and monoclonal gammopathy. Timely investigation with kidney biopsy to differentiate this condition from light chain cast nephropathy and initiation of appropriate treatment can lead to remission of disease and excellent recovery of kidney function.

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

The evolution of genetic research over the past two decades has greatly improved the understanding of pheochromocytomas and paragangliomas. It is now accepted that more than one third of pheochromocytoma and paragangliomas arise in the context of syndromic disease, usually hereditary. The genetic profile of these tumors also has important prognostic implications which may help guide treatment. Accompanying the changing molecular landscape is the development of new immunohistochemical markers. Initially used in assisting with diagnosis, immunohistochemical markers have now become an important adjunct to screening programs for inherited conditions and subsequently as prognostic markers. The accessibility and efficiency of immunohistochemistry bring pathologists to the forefront in triaging patients based on tumor genotype-phenotype. In this review, we provide an update on the role of immunohistochemistry in the diagnosis of pheochromocytomas and paragangliomas, as an adjunct to assessment for hereditary disease and finally as a potential tool to assist risk stratification.

C cell hyperplasia is considered a precursor lesion for hereditary forms of medullary thyroid carcinoma. It has therefore been suggested as a morphological marker to distinguish hereditary from sporadic medullary thyroid carcinoma and to triage genetic testing in resource poor settings. However, numerous definitions for C cell hyperplasia have been suggested, and there is surprisingly little data regarding the number of C cells present in thyroid glands removed for conditions other than medullary carcinoma. We therefore sought to investigate the specificity of different criteria for C cell hyperplasia. We examined the number of C cells and solid cell nests (ultimobranchial body remnants) present in 118 completion thyroidectomy specimens from patients without medullary carcinoma and with no risk factors for MEN2. Morphological review was performed on all H&E-stained slides, and immunohistochemistry for calcitonin was performed on one block from each case. Solid cell nests were found in 4 (3.3%) of thyroids. Increased numbers of C cells sufficient to fulfil criteria for C cell hyperplasia were found in 5 (4.2%) to 36 (30.5%) cases depending on the criteria used. We conclude that large numbers of C cells are commonly found in thyroids not associated with medullary carcinoma. Therefore, regardless of which criteria are used, the presence of C cell hyperplasia is not a specific marker for hereditary medullary thyroid carcinoma.

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

Pathogenic ALK translocations have been reported in papillary thyroid carcinoma (PTC). We developed and validated a screening algorithm based on immunohistochemistry (IHC), followed by fluorescence in situ hybridization (FISH) in IHC-positive cases to identify ALK-rearranged PTC. IHC and FISH were performed in a cohort of 259 thyroid carcinomas enriched for aggressive variants. IHC was positive in 8 cases, 6 confirmed translocated by FISH (specificity 75%). All 251 IHC-negative cases were FISH negative (sensitivity 100%). Having validated this approach, we performed screening IHC, followed by FISH in IHC-positive cases in an expanded cohort. ALK translocations were identified in 11 of 498 (2.2%) of all consecutive unselected PTCs and 3 of 23 (13%) patients with diffuse sclerosing variant PTCs. No ALK translocations were identified in 36 PTCs with distant metastases, 28 poorly differentiated (insular) carcinomas, and 20 anaplastic carcinomas. All 14 patients with ALK translocations were female (P=0.0425), and translocations occurred at a younger age (mean 38 vs. 48 y, P=0.0289 in unselected patients). ALK translocation was an early clonal event present in all neoplastic cells and mutually exclusive with BRAFV600E mutation. ALK translocation was not associated with aggressive clinicopathologic features (size, stage, metastasis, vascular invasion, extrathyroidal extension, multifocality, risk for recurrence, radioiodine resistance). We conclude that 2.2% of PTCs are ALK-translocated and can be identified by screening IHC followed by FISH. ALK translocations may be more common in young females and diffuse sclerosing variant PTC but do not connote more aggressive disease.

Crizotinib, a small molecule tyrosine kinase inhibitor, has shown tremendous promise in the treatment of lung adenocarcinomas harboring either ALK or ROS1 rearrangements. Recently, small studies of colorectal carcinomas (CRCs) have suggested an incidence of EML4-ALK translocations of 0.4% to 2.4% and FIG-ROS1 translocations of 0.8%. In lung cancer, screening immunohistochemical staining for ALK and ROS1 has been validated as highly sensitive for these translocations, but this has not been investigated in CRC. We therefore sought to investigate the incidence of ALK and ROS1 overexpression as detected by immunohistochemical staining in a large cohort of CRCs. Of the 1889 CRCs, only 1 case (0.05%) demonstrated diffuse strong positive staining for ALK, whereas 14 (0.7%) showed weak nonspecific staining; the remainder were negative. The 1 positive case was confirmed to harbor an ALK rearrangement by fluorescent in situ hybridization (FISH), whereas the 14 tumors with weak staining were FISH-negative. The ALK positive case demonstrated positive expression in all dysplastic and malignant cells indicating that the translocation was an early clonal event. No cases were positive for ROS1 by immunohistochemical staining, although 2 cases did show some nonspecific staining and were shown to be negative for ROS1 translocation by FISH. We conclude that although diffuse strong positive staining for ALK is likely to be highly specific for ALK rearrangement in CRC, both ALK and ROS1 immunohistochemical staining are very low-yield tests and difficult to justify in the routine clinical setting.

Aims Mesothelioma is a relatively uncommon but highly malignant neoplasm. Most patients die of disease within one year of diagnosis, but some have prolonged survival. Prospective identification of these longer-term survivors may help guide treatment. We therefore sought to investigate the role of p16 immunohistochemistry (IHC) both alone and in combination with other markers as a potential predictor of prolonged survival in mesothelioma. Methods and Results P16 IHC was performed on unselected pleural mesotheliomas biopsied from 1991 to 2014. 153 of 208 (74%) cases were p16-negative which significantly correlated with poor overall survival in both univariate (median survival 7.6 v 13.6 months; p = 0.001) and multivariate analysis (HR 1.632; 95% CI 1.103-2.415; p = 0.014). Other independent factors associated with prolonged survival included loss of expression of BAP1, and epithelioid morphology. We therefore further stratified patients based on these three independent prognostic variables and demonstrated an unusually prolonged survival in mesotheliomas which were epithelioid, BAP1 IHC negative and p16 IHC positive (12% of cases, median survival 31.7 months, p

Pancreatic cancer is molecularly diverse, with few effective therapies. Increased mutation burden and defective DNA repair are associated with response to immune checkpoint inhibitors in several other cancer types. We interrogated 385 pancreatic cancer genomes to define hypermutation and its causes. Mutational signatures inferring defects in DNA repair were enriched in those with the highest mutation burdens. Mismatch repair deficiency was identified in 1% of tumors harboring different mechanisms of somatic inactivation of MLH1 and MSH2. Defining mutation load in individual pancreatic cancers and the optimal assay for patient selection may inform clinical trial design for immunotherapy in pancreatic cancer.

Pancreatic ductal adenocarcinoma is a lethal cancer with fewer than 7% of patients surviving past 5 years. T-cell immunity has been linked to the exceptional outcome of the few long-term survivors, yet the relevant antigens remain unknown. Here we use genetic, immunohistochemical and transcriptional immunoprofiling, computational biophysics, and functional assays to identify T-cell antigens in long-term survivors of pancreatic cancer. Using whole-exome sequencing and in silico neoantigen prediction, we found that tumours with both the highest neoantigen number and the most abundant CD8+ T-cell infiltrates, but neither alone, stratified patients with the longest survival. Investigating the specific neoantigen qualities promoting T-cell activation in long-term survivors, we discovered that these individuals were enriched in neoantigen qualities defined by a fitness model, and neoantigens in the tumour antigen MUC16 (also known as CA125). A neoantigen quality fitness model conferring greater immunogenicity to neoantigens with differential presentation and homology to infectious disease-derived peptides identified long-term survivors in two independent datasets, whereas a neoantigen quantity model ascribing greater immunogenicity to increasing neoantigen number alone did not. We detected intratumoural and lasting circulating T-cell reactivity to both high-quality and MUC16 neoantigens in long-term survivors of pancreatic cancer, including clones with specificity to both high-quality neoantigens and predicted cross-reactive microbial epitopes, consistent with neoantigen molecular mimicry. Notably, we observed selective loss of high-quality and MUC16 neoantigenic clones on metastatic progression, suggesting neoantigen immunoediting. Our results identify neoantigens with unique qualities as T-cell targets in pancreatic ductal adenocarcinoma. More broadly, we identify neoantigen quality as a biomarker for immunogenic tumours that may guide the application of immunotherapies.

Mutations in EGFR guide treatment in non-small cell lung cancer (NSCLC). The most common mutations, exon 19 (delE746-A750) and exon 21 (L858R), can be identified by mutation specific immunohistochemistry (IHC). We present our prospective experience of universal reflex IHC and molecular testing in non-squamous NSCLC in the routine clinical setting.A total of 411 specimens from 332 patients were encountered over two years. Of these, 326 (98%) patients underwent EGFR IHC, 15 (5%) were positive for exon 19 deletions and 27 (8%) for exon 21 (L858R); 244 (73%) patients underwent molecular testing. Seventy-six mutations in 64 patients (19% of all patients encountered; 26% with sufficient material for testing) were identified. These comprised nine exon 18 (G719X) mutations, three also with exon 20 mutations; 24 exon 19 deletions, six also with exon 20 mutations; 23 exon 21 (L858R), three also with exon 20 mutations; and 8 exon 20 alone.All 15 exon 19 IHC positive patients were proven mutated (100% specificity, 63% sensitivity). Twenty-two of 27 exon 21 IHC positive cases were proven mutated while three patients had insufficient material for molecular testing (92% specificity, 96% sensitivity). The overall specificity and sensitivity of IHC for any EGFR mutation was 95% and 58%. Five patients initially thought to be wild type for EGFR but IHC positive underwent repeat molecular testing because of the discrepancy which confirmed the IHC result in three cases (60%).We conclude IHC is very specific but not sensitive. Whilst IHC cannot replace molecular testing, it is a useful adjunct which requires minimal tissue and identifies false negative molecular results which occurred in 5% of our patients with eventually confirmed EGFR mutations.

Germline mutations in the succinate dehydrogenase genes (SDHA, SDHB, SDHC, and SDHD) are established as causes of pheochromocytoma/paraganglioma, renal carcinoma, and gastrointestinal stromal tumor. It has recently been suggested that pituitary adenomas may also be a component of this syndrome. We sought to determine the incidence of SDH mutation in pituitary adenomas. We performed screening immunohistochemistry for SDHB and SDHA on all available pituitary adenomas resected at our institution from 1998 to 2012. In those patients with an abnormal pattern of staining, we then performed SDH mutation analysis on DNA extracted from paraffin-embedded tissue, fresh frozen tissue, and peripheral blood. One of 309 adenomas (0.3%) demonstrated an abnormal pattern of staining, a 30 mm prolactin-producing tumor from a 62-year-old man showing loss of staining for both SDHA and SDHB. Examination of paraffin-embedded and frozen tissues confirmed double-hit inactivating somatic SDHA mutations (c.725_736del and c.989_990insTA). Neither of these mutations was present in the germline. We conclude that, although pathogenic SDH mutation may occur in pituitary adenomas and can be identified by immunohistochemistry, it appears to be a very rare event and can occur in the absence of germline mutation. SDH-deficient pituitary adenomas may be larger and more likely to produce prolactin than other pituitary adenomas. Unless suggested by family history and physical examination, it is difficult to justify screening for SDH mutations in pituitary adenomas. Surveillance programs for patients with SDH mutation may be tailored to include the possibility of pituitary neoplasia; however, this is likely to be a low-yield strategy.

Summary The use of frozen section to assess resection margins intrao-peratively during pancreaticoduodenectomy facilitates further resection. However, it is unclear whether this actually improves patient survival. We reviewed the overall survival and resection margin status in consecutive pancreaticoduodenectomies performed for carcinoma. An R1 resection was defined as an incomplete excision (≤1 mm margin); R0(p) resection as complete excision without re-resection and R0(s) resection as an initially positive neck margin which was converted to R0 resection after re-resection. Between 2007 and 2012, 116 pancreatoduodenectomies were performed for adenocarcinoma; 101 (87%) underwent frozen section of the neck margin which was positive in 19 (19%). Sixteen of these patients had negative neck margins after re-excision but only seven patients had no other involved margins [true R0(s) resections]. Median survival for the R0(p), R0(s) and R1 groups were 29, 16, 23 months, respectively ( p = 0.049; R0(p) versus R0(s) p = 0.040). Intra-operative frozen section increased the overall R0 rate by 7% but this did not improve survival. Our findings question the clinical benefit of intraoperative margin assessment, particularly if re-excision cannot be performed easily and safely.

background: Adjuvant chemotherapy improves survival for patients with resected pancreatic cancer. Elderly patients are under-represented in Phase III clinical trials, and as a consequence the efficacy of adjuvant therapy in older patients with pancreatic cancer is not clear. We aimed to assess the use and efficacy of adjuvant chemotherapy in older patients with pancreatic cancer.\ud methods: We assessed a community cohort of 439 patients with a diagnosis of pancreatic ductal adenocarcinoma who underwent operative resection in centres associated with the Australian Pancreatic Cancer Genome Initiative.\ud results: The median age of the cohort was 67 years. Overall only 47% of all patients received adjuvant therapy. Patients who received adjuvant chemotherapy were predominantly younger, had later stage disease, more lymph node involvement and more evidence of perineural invasion than the group that did not receive adjuvant treatment. Overall, adjuvant chemotherapy was associated with prolonged survival (median 22.1 vs 15.8 months; P\ud conclusion: Patients aged greater than or equal to70 are less likely to receive adjuvant therapy although it is associated with improved outcome. Increased use of adjuvant therapy in older individuals is encouraged as they constitute a large proportion of patients with pancreatic cancer.

Immunohistochemistry has recently been validated for the detection of the BRAFV600E mutation across a range of tumor types. In colorectal carcinoma, the presence of the BRAFV600E mutation can be used to virtually exclude Lynch syndrome in mismatch repair-deficient tumors. In mismatch repair-proficient tumors, BRAFV600E mutation assessed by molecular methods has been proposed as a poor prognostic factor. We investigated whether combined BRAFV600E and mismatch repair status assessment by immunohistochemistry alone can be used as a prognostic marker in the routine clinical setting. We performed immunohistochemistry for BRAFV600E, MLH1, PMS2, MSH2, and MSH6 on 1426 consecutive unselected colorectal carcinomas. Ninety-one (6.4%) carcinomas were mismatch repair-proficient and BRAFV600E mutant, and these tumors demonstrated a significantly worse 5-year survival of 49.7% compared with mismatch repair-proficient BRAF wild type (74.1% of tumors, 65.4% survival), mismatch repair-deficient BRAFV600E mutant (12.9% of tumors, 70.1% survival), and mismatch repair-deficient BRAF wild type (6.6% of tumors, 73.6% survival). The poor survival was confirmed by univariate analysis (P

BRAFV600E mutation in microsatellite-unstable (MSI) colorectal carcinomas (CRCs) virtually excludes Lynch syndrome (LS). In microsatellite-stable (MSS) CRCs it predicts poor prognosis. We propose a universal CRC LS screening algorithm using concurrent reflex immunohistochemistry (IHC) for BRAFV600E and mismatch-repair (MMR) proteins. We compared BRAFV600E IHC with multiplex polymerase chain reaction (PCR) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry in 216 consecutive CRCs from 2011. Discordant cases were resolved with real-time PCR. BRAFV600E IHC was performed on 51 CRCs from the Australasian Colorectal Cancer Family Registry (ACCFR), which were fully characterized for BRAF mutation by allele-specific PCR, MMR status (MMR IHC and MSI), MLH1 promoter methylation, and germline MLH1 mutation. We then assessed MMR and BRAFV600E IHC on 1403 consecutive CRCs. By matrix-assisted laser desorption/ionization-time of flight mass spectrometry 15 cases did not yield a BRAF result, whereas 38/201 (19%) were positive. By IHC 45/216 (20%) were positive. Of the 7 discordant cases, real-time PCR confirmed the IHC result in 6. In the 51 CRCs from the ACCFR, IHC was concordant with allele-specific PCR in 50 cases. BRAFV600E and MSI IHC on 1403 CRCs demonstrated the following phenotypes: BRAF/MSS (1029 cases, 73%), BRAF/MSS (98, 7%), BRAF/MSI (183, 13%), and BRAF/MSI (93, 7%). All 11/1403 cancers associated with proven LS were BRAF/MSI. We conclude that BRAF IHC is highly concordant with 2 commonly used PCR-based BRAFV600E assays; it performed well in identifying MLH1 mutation carriers from the ACCFR and identified all cases of proven LS among the 1403 CRCs. Reflex BRAFV600E and MMR IHC are simple cheap tests that facilitate universal LS screening and identify the poor prognosis of the BRAFV600E-mutant MSS CRC phenotype.

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

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

Identification of BRAFV600E in thyroid neoplasia may be useful because it is specific for malignancy, connotes a worse prognosis, and is the target of novel therapies currently under investigation. Sanger sequencing is the ‘gold standard’ for mutation detection but is subject to sampling error and requires resources beyond many diagnostic pathology laboratories. In this study, we compared immunohistochemistry (IHC) using a BRAFV600E mutation-specific MAB to Sanger sequencing on DNA from formalin-fixed paraffin-embedded tissue, in a well-characterized cohort of 101 papillary thyroid carcinoma (PTC) patients. For all cases, an IHC result was available; however, five cases failed Sanger sequencing. Of the 96 cases with molecular data, 68 (71%) were BRAFV600E positive by IHC and 59 (61%) were BRAFV600E positive by sequencing. Eleven cases were discordant. One case was negative by IHC and initially positive by sequencing. Repeat sequencing of that sample and sequencing of a macrodissected sample were negative for BRAFV600E. Of ten cases positive by IHC but negative by sequencing on whole sections, repeat sequencing on macrodissected tissue confirmed the IHC result in seven cases (suggesting that these were false negatives of sequencing on whole sections). In three cases, repeat sequencing on recut tissue remained negative (including using massive parallel sequencing), but these cases demonstrated relatively low neoplastic cellularity. We conclude that IHC for BRAFV600E is more sensitive and specific than Sanger sequencing in the routine diagnostic setting and may represent the new gold standard for detection of BRAFV600E mutation in PTC.

Succinate dehydrogenase-deficient gastrointestinal stromal tumors (GISTs) demonstrate unique pathological and clinical features, including the absence of activating mutations of KIT and PDGFRA, and primary resistance to imatinib. They arise exclusively in the stomach and account for 5-7.5% of all adult stomach GISTs and the great majority of these tumors in childhood. Insulin-like growth factor 1 receptor (IGF1R) overexpression has been associated with wild-type and pediatric GISTs. We propose that IGF1R overexpression is a feature of succinate dehydrogenase-deficient GISTs as a group. We assessed succinate dehydrogenase complex subunit B (SDHB) and IGF1R expression by immunohistochemistry in eight known succinate dehydrogenase-deficient GISTs, three GISTs arising in the setting of neurofibromatosis type 1 syndrome and 40 unselected GISTs. Selected KIT and PDGFRA exons were amplified and sequenced from formalin-fixed paraffin-embedded tumor samples. All eight succinate dehydrogenase-deficient tumors were wild-type for KIT and PDGFRA, succinate dehydrogenase B negative and demonstrated IGF1R overexpression. The three neurofibromatosis-related tumors were succinate dehydrogenase B positive and IGF1R negative. Of the 40 unselected upper GISTs, five were wild-type for KIT and PDGFRA in the selected exons. Two of the wild-type GISTs were succinate dehydrogenase B negative and showed IGF1R overexpression and three were succinate dehydrogenase B positive and IGF1R negative. We conclude that IGF1R overexpression is a feature of succinate dehydrogenase deficient GIST as a group, rather than pediatric or wild-type GIST per se. Therefore, IGF1R inhibition represents a potential rational therapeutic approach in this recently recognized subgroup of GIST.

Do significant TFE3 gene rearrangements occur in succinate dehydrogenase-deficient renal cell carcinoma? Borderline FISH results should be interpreted with caution

Germline succinate dehydrogenase B (SDHB) mutation causes pheochromocytoma/paraganglioma syndrome type 4 (PGL4). PGL4 is characterized by pheochromocytoma and paraganglioma, type 2 (SDHB negative) gastrointestinal stromal tumors and renal tumors, which are usually classified as carcinoma. We report 4 kindreds with 5 PGL4-associated renal tumors. Four of the tumors occurred before the age of 30 years, 4 were in the left kidney, 3 were in female patients, and 4 demonstrated consistent but previously unrecognized morphology. The tumors were composed of cuboidal cells with bubbly eosinophilic cytoplasm and indistinct cell borders. Many of the cells displayed distinctive cytoplasmic inclusions, which were vacuolated or contained eosinophilic fluid-like material. The cells were arranged in solid nests or in tubules surrounding central spaces. The tumors were well circumscribed or lobulated and frequently showed cystic change. Benign tubules or glomeruli were often entrapped at the edges of the tumors. The fifth tumor lacked these features but displayed sarcomatoid dedifferentiation. Immunohistochemistry for SDHB was completely negative in all 4 available tumors. Death from metastatic disease occurred in the patient with dedifferentiated tumor 1 year after diagnosis, whereas the other 4 tumors were cured by local excision alone (mean follow-up, 11 y; range, 2 to 30 y). We conclude that morphology supported by negative immunohistochemistry for SDHB can be used to identify kindreds with germline SDHB mutations (PGL4 syndrome) presenting with this unique type of renal tumor. These renal tumors appear to have a good prognosis after complete excision unless there is sarcomatoid dedifferentiation.

Background The role of the B-isoform of the Raf kinase (BRAF) mutation BRAFV600E as an independent prognostic factor in papillary thyroid cancer (PTC) remains controversial. Some studies suggest that tumors containing BRAFV600E have decreased radioiodine avidity and present a greater risk of nodal recurrence and distant metastases. Methods Paraffin-embedded specimens from consecutive patients who underwent surgery for PTC before 2003 were independently reviewed by an endocrine pathologist. DNA was extracted, amplified by polymerase chain reaction, and the presence of the BRAFV600E mutation was determined by restriction digest. Tumor characteristics and long-term disease outcomes were analyzed according to BRAFV600E status. Results BRAFV600E was identified in 60 (59%) of 101 patients. At a median follow-up of 106 months, the overall disease-free survival was 78%. Clinically evident nodal recurrence occurred in 11% of BRAFV600E-positive patients, and all patients required lateral neck dissection (P = .02). In contrast, subclinical nodal recurrence occurred in 7% of BRAFV600E-negative patients, and all recurrences were successfully ablated with radioactive iodine. There was a trend toward poorer disease-free survival among patients with stage III/IV PTC and BRAFV600E mutation (P = .08). All 5 disease-related deaths occurred in patients with BRAFV600E-positive primary tumors (P = .06). Conclusion The BRAFV600E mutation in PTC is associated with an increased risk of palpable nodal recurrence and the need for reoperative surgery.

Pancreatic cancer is the third leading cause of cancer-related deaths, characterised by poor survival, marked molecular heterogeneity and high intrinsic and acquired chemoresistance. Only 10–20% of pancreatic cancer patients present with surgically resectable disease and even then, 80% die within 5 years. Our increasing understanding of the genomic heterogeneity of cancer suggests that the failure of definitive clinical trials to demonstrate efficacy in the majority of cases is likely due to the low proportion of responsive molecular subtypes. As a consequence, novel treatment strategies to approach this disease are urgently needed. Significant developments in the field of precision oncology have led to increasing molecular stratification of cancers into subtypes, where individual cancers are selected for optimal therapy depending on their molecular or genomic fingerprint. This review provides an overview of the current status of clinically used and emerging treatment strategies, and discusses the advances in and the potential for the implementation of precision medicine in this highly lethal malignancy, for which there are currently no curative systemic therapies.

Introduction Pancreatic cancer remains a highly lethal cancer where response is limited by both intrinsic and acquired chemoresistance. Understanding resistance mechanisms may therefore lead to improved therapeutic strategies. We have recently defined specific molecular subgroups of pancreatic cancer associated with pre-clinical and clinical response to select tailored treatment strategies.1–3 Material and methods Using robust patient-derived xenografts (PDXs) of pancreatic cancer, here we generated novel in vivo models for the study of intrinsic and acquired chemoresistance mechanisms to clinically-used agents, gemcitabine, mitomycin C, and cisplatin. Here, we used whole genome sequencing (WGS) and microarray analysis to compare gemcitabine-resistant and gemcitabine-sensitive pancreatic tumours to identify relevant resistance mechanisms. Results and discussions Integrative analysis of WGS and microarray profiling of gemcitabine-resistant tumours revealed complex but potentially targetable resistance mechanisms, including increased DNA repair through activation of PARP1, MCM genes and RRM1, and changes within the tumour microenvironment. Importantly, acquired resistance to gemcitabine was effectively reversed by a novel PARP inhibitor, rucaparib, indicating that combination therapy involving this low toxicity agent may be useful in treating gemcitabine-resistant tumours defined by high genomic instability. Similarly, modulation of key components of the tumour microenvironment with fasudil, as recently achieved,2 provided another effective way of reversing gemcitabine resistance. Conclusion Significance our findings demonstrate the promise of patient-derived xenograft models for the study of in vivo mechanisms of chemotherapy resistance and efficacy testing of novel agents for the treatment of human pancreatic cancer. References Waddell N, et al. Nature (2015) 518(7540):495 Vennin C, et al. Science Translational Medicine (2017) pii: eaai8504 Chou A, et al. Gut (2017) pii: gutjnl-2017-315144 [epub ahead of print]