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Altered RNA expression of repetitive sequences and retrotransposition are frequently seen in colorectal cancer, implicating a functional importance of repeat activity in cancer progression. We show the nucleoside reverse transcriptase inhibitor 3TC targets activities of these repeat elements in colorectal cancer preclinical models with a preferential effect in p53-mutant cell lines linked with direct binding of p53 to repeat elements. We translate these findings to a human phase II trial of single-agent 3TC treatment in metastatic colorectal cancer with demonstration of clinical benefit in 9 of 32 patients. Analysis of 3TC effects on colorectal cancer tumorspheres demonstrates accumulation of immunogenic RNA:DNA hybrids linked with induction of interferon response genes and DNA damage response. Epigenetic and DNA-damaging agents induce repeat RNAs and have enhanced cytotoxicity with 3TC. These findings identify a vulnerability in colorectal cancer by targeting the viral mimicry of repeat elements.Significance:Colorectal cancers express abundant repeat elements that have a viral-like life cycle that can be therapeutically targeted with nucleoside reverse transcriptase inhibitors (NRTI) commonly used for viral diseases. NRTIs induce DNA damage and interferon response that provide a new anticancer therapeutic strategy.This article is highlighted in the In This Issue feature, p. 1397

Supplementary Figure from Reverse Transcriptase Inhibition Disrupts Repeat Element Life Cycle in Colorectal Cancer

Supplementary Data from Reverse Transcriptase Inhibition Disrupts Repeat Element Life Cycle in Colorectal Cancer

Figure S3. Illustration of modeling and correcting for dependence of each marker through the expression level of housekeeping genes.

Supplemental Methods, Tables S1-2, Table Legends and Figure Legends Table S1.List of qRT-PCR primers used for epithelial, mesenchymal, inducer and housekeeping genes. Table S2. RNA-ISH quantitation of expression scores used in Figure 5.

Purpose: The androgen receptor (AR) mRNA splice variant AR-V7 has emerged as a predictive biomarker for response to AR-targeted therapies. There are currently no commercially available assays to detect AR splice variants. The branched chain RNA in situ hybridization (ISH) platform enables the highly sensitive detection of RNA transcripts in formalin-fixed, paraffin-embedded (FFPE) tissues.Experimental design: We designed a branched chain RNA ISH probe to target the unique cryptic exon CE3 of AR-V7 using multiple tiling probes. This automated ISH assay was applied to tumor tissue from two distinct clinical cohorts that we hypothesized would differ in AR-V7 status.Results: We detected AR-V7 in all tumor samples from men with metastatic castration-resistant prostate cancer with tissue obtained after disease progression despite at least one subsequent line of hormonal therapy (abiraterone, enzalutamide, or bicalutamide; n = 12). We detected AR-V7 in just one tumor from men who had undergone prostatectomy for localized adenocarcinoma (n = 30; Gleason 4 + 5 = 9 in the AR-V7–positive sample). Given the apparent distinction between the above groups by AR-V7 signal, we analyzed pretreatment AR-V7 status as a predictive and prognostic biomarker in men with treatment-naïve metastatic disease. Patients with metastases but without detectable AR-V7 RNA at baseline had significantly longer overall survival (log-rank P = 0.044) and a trend toward superior progression-free survival (log-rank P = 0.055).Conclusions: Within an institutional cohort, the RNA ISH assay identified AR-V7 within FFPE tissue and may have prognostic value in metastatic castration-sensitive prostate cancer. These preliminary findings warrant further study in larger cohorts. Clin Cancer Res; 23(2); 363–9. ©2016 AACR.

Control cell line validations.

Supplementary Figure Legend

Altered RNA expression of repetitive sequences and retrotransposition are frequently seen in colorectal cancer, implicating a functional importance of repeat activity in cancer progression. We show the nucleoside reverse transcriptase inhibitor 3TC targets activities of these repeat elements in colorectal cancer preclinical models with a preferential effect in p53-mutant cell lines linked with direct binding of p53 to repeat elements. We translate these findings to a human phase II trial of single-agent 3TC treatment in metastatic colorectal cancer with demonstration of clinical benefit in 9 of 32 patients. Analysis of 3TC effects on colorectal cancer tumorspheres demonstrates accumulation of immunogenic RNA:DNA hybrids linked with induction of interferon response genes and DNA damage response. Epigenetic and DNA-damaging agents induce repeat RNAs and have enhanced cytotoxicity with 3TC. These findings identify a vulnerability in colorectal cancer by targeting the viral mimicry of repeat elements.Significance:Colorectal cancers express abundant repeat elements that have a viral-like life cycle that can be therapeutically targeted with nucleoside reverse transcriptase inhibitors (NRTI) commonly used for viral diseases. NRTIs induce DNA damage and interferon response that provide a new anticancer therapeutic strategy.This article is highlighted in the In This Issue feature, p. 1397

Improved biomarkers are needed for early cancer detection, risk stratification, treatment selection, and monitoring treatment response. While proteins can be useful blood-based biomarkers, many have limited sensitivity or specificity for these applications. Long INterspersed Element-1 (LINE-1, L1) open reading frame 1 protein (ORF1p) is a transposable element protein overexpressed in carcinomas and high-risk precursors during carcinogenesis with negligible detectable expression in corresponding normal tissues, suggesting ORF1p could be a highly specific cancer biomarker. To explore the potential of ORF1p as a blood-based biomarker, we engineered ultrasensitive digital immunoassays that detect mid-attomolar (10-17M) ORF1p concentrations in patient plasma samples across multiple cancers with high specificity. Plasma ORF1p shows promise for early detection of ovarian cancer, improves diagnostic performance in a multi-analyte panel, and provides early therapeutic response monitoring in gastric and esophageal cancers. Together, these observations nominate ORF1p as a multi-cancer biomarker with potential utility for disease detection and monitoring.Statement of SignificanceLINE-1 ORF1p transposon protein is pervasively expressed in many cancers and a highly specific biomarker of multiple common, lethal carcinomas and their high-risk precursors in tissue and blood. Ultrasensitive ORF1p assays from as little as 25 μL plasma are novel, rapid, cost-effective tools in cancer detection and monitoring.

Inflammatory pseudotumor is a term used to designate inflammation-rich tumefactive lesions. Following the exclusion of specific entities such as IgG4-related disease and other neoplastic entities previously included in this entity, the majority of hepatic pseudotumors show a prominent fibrohistiocytic inflammatory reaction and have been previously categorized as fibrohistiocytic variant of hepatic pseudotumor (FHVHPT). The goal of this study was to examine the clinical, radiologic, histologic, and etiologic aspects of this entity. After excluding neoplastic diseases, we identified 30 patients with FHVHPT from 3 institutions between 2009 and 2019. We extracted demographic and clinical data, liver function tests as well as culture results and radiologic information. Hematoxylin and eosin-stained slides were reviewed for pattern of inflammation as well as its cellular composition. Immunohistochemistry for IgG4 and IgG was performed in all cases. The mean age of the 30 lesions characterized as FHVHPT was 56 years (range: 23 to 79 y). Nineteen patients showed solitary lesions; 11 were multiple. The mean size of the lesion was 3.8 cm (range: 1 to 7.5 cm). On imaging, a neoplastic process or metastasis was the leading diagnostic consideration (n=15, 50%). The most common symptom was abdominal pain (n=14/30); 8 patients presented with symptoms compatible with an infectious process, including fever. The inflammatory infiltrate was dominated by lymphocytes and plasma cells, and in most cases, a prominent histiocytic infiltrate (22/30). Neutrophils were identified in 12 cases, with microabscess noted in 8. Storiform pattern of fibrosis was seen in 14/30 cases; obliterative phlebitis was not identified. Culture identified a microorganism in 4 of 9 cases evaluated. The mean IgG4 count was 9.3 per HPF (range: 0 to 51) with 9 of the 26 (35%) biopsies showing >10 IgG4 positive plasma cells per HPF. The mean IgG4 to IgG ratio was 8% (range: 8% to 46%). A hepatectomy was performed in 4 cases. On broad spectrum antibiotics (n=14) the lesions either resolved or decreased in size. Eight patients did not receive specific therapy, nevertheless, the lesion(s) resolved spontaneously in 6 cases, remained stable or decreased in size in 2 cases. Notably, none of these patients showed evidence of a hepatic recurrence. FHVHPT, a tumefactive lesion that mimics hepatic neoplasia, is histologically characterized by a fibrohistiocytic infiltrate. In the majority of patients FHVHPT represents the organizing phase of hepatic abscess and can be successfully managed with antibiotic therapy.

Immune checkpoint inhibitors (ICIs) are becoming the standard of care treatment for many malignancies. ICIs are associated with a unique spectrum of immune-related adverse events (irAEs) due to the blockade of inhibitory signals of immune activation. The main objective of this study is to review the characteristic histological features and pathologic differential diagnosis of ICI-related injury of the gastrointestinal (GI) tract and hepatobiliary system. Diarrhea and hepatitis are some of the more common irAEs. The pathology of ICI-related injury is both diverse and largely non-specific, with various site-specific findings to become familiar with. Early and accurate recognition of an irAE is important in order to initiate proper management. This generally includes withdrawal of ICI therapy, and possibly the administration of a corticosteroid or other immunosuppressives depending on the severity of injury.

Immunoglobulin 4-related disease (IgG4-RD) is a multisystem disease, characterised by tumefactive lesions and a swift response to immunosuppressive therapy. Although elevated serum and tissue IgG4 are characteristic, T cells appear to be the primary driver of this immunologically mediated disease. The overarching goal was to examine the role of immunomodulatory cells in IgG4-RD.Biopsies from patients with IgG4-RD (n = 39) and mimics of this disease (n = 78) were evaluated for IgG4, IgG, CD8, programmed cell death ligand 1 (PD-L1) and a subset (n = 18) evaluated for CD4, purine rich box 1 (PU.1), forkhead box protein 3 (FoxP3), PD-L1, programmed cell death 1 (PD-1), indoleamine 2,3-dioxygenase 1 (IDO1) and lymphocyte-activation gene 3 (LAG3). Data pertaining to demographics and laboratory findings at baseline evaluation was extracted from electronic medical records. When compared to mimics, IgG4-RD showed increased numbers of PD-L1- (P = 0.0001), PD-1- (P = 0.001), IDO1- (P = 0.03), LAG3- (P = 0.04) and FoxP3- (P = 0.04)-positive immune cells. The PD-L1-positive cells were enriched within aggregates of CD4 and CD8-positive T cells. Thirty-one of 39 (80%) IgG4-RD cases showed greater than five PD-L1-positive cells per high-power field (HPF), while four of 78 (5%) mimics of this disease exceeded this cut-point. In IgG4-RD, PD-L1-positive macrophages correlated with PD-1- (P = 0.002), LAG3- (P = 0.001) and IDO1-positive cells (P = 0.001); a-positive correlation was also noted between IgG4/IgG ratio and PD-L1-, PD-1- and IDO1-positive cells.IgG4-RD shows expansion of mechanisms that maintain peripheral tolerance. The spatial and temporal relationship between T cells and the PD-L1-PD-1 axis and the up-regulation of multiple immunomodulatory proteins suggests that these immunoregulatory mechanisms play a significant role in IgG4-RD.

AimsThe histological distinction of intrahepatic cholangiocarcinoma (ICC) from metastatic adenocarcinoma remains a challenge. The primary goal was to evaluate the diagnostic value of morphology and albumin expression in the diagnosis of ICC.MethodsWe evaluated morphological patterns in 120 ICCs and 677 non-hepatic adenocarcinomas and performed in situ hybridisation (ISH) stain for albumin in the former cohort (retrospective cohort). We also identified 119 samples from primary and metastatic lesions, the validation cohort, in which albumin ISH was performed as part of the diagnostic workup. Targeted sequencing was performed on selected cases. We also mined existing expression profiling data including cases from The Cancer Genome Atlas (TCGA) (41 760 unique samples).ResultsIn the retrospective cohort, 45% of ICCs and IDH mutations and FGFR2 fusions) were identified in 31% of ICCs (10 of 32). An analysis of the TCGA data validated the specificity of the albumin assay.ConclusionsThe cholangiolar pattern and albumin RNA ISH distinguishes ICC from metastatic adenocarcinoma with high specificity. Given the high prevalence of targetable mutations in ICC, albumin RNA ISH is an essential component in the workup of tumours of uncertain origin. A specific diagnosis of ICC could trigger molecular testing and uncover targetable genetic alterations.

Purpose: This study evaluated the potential role of immune cells and molecules in the pathogenesis and clinical course of pancreatic neuroendocrine tumors (PanNET). Experimental Design: Surgically resected PanNETs (N = 104) were immunohistochemically analyzed for Ki67 index, mitotic rate, macrophage, CD4+ cells, and CD8+ T-cell infiltration, as well as HLA class I, PD-L1, and B7-H3 expression. Results were correlated with clinicopathologic characteristics as well as with disease-free (DFS) and disease-specific (DSS) survival. Results: The median age of the 57 WHO grade 1 and 47 WHO grade 2 patients was 55 years. High intratumoral CD8+ T-cell infiltration correlated with prolonged DFS (P = 0.05), especially when the number of tumor-associated macrophages (TAM) was low. In contrast, high peritumoral CD4+ cell and TAM infiltration was associated with a worse DFS and DSS. PD-L1 and B7-H3 were expressed in 53% and 78% PanNETs, respectively. HLA class I expression was defective in about 70% PanNETs. HLA-A expression correlated with favorable DSS in PD-L1–negative tumors (P = 0.02). TAM infiltration (P = 0.02), WHO grade (P = 0.04), T stage (P = 0.01), and lymph node positivity (P = 0.04) were independent predictors of DFS. TAM infiltration (P = 0.026) and T stage (P = 0.012) continued to be predictors of DFS in WHO grade 1 PanNET patients. TAM infiltration was the sole independent predictor of DSS for WHO grade 1 and 2 patients (P = 0.02). Therefore, this biomarker may contribute to identifying WHO grade 1 patients with poor prognosis. Conclusions: TAM infiltration appears to be the most informative prognostic biomarker in PanNET. It may represent a useful immunotherapeutic target in patients with PanNET.

Aims The pathological diagnosis of IgG4-related disease (IgG4-RD) relies on histology, IgG4-positive cells, and an increased IgG4/IgG ratio. Small biopsies from patients with a presumptive diagnosis of IgG4-RD often fail to meet consensus histological criteria. The aims of this study were to evaluate consecutive small biopsies from patients with a presumptive diagnosis of IgG4-RD, and to assess the significance of the pathological findings. Methods and results We evaluated 55 small biopsies from patients with a presumptive diagnosis of IgG4-RD. The retrospective cohort comprised 71 patients with IgG4-RD and 57 mimics. We performed immunohistochemistry (IHC) and in-situ hybridisation (ISH) for IgG4 and IgG. Twenty-six patients from the prospective cohort met the histological criteria for IgG4-RD (definite); 29 patients lacked one or more pathological features (borderline). Twenty biopsies (36%) lacked both storiform fibrosis and obliterative phlebitis, and nine (16%) lacked an increase in the number of IgG4-positive plasma cells. Ninety-three per cent of patients showed an IgG4/total IgG ratio of >40% (>30% by ISH). There were no differences in the incidence of multiorgan disease (P = 0.9), serum IgG4 levels (P = 0.6) and response to therapy between the definite and borderline groups. A strong correlation (Pearson 0.77) between the IHC and ISH platforms was noted with regard to the IgG4/total IgG ratio. Conclusion Patients with a presumptive diagnosis of IgG4-RD but lacking the characteristic pathological features of this disease appear to be clinically similar to those who meet the current pathological criteria. An elevated IgG4/total IgG ratio is the most sensitive pathological feature, and ISH provides a robust quantification platform. We recommend evaluating tumefactive lymphoplasmacytic infiltrates with an increased IgG4/IgG ratio, regardless of histological features, for IgG4-RD.

SUMMARY It has been posited that anti-tumoral innate activation is driven by derepression of endogenous repeats. We compared RNA sequencing protocols to assess repeat transcriptomes in The Cancer Genome Atlas (TCGA). Although poly(A) selection efficiently detects coding genes, most non-coding genes, and limited subsets of repeats, it fails to capture overall repeat expression and co-expression. Alternatively, total RNA expression reveals distinct repeat co-expression subgroups and delivers greater dynamic changes, implying they may serve as better biomarkers of clinical outcomes. We show that endogenous retrovirus expression predicts immunotherapy response better than conventional immune signatures in one cohort yet is not predictive in another. Moreover, we find that global repeat derepression, including the HSATII satellite repeat, correlates with an immunosuppressive phenotype in colorectal and pancreatic tumors and validate in situ. In conclusion, we stress the importance of analyzing the full spectrum of repeat transcription to decode their role in tumor immunity., In Brief Solovyov et al. compare protocols used in tumor transcriptional profiling. They show the most widely used poly(A) protocol fails to detect several classes of repeat RNAs. In contrast, repeat expression in total RNA sequencing can correlate with the cancer-immune phenotypes and patient responses to immunotherapy.

// Trevor J. Grant 1 , Anita K. Mehta 1 , Anamika Gupta 1 , Ahmad A.D. Sharif 2 , Kshitij S. Arora 3 , Vikram Deshpande 3 , David T. Ting 3 , Nabeel Bardeesy 3 , Neil J. Ganem 1 , Alexander Hergovich 2 and Anurag Singh 1 1 Department of Pharmacology and Experimental Therapeutics, Center for Cancer Research, Boston University Graduate School of Medicine, Boston, MA, USA 2 University College London, Cancer Institute, London, United Kingdom 3 Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA, USA Correspondence to: Anurag Singh, email: asingh3@bu.edu Keywords: KRAS, oncogene dependency, Hippo pathway, pancreatic cancer, kinase Received: July 12, 2017 Accepted: August 27, 2017 Published: September 11, 2017 ABSTRACT Pancreatic ductal adenocarcinomas (PDACs) are highly aggressive malignancies, associated with poor clinical prognosis and limited therapeutic options. Oncogenic KRAS mutations are found in over 90% of PDACs, playing a central role in tumor progression. Global gene expression profiling of PDAC reveals 3-4 major molecular subtypes with distinct phenotypic traits and pharmacological vulnerabilities, including variations in oncogenic KRAS pathway dependencies. PDAC cell lines of the aberrantly differentiated endocrine exocrine (ADEX) subtype are robustly KRAS-dependent for survival. The KRAS gene is located on chromosome 12p11-12p12, a region amplified in 5-10% of primary PDACs. Within this amplicon, we identified co-amplification of KRAS with the STK38L gene in a subset of primary human PDACs and PDAC cell lines. Therefore, we determined whether PDAC cell lines are dependent on STK38L expression for proliferation and viability. STK38L encodes a serine/threonine kinase, which shares homology with Hippo pathway kinases LATS1/2. We show that STK38L expression is elevated in a subset of primary PDACs and PDAC cell lines displaying ADEX subtype characteristics, including overexpression of mutant KRAS. RNAi-mediated depletion of STK38L in a subset of ADEX subtype cell lines inhibits cellular proliferation and induces apoptosis. Concomitant with these effects, STK38L depletion causes increased expression of the LATS2 kinase and the cell cycle regulator p21. LATS2 depletion partially rescues the cytostatic and cytotoxic effects of STK38L depletion. Lastly, high STK38L mRNA expression is associated with decreased overall patient survival in PDACs. Collectively, our findings implicate STK38L as a candidate targetable vulnerability in a subset of molecularly-defined PDACs.

Early detection strategies for pancreatic ductal adenocarcinoma are needed. This article describes a high‐sensitivity platform for detection of epithelial cells shed from preneoplastic lesions at a high risk of becoming malignant and a combination of technologies that could be used for early detection of disease in high‐risk patients., Background. Recent work has demonstrated early shedding of circulating epithelial cells (CECs) from premalignant intraductal papillary mucinous neoplasms (IPMNs). However, the potential use of CECs as a “liquid biopsy” for patients with IPMNs has been limited by antigen dependence of CEC isolation devices and the lack of robust detection biomarkers across CEC phenotypes. Materials and Methods. We utilized a negative depletion microfluidic platform to purify CECs from contaminating leukocytes and coupled this platform with immunofluorescence, RNA in situ hybridization, and RNA sequencing (RNA‐seq) detection and enumeration. Results. Using established protein (EpCAM, cytokeratins) and novel noncoding RNA (HSATII, cytokeratins) biomarkers, we detected CECs in 88% of patients bearing IPMN lesions. RNA‐seq analysis for MUC genes confirm the likely origin of these CECs from pancreatic lesions. Conclusion. Our findings increase the sensitivity of detection of these cells and therefore could have clinical implications for cancer risk stratification. Implications for Practice. This work describes a high‐sensitivity platform for detection of epithelial cells shed from preneoplastic lesions at high risk of malignant transformation. Further research efforts are underway to define the transcriptional programs that might allow discrimination between circulating cells released from tumors that will become malignant and cells released from tumors that will not. After further refinement, this combination of technologies could be deployed for monitoring and early detection of patients at high risk for developing new or recurrent pancreatic malignancies.

The large number of manual steps involved in processing tissue creates many opportunities for specimen cross-contamination and carry-over defects, which may have serious medicolegal ramifications.1 In one study extraneous tissue was identified in 0.6% of slides, causing severe diagnostic difficulties in 0.4% of cases.1 As these events are not fully preventable, a quick, reliable, easily accessible method to resolve suspected contamination events is crucial. This article is protected by copyright. All rights reserved.

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

The distinction of atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) from its benign counterpart, lipoma, may represent a challenge. MDM2 DNA amplification is used as the gold standard as MDM2 immunohistochemistry lacks specificity and sensitivity. Herein, we investigate the diagnostic utility of MDM2 RNA in situ hybridization (RNA-ISH) and compare the test with MDM2 immunohistochemistry and MDM2 DNA fluorescence in situ hybridization (FISH) in benign and malignant lipomatous neoplasms. We evaluated 109 neoplasms including 27 lipomas, 25 spindle cell lipomas, 32 ALTs/WDLs, and 25 dedifferentiated liposarcomas (DDL). The validation cohort included 14 lipoma-like neoplasms that lacked unequivocal features of ALT/WDL and in which MDM2 immunohistochemistry was either equivocal, negative or falsely positive. Immunohistochemistry, automated RNA-ISH and DNA-FISH for MDM2 were performed. Tumors with diffuse nuclear staining or >50 dots per cell on RNA-ISH were considered positive. All lipomas and lipoma variants were negative for RNA-ISH while all ALTs/WDLs and DDLs were positive. Eighty percent (24/30) and 92% (22/24) of ALTs/WDLs and DDLs were positive for MDM2 immunohistochemistry. Lipomas and its variants were negative for MDM2 amplification; 92% and 100% of ALTs/WDLs and DDLs showed MDM2 DNA amplification. The mean percentage of ALT/WDL tumor cells showing MDM2 RNA-ISH positivity was 73% compared with 24% on MDM2 immunohistochemistry. RNA-ISH correctly classified all 10 ALTs/WDLs and all 4 lipomas in the validation cohort. The performance of MDM2 RNA-ISH and MDM2 DNA-FISH are equivalent. MDM2 RNA-ISH can be of diagnostic value in histologically challenging lipomatous neoplasms. The automated MDM2 RNA-ISH assay should allow for more widespread use of MDM2 testing and for a more sensitive and specific diagnosis of ALT/WDL.

INTRODUCTION: The expression of LGR5, a known stem cell marker, is poorly understood in Barrett's esophagus (BE) and related neoplasia. The aim of this study was to evaluate LGR5 in BE and related neoplasia and to evaluate its utility as a potential biomarker of progression to advanced neoplasia. METHODS: We evaluated total 137 patients, including 119 with BE and 18 with normal gastroesophageal mucosa for expression of LGR5 using RNA in situ hybridization; this also included 28 progressors and 30 nonprogressors. The LGR5 stain was evaluated using 1 qualitative and 2 quantitative parameters, using manual and automated platforms. RESULTS: Surface LGR5 expression was mainly seen in high-grade dysplasia (12/18) compared with low-grade dysplasia (1/8) and nondysplastic BE (0/17) (P < 0.0001). In contrast to nondysplastic BE, low- and high-grade dysplasia showed a higher percentage of mean number of LGR5-positive crypts per patient (P < 0.0001) and an increase in the mean number of LGR5 transcripts per cell (P < 0.0001). The mean percentage of LGR5-positive crypts per patient and the mean number of LGR5 transcripts per cell were also significantly higher in nondysplastic BE from progressor compared with nonprogressor (P < 0.0001, P = 0.014). The sensitivity and specificity of LGR5 for distinguishing progressor from nonprogressor were 50% and 87%, respectively. DISCUSSION: BE-related advanced neoplasia shows an expansion of the LGR5-positive cellular compartment, supporting its role as a stem cell marker in this disease. Quantitative LGR5 expression and surface epithelial reactivity are novel biomarkers of increased risk of progression to advanced neoplasia in BE.

High-risk human papillomavirus (HR-HPV) is a major etiologic agent in a subset of head and neck squamous cell carcinomas (HNSCCs), and its recognition has prognostic and predictive implications. The availability of a sensitive and specific test to assess HR-HPV status is limited. We evaluate an RNA in situ hybridization (ISH) method using branch chain technology to detect HR-HPV and compare its results with DNA ISH, p16 immunohistochemistry, and polymerase chain reaction (PCR). Tissue sections from 54 patients were stained with a manual RNA ISH assay (ViewRNA), which detects 14 HR-HPV types, an automated DNA ISH assay, and p16 immunohistochemistry. Most cases (83%, n=45) were also tested on an automated platform for 14 HR-HPV types and 1 limited to HPV 16/18. PCR was performed in all cases and was successful in 93% (n=50). The RNA ISH assay produced results in 96% of the cases with strong signals and was easily interpreted. HR-HPV was detected in more cases (63%, n=34) by RNA ISH than by DNA ISH (39%, n=21). Compared with PCR, both ISH platforms were 94% specific. RNA ISH was more sensitive (91%) than DNA ISH (65%), and RNA ISH correlated more strongly with p16 immunostaining. HPV 16 represented 89% of HR-HPV detected. The cocktail HPV 16/18 platform was concordant with the pooled HR-HPV assay in all expected cases. The automated assay demonstrated high concordance (96%) with the manual version, showed decreased background, and should allow for easy implementation into the workflow of the diagnostic pathology laboratory.

Metastasis following surgical resection is a leading cause of mortality in pancreatic ductal adenocarcinoma. Epithelial-mesenchymal transition is thought to play an important role in metastasis, although its clinical relevance in metastasis remains uncertain. We evaluated a panel of RNA in-situ hybridization probes for epithelial-mesenchymal transition-related genes expressed in circulating tumor cells. We assessed the predictive value of this panel for metastasis in pancreatic ductal adenocarcinoma and, to determine if the phenotype is generalizable between cancers, in colonic adenocarcinoma. One hundred fifty-eight pancreatic ductal adenocarcinomas and 205 colonic adenocarcinomas were classified as epithelial or quasimesenchymal phenotype using dual colorimetric RNA-in-situ hybridization. SMAD4 expression on pancreatic ductal adenocarcinomas was assessed by immunohistochemistry. Pancreatic ductal adenocarcinomas with quasimesenchymal phenotype had a significantly shorter disease-specific survival (P = 0.031) and metastasis-free survival (P = 0.0001) than those with an epithelial phenotype. Pancreatic ductal adenocarcinomas with SMAD4 loss also had lower disease-specific survival (P = 0.041) and metastasis-free survival (P = 0.001) than those with intact SMAD4. However, the quasimesenchymal phenotype proved a more robust predictor of metastases—area under the curve for quasimesenchymal = 0.8; SMAD4 = 0.6. The quasimesenchymal phenotype also predicted metastasis-free survival (P = 0.004) in colonic adenocarcinoma. Epithelial-mesenchymal transition defined two phenotypes with distinct metastatic capabilities—epithelial phenotype tumors with predominantly organ-confined disease and quasimesenchymal phenotype with high risk of metastatic disease in two epithelial malignancies. Collectively, this work validates the relevance of epithelial-mesenchymal transition in human gastrointestinal tumors.

Single cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single cell phenotypes have not been well defined. Combining single cell RNA and protein analytics in pancreatic cancer (PDAC) model systems, we demonstrated the role of stromal fibroblasts in shaping PDAC single cell heterogeneity towards invasive (EMT) and proliferative (PRO) phenotypes. Using highcontent digital imaging of RNA in situ hybridization in 195 tumors, we observed these EMT and PRO subpopulations in 319,626 individual cancer cells. Interestingly, we found these EMT and PRO subpopulations form distinct tumor gland “units” associated with differences in stromal abundance and patient survival. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

BACKGROUND Current immunohistochemical and in situ hybridization (ISH) assays are generally inconclusive for clonality unless plasmacytic differentiation is present. This study examined a series of cytology specimens and explored the ability of a branched-chain RNA (bRNA) ISH assay for immunoglobulin κ constant (IGKC) and immunoglobulin λ constant (IGLC) to detect a clonal population of B lymphocytes. METHODS Pathology databases were used to identify fine-needle aspiration biopsies (n = 28) and exfoliative cytology samples (n = 20). Demographic, flow cytometry, and excision biopsy results were recorded. bRNA ISH was performed on the Leica Bond platform with the following probes: IGKC, IGLC, immunoglobulin λ-like polypeptide 5 (IGLL5), and a housekeeping gene (HKG). RESULTS The bRNA ISH assay was validated with 30 surgical biopsies. On bRNA ISH, a clonal B-cell population (light-chain ratio > 10:1) was detected in 22 of 28 cases with a final diagnosis of lymphoma. In 2 cases, a κ predominance was present, although the ratio was

Circulating signals of drug resistance Cancer drugs often lose their effectiveness because tumors acquire genetic changes that confer drug resistance. Ideally, patients would be switched to a different drug before tumor growth resumes, but this requires early knowledge of how resistance arose. Miyamoto et al. have developed a non-invasive method to spot resistance by sequencing RNA transcripts in single circulating tumor cells (CTCs) (see the Perspective by Nanus and Giannakakou). For example, in prostate cancer patients, drug resistance was triggered by activation of the Wnt signaling pathway. But CTCs are rare and fragile, and the technology needs further development before it is used in clinical practice. Science , this issue p. 1351 ; see also p. 1283

Intrahepatic cholangiocarcinoma (ICC) often is a diagnosis determined by exclusion. Distinguishing ICC from other metastatic adenocarcinomas based on histopathologic or immunohistochemical analysis often is difficult and requires an extensive workup. This study aimed to determine whether albumin, whose expression is restricted to the liver, has potential as a biomarker for ICC using a novel and highly sensitive RNA in situ hybridization (ISH) platform. Modified branched DNA probes were developed for albumin RNA ISH. The study evaluated 467 patient samples of primary and metastatic lesions. Of the 467 samples evaluated, 83 were ICCs, 42 were hepatocellular carcinomas (HCCs), and 332 were nonhepatic carcinomas including tumors arising from the perihilar region and bile duct, pancreas, stomach, esophagus, colon, breast, ovary, endometrium, kidney, and urinary bladder. Albumin RNA ISH was highly sensitive for cancers of liver origin, staining positive in 82 (99 %) of 83 ICCs and in 42 HCCs (100 %). Perihilar and distal bile duct carcinomas as well as carcinomas arising at other sites tested negative for albumin. Notably, 6 (22 %) of 27 intrahepatic tumors previously diagnosed as carcinomas of undetermined origin tested positive for albumin. Albumin RNA ISH is a sensitive and highly specific diagnostic tool for distinguishing ICC from metastatic adenocarcinoma to the liver or carcinoma of unknown origin. Albumin RNA ISH could replace the extensive diagnostic workup, leading to timely confirmation of the ICC diagnosis. Additionally, the assay could serve as a guide to distinguish ICC from perihilar adenocarcinoma.

BACKGROUND. Melanoma is a heterogeneous malignancy. We set out to identify the molecular underpinnings of high-risk melanomas, those that are likely to progress rapidly, metastasize, and result in poor outcomes. METHODS. We examined transcriptome changes from benign states to early-, intermediate-, and late-stage tumors using a set of 78 treatment-naive melanocytic tumors consisting of primary melanomas of the skin and benign melanocytic lesions. We utilized a next-generation sequencing platform that enabled a comprehensive analysis of protein-coding and -noncoding RNA transcripts. RESULTS. Gene expression changes unequivocally discriminated between benign and malignant states, and a dual epigenetic and immune signature emerged defining this transition. To our knowledge, we discovered previously unrecognized melanoma subtypes. A high-risk primary melanoma subset was distinguished by a 122-epigenetic gene signature (“epigenetic” cluster) and TP53 family gene deregulation (TP53, TP63, and TP73). This subtype associated with poor overall survival and showed enrichment of cell cycle genes. Noncoding repetitive element transcripts (LINEs, SINEs, and ERVs) that can result in immunostimulatory signals recapitulating a state of “viral mimicry” were significantly repressed. The high-risk subtype and its poor predictive characteristics were validated in several independent cohorts. Additionally, primary melanomas distinguished by specific immune signatures (“immune” clusters) were identified. CONCLUSION. The TP53 family of genes and genes regulating the epigenetic machinery demonstrate strong prognostic and biological relevance during progression of early disease. Gene expression profiling of protein-coding and -noncoding RNA transcripts may be a better predictor for disease course in melanoma. This study outlines the transcriptional interplay of the cancer cell’s epigenome with the immune milieu with potential for future therapeutic targeting. FUNDING. National Institutes of Health ({"type":"entrez-nucleotide","attrs":{"text":"CA154683","term_id":"35062023","term_text":"CA154683"}}CA154683, {"type":"entrez-nucleotide","attrs":{"text":"CA158557","term_id":"35069983","term_text":"CA158557"}}CA158557, {"type":"entrez-nucleotide","attrs":{"text":"CA177940","term_id":"35109217","term_text":"CA177940"}}CA177940, CA087497-13), Tisch Cancer Institute, Melanoma Research Foundation, the Dow Family Charitable Foundation, and the Icahn School of Medicine at Mount Sinai.

AimsThis multi-institutional study and a re-evaluation of the TCGA cohort explores the morphological spectrum, genetics and outcome of GI (gastrointestinal) hepatoid tumours, tumours expressing alpha-fetoprotein (AFP) and fetal-type (FT) GI adenocarcinomas.Methods44 tumours with evidence of hepatocellular differentiation were evaluated for morphology as well as by immunohistochemistry for AFP, HepPar1, glypican-3 and arginase-1 and by in situ hybridisation for albumin. Three categories were defined: type I (hepatoid: morphological evidence of hepatocellular differentiation), type II (FT GI adenocarcinoma: tubular profiles and subnuclear vacuolisation, resembling fetal intestine) and type III: positive for at least two hepatocyte-specific markers but lacking morphological evidence of hepatocellular differentiation. GI adenocarcinomas in the TCGA cohort were also evaluated (n=829).Results18 cases were classified as type I, 19 as FT GI adenocarcinomas and 7 as type III (resembling conventional gastrointestinal carcinomas). Serum AFP was elevated in 92% of cases. 93% of tumours were positive for glypican-3, 90% for albumin and 89% for AFP. Arginase-1 was restricted to 35% of type 1 tumours. TCGA gastric tumours with elevated AFP expression showed morphological features of FT GI adenocarcinoma (70%) and were exclusively MSI stable. TCGA gastric adenocarcinomas with high AFP expression showed inferior survival on univariate and multivariate analysis.ConclusionsFT GI adenocarcinomas show a distinctive morphological and immunohistochemical profile. Gastric adenocarcinomas with elevated expression of AFP morphologically resemble FT GI adenocarcinomas, demonstrate aggressive behaviour, independent of grade and stage, and a distinct genetic profile.

SUMMARY Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.

Chromatin remodeling proteins are frequently dysregulated in human cancer, yet little is known about how they control tumorigenesis. Here, we uncover an epigenetic program mediated by the NAD(+)-dependent histone deacetylase Sirtuin 6 (SIRT6) that is critical for suppression of pancreatic ductal adenocarcinoma (PDAC), one of the most lethal malignancies. SIRT6 inactivation accelerates PDAC progression and metastasis via upregulation of Lin28b, a negative regulator of the let-7 microRNA. SIRT6 loss results in histone hyperacetylation at the Lin28b promoter, Myc recruitment, and pronounced induction of Lin28b and downstream let-7 target genes, HMGA2, IGF2BP1, and IGF2BP3. This epigenetic program defines a distinct subset with a poor prognosis, representing 30%-40% of human PDAC, characterized by reduced SIRT6 expression and an exquisite dependence on Lin28b for tumor growth. Thus, we identify SIRT6 as an important PDAC tumor suppressor and uncover the Lin28b pathway as a potential therapeutic target in a molecularly defined PDAC subset. PAPERCLIP.

AIMS Bile duct adenomas may be difficult to distinguish from metastatic carcinomas, particularly well-differentiated pancreatic ductal adenocarcinoma. Prior studies have evaluated the utility of various immunohistochemical markers, although these markers are notable for low sensitivity and/or specificity. The aim of this study was to investigate the utility of albumin and BRAFV600E expression in distinguishing between metastatic pancreatic adenocarcinoma and bile duct adenoma. METHODS AND RESULTS We studied 26 bile duct adenomas, three bile duct hamartomas, and 158 pancreatic ductal adenocarcinomas. Branched-chain in-situ hybridization (bISH) for albumin was performed; bISH is based on the branched DNA technology, wherein signal amplification is achieved via a series of sequential steps. Additionally, BRAFV600E immunohistochemistry (IHC) was performed on a subset of cases. Twenty-three of 25 (92%) bile duct adenomas were positive for albumin; 18 (72%) showed diffuse staining, and five showed focal staining (20%), including two challenging examples. Two bile duct hamartomas also stained positively. All pancreatic adenocarcinomas were negative for albumin. Seven of 16 (44%) bile duct adenomas and five of 106 (5%) pancreatic ductal adenocarcinomas were positive for BRAFV600E by IHC. The sensitivity and specificity of expression of albumin, as detected by bISH, for distinguishing bile duct adenomas from metastatic pancreatic adenocarcinomas were 92% and 100%, respectively; the sensitivity and specificity of BRAFV600E IHC for distinguishing bile duct adenomas from metastatic pancreatic adenocarcinomas were 43.8% and 95.3%, respectively. CONCLUSIONS Diagnostically challenging examples of bile duct adenoma may be distinguished from metastatic pancreatic adenocarcinoma by the use of albumin bISH.

Epithelial-to-mesenchymal transition (EMT) has been implicated in models of tumor cell migration, invasion, and metastasis. In a search for candidate therapeutic targets to reverse this process, nontumorigenic MCF10A breast epithelial cells were infected with an arrayed lentiviral kinome shRNA library and screened for either suppression or enhancement of a 26-gene EMT RNA signature. No individual kinase gene knockdown was sufficient to induce EMT. In contrast, grouped epithelial markers were induced by knockdown of multiple kinases, including mitogen activated protein kinase 7 (MAPK7). In breast cancer cells, suppression of MAPK7 increased E-cadherin (CDH1) expression and inhibited cell migration. In an orthotopic mouse model, MAPK7 suppression reduced the generation of circulating tumor cells and the appearance of lung metastases. Together, these observations raise the possibility that targeting kinases that maintain mesenchymal cell properties in cancer cells, such as MAPK7, may lessen tumor invasiveness. Implications: Suppression of MAPK7 induces epithelial markers, reduces generation of circulating tumor cells and appearance of lung metastases. Mol Cancer Res; 13(5); 934–43. ©2015 AACR.

s: Abstracts: AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; May 12-15, 2016; Orlando, FL Chromatin remodeling proteins are frequently dysregulated in human cancer; however, little is known about how they control tumorigenesis. The NAD+ dependent histone deacetylase Sirtuin 6 (SIRT6) is a nutrient sensor that reprograms the epigenome in response to nutrient stress and shows reduced expression in human pancreatic ductal adenocarcinoma (PDAC) relative to normal tissue. Thus, we aimed to determine the role of SIRT6 in PDAC and to determine how the loss of this chromatin modifier could influence PDAC formation, progression and metastasis. Here, we uncover an epigenetic program mediated by SIRT6 that is critical for the suppression of PDAC. We demonstrate that SIRT6 inactivation cooperates with oncogenic KRAS to greatly accelerate the development of lethal pancreatic tumors regardless of p53 status. In addition to developing PDAC and high-grade pancreatic intraepithelial neoplasia (PanIN) at an earlier age, SIRT6-deficient tumors had a greater propensity to metastasize to the lung, compared to their SIRT6 wild-type (WT) counterparts. Through genome-wide profiling of SIRT6 WT and knock-out (KO) PDAC cell lines, we identified the oncofetal RNA-binding protein Lin28b as a target of Sirt6-mediated transcriptional repression. Loss of SIRT6 in both murine and human PDAC cell lines resulted in hyperacetylation of histone 3 lysine 9 and lysine 56 at the Lin28b promoter, MYC recruitment, and pronounced induction of Lin28b expression. Knocking down Lin28b resulted in potent suppression of cell proliferation and tumor sphere formation in Sirt6null/low murine and human cell lines, while murine and human Sirt6WT/high PDAC lines were completely insensitive to the same treatment. Functionally, Lin28b binds to and induces the degradation of the let-7 family of microRNA, thus resulting in increased expression of let-7 target genes such as the IGF2BPs and HMGA2, which have been correlated with increased agressiveness and metastasis in pancreatic tumors. Furthermore, Gene Set Enrichment Analysis (GSEA) comparing PDAC tumors and cell lines with high versus low expression of LIN28B revealed that LIN28Bhigh tumors were strongly enriched for the expression of let-7 targets. Finally, either loss of SIRT6 or the reactivation of Lin28b or the let-7 target genes IGF2BP3 and HMGA2 correlated with a worse overall survival in human PDAC patients. Thus, our critical findings define SIRT6 as a tumor suppressor in PDAC, working as a transcriptional repressor of the oncofetal RNA-binding protein Lin28b. We demonstrate that loss of Sirt6 creates a permissive hyperacetylated chromatin state, allowing for MYC-dependent transactivation of Lin28b. This work not only provides new insights into the epigenetic mechanisms governing the reactivation of oncofetal proteins in cancer but also demonstrates that this pathway is well-conserved in human PDAC, where reduced SIRT6 expression defines a specific subset of tumors which may benefit from novel therapeutic approaches aimed at targeting components of the LIN28B/let-7 pathway. This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section PR09 of the Conference Proceedings. Citation Format: Sita Kugel, Carlos Sebastian, Julien Fitamant, Kenneth N. Ross, Supriya K. Saha, Esha Jain, Adrianne Gladden, Kshitij S. Arora, Yasutaka Kato, Miguel N. Rivera, Sridhar Ramaswamy, Ruslan I. Sadreyev, Alon Goren, Vikram Deshpande, Nabeel Bardeesy, Raul Mostoslavsky.{Authors}. Loss of SIRT6 reactivates the RNA-binding protein Lin28b to drive pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B16.

Introduction: Androgen receptor (AR) mRNA splice variants have emerged as predictive biomarkers for response to AR targeted therapies. There are no commercially available assays to detect AR splice variants. The branched chain RNA in situ hybridization (ISH) platform enables the highly sensitive detection of RNA transcripts in formalin-fixed, paraffin-embedded (FFPE) tissues. In this pilot study, we developed an RNA ISH assay to detect the constitutively active AR-V7 splice variant, and we correlated AR-V7 expression with clinical outcomes after first-line androgen deprivation therapy (ADT) for metastatic prostate cancer. Methods: We designed a branched chain RNA ISH probe to target the unique cryptic exon CE3 of AR-V7 using multiple tiling probes (Affymetrix). Automated ISH assays were performed using ViewRNA eZl Detection Kit on the BOND RX IHC and ISH Staining System. A semiquantitative scoring method was used to score the RNA ISH signal in FFPE tissue. We analyzed AR-V7 and full length AR within two prostate cancer cohorts that we hypothesized to represent “low likelihood” and “high likelihood” to have detectable AR-V7. “Low likelihood” men had undergone prostatectomy for Gleason 6 adenocarcinoma, and “high likelihood” men had metastatic castration resistant prostate cancer with tumor tissue obtained after disease progression despite at least one subsequent line of hormonal therapy (abiraterone, enzalutamide, or bicalutamide). We then analyzed two additional cohorts who experienced a “sustained response” (>2.5 years; n = 13) or “brief response” ( Results: “High likelihood” cohort samples had detectable AR-V7 with a score of 1+ to 3+ in all samples (n = 12). “Low likelihood” cohort samples had no detectable AR-V7 (n = 10). Given the apparent binary distinction of AR-V7 signal among the above groups, we analyzed pre-treatment AR-V7 status as a predictive and prognostic biomarker in men with treatment-naive metastatic disease. Detectable AR-V7 was more common among “brief response” samples (6 of 9) than among “sustained response” samples (4 of 13) (not significant; P = 0.19). Patients without detectable AR-V7 RNA had significantly longer overall survival (OS, logrank P = 0.044), with a non-significant trend toward superior progression-free survival (PFS, logrank P = 0.055). Conclusions: Within an institutional cohort, the RNA ISH assay identified AR-V7 in all tested samples of high clinical likelihood for the splice variant RNA and no tested samples of low clinical likelihood. AR-V7 RNA was detectable in some pretreatment samples, and its presence was associated with significantly shorter overall survival. The potential prognostic and predictive utility of AR-V7 status as determined by RNA ISH from conventionally prepared FFPE tissue warrants further study in larger cohorts. Citation Format: Philip J. Saylor, Richard J. Lee, Kshitij S. Arora, David T. Ting, Vikram Deshpande, Miguel N. Rivera, Rong Hu, Chin-Lee Wu, David T. Miyamoto. Branched chain RNA in situ hybridization for the detection of androgen receptor splice variants within archival prostate cancer tissue. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4947.

Purpose: The androgen receptor (AR) mRNA splice variant AR-V7 has emerged as a predictive biomarker for response to AR-targeted therapies. There are currently no commercially available assays to detect AR splice variants. The branched chain RNA in situ hybridization (ISH) platform enables the highly sensitive detection of RNA transcripts in formalin-fixed, paraffin-embedded (FFPE) tissues. Experimental design: We designed a branched chain RNA ISH probe to target the unique cryptic exon CE3 of AR-V7 using multiple tiling probes. This automated ISH assay was applied to tumor tissue from two distinct clinical cohorts that we hypothesized would differ in AR-V7 status. Results: We detected AR-V7 in all tumor samples from men with metastatic castration-resistant prostate cancer with tissue obtained after disease progression despite at least one subsequent line of hormonal therapy (abiraterone, enzalutamide, or bicalutamide; n = 12). We detected AR-V7 in just one tumor from men who had undergone prostatectomy for localized adenocarcinoma (n = 30; Gleason 4 + 5 = 9 in the AR-V7–positive sample). Given the apparent distinction between the above groups by AR-V7 signal, we analyzed pretreatment AR-V7 status as a predictive and prognostic biomarker in men with treatment-naïve metastatic disease. Patients with metastases but without detectable AR-V7 RNA at baseline had significantly longer overall survival (log-rank P = 0.044) and a trend toward superior progression-free survival (log-rank P = 0.055). Conclusions: Within an institutional cohort, the RNA ISH assay identified AR-V7 within FFPE tissue and may have prognostic value in metastatic castration-sensitive prostate cancer. These preliminary findings warrant further study in larger cohorts. Clin Cancer Res; 23(2); 363–9. ©2016 AACR.

Isolation of circulating tumor cells (CTCs) in the blood allows noninvasive tumor sampling from patients with cancer. Molecular analysis of single CTCs may uncover heterogeneous cellular pathways that underlie disease progression and resistance to therapy. Using a microfluidic device, we isolated individual CTCs from patients with metastatic prostate cancer. Single candidate CTCs were micromanipulated after cell surface staining for epithelial (EpCAM) and mesenchymal (CDH11) markers, and then subjected to single cell RNA-sequencing. Digital gene expression profiles of lineage-confirmed CTCs were compared with each other, with primary prostate tumors, and with annotated markers of cellular signaling pathways. Single prostate CTCs displayed considerable heterogeneity in transcriptional profiles, but clustered according to patient of origin, indicating higher diversity in CTCs across different individuals (mean correlation 0.10 for CTCs within patients vs. 0.0014 for CTCs across patients, P=2.0x10E-11). Compared to primary tumors, CTCs were significantly enriched in 37 molecular pathways (FDR Citation Format: David T. Miyamoto, Yu Zheng, Ben S. Wittner, Richard J. Lee, Huili Zhu, Katherine T. Broderick, Rushil Desai, Brian W. Brannigan, Kshitij S. Arora, Douglas M. Dahl, Lecia V. Sequist, Matthew R. Smith, Ravi Kapur, Chin-Lee Wu, Toshi Shioda, Sridhar Ramaswamy, David T. Ting, Mehmet Toner, Shyamala Maheswaran, Daniel A. Haber. Single cell RNA-sequencing of circulating tumor cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr IA09.

Pancreatic adenocarcinoma (PDAC) is a lethal disease with a rising incidence, expected to become the second cause of cancer death in the next 5 years. Despite advances in novel therapies for other malignancies, PDAC has remained a highly chemorefractory disease leading to inevitable disease recurrence in the vast majority of patients. The identification of the cellular mechanisms of chemoresistance unique to PDAC is of particular importance. Our prior work using single cell RNA-sequencing in pancreatic circulating tumor cells (CTCs) identified 878 genes enriched in CTCs compared to matched primary tumor cells. CTCs are thought to be enriched for cells with high metastatic potential, which we predict to have intrinsic chemoresistant behavior. We hypothesized that CTC enriched genes may identify a subpopulation of cells resistant to chemotherapy that could be used as a biomarker and novel therapeutic target. The neuroendocrine marker SV2 was found in both mouse and human pancreatic CTCs and was selected given its presence on cell membranes, providing a potential therapeutic target. We evaluated a panel of patient derived cell lines and identified an SV2 positive subpopulation comprising ∼1% of cells in 3 of 5 cell lines. Notably, only differentiated epithelioid PDAC cell lines contained an SV2 subpopulation, while this population is not present in poorly differentiated quasi-mesenchymal cell lines. We performed RNA-ISH for SV2 isoforms in an annotated PDAC tissue microarray demonstrating positive staining in 77% of samples (245/317). Interestingly, SV2 positivity was often focally positive in a subpopulation of cells at the basolateral region of ductal adenocarcinoma and was found in more differentiated morphology (69% well/moderate vs 31% poor/undifferentiated; p = 0.047). The presence of SV2 cells was associated with an improved disease free survival (HR: 0.49 p = 0.009) and overall survival (HR: 0.54 p = 0.018) even after correction in multivariate analysis. Analysis in both cell lines and tissue revealed SV2 positive cells were also found to have higher levels of the ALDH1 stem cell marker (Pearson coefficient = 0.92 in tissue). Initial in vivo treatment of human orthotopic xenografts with gemcitabine, reveal an enrichment of SV2 positive cells pointing to their potential role as the recalcitrant cancer cells treated with cytotoxic chemotherapy population. Together, this is consistent with prior work demonstrating classical epithelial PDAC has improved survival compared to quasi-mesenchymal tumors. However, almost all patients with classical epithelial PDAC will die from recurrent disease, and we have now shown SV2 as a potential marker that identifies the chemoresistant pancreatic cancer cells in this patient population. Ultimately, a more detailed characterization of the SV2 subpopulation in classical PDAC will help us develop novel targeted therapies to overcome chemoresistance. Citation Format: Daniela Dias-Santos, Matteo Ligorio, Kshitij Arora, Vishal Thapar, Olivia C. MacKenzie, Srinjoy Sil, Niyati Desai, Vikram Deshpande, Miguel N. Rivera, Cristina R. Ferrone, David T. Ting. Molecular subtyping of pancreatic adenocarcinoma identifies SV2 positive subpopulation in classical PDAC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 609. doi:10.1158/1538-7445.AM2015-609