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Background The impact of transarterial radioembolization (TARE) of breast cancer liver metastasis (BCLM) on antitumor immunity is unknown, which hinders the optimal selection of candidates for TARE. Purpose To determine whether response to TARE at PET/CT in participants with BCLM is associated with specific immune markers (cytokines and immune cell populations). Materials and Methods This prospective pilot study enrolled 23 women with BCLM who planned to undergo TARE (June 2018 to February 2020). Peripheral blood and liver tumor biopsies were collected at baseline and 1-2 months after TARE. Monocyte, myeloid-derived suppressor cell (MDSC), interleukin (IL), and tumor-infiltrating lymphocyte (TIL) levels were assessed with use of gene expression studies and flow cytometry, and immune checkpoint and cell surface marker levels with immunohistochemistry. Modified PET Response Criteria in Solid Tumors was used to determine complete response (CR) in treated tissue. After log-transformation, immune marker levels before and after TARE were compared using paired

Small cell lung cancer (SCLC) is an aggressive malignancy characterized by early metastasis and extreme lethality. The backbone of SCLC treatment over the past several decades has been platinum-based doublet chemotherapy, with the recent addition of immunotherapy providing modest benefits in a subset of patients. However, nearly all patients treated with systemic therapy quickly develop resistant disease, and there is an absence of effective therapies for recurrent and progressive disease. Here we conducted CRISPR-Cas9 screens using a druggable genome library in multiple SCLC cell lines representing distinct molecular subtypes. This screen nominated exportin-1, encoded by XPO1, as a therapeutic target. XPO1 was highly and ubiquitously expressed in SCLC relative to other lung cancer histologies and other tumor types. XPO1 knockout enhanced chemosensitivity, and exportin-1 inhibition demonstrated synergy with both first- and second-line chemotherapy. The small molecule exportin-1 inhibitor selinexor in combination with cisplatin or irinotecan dramatically inhibited tumor growth in chemonaïve and chemorelapsed SCLC patient-derived xenografts, respectively. Together these data identify exportin-1 as a promising therapeutic target in SCLC, with the potential to markedly augment the efficacy of cytotoxic agents commonly used in treating this disease.Significance:CRISPR-Cas9 screening nominates exportin-1 as a therapeutic target in SCLC, and exportin-1 inhibition enhances chemotherapy efficacy in patient-derived xenografts, providing a novel therapeutic opportunity in this disease.

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Evasion of apoptosis is a characteristic feature of pancreatic cancer, a prototypic cancer that is refractory to current treatment approaches. Hence, there is an urgent need to design rational strategies that counter apoptosis resistance. To explore X-linked inhibitor of apoptosis (XIAP) as a therapeutic target in pancreatic cancer, we analyzed the expression of XIAP in pancreatic tumor samples and evaluated the effect of small molecule XIAP inhibitors alone and in combination with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) against pancreatic carcinoma in vitro and in vivo. Here, we report that XIAP is highly expressed in pancreatic adenocarcinoma samples compared with normal pancreatic ducts. Small molecule XIAP inhibitors synergize with TRAIL to induce apoptosis and to inhibit long-term clonogenic survival of pancreatic carcinoma cells. In contrast, they do not reverse the lack of toxicity of TRAIL on nonmalignant cells in vitro or normal tissues in vivo, pointing to a therapeutic index. Most importantly, XIAP inhibitors cooperate with TRAIL to trigger apoptosis and suppress pancreatic carcinoma growth in vivo in two preclinical models, i.e., the chorioallantoic membrane model and a mouse xenograft model. Parallel immunohistochemical analysis of tumor tissue under therapy reveals that the XIAP inhibitor acts in concert with TRAIL to cause caspase-3 activation and apoptosis. In conclusion, our findings provide, for the first time, evidence in vivo that XIAP inhibitors prime pancreatic carcinoma cells for TRAIL-induced apoptosis and potentiate the antitumor activity of TRAIL against established pancreatic carcinoma. These findings build the rationale for further (pre)clinical development of XIAP inhibitors and TRAIL against pancreatic cancer. [Cancer Res 2009;69(6):2425–34]

Supplementary Figure and Table Legends from Small Molecule XIAP Inhibitors Enhance TRAIL-Induced Apoptosis and Antitumor Activity in Preclinical Models of Pancreatic Carcinoma

Supplementary Figures 1-3, Table 1 from Small Molecule XIAP Inhibitors Enhance TRAIL-Induced Apoptosis and Antitumor Activity in Preclinical Models of Pancreatic Carcinoma

Small cell lung cancer (SCLC) is an aggressive malignancy characterized by early metastasis and extreme lethality. The backbone of SCLC treatment over the past several decades has been platinum-based doublet chemotherapy, with the recent addition of immunotherapy providing modest benefits in a subset of patients. However, nearly all patients treated with systemic therapy quickly develop resistant disease, and there is an absence of effective therapies for recurrent and progressive disease. Here we conducted CRISPR-Cas9 screens using a druggable genome library in multiple SCLC cell lines representing distinct molecular subtypes. This screen nominated exportin-1, encoded by XPO1, as a therapeutic target. XPO1 was highly and ubiquitously expressed in SCLC relative to other lung cancer histologies and other tumor types. XPO1 knockout enhanced chemosensitivity, and exportin-1 inhibition demonstrated synergy with both first- and second-line chemotherapy. The small molecule exportin-1 inhibitor selinexor in combination with cisplatin or irinotecan dramatically inhibited tumor growth in chemonaïve and chemorelapsed SCLC patient-derived xenografts, respectively. Together these data identify exportin-1 as a promising therapeutic target in SCLC, with the potential to markedly augment the efficacy of cytotoxic agents commonly used in treating this disease. Significance: CRISPR-Cas9 screening nominates exportin-1 as a therapeutic target in SCLC, and exportin-1 inhibition enhances chemotherapy efficacy in patient-derived xenografts, providing a novel therapeutic opportunity in this disease.

Lineage plasticity is implicated in treatment resistance in multiple cancers. In lung adenocarcinomas (LUAD) amenable to targeted therapy, transformation to small cell lung cancer (SCLC) is a recognized resistance mechanism. Defining molecular mechanisms of neuroendocrine (NE) transformation in lung cancer has been limited by a paucity of pre/posttransformation clinical samples. Detailed genomic, epigenomic, transcriptomic, and protein characterization of combined LUAD/SCLC tumors, as well as pre/posttransformation samples, supports that NE transformation is primarily driven by transcriptional reprogramming rather than mutational events. We identify genomic contexts in which NE transformation is favored, including frequent loss of the 3p chromosome arm. We observed enhanced expression of genes involved in the PRC2 complex and PI3K/AKT and NOTCH pathways. Pharmacologic inhibition of the PI3K/AKT pathway delayed tumor growth and NE transformation in an EGFR-mutant patient-derived xenograft model. Our findings define a novel landscape of potential drivers and therapeutic vulnerabilities of NE transformation in lung cancer. Significance: The difficulty in collection of transformation samples has precluded the performance of molecular analyses, and thus little is known about the lineage plasticity mechanisms leading to LUAD-to-SCLC transformation. Here, we describe biological pathways dysregulated upon transformation and identify potential predictors and potential therapeutic vulnerabilities of NE transformation in the lung. See related commentary by Meador and Lovly, p. 2962. This article is highlighted in the In This Issue feature, p. 2945

Multiple large-scale tumor genomic profiling efforts have been undertaken in osteosarcoma, however, little is known about the spatial and temporal intratumor heterogeneity and how it may drive treatment resistance. We performed whole-genome sequencing of 37 tumor samples from eight patients with relapsed or refractory osteosarcoma. Each patient had at least one sample from a primary site and a metastatic or relapse site. We identified subclonal copy number alterations in all but one patient. We observed that in five patients, a subclonal copy number clone from the primary tumor emerged and dominated at subsequent relapses.MYCgain/amplification was enriched in the treatment-resistant clone in 6 out of 7 patients with more than one clone. Amplifications in other potential driver genes, such asCCNE1,RAD21,VEGFA, andIGF1R, were also observed in the resistant copy number clones. Our study sheds light on intratumor heterogeneity and the potential drivers of treatment resistance in osteosarcoma.SignificanceSubclonal copy number clones emerged and dominated in relapsed osteosarcoma, withMYCgain/amplification being the defining characteristic in our cohort. Selective pressure from neoadjuvant chemotherapy revealed this clone at the time of primary resection, highlighting that genomic profiling at this time may identify clones that are selected for, or determine innate resistance to primary chemotherapy.

The impact of the intratumoral microbiome on immune checkpoint inhibitor (ICI) efficacy in patients (pts) with non-small cell lung cancer (NSCLC) is unknown. In preclinical studies, the presence of lung intratumoral Escherichia was associated with a proinflammatory tumor microenvironment and decreased metastases within lung tissue. We sought to detect intratumoral bacteria in pts with advanced NSCLC using hybrid capture-based, next generation sequencing (NGS). We studied 849 pts treated with ICI-based therapy who underwent NGS at our center. We extracted unmapped reads from BAM files, and these were queried for bacteria (blastn alignment using the NCBI database). Putative environmental contaminants were subtracted from the analysis using “no template” controls (n=2,539) to exclude possible artifactual false positives. A custom E.Coli fluorescence in situ hybridization (FISH) probe was used to visualize Escherichia within the tumors after co-registration with H&E. In 849 pts, a median of 30 bacterial reads was detected per sample (inter-quartile range (18-85)). Among 68 pts with paired primary/metastatic samples, the bacterial spectra were similar in both sites, suggesting that tumor resident bacteria might travel with cancer cells to distant sites. Antibiotic use within 30 days of tumor sampling was associated with decreased intratumoral bacterial diversity (p=0.023 by Inverse Simpson, p=0.038 by Shannon). Intratumoral Escherichia was associated with better PFS (HR 0.78, 95% CI 0.62-0.98, p=0.036), and OS (HR 0.74, 95% CI 0.58-0.95, p=0.017) in pts treated with single-agent ICI, but not combination Chemo/ICI. In a multivariable model adjusting for prognostic features in NSCLC including PD-L1 tumor proportion score, the presence of intratumoral Escherichia was associated with better PFS (p=0.040) and OS (p=0.045) upon single-agent ICI therapy. Escherichia appeared to be intracellular based on co-registration of FISH staining and serial H&E sections. These findings warrant further investigation of the possible inter-relationships between intratumoral Escherichia, tumor immune micro-environment, and ICI therapeutic outcomes. Citation Format: Arielle Elkrief, Anita S. Bowman, Ayyuce Begum Bektas, Wenfei Kang, Katia Manova-Todorova, Jacklynn V. Egger, Hira Rizvi, Daniel Kelly, Eric Chan, Eric Rosiek, Fan Ning, Gregory J. Riely, Álvaro Quintanal Villalonga, Snjezana Dogan, Umesh Bhanot, Mithat Gonen, Matthew D. Hellmann, Adam J. Schoenfeld, Charles M. Rudin, Marc Ladanyi, Chad M. Vanderbilt. Intratumoral Escherichia is associated with response to single-agent immune checkpoint inhibition in patients with advanced non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5185.

Background Lineage plasticity, the ability to transdifferentiate among distinct phenotypic identities, facilitates therapeutic resistance in cancer. In lung adenocarcinomas (LUADs), this phenomenon includes small cell and squamous cell (LUSC) histologic transformation in the context of acquired resistance to targeted inhibition of driver mutations. LUAD-to-LUSC transdifferentiation, occurring in up to 9% of EGFR-mutant patients relapsed on osimertinib, is associated with notably poor prognosis. We hypothesized that multi-parameter profiling of the components of mixed histology (LUAD/LUSC) tumors could provide insight into factors licensing lineage plasticity between these histologies. Methods We performed genomic, epigenomics, transcriptomics and protein analyses of microdissected LUAD and LUSC components from mixed histology tumors, pre-/post-transformation tumors and reference non-transformed LUAD and LUSC samples. We validated our findings through genetic manipulation of preclinical models in vitro and in vivo and performed patient-derived xenograft (PDX) treatments to validate potential therapeutic targets in a LUAD PDX model acquiring LUSC features after osimertinib treatment. Results Our data suggest that LUSC transdifferentiation is primarily driven by transcriptional reprogramming rather than mutational events. We observed consistent relative upregulation of PI3K/AKT, MYC and PRC2 pathway genes. Concurrent activation of PI3K/AKT and MYC induced squamous features in EGFR-mutant LUAD preclinical models. Pharmacologic inhibition of EZH1/2 in combination with osimertinib prevented relapse with squamous-features in an EGFR-mutant patient-derived xenograft model, and inhibition of EZH1/2 or PI3K/AKT signaling re-sensitized resistant squamous-like tumors to osimertinib. Conclusions Our findings provide the first comprehensive molecular characterization of LUSC transdifferentiation, suggesting putative drivers and potential therapeutic targets to constrain or prevent lineage plasticity.

Summary Small cell lung cancer (SCLC) is an aggressive malignancy that includes subtypes defined by differential expression of ASCL1, NEUROD1, and POU2F3 (SCLC-A, -N, and -P, respectively). To define the heterogeneity of tumors and their associated microenvironments across subtypes, we sequenced 155,098 transcriptomes from 21 human biospecimens, including 54,523 SCLC transcriptomes. We observe greater tumor diversity in SCLC than lung adenocarcinoma, driven by canonical, intermediate, and admixed subtypes. We discover a PLCG2-high SCLC phenotype with stem-like, pro-metastatic features that recurs across subtypes and predicts worse overall survival. SCLC exhibits greater immune sequestration and less immune infiltration than lung adenocarcinoma, and SCLC-N shows less immune infiltrate and greater T cell dysfunction than SCLC-A. We identify a profibrotic, immunosuppressive monocyte/macrophage population in SCLC tumors that is particularly associated with the recurrent, PLCG2-high subpopulation.

Group 2 innate lymphoid cells (ILC2s) regulate inflammation and immunity in mammalian tissues1,2. Although ILC2s are found in cancers of these tissues3, their roles in cancer immunity and immunotherapy are unclear. Here we show that ILC2s infiltrate pancreatic ductal adenocarcinomas (PDACs) to activate tissue-specific tumour immunity. Interleukin-33 (IL33) activates tumour ILC2s (TILC2s) and CD8+ T cells in orthotopic pancreatic tumours but not heterotopic skin tumours in mice to restrict pancreas-specific tumour growth. Resting and activated TILC2s express the inhibitory checkpoint receptor PD-1. Antibody-mediated PD-1 blockade relieves ILC2 cell-intrinsic PD-1 inhibition to expand TILC2s, augment anti-tumour immunity, and enhance tumour control, identifying activated TILC2s as targets of anti-PD-1 immunotherapy. Finally, both PD-1+ TILC2s and PD-1+ T cells are present in most human PDACs. Our results identify ILC2s as anti-cancer immune cells for PDAC immunotherapy. More broadly, ILC2s emerge as tissue-specific enhancers of cancer immunity that amplify the efficacy of anti-PD-1 immunotherapy. As ILC2s and T cells co-exist in human cancers and share stimulatory and inhibitory pathways, immunotherapeutic strategies to collectively target anti-cancer ILC2s and T cells may be broadly applicable.

Although cancer immunotherapy with PD-(L)1 blockade is now routine treatment for patients with lung cancer, remarkably little is known about acquired resistance. We examined 1,201 patients with NSCLC treated with PD-(L)1 blockade to clinically characterize acquired resistance, finding it to be common (occurring in more than 60% of initial responders), with persistent but diminishing risk over time, and with distinct metastatic and survival patterns compared to primary resistance. To examine the molecular phenotype and potential mechanisms of acquired resistance, we performed whole transcriptome and exome tumor profiling in a subset of NSCLC patients (n=29) with acquired resistance. Systematic immunogenomic analysis revealed that tumors with acquired resistance generally had enriched signals of inflammation (including IFNγ signaling and inferred CD8+ T cells) and could be separated into IFNγ upregulated and stable subsets. IFNγ upregulated tumors had putative routes of resistance with signatures of dysfunctional interferon signaling and mutations in antigen presentation genes. Transcriptomic profiling of cancer cells from a murine model of acquired resistance to PD-(L)1 blockade also showed evidence of dysfunctional interferon signaling and acquired insensitivity to in vitro interferon gamma treatment. In summary, we characterized clinical and molecular features of acquired resistance to PD-(L)1 blockade in NSCLC and found evidence of ongoing but dysfunctional IFN response. The persistently inflamed, rather than excluded or deserted, tumor microenvironment of acquired resistance informs therapeutic strategies to effectively reprogram and reverse acquired resistance.

e21166 Background: In lung adenocarcinomas (LUADs), lineage plasticity drives neuroendocrine (NE) and squamous cell (LUSC) transdifferentiation in the context of acquired resistance to targeted inhibition of driver mutations, with up to 14% and 9% incidences in EGFR-mutant tumors relapsed on EGFR inhibitors, respectively. Notably, survival of patients with NE- or LUSC-transdifferentiated tumors is remarkably lower than those of LUAD or de novo LUSC patients. The paucity of transforming clinical specimens amenable for molecular analyses has hindered the identification of histological transformation drivers, and to date no specific therapies aimed to prevent or delay transdifferentiation-led therapy relapse are available for patients at high risk of transformation. Methods: We performed multi-omic profiling of LUAD-to-LUSC and LUAD-to-NE transdifferentiating clinical samples, including comprehensive and integrative genomic (whole exome sequencing), epigenomic (bisulfite sequencing), transcriptomic (RNAseq) and protein (antibody arrays) characterization. Clinical findings were validated in preclinical models including cell lines as well as LUSC- and NE-transdifferentiation patient-derived xenograft models. Results: Our data supports that histological transdifferentiation from LUAD to LUSC or NE tumors is driven by epigenetic remodeling rather than by mutational events, and indicate that transdifferentiated tumors retain epigenomic features of their previous LUAD state. Integrative epigenomic, transcriptomic and protein analysis revealed divergent biological pathways dysregulated for each histological outcome, such as downregulation of RTK signaling and Notch-related genes in NE-transformed tumors, and upregulation of genes involved in Hedgehog and Notch signaling and MYC targets in LUSC-transdifferentiated tumors. Most interestingly, these analyses identified commonly dysregulated pathways in both NE- and LUSC-transdifferentiating tumors, including remarkable downregulation of a variety of immune-related pathways and upregulation of genes involved in AKT signaling and in the PRC2 epigenetic remodeling complex. Concurrent activation of AKT and MYC overexpression induced a squamous phenotype in EGFR-mutant LUAD preclinical models, further accentuated by EGFR inhibition. Pharmacological targeting of AKT in combination with osimertinib delayed both squamous and NE transformation in different EGFR-mutant patient-derived xenograft transdifferentiation models. Conclusions: These results identify common and divergent dysregulated pathways in NE and LUSC transdifferentiation, and nominate AKT as a therapeutic target to prevent the acquisition of resistance to EGFR-targeted therapies through histological transdifferentiation.

e20613 Background: Small cell lung cancer (SCLC) is an extremely aggressive disease comprising around 13% of lung cancer cases and responsible for approximately 250,000 deaths globally per year. A major cause of SCLC poor prognosis is the sparse treatment options available, typically resulting in only transient responses. The backbone of SCLC treatment over the past several decades has been platinum-based doublet chemotherapy, with the recent addition of immunotherapy to first-line chemotherapy showing only limited benefit in a small subset of patients. Major hurdles to improving SCLC treatment include development of rapid chemoresistance and ineffective second line therapies. The identification of more durably effective therapeutic strategies is a major unmet clinical need. Methods: We performed an in vitro CRISPR screen in SCLC cell lines to identify potential therapeutic targets to sensitize to chemotherapy, including short-term cultured cell lines derived from patient-derived xenografts (PDXs). Candidate hits were validated genetically and pharmacologically with in vitro and in vivo. Signaling pathways involved in phenotypes observed were studied by RNA sequencing and western blot, and toxicity studies were performed in vivo to assess the safety of the agents at pharmacologically effective doses. We performed immunohistochemistry (IHC) to assess expression of candidate targets in tissue microarrays (TMAs). Results: Our CRISPR screen revealed the nuclear exporter XPO1 (Exportin 1) as a promising target sensitizing to chemotherapy, independently of the SCLC subtype. Importantly, exportin 1 is a therapeutic target in hematological malignancies, counting with a potent and selective inhibitor, selinexor, approved for clinical use. Combination of selinexor with cisplatin or irinotecan demonstrated high synergy in vitro and exquisite efficacy i n vivo in an array of chemonäive and chemoresistant SCLC PDXs, respectively, including all major SCLC subtypes. These chemotherapy-sensitizing effects were associated with the ability of Exportin 1 to impair chemotherapy-induced AKT overactivation, potentially occurring as a cytoprotective/anti-apoptotic mechanism in response to chemotherapy. The combinations were well tolerated in mice. We found that XPO1 mRNA expression was higher in SCLC than in any other solid tumor type or hematological malignancies, which we were able to confirm at the protein level in clinical TMAs. Conclusions: Exportin 1 inhibition strongly enhances sensitivity of SCLC tumors to cisplatin and irinotecan, used in first line and second line treatment of SCLC tumors, respectively. These results provide preclinical rationale for the combination of selinexor with first line and second line chemotherapy in SCLC. The clinical availability of selinexor will allow immediate clinical translation of these results in a disease setting with extremely limited therapeutic options.

Small cell lung cancer (SCLC) is an aggressive malignancy that includes subtypes defined by differential expression of ASCL1, NEUROD1, and POU2F3 (SCLC-A, -N, and -P, respectively), which are associated with distinct therapeutic vulnerabilities. To define the heterogeneity of tumors and their associated microenvironments across subtypes, we sequenced 54,523 cellular transcriptomes from 21 human biospecimens. Our single-cell SCLC atlas reveals tumor diversity exceeding lung adenocarcinoma, driven by canonical, intermediate, and admixed subtypes. We discovered a PLCG2-high tumor cell population with stem-like, pro-metastatic features that recurs across subtypes and predicts worse overall survival, and manipulation of PLCG2 expression in cells confirms correlation with key metastatic markers. Treatment and subtype are associated with substantial phenotypic changes in the SCLC immune microenvironment, with greater T-cell dysfunction in SCLC-N than SCLC-A. Moreover, the recurrent, PLCG2-high subclone is associated with exhausted CD8+ T-cells and a pro-fibrotic, immunosuppressive monocyte/macrophage population, suggesting possible tumor-immune coordination to promote metastasis.

// Benjamin A. Farber 1, 2 , Gadi Lalazar 1 , Elana P. Simon 1 , William J. Hammond 1, 2 , David Requena 1 , Umesh K. Bhanot 3 , Michael P. La Quaglia 2 and Sanford M. Simon 1 1 Laboratory of Cellular Biophysics, The Rockefeller University, New York, 10065 NY, USA 2 Division of Pediatric Surgery, Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, 10065 NY, USA 3 Pathology Core Facility Memorial Sloan-Kettering Cancer Center, New York, 10065 NY, USA Correspondence to: Sanford M. Simon, email: simon@rockefeller.edu Keywords: pediatric cancer; fusion protein; microRNA; lncRNAs; liver cancer Received: September 11, 2017 Accepted: December 08, 2017 Published: December 15, 2017 ABSTRACT Fibrolamellar hepatocellular carcinoma (FLC) is a rare primary liver cancer found in adolescents and young adults without underlying liver disease. A deletion of ~400 kD has been found in one copy of chromosome 19 in the tumor tissue of all patients tested. This produces a fusion of the genes DNAJB1 and PRKACA which, in turn, produces a chimeric transcript and protein. Transcriptomic analysis of the tumor has shown upregulation of various oncologically relevant pathways, including EGF/ErbB, Aurora Kinase A, pak21 and wnt. To explore other factors that may contribute to oncogenesis, we examined the microRNA (miRNA) and long non-coding RNA (lncRNA) expression in FLC. The non-coding RNA expression profile in tumor tissue samples is distinctly different from the adjacent normal liver and from other liver tumors. Furthermore, miRZip knock down or over expression of certain miRNAs led to changes in the levels of coding genes that recapitulated changes observed in FLC, suggesting mechanistically that the changes in the cellular levels of miRNA are not merely correlative. Thus, in addition to serving as diagnostic tools for FLC, non-coding RNAs may serve as therapeutic targets.

Background Metastatic renal cell carcinoma (RCC) patients are commonly treated with vascular endothelial growth factor (VEGF) inhibitors or mammalian target of rapamycin inhibitors. Correlations between somatic mutations and first-line targeted therapy outcomes have not been reported on a randomized trial. Objective To evaluate the relationship between tumor mutations and treatment outcomes in RECORD-3, a randomized trial comparing first-line everolimus (mTOR inhibitor) followed by sunitinib (VEGF inhibitor) at progression with the opposite sequence in 471 metastatic RCC patients. Design, setting, and participants Targeted sequencing of 341 cancer genes at ∼540× coverage was performed on available tumor samples from 258 patients; 220 with clear cell histology (ccRCC). Outcome measurements and statistical analysis Associations between somatic mutations and median first-line progression free survival (PFS1L) and overall survival were determined in metastatic ccRCC using Cox proportional hazards models and log-rank tests. Results and limitations Prevalent mutations (≥ 10%) were VHL (75%), PBRM1 (46%), SETD2 (30%), BAP1 (19%), KDM5C (15%), and PTEN (12%). With first-line everolimus, PBRM1 and BAP1 mutations were associated with longer (median [95% confidence interval {CI}] 12.8 [8.1, 18.4] vs 5.5 [3.1, 8.4] mo) and shorter (median [95% CI] 4.9 [2.9, 8.1] vs 10.5 [7.3, 12.9] mo) PFS1L, respectively. With first-line sunitinib, KDM5C mutations were associated with longer PFS1L (median [95% CI] of 20.6 [12.4, 27.3] vs 8.3 [7.8, 11.0] mo). Molecular subgroups of metastatic ccRCC based on PBRM1 , BAP1 , and KDM5C mutations could have predictive values for patients treated with VEGF or mTOR inhibitors. Most tumor DNA was obtained from primary nephrectomy samples (94%), which could impact correlation statistics. Conclusions PBRM1 , BAP1 , and KDM5C mutations impact outcomes of targeted therapies in metastatic ccRCC patients. Patient summary Large-scale genomic kidney cancer studies reported novel mutations and heterogeneous features among individual tumors, which could contribute to varied clinical outcomes. We demonstrated correlations between somatic mutations and treatment outcomes in clear cell renal cell carcinoma, supporting the value of genomic classification in prospective studies.

We developed and validated a clinical whole-genome and transcriptome sequencing (WGTS) assay that provides a comprehensive genomic profile of a patient's tumor. The ability to fully capture the mappable genome with sufficient sequencing coverage to precisely call DNA somatic single nucleotide variants, insertions/deletions, copy number variants, structural variants, and RNA gene fusions was analyzed. New York State's Department of Health next-generation DNA sequencing guidelines were expanded for establishing performance validation applicable to whole-genome and transcriptome sequencing. Whole-genome sequencing laboratory protocols were validated for the Illumina HiSeq X Ten platform and RNA sequencing for Illumina HiSeq2500 platform for fresh or frozen and formalin-fixed, paraffin-embedded tumor samples. Various bioinformatics tools were also tested, and CIs for sensitivity and specificity thresholds in calling clinically significant somatic aberrations were determined. The validation was performed on a set of 125 tumor normal pairs. RNA sequencing was performed to call fusions and to confirm the DNA variants or exonic alterations. Here, we present our results and WGTS standards for variant allele frequency, reproducibility, analytical sensitivity, and present limit of detection analysis for single nucleotide variant calling, copy number identification, and structural variants. We show that The New York Genome Center WGTS clinical assay can provide a comprehensive patient variant discovery approach suitable for directed oncologic therapeutic applications.

Intraductal tubulopapillary neoplasm is a relatively recently described member of the pancreatic intraductal neoplasm family. The more common member of this family, intraductal papillary mucinous neoplasm, often carries genetic alterations typical of pancreatic infiltrating ductal adenocarcinoma (KRAS, TP53, and CDKN2A) but additionally has mutations in GNAS and RNF43 genes. However, the genetic characteristics of intraductal tubulopapillary neoplasm have not been well characterized. Twenty-two intraductal tubulopapillary neoplasms were analyzed by either targeted next-generation sequencing, which enabled the identification of sequence mutations, copy number alterations, and selected structural rearrangements involving all targeted (≥300) genes, or whole-exome sequencing. Three of these intraductal tubulopapillary neoplasms were also subjected to whole-genome sequencing. All intraductal tubulopapillary neoplasms revealed the characteristic histologic (cellular intraductal nodules of back-to-back tubular glands lined by predominantly cuboidal cells with atypical nuclei and no obvious intracellular mucin) and immunohistochemical (immunolabeled with MUC1 and MUC6 but were negative for MUC2 and MUC5AC) features. By genomic analyses, there was loss of CDKN2A in 5/20 (25%) of these cases. However, the majority of the previously reported intraductal papillary mucinous neoplasm-related alterations were absent. Moreover, in contrast to most ductal neoplasms of the pancreas, MAP-kinase pathway was not involved. In fact, 2/22 (9%) of intraductal tubulopapillary neoplasms did not reveal any mutations in the tested genes. However, certain chromatin remodeling genes (MLL1, MLL2, MLL3, BAP1, PBRM1, EED, and ATRX) were found to be mutated in 7/22 (32%) of intraductal tubulopapillary neoplasms and 27% harbored phosphatidylinositol 3-kinase (PI3K) pathway (PIK3CA, PIK3CB, INPP4A, and PTEN) mutations. In addition, 4/18 (18%) of intraductal tubulopapillary neoplasms had FGFR2 fusions (FGFR2-CEP55, FGFR2-SASS6, DISP1-FGFR2, FGFR2-TXLNA, and FGFR2-VCL) and 1/18 (5.5%) had STRN-ALK fusion. Intraductal tubulopapillary neoplasm is a distinct clinicopathologic entity in the pancreas. Although its intraductal nature and some clinicopathologic features resemble those of intraductal papillary mucinous neoplasm, our results suggest that intraductal tubulopapillary neoplasm has distinguishing genetic characteristics. Some of these mutated genes are potentially targetable. Future functional studies will be needed to determine the consequences of these gene alterations.

441 Background: Clinical factors associated with pathologic response (PResp) following neoadjuvant chemotherapy (NCT) in locally advanced gastric cancer (LAGC) are well studied; however, genomic correlates of such response have not been previously investigated. Methods: Evaluable pre-NCT tumor samples from patients with LAGC who underwent resection and demonstrated extreme pathologic response (EPR; ≤10% PResp: n = 21, ≥80% PResp: n = 19) were sequenced using a targeted exome capture platform. Gene- and signaling pathway-level correlates of EPR and disease-specific survival (DSS) were examined. Results: Of 40 patients, a majority had ≥cT2/N+ disease and were treated with predominantly platinum (98%) or 5-FU (88%) based NCT regimens. Two patients with MSI-high tumors had ≤10% PResp and were excluded from analysis. The EPR cohorts did not differ significantly in demographic or clinical (i.e., tumor location, cT/N status, NCT regimen, extent of gastrectomy, number of lymph nodes examined, or margin status) characteristics. Although EPR cohorts did not differ with respect to tumor differentiation/grade, Lauren classification, proportions of TCGA consensus CIN or GS subtypes, tumors with ≤10% PResp were more likely to have vascular (P < 0.001) and perineural (P = 0.007) invasion. At median follow-up of 31m (IQR 21-57), ≥80% PResp was associated with improved DSS compared with ≤10% PResp (median NR vs. 32m, P = 0.04). On gene-level analysis, tumors with ≤10% PResp were significantly more likely to be ERBB2-altered (32% vs 5%, P = 0.04) compared with ≥80% PResp tumors. Conversely, ARID1A truncating mutations were enriched in tumors with ≥80% vs ≤10% PResp (32% vs 5%, P = 0.04). There was no difference in pathway-level alteration frequency between EPR cohorts. While frequency of oncogenic TP53 alterations was similar between EPR cohorts, TP53-altered tumors were associated with worse DSS vs TP53-wildtype tumors (median 80m vs 24m, P = 0.005) in patients demonstrating ≤10%, but not ≥80%, PResp. Conclusions: Genomic comparison of cohorts demonstrating EPR after NCT in LAGC reveal molecular vulnerabilities with distinct prognostic and therapeutic implications.

Phacomatosis pigmentokeratotica (PPK) is characterized by the co-existence of epidermal nevi and large segmental speckled lentiginous nevi of the papulosa type. PPK, previously explained as 'twin spot' mosaicism due to the postzygotic crossing-over of two homozygous recessive mutations, has recently been shown to derive from one postzygotic activating RAS mutation. Epidermal nevi, including those in PPK, are known to give rise to neoplasms such as trichoblastoma and basal cell carcinoma. Within speckled lentiginous nevi, Spitz nevi and melanoma have been well documented. We report a case of PPK with a combined melanocytic and adnexal neoplasm presenting where the nevi conjoined. Using next-generation sequencing techniques, we were able to identify the same HRAS G13R mutation within both components of the tumor, and to show the absence of additional mutated modifier genes in a panel of 300 cancer-related genes. Given the genetic findings in this rare tumor-type, we suggest that this case may be used as a model for understanding the development of biphenotypic neoplasia or intratumoral heterogeneity in some cases.

Pancreatic ductal adenocarcinoma (PDAC) is the classic cold tumor, with few tumor-infiltrating activated CD8+ T cells, a major barrier to immunotherapy. Strategies to convert cold PDACs to hot can enable more effective immunotherapies. To identify targets that can convert cold PDACs to hot, we compared a large cohort of rare long-term PDAC survivors with hot tumors to short-term survivors with cold tumors. Surprisingly, hot PDACs were enriched not only in activated CD8+ T cells, but also in group-2 innate lymphoid cells (ILC2s), a class of largely tissue-resident, cytokine-producing innate lymphocyte that potentiates T cell immunity in local tissues. We identified that tumor ILC2s (TILC2s) infiltrate human and mouse PDACs, and TILC2 frequency and tumor expression of the ILC2-activating ligand interleukin (IL)-33 positively correlate with tumor immune cytolytic activity, and long-term patient survival. Using PDAC mouse models, we discovered that the IL33-ILC2 axis activates pancreatic tissue-specific antitumor T-cell immunity. Host IL33 deficiency attenuated TILC2 expansion and CD8+ T-cell activation, accelerating tumor growth in orthotopic PDACs but not heterotopic skin implanted PDACs where TILC2s lack the IL33 receptor. Acute ILC2 depletion partially phenocopied the tissue-specific phenotype of IL33 deficiency, with impaired rejection of immunogenic orthotopic but not heterotopic skin PDACs. Treatment with recombinant IL33 (rIL33), but not with the ILC1-activating ligand rIL18, increased TILC2 frequencies, activated CD8+ T cells, and abrogated orthotopic but not heterotopic PDAC establishment in >70% of mice. Taken together, these studies identify ILC2s as novel anticancer immune cells for PDAC immunotherapy. Strategies to activate tissue-specific ILC2s may be a promising immunotherapeutic approach for PDAC. Citation Format: Joanne Leung, Luis A Rojas, Jennifer Ruan, Julia Zhao, Billel Gasmi, Zachary Sethna, Anita Ramnarain, Umesh Bhanot, Gokce Askan, Murali Gururajan, Ela Elyada, Youngkyu Park, David A. Tuveson, Mithat Gonen, Steven D. Leach, Jedd D. Wolchok, Ronald P. DeMatteo, Taha Merghoub, Vinod P. Balachandran, John Alec Moral. Tissue-specific innate lymphoid cells are novel targets for pancreatic cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B02.

Oncogenic Suspect Exposed It can be difficult logistically to study the genomics of rare variants of common cancers. Nevertheless, Honeyman et al. (p. 1010 ) studied fibrolamellar hepatocellular carcinoma (FL-HCC), a rare and poorly understood liver tumor that affects adolescents and young adults and for which there is no effective treatment. FL-HCCs from 15 patients all expressed a chimeric RNA transcript and protein containing sequences from a molecular chaperone fused in frame with sequences from the catalytic domain of protein kinase A. The chimeric protein retained kinase activity in vitro. Such recurrent gene fusions in cancer may signal a role in pathogenesis and provide an opportunity for therapeutic intervention.

Synonymous mutations, which do not alter the protein sequence, have been shown to affect protein function [Sauna ZE, Kimchi-Sarfaty C (2011) Nat Rev Genet 12(10):683–691]. However, synonymous mutations are rarely investigated in the cancer genomics field. We used whole-genome and -exome sequencing to identify somatic mutations in 29 melanoma samples. Validation of one synonymous somatic mutation in BCL2L12 in 285 samples identified 12 cases that harbored the recurrent F17F mutation. This mutation led to increased BCL2L12 mRNA and protein levels because of differential targeting of WT and mutant BCL2L12 by hsa-miR-671–5p. Protein made from mutant BCL2L12 transcript bound p53, inhibited UV-induced apoptosis more efficiently than WT BCL2L12 , and reduced endogenous p53 target gene transcription. This report shows selection of a recurrent somatic synonymous mutation in cancer. Our data indicate that silent alterations have a role to play in human cancer, emphasizing the importance of their investigation in future cancer genome studies.

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.

The pathobiology of chronic pancreatitis (CP) remains enigmatic despite remarkable progress made recently in uncovering key mechanisms involved in the initiation and progression of the disease. CP is increasingly thought of as a multifactorial disorder. Apoptosis plays a role in parenchymal destruction, the pathological hallmark of CP. The apoptotic mechanisms preferentially target the exocrine compartment, leaving endocrine islets relatively intact for a prolonged period. Exocrine cells shed their 'immunoprivileged' status, express death receptors, and are rendered susceptible to apoptosis induced by death ligands on infiltrating lymphocytes, and released locally by activated pancreatic stellate cells. Islet cells retain their 'immunoprivileged' status and activate anti-apoptotic programs through NF-kappaB. Ductal changes, including distortion, dilatation, and pancreatic ductal hypertension in the setting of CP, induce genomic damage and increased cell turnover. In addition, signaling mechanisms that play a role in the development of embryonic pancreas are reinstated, thus, playing a role in repair, regeneration, and transformation. This, in turn, leads to acino-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN). Some of these pathways are activated in pancreatic cancer. We attempt to integrate the current knowledge and major concepts in the pathogenesis of CP and to explain the mechanism of differential cell loss. We also discuss the possible implications of signaling pathway activation in pancreatic inflammation, relevant to the cellular transformation that leads to pancreatic neoplasia.

Evasion of apoptosis is a characteristic feature of pancreatic cancer, a prototypic cancer that is refractory to current treatment approaches. Hence, there is an urgent need to design rational strategies that counter apoptosis resistance. To explore X-linked inhibitor of apoptosis (XIAP) as a therapeutic target in pancreatic cancer, we analyzed the expression of XIAP in pancreatic tumor samples and evaluated the effect of small molecule XIAP inhibitors alone and in combination with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) against pancreatic carcinoma in vitro and in vivo. Here, we report that XIAP is highly expressed in pancreatic adenocarcinoma samples compared with normal pancreatic ducts. Small molecule XIAP inhibitors synergize with TRAIL to induce apoptosis and to inhibit long-term clonogenic survival of pancreatic carcinoma cells. In contrast, they do not reverse the lack of toxicity of TRAIL on nonmalignant cells in vitro or normal tissues in vivo, pointing to a therapeutic index. Most importantly, XIAP inhibitors cooperate with TRAIL to trigger apoptosis and suppress pancreatic carcinoma growth in vivo in two preclinical models, i.e., the chorioallantoic membrane model and a mouse xenograft model. Parallel immunohistochemical analysis of tumor tissue under therapy reveals that the XIAP inhibitor acts in concert with TRAIL to cause caspase-3 activation and apoptosis. In conclusion, our findings provide, for the first time, evidence in vivo that XIAP inhibitors prime pancreatic carcinoma cells for TRAIL-induced apoptosis and potentiate the antitumor activity of TRAIL against established pancreatic carcinoma. These findings build the rationale for further (pre)clinical development of XIAP inhibitors and TRAIL against pancreatic cancer. [Cancer Res 2009;69(6):2425–34]

In 2010, the World Health Organization reclassified the entity originally described as intraductal oncocytic papillary neoplasm as the ‘oncocytic subtype’ of intraductal papillary mucinous neoplasm. Although several key molecular alterations of other intraductal papillary mucinous neoplasm subtypes have been discovered, including common mutations in KRAS, GNAS, and RNF3, those of oncocytic subtype have not been well characterized. We analyzed 11 pancreatic ‘oncocytic subtype’ of intraductal papillary mucinous neoplasms. Nine pancreatic ‘oncocytic subtype’ of intraductal papillary mucinous neoplasms uniformly exhibited typical entity-defining morphology of arborizing papillae lined by layers of cells with oncocytic cytoplasm, prominent, nucleoli, and intraepithelial lumina. The remaining two were atypical. One lacked the arborizing papilla and had flat oncocytic epithelium only; the other one had focal oncocytic epithelium in a background of predominantly intestinal subtype intraductal papillary mucinous neoplasm. Different components of this case were analyzed separately. Formalin-fixed, paraffin-embedded specimens of all cases were microdissected and subjected to high-depth-targeted next-generation sequencing for a panel of 300 key cancer-associated genes in a platform that enabled the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Fresh frozen specimens of two cases were also subjected to whole-genome sequencing. For the nine typical pancreatic ‘oncocytic subtype’ of intraductal papillary mucinous neoplasms, the number of mutations per case, identified by next-generation sequencing, ranged from 1 to 10 (median = 4). None of these cases had KRAS or GNAS mutations and only one had both RNF43 and PIK3R1 mutations. ARHGAP26, ASXL1, EPHA8, and ERBB4 genes were somatically altered in more than one of these typical ‘oncocytic subtype’ of intraductal papillary mucinous neoplasms but not in the other two atypical ones. In the neoplasm with flat oncocytic epithelium, the only mutated gene was KRAS. All components of the intestinal subtype intraductal papillary mucinous neoplasms with focal oncocytic epithelium manifested TP53, GNAS, and RNF43 mutations. In conclusion, this study elucidates that ‘oncocytic subtype’ of intraductal papillary mucinous neoplasm is not only morphologically distinct but also genetically distinct from other intraductal papillary mucinous neoplasm subtypes. Considering that now its biologic behavior is also being found to be different than other intraductal papillary mucinous neoplasm subtypes, ‘oncocytic subtype’ of intraductal papillary mucinous neoplasm warrants being recognized separately.

Ductal concretions in chronic pancreatitis (CP) are one of the causes of ductal obstruction, resulting in pancreatic ductal hypertension (PDH) and duct ectasia. Ductal epithelium subjected to chronic stress by PDH may undergo molecular alterations, thereby not only initiating and sustaining the inflammatory process but also activating molecules that have transforming potential. Acino-ductal metaplasia and pancreatic intraepithelial neoplasia (PanIN) are frequently seen in CP. Using laser capture microdissection, cDNA microarrays and Ingenuity Pathways Analysis, we found an altered Notch pathway in the ectatic ducts of CP. The microarray data was further validated by real-time PCR. We also found elevated transcripts of Notch receptors, Notch1 and Notch3 in microdissected ectatic ducts of CP. The Notch pathway ligands, Jagged/Delta-like and a Notch target, HES-related repressor protein (HERP), were up-regulated in ectatic compared to normal pancreatic ducts, while another target of Notch, hairy/enhancer of split (HES), was down-regulated. The transcripts of Delta-like1 and Jagged1 were increased 3.7-fold and 1.3-fold, respectively, while those of HERP1 were elevated 2.4-fold in the ectatic ducts of CP, compared to normal ducts. Immunohistochemistry showed that Jagged1 was not expressed in normal pancreatic ducts, while it was highly expressed in ectatic ducts. This pattern of Notch component alteration in ectatic ducts was mimicked to some extent in vitro in a human pancreatic duct epithelial (HPDE) cell line, when subjected to a pressure of 200 mmHg for 24 h. Therefore, we conclude that in the ectatic ducts of CP, PDH activates signalling pathways such as Notch, which have transforming potential.

Fibrolamellar hepatocellular carcinoma (FLHCC) tumors all carry a deletion of ∼ 400 kb in chromosome 19, resulting in a fusion of the genes for the heat shock protein, DNAJ (Hsp40) homolog, subfamily B, member 1, DNAJB1, and the catalytic subunit of protein kinase A, PRKACA. The resulting chimeric transcript produces a fusion protein that retains kinase activity. No other recurrent genomic alterations have been identified. Here we characterize the molecular pathogenesis of FLHCC with transcriptome sequencing (RNA sequencing). Differential expression (tumor vs. adjacent normal tissue) was detected for more than 3,500 genes (log2 fold change ≥ 1, false discovery rate ≤ 0.01), many of which were distinct from those found in hepatocellular carcinoma. Expression of several known oncogenes, such as ErbB2 and Aurora Kinase A, was increased in tumor samples. These and other dysregulated genes may serve as potential targets for therapeutic intervention.

Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive gastrointestinal cancers and is thought to arise from noninvasive precursors-pancreatic intraepithelial neoplasia (PanIN). Aberrantly prolonged cell survival due to apoptosis suppression is likely to contribute to carcinogenesis and carcinoma progression where the inhibitor of apoptosis proteins (IAPs) may play an important role. IAPs specifically inhibit caspases 3, 7, and 9 and prevent apoptosis. Survivin is a unique member of the IAPs family that is expressed in most human cancers including PDA but is not expressed in most normal adult tissues. To measure survivin transcript levels in normal pancreatic ducts, PanINs, and PDA, we used laser capture microdissection and real-time polymerase chain reaction. Survivin protein expression in normal pancreatic ducts, PanINs, PDA, and its metastases to lymph nodes were evaluated by immunohistochemistry. In microdissected tissues, we found a steady and close to exponential increase in survivin transcript levels from low-grade lesions (PanINs-1) to high-grade lesions (PanINs-2 and 3) and further to PDA. This observation was strictly mirrored by survivin protein expression. In addition, survivin was localized to the nucleus in high-grade lesions (starting at PanIN-2 stage), PDA, and nodal metastases, suggesting that nuclear translocation of survivin may be an early event in transformation to malignancy.

We describe cyclic hydrostatic pressure of 200/100 mmHg with a frequency of 85/min as a hemodynamically relevant pathological condition enforcing apoptosis in endothelial cells (EC) after 24 h of treatment. This went along with an increase of CD95 and CD95L surface expression, shedding of CD95L into the supernatant, cleavage of caspase-3 and caspase-8, and elevated JNK-2, c-Jun, and CD95L mRNA expression. Furthermore, increased DNA-binding activity of the AP-1 transcription factor family members FRA-1 and c-Jun was observed. This activation was reduced by inhibition of JNK, which subsequently prevented elevated CD95L mRNA expression. Caspase inhibitors and a CD95L-neutralizing antibody also reduced EC apoptosis. Most of the pressure-induced events were most prominent at 24 and 48 h. However, after 48 h, the CD95/CD95L expression pattern switched back to CD95−/CD95L+ and the specific death rate decreased. Cyclic pathological hydrostatic pressure is a novel type of stress to EC that renders them susceptible to CD95/CD95L-mediated autoapoptosis and/or paracrine apoptosis accompanied by upregulation of intracellular molecules known to trigger both apoptosis and survival.

Next Generation DNA Sequencing (NGS) technologies are currently being applied in the clinical setting for the treatment of disease. The goal is to use high-throughput sequencing to identify specific variants within each tumor and recommend personalized treatment approaches or clinical trials tailored to the individual’s disease and genomic profile. These assays are comprised of either predefined sequencing panels, where a handpicked set of clinically significant genes are examined within each patient, or are cancer type specific targeted sequencing protocols or whole exome platforms covering only the coding region of the patient’s genome. Whole genome sequencing allows hypothesis-free interrogation of both coding and non-coding regions of the genome revealing more potential therapeutic options than examining a small set of genes or genomic loci. The protocol eliminates sequence capture related bias observed in whole exome or panel sequencing. The New York Genome Center therefore has performed analytical validation of whole genome and transcriptome sequencing (WGTS) of patient derived tumors and matched normals for the purposes of clinical testing and have devised a clinical reporting strategy of significant driver and therapeutic associated mutations. Many clinical NGS guidelines are directed toward targeted panel or exome sequencing validation. Here, we expanded on New York State’s Department of Health NGS guidelines developing them into novel standards applicable to WGTS for the purposes of clinical test validation. We first sequenced a virtual tumor at very high coverage (300x) and downsampled to determine the optimum depth of sequencing necessary for high confidence somatic variant calling across the entire genome. We then validated whole genome sequencing laboratory protocols for DNA and RNA sequencing on a total of 50 specimens derived from fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tumor samples. We performed a series of experiments to assess the accuracy and reliability of the results based on our laboratory and bioinformatics protocols. We performed our validation on the 50 tumor normal pairs, a subset of which had known genomic profiles. Comparisons were also made for variant calling concordance and reproducibility between matched FF and FFPE tumors. Here, we present our validation results and clinical WGTS standards for depth of sequencing, reproducibility, sensitivity, and present limit of detection analysis for SNV calling, copy number identification and structural variants. RNA sequencing is performed to call fusion or exon skipping events and to confirm the DNA variants. The New York Genome Center WGTS clinical assay is intended to provide a more comprehensive patient variant discovery approach suitable for directed oncological therapeutic applications. Citation Format: Kazimierz O. Wrzeszczynski, Avinash Abhyankar, Vanessa Felice, Esra Dikoglu, Lukasz Kozon, Nicolas Robine, Anne-Katrin Emde, Olca Basturk, Umesh K. Bhanot, Alex Kentsis, Mahesh Mansukhani, Govind Bhagat, Vaidehi Jobanputra. Analytical validation of clinical whole genome and transcriptome sequencing of patient derived tumors: clinical application of whole genome sequencing for reporting targetable variants in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2714. doi:10.1158/1538-7445.AM2017-2714

// David G. Darcy 1, 2 , Rachel Chiaroni-Clarke 1 , Jennifer M. Murphy 1, 2 , Joshua N. Honeyman 1, 2 , Umesh Bhanot 3 , Michael P. LaQuaglia 2 , Sanford M. Simon 1 1 Laboratory of Cellular Biophysics, The Rockefeller University, New York, NY 10065, USA 2 Division of Pediatric Surgery, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA 3 Pathology Core, Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA Correspondence to: Sanford M. Simon, e-mail: simon@rockefeller.edu Keywords: Fibrolamellar, hepatocellular carcinoma, cancer, genome, sequencing Received: October 13, 2014 Accepted: November 11, 2014 Published: December 22, 2014 ABSTRACT Fibrolamellar hepatocellular carcinoma is a rare, malignant liver tumor that often arises in the otherwise normal liver of adolescents and young adults. Previous studies have focused on biomarkers and comparisons to traditional hepatocellular carcinoma, and have yielded little data on the underlying pathophysiology. We performed whole genome sequencing on paired tumor and normal samples from 10 patients to identify recurrent mutations and structural variations that could predispose to oncogenesis. There are relatively few coding, somatic mutations in this cancer, putting it on the low end of the mutational spectrum. Aside from a previously described heterozygous deletion on chromosome 19 that encodes for a functional, chimeric protein, there were no other recurrent structural variations that contribute to the tumor genotype. The lack of a second-hit mutation in the genomic landscape of fibrolamellar hepatocellular carcinoma makes the DNAJB1-PRKACA fusion protein the best target for diagnostic and therapeutic advancements. The mutations, altered pathways and structural variants that characterized fibrolamellar hepatocellular carcinoma were distinct from those in hepatocellular carcinoma, further defining it as a distinct carcinoma.

Much emphasis has been placed on the identification, functional characterization, and therapeutic potential of somatic variants in tumor genomes. However, the majority of somatic variants lie outside coding regions and their role in cancer progression remains to be determined. In order to establish a system to test the functional importance of non-coding somatic variants in cancer, we created a low-passage cell culture of a metastatic melanoma tumor sample. As a foundation for interpreting functional assays, we performed whole-genome sequencing and analysis of this cell culture, the metastatic tumor from which it was derived, and the patient-matched normal genomes. When comparing somatic mutations identified in the cell culture and tissue genomes, we observe concordance at the majority of single nucleotide variants, whereas copy number changes are more variable. To understand the functional impact of non-coding somatic variation, we leveraged functional data generated by the ENCODE Project Consortium. We analyzed regulatory regions derived from multiple different cell types and found that melanocyte-specific regions are among the most depleted for somatic mutation accumulation. Significant depletion in other cell types suggests the metastatic melanoma cells de-differentiated to a more basal regulatory state. Experimental identification of genome-wide regulatory sites in two different melanoma samples supports this observation. Together, these results show that mutation accumulation in metastatic melanoma is nonrandom across the genome and that a de-differentiated regulatory architecture is common among different samples. Our findings enable identification of the underlying genetic components of melanoma and define the differences between a tissue-derived tumor sample and the cell culture created from it. Such information helps establish a broader mechanistic understanding of the linkage between non-coding genomic variations and the cellular evolution of cancer., Author Summary Here we investigate the relationship between somatic variants and non-coding regulatory regions. To do this, we develop a new algorithm for identifying single nucleotide somatic variants in whole-genome sequencing data and apply it to a metastatic melanoma sample and a cell culture derived from this sample. Our results show that the two genomes are similar at the level of single nucleotide changes and more variable at larger copy number changes. We further observe that patterns of somatic mutation accumulation in non-coding regulatory regions suggests that the metastatic melanoma cells de-differentiated into a more basal regulatory state. That is, by simply looking at mutation accumulation across cell-type-specific non-coding functional regions, one can clearly see patterns that are indicative of cell state de-differentiation. Results from genome-wide functional regulatory region experimental mapping support this observation.

Tissue banking has become a major initiative at many oncology centers. The influence of warm ex-vivo ischemia times, storage times, and biobanking protocols on RNA integrity and subsequent microarray data is not well documented.A prospective institutional review board-approved protocol for the banking of abdominal neoplasms was initiated at Memorial Sloan-Kettering Cancer Center in 2001. Sixty-four representative pancreas cancer specimens snap-frozen at various ex-vivo procurement times (or =10 min, 11-30 min, 31-60 min,1 h) and banked during three time periods (2001-2004, 2004-2006, 2006-2008) were processed. RNA integrity was determined by microcapillary electrophoresis using the RNA integrity number (RIN) algorithm and by results of laser-capture microdissection (LCM).Overall, 42% of human pancreas cancer specimens banked under a dedicated protocol yielded RNA with a RIN ofor =7. Limited warm ex-vivo ischemia times did not negatively impact RNA quality (percentage of tissue with total RNA with RIN ofor =7 foror =10 min, 42%; 11-30 min, 58%; 31-60 min, 33%;60 min, 42%), and long-term storage of banked pancreas cancer biospecimens did not negatively influence RNA quality (total RNA with RIN ofor =7 banked 2001-2004, 44%; 2004-2006, 38%; 2006-2008, 50%). RNA retrieved from pancreatic cancer samples with RIN ofor =7 subject to LCM yielded RNA suitable for further downstream applications.Fresh-frozen pancreas tissue banked within a standardized research protocol yields high-quality RNA in approximately 50% of specimens and can be used for enrichment by LCM. Quality of tissues of the biobank were not adversely impacted by limited variations of warm ischemia times or different storage periods. This study shows the challenges and investments required to initiate and maintain high-quality tissue repositories.

Chronic pancreatitis is now thought to have a multifactorial etiology. New concepts integrating cellular, molecular and genetic knowledge of the disease have been proposed to explain its pathogenesis. However, the mechanisms responsible for early exocrine parenchymal destruction and preservation of endocrine islets were unexplored until recently. In the course of chronic inflammation, pancreatic acini lose their "immunoprotective" status by neo-expressing death receptors. Therefore, they become susceptible to apoptosis that is triggered by their respective ligands expressed on lymphocytes and released by pancreatic stellate cells. By contrast, islets retain their immunoprotective status and activate nuclear factor-kappaB (NF-kappaB)-induced anti-apoptotic factors, thus enabling survival. This knowledge might be exploited for devising therapeutic approaches to retard acinar loss and to prolong islet survival.

In advanced chronic pancreatitis (CP), islets are preserved even in the midst of scarring. We recently showed in CP local production of interferon (IFN)gamma, transforming growth factor (TGF)beta and death receptor ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), along with functional death receptor neoexpression and apoptosis in exocrine but not in endocrine cells. However, islets are strongly induced for TRAIL-receptor (R)-4 lacking the functional death domain. TRAIL-R4 signaling in T cells induces NFkappaB, which activates antiapoptotic programs. Here, we demonstrate that in insulinoma cells CM, TGFbeta/IFNgamma/TRAIL in combination induced TRAIL-R4 surface expression. TRAIL/IFNgamma upregulated NFkappaB subunits and its target gene survivin while downmodulating IkappaB alpha mRNA. RelA transcriptional activity increased upon stimulation with IFNgamma and IFNgamma/TRAIL. In situ, normal pancreatic epithelia had low mRNA levels of NFkappaB subunits. These were higher in parenchymal areas of CP with severe fibrosis and highest in islets. NFkappaB-regulated proteins IkappaB alpha, survivin and another apoptosis inhibitor, cIAP1, were found in corresponding sites, again at highest levels in islets surrounded by fibrosis. In conclusion, islets in CP not only evade immune attack by nonexposure of functional death receptors in the presence of TRAIL-R4 but also additionally neoexpress NFkappaB and its target genes, survivin and cIAP1, to protect themselves from apoptosis.

Background: Loss of CD95 expression in tumour cells occurs frequently in colon carcinoma and may be associated with disease progression. On the other hand, neo-expression of CD95L in tumour cells may contribute to immune evasion. Aims: We aimed at further exploring the functional role and prognostic significance of the CD95/CD95L death inducing system in colon carcinomas. Patients and methods: CD95 and CD95L expression was examined by immunohistochemistry in 128 R0 resected UICC (International Union against Cancer) stage II/III colon carcinomas and correlated with disease free survival. Results: CD95 expression in tumour cells was observed in only 30 carcinomas (23.4%) whereas the others had at least a minor subpopulation of CD95 negative cells. Loss of CD95 in tumour cells was related to adverse prognosis in uni- and multivariate analysis (p = 0.046 and p = 0.036, respectively). Tumour infiltrating lymphocytes (TIL) were the major source of CD95L in colon carcinomas. CD95L+TIL were present in 83% of cases whereas CD95L was found in tumour cells in only 12% of cases. Moreover, a high rate of CD95L+TIL correlated with prolonged disease free survival in patients with UICC stage II (p = 0.05) but not in those with stage III. Conclusions: Loss of CD95 in tumour cells may be an independent prognostic factor in colon carcinomas. The CD95L counterattack is not a relevant feature in colon carcinoma but CD95L+TIL may contribute to tumour control in the early stages of the disease, exerting a concurrent selection pressure in the direction of CD95 abrogation/resistance.

561 Background: PBRM1 and BAP1, both of which encode chromatin modulating proteins, have recently been identified as frequently mutated tumor suppressor genes in RCC. However, their correlation with outcomes of targeted therapies is unknown. We explored correlations between overall survival (OS) and mutations in archival tumor samples from RECORD-3, a randomized phase 2 trial comparing first-line everolimus (EVE) then sunitinib (SUN) to first-line SUN then EVE at progression in 471 treatment-naïve mRCC patients (J Clin Oncol 2014; 32:2764). Methods: Somatic mutations in exons of 341 cancer related genes were identified by a next generation sequencing (NGS) assay (MSK IMPACT). Association between genotypes and OS was assessed by Cox PH models and log-rank tests. Results: DNA of 258 archival tumor and 181 matched germline samples was successfully analyzed (median coverage 530X). Three molecular subgroups Gr1 (BAP1 MT [mutant], PBRM1 WT [wild-type]/MT; 17.4%), Gr2 (PBRM1 MT, BAP1 WT; 38.4%), and Gr3 (PBRM1 WT, BAP1 WT; 44.2%) with different OS outcomes after sequential EVE-SUN or SUN-EVE were identified (Table). In the sequential EVE-SUN arm, median OS (months) was shortest with Gr1 (9.8; 95% CI, 7.8–20.0), longest with Gr2 (39.6; 95% CI, 31.7–not estimable), and intermediate with Gr3 (18.1; 13.7–30.0), whereas in the SUN-EVE arm there was no significant difference among groups. When comparing OS between treatment sequences within molecular groups, a trend suggested differences for EVE-SUN vs SUN-EVE in Gr1 (HR 1.5), Gr2 (0.8), and Gr3 (1.2), although comparisons were not statistically significant. Conclusions: Our results suggest that these genotypes may represent distinct RCC molecular subtypes with potentially different predictive/prognostic values on targeted therapies. Different treatment sequences may be considered depending on the genotypes. Clinical trial information: NCT00903175. [Table: see text]