Clinical and molecular features of acquired resistance to immunotherapy in non-small cell lung cancer.

Although immunotherapy with PD-(L)1 blockade is routine for lung cancer, little is known about acquired resistance. Among 1,201 patients with non-small cell lung cancer (NSCLC) treated with PD-(L)1 blockade, acquired resistance is common, occurring in >60% of initial responders. Acquired resistance shows differential expression of inflammation and interferon (IFN) signaling. Relapsed tumors can be separated by upregulated or stable expression of IFNγ response genes. Upregulation of IFNγ response genes is associated with putative routes of resistance characterized by signatures of persistent IFN signaling, immune dysfunction, and mutations in antigen presentation genes which can be recapitulated in multiple murine models of acquired resistance to PD-(L)1 blockade after in vitro IFNγ treatment. Acquired resistance to PD-(L)1 blockade in NSCLC is associated with an ongoing, but altered IFN response. The persistently inflamed, rather than excluded or deserted, tumor microenvironment of acquired resistance may inform therapeutic strategies to effectively reprogram and reverse acquired resistance.

Subclonal Somatic Copy-Number Alterations Emerge and Dominate in Recurrent Osteosarcoma.

Multiple large-scale genomic profiling efforts have been undertaken in osteosarcoma to define the genomic drivers of tumorigenesis, therapeutic response, and disease recurrence. The spatial and temporal intratumor heterogeneity could also play a role in promoting tumor growth and treatment resistance. We conducted longitudinal whole-genome sequencing of 37 tumor samples from 8 patients with relapsed or refractory osteosarcoma. Each patient had at least one sample from a primary site and a metastatic or relapse site. Subclonal copy-number alterations were identified in all patients except one. In 5 patients, subclones from the primary tumor emerged and dominated at subsequent relapses. MYC gain/amplification was enriched in the treatment-resistant clones in 6 of 7 patients with multiple clones. Amplifications in other potential driver genes, such as CCNE1, RAD21, VEGFA, and IGF1R, were also observed in the resistant copy-number clones. A chromosomal duplication timing analysis revealed that complex genomic rearrangements typically occurred prior to diagnosis, supporting a macroevolutionary model of evolution, where a large number of genomic aberrations are acquired over a short period of time followed by clonal selection, as opposed to ongoing evolution. A mutational signature analysis of recurrent tumors revealed that homologous repair deficiency (HRD)-related SBS3 increases at each time point in patients with recurrent disease, suggesting that HRD continues to be an active mutagenic process after diagnosis. Overall, by examining the clonal relationships between temporally and spatially separated samples from patients with relapsed/refractory osteosarcoma, this study sheds light on the intratumor heterogeneity and potential drivers of treatment resistance in this disease. SIGNIFICANCE: The chemoresistant population in recurrent osteosarcoma is subclonal at diagnosis, emerges at the time of primary resection due to selective pressure from neoadjuvant chemotherapy, and is characterized by unique oncogenic amplifications.

Exportin 1 inhibition prevents neuroendocrine transformation through SOX2 down-regulation in lung and prostate cancers.

In lung and prostate adenocarcinomas, neuroendocrine (NE) transformation to an aggressive derivative resembling small cell lung cancer (SCLC) is associated with poor prognosis. We previously described dependency of SCLC on the nuclear transporter exportin 1. Here, we explored the role of exportin 1 in NE transformation. We observed up-regulated exportin 1 in lung and prostate pretransformation adenocarcinomas. Exportin 1 was up-regulated after genetic inactivation of TP53 and RB1 in lung and prostate adenocarcinoma cell lines, accompanied by increased sensitivity to the exportin 1 inhibitor selinexor in vitro. Exportin 1 inhibition prevented NE transformation in different TP53/RB1-inactivated prostate adenocarcinoma xenograft models that acquire NE features upon treatment with the aromatase inhibitor enzalutamide and extended response to the EGFR inhibitor osimertinib in a lung cancer transformation patient-derived xenograft (PDX) model exhibiting combined adenocarcinoma/SCLC histology. Ectopic SOX2 expression restored the enzalutamide-promoted NE phenotype on adenocarcinoma-to-NE transformation xenograft models despite selinexor treatment. Selinexor sensitized NE-transformed lung and prostate small cell carcinoma PDXs to standard cytotoxics. Together, these data nominate exportin 1 inhibition as a potential therapeutic target to constrain lineage plasticity and prevent or treat NE transformation in lung and prostate adenocarcinoma.

Determinants of Survival with Combined HER2 and PD-1 Blockade in Metastatic Esophagogastric Cancer.

PURPOSE: We report updated clinical outcomes from a phase II study of pembrolizumab, trastuzumab, and chemotherapy (PTC) in metastatic esophagogastric cancer in conjunction with outcomes from an independent Memorial Sloan Kettering (MSK) cohort. PATIENTS AND METHODS: The significance of pretreatment 89Zr-trastuzumab PET, plasma circulating tumor DNA (ctDNA) dynamics, and tumor HER2 expression and whole exome sequencing was evaluated to identify prognostic biomarkers and mechanisms of resistance in patients treated on-protocol with PTC. Additional prognostic features were evaluated using a multivariable Cox regression model of trastuzumab-treated MSK patients (n = 226). Single-cell RNA sequencing (scRNA-seq) data from MSK and Samsung were evaluated for mechanisms of therapy resistance. RESULTS: 89Zr-trastuzumab PET, scRNA-seq, and serial ctDNA with CT imaging identified how pre-treatment intrapatient genomic heterogeneity contributes to inferior progression-free survival (PFS). We demonstrated that the presence of intensely avid lesions by 89Zr-trastuzumab PET declines in tumor-matched ctDNA by 3 weeks, and clearance of tumor-matched ctDNA by 9 weeks were minimally invasive biomarkers of durable PFS. Paired pre- and on-treatment scRNA-seq identified rapid clearance of HER2-expressing tumor clones with expansion of clones expressing a transcriptional resistance program, which was associated with MT1H, MT1E, MT2A, and MSMB expression. Among trastuzumab-treated patients at MSK, ERBB2 amplification was associated with improved PFS, while alterations in MYC and CDKN2A/B were associated with inferior PFS. CONCLUSIONS: These findings highlight the clinical relevance of identifying baseline intrapatient heterogeneity and serial ctDNA monitoring of HER2-positive esophagogastric cancer patients to identify early evidence of treatment resistance, which could guide proactive therapy escalation or deescalation.

Combination Therapy With MDM2 and MEK Inhibitors Is Effective in Patient-Derived Models of Lung Adenocarcinoma With Concurrent Oncogenic Drivers and MDM2 Amplification.

INTRODUCTION: Although targeted therapies have revolutionized the therapeutic landscape of lung adenocarcinomas (LUADs), disease progression on single-agent targeted therapy against known oncogenic drivers is common, and therapeutic options after disease progression are limited. In patients with MDM2 amplification (MDM2amp) and a concurrent oncogenic driver alteration, we hypothesized that targeting of the tumor-suppressor pathway (by means of restoration of p53 using MDM2 inhibition) and simultaneous targeting of co-occurring MAPK oncogenic pathway might represent a more durably effective therapeutic strategy. METHODS: We evaluated genomic next-generation sequencing data using the Memorial Sloan Kettering Cancer Center-Integrated Mutation Profiling of Actionable Cancer Targets platform to nominate potential targets for combination therapy in LUAD. We investigated the small molecule MDM2 inhibitor milademetan in cell lines and patient-derived xenografts of LUAD with a known driver alteration and MDM2amp. RESULTS: Of 10,587 patient samples from 7121 patients with LUAD profiled by next-generation sequencing, 6% (410 of 7121) harbored MDM2amp. MDM2amp was significantly enriched among tumors with driver alterations in METex14 (36%, p < 0.001), EGFR (8%, p < 0.001), RET (12%, p < 0.01), and ALK (10%, p < 0.01). The combination of milademetan and the MEK inhibitor trametinib was synergistic in growth inhibition of ECLC5-GLx (TRIM33-RET/MDM2amp), LUAD12c (METex14/KRASG12S/MDM2amp), SW1573 (KRASG12C, TP53 wild type), and A549 (KRASG12S) cells and in increasing expression of proapoptotic proteins PUMA and BIM. Treatment of ECLC5-GLx and LUAD12c with single-agent milademetan increased ERK phosphorylation, consistent with previous data on ERK activation with MDM2 inhibition. This ERK activation was effectively suppressed by concomitant administration of trametinib. In contrast, ERK phosphorylation induced by milademetan was not suppressed by concurrent RET inhibition using selpercatinib (in ECLC5-GLx) or MET inhibition using capmatinib (in LUAD12c). In vivo, combination milademetan and trametinib was more effective than either agent alone in ECLC5-GLx, LX-285 (EGFRex19del/MDM2amp), L13BS1 (METex14/MDM2amp), and A549 (KRASG12S, TP53 wild type). CONCLUSIONS: Combined MDM2/MEK inhibition was found to have efficacy across multiple patient-derived LUAD models harboring MDM2amp and concurrent oncogenic drivers. This combination, potentially applicable to LUADs with a wide variety of oncogenic driver mutations and kinase fusions activating the MAPK pathway, has evident clinical implications and will be investigated as part of a planned phase 1/2 clinical trial.

Lineage tracing reveals clonal progenitors and long-term persistence of tumor-specific T cells during immune checkpoint blockade.

Paired single-cell RNA and T cell receptor sequencing (scRNA/TCR-seq) has allowed for enhanced resolution of clonal T cell dynamics in cancer. Here, we report a scRNA/TCR-seq analysis of 187,650 T cells from 31 tissue regions, including tumor, adjacent normal tissues, and lymph nodes (LN), from three patients with non-small cell lung cancer after immune checkpoint blockade (ICB). Regions with viable cancer cells are enriched for exhausted CD8+ T cells, regulatory CD4+ T cells (Treg), and follicular helper CD4+ T cells (TFH). Tracking T cell clonotypes across tissues, combined with neoantigen specificity assays, reveals that TFH and tumor-specific exhausted CD8+ T cells are clonally linked to TCF7+SELL+ progenitors in tumor draining LNs, and progressive exhaustion trajectories of CD8+ T, Treg, and TFH cells with proximity to the tumor microenvironment. Finally, longitudinal tracking of tumor-specific CD8+ and CD4+ T cell clones reveals persistence in the peripheral blood for years after ICB therapy.

Subclonal somatic copy number alterations emerge and dominate in recurrent osteosarcoma.

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. MYC gain/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 as CCNE1, RAD21, VEGFA, and IGF1R, 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.

Prospective Evaluation of Immune Activation Associated with Response to Radioembolization Assessed with PET/CT in Women with Breast Cancer Liver Metastasis.

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 t tests. Association with CR was assessed with Wilcoxon rank-sum or unpaired t tests. Results Twenty women were included. After TARE, peripheral IL-6 (geometric mean, 1.0 vs 1.6 pg/mL; P = .02), IL-10 (0.2 vs 0.4 pg/mL; P = .001), and IL-15 (1.9 vs 2.4 pg/mL; P = .01) increased. In biopsy tissue, lymphocyte activation gene 3-positive CD4+ TILs (15% vs 31%; P < .001) increased. Eight of 20 participants (40% [exact 95% CI: 19, 64]) achieved CR. Participants with CR had lower baseline peripheral monocytes (10% vs 29%; P < .001) and MDSCs (1% vs 5%; P < .001) and higher programmed cell death protein (PD) 1-positive CD4+ TILs (59% vs 26%; P = .006) at flow cytometry and higher PD-1+ staining in tumor (2% vs 1%; P = .046). Conclusion Complete response to transarterial radioembolization was associated with lower baseline cytokine, monocyte, and myeloid-derived suppressor cell levels and higher programmed cell death protein 1-positive tumor-infiltrating lymphocyte levels. © RSNA, 2022 Online supplemental material is available for this article.

Inhibition of XPO1 Sensitizes Small Cell Lung Cancer to First- and Second-Line Chemotherapy.

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.

Comprehensive molecular characterization of lung tumors implicates AKT and MYC signaling in adenocarcinoma to squamous cell transdifferentiation.

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.

Signatures of plasticity, metastasis, and immunosuppression in an atlas of human small cell lung cancer.

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.

Multiomic Analysis of Lung Tumors Defines Pathways Activated in Neuroendocrine Transformation.

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.

ILC2s amplify PD-1 blockade by activating tissue-specific cancer immunity.

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.

Analytical Validation of Clinical Whole-Genome and Transcriptome Sequencing of Patient-Derived Tumors for Reporting Targetable Variants in Cancer.

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.

Non coding RNA analysis in fibrolamellar hepatocellular carcinoma.

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.

Identification of unique neoantigen qualities in long-term survivors of pancreatic cancer.

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

Pancreatic intraductal tubulopapillary neoplasm is genetically distinct from intraductal papillary mucinous neoplasm and ductal adenocarcinoma.

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.

Genomic Biomarkers of a Randomized Trial Comparing First-line Everolimus and Sunitinib in Patients with Metastatic Renal Cell Carcinoma.

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.

The oncocytic subtype is genetically distinct from other pancreatic intraductal papillary mucinous neoplasm subtypes.

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.

Transcriptomic characterization of fibrolamellar hepatocellular carcinoma.

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.