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Comparison of differences in clonal mutations between pre-treatment and resistant tumors

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Immune checkpoint inhibitors are promising treatments for patients with a variety of malignancies. Toward understanding the determinants of response to immune checkpoint inhibitors, it was previously demonstrated that the presence of somatic mutations is associated with benefit from checkpoint inhibition. A hypothesis was posited that neoantigen homology to pathogens may in part explain the link between somatic mutations and response. To further examine this hypothesis, we reanalyzed cancer exome data obtained from our previously published study of 64 melanoma patients treated with CTLA-4 blockade and a new dataset of RNA-Seq data from 24 of these patients. We found that the ability to accurately predict patient benefit did not increase as the analysis narrowed from somatic mutation burden, to inclusion of only those mutations predicted to be MHC class I neoantigens, to only including those neoantigens that were expressed or that had homology to pathogens. The only association between somatic mutation burden and response was found when examining samples obtained prior to treatment. Neoantigen and expressed neoantigen burden were also associated with response, but neither was more predictive than somatic mutation burden. Neither the previously described tetrapeptide signature nor an updated method to evaluate neoepitope homology to pathogens was more predictive than mutation burden. Cancer Immunol Res; 5(1); 84–91. ©2016 AACR.

In vitro kinase inhibitory activity of Compound 1

Comparison of mRNA expression of immune-related genes in tumors derived from control and Fbxw7-deficient tumors

Gene set enrichment analysis of FBXW7 wild-type or mutated melanoma samples from TCGA

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1. Mutation burden and survival using updated mutation calling system. 2. Similarity scores between samples and viral or non-viral epitopes. 3. Deconvolution of immune infiltrates. 4. Gene set enrichment analysis.

The molecular mechanisms leading to resistance to PD-1 blockade are largely unknown. Here, we characterize tumor biopsies from a patient with melanoma who displayed heterogeneous responses to anti–PD-1 therapy. We observe that a resistant tumor exhibited a loss-of-function mutation in the tumor suppressor gene FBXW7, whereas a sensitive tumor from the same patient did not. Consistent with a functional role in immunotherapy response, inactivation of Fbxw7 in murine tumor cell lines caused resistance to anti–PD-1 in immunocompetent animals. Loss of Fbxw7 was associated with altered immune microenvironment, decreased tumor-intrinsic expression of the double-stranded RNA (dsRNA) sensors MDA5 and RIG-I, and diminished induction of type I IFN and MHC-I expression. In contrast, restoration of dsRNA sensing in Fbxw7-deficient cells was sufficient to sensitize them to anti–PD-1. Our results thus establish a new role for the commonly inactivated tumor suppressor FBXW7 in viral sensing and sensitivity to immunotherapy.Significance:Our findings establish a role of the commonly inactivated tumor suppressor FBXW7 as a genomic driver of response to anti–PD-1 therapy. Fbxw7 loss promotes resistance to anti–PD-1 through the downregulation of viral sensing pathways, suggesting that therapeutic reactivation of these pathways could improve clinical responses to checkpoint inhibitors in genomically defined cancer patient populations.This article is highlighted in the In This Issue feature, p. 1241

List of all identified somatic mutations in pre-treatment lesion and PD-1 resistant tumors by ABSOLUTE analysis

Supplementary Figure Legends and Figures

Sequences of primers

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Supplementary Figures 1-10, Supplementary Legends 1-10, Supplementary Legends to Tables 1-7

Spearman correlation of FBXW7 mRNA levels with CD8A mRNA levels across TCGA cancers

Antibodies used in this manuscript

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Supplementary Methods, Tables, and Figures

Purpose: Squamous cell carcinoma of the anal canal (ASCC) accounts for 2% to 4% of gastrointestinal malignancies in the United States and is increasing in incidence; however, genomic features of ASCC are incompletely characterized. Primary treatment of ASCC involves concurrent chemotherapy and radiation (CRT), but the mutational landscape of resistance to CRT is unknown. Here, we aim to compare mutational features of ASCC in the pre- and post-CRT setting.Experimental Design: We perform whole-exome sequencing of primary (n = 31) and recurrent (n = 30) ASCCs and correlate findings with clinical data. We compare genomic features of matched pre- and post-CRT tumors to identify genomic features of CRT response. Finally, we investigate the mutational underpinnings of an extraordinary ASCC response to immunotherapy.Results: We find that both primary and recurrent ASCC tumors harbor mutations in genes, such as PIK3CA and FBXW7, that are also mutated in other HPV-associated cancers. Overall mutational burden was not significantly different in pre- versus post-CRT tumors, and several examples of shared clonal driver mutations were identified. In two cases, clonally related pre- and post-CRT tumors harbored distinct oncogenic driver mutations in the same cancer gene (KRAS or FBXW7). A patient with recurrent disease achieved an exceptional response to anti-programmed death (PD-1) therapy, and genomic dissection revealed high mutational burden and predicted neoantigen load.Conclusions: We perform comprehensive mutational analysis of ASCC and characterize mutational features associated with CRT. Although many primary and recurrent tumors share driver events, we identify several unique examples of clonal evolution in response to treatment. Clin Cancer Res; 23(12); 3214–22. ©2016 AACR.

The molecular mechanisms leading to resistance to PD-1 blockade are largely unknown. Here, we characterize tumor biopsies from a patient with melanoma who displayed heterogeneous responses to anti–PD-1 therapy. We observe that a resistant tumor exhibited a loss-of-function mutation in the tumor suppressor gene FBXW7, whereas a sensitive tumor from the same patient did not. Consistent with a functional role in immunotherapy response, inactivation of Fbxw7 in murine tumor cell lines caused resistance to anti–PD-1 in immunocompetent animals. Loss of Fbxw7 was associated with altered immune microenvironment, decreased tumor-intrinsic expression of the double-stranded RNA (dsRNA) sensors MDA5 and RIG-I, and diminished induction of type I IFN and MHC-I expression. In contrast, restoration of dsRNA sensing in Fbxw7-deficient cells was sufficient to sensitize them to anti–PD-1. Our results thus establish a new role for the commonly inactivated tumor suppressor FBXW7 in viral sensing and sensitivity to immunotherapy. Significance: Our findings establish a role of the commonly inactivated tumor suppressor FBXW7 as a genomic driver of response to anti–PD-1 therapy. Fbxw7 loss promotes resistance to anti–PD-1 through the downregulation of viral sensing pathways, suggesting that therapeutic reactivation of these pathways could improve clinical responses to checkpoint inhibitors in genomically defined cancer patient populations. This article is highlighted in the In This Issue feature, p. 1241

Objective Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) and SMARCA4-deficient undifferentiated uterine sarcoma (SMARCA4-DUS) are rare and aggressive tumors, primarily affecting pre- and perimenopausal women. Inactivating SMARCA4 mutations are thought to be the driving molecular events in the majority of these tumors. Here, we report the clinical course of a family with germline SMARCA4 mutation and compare large cohorts of these rare tumor types. Methods We extracted clinico-pathological medical record data for the family with germline SMARCA4 mutation. Clinico-genomic data from SCCOHT and SMARCA4-DUS cohorts were retrospectively extracted from the archives of a large CLIA-certified reference molecular laboratory. Results We identified a single family with an inherited germline SMARCA4 mutation, in which two different family members developed either SCCOHT or SMARCA4-DUS, both of whom died within one year of diagnosis, despite aggressive surgical, chemotherapy and immunotherapy treatment. Retrospective comparative analysis of large SCCOHT (n = 48) and SMARCA4-DUS (n = 17) cohorts revealed that SCCOHT patients were younger (median age: 28.5 vs. 49.0) and more likely to have germline SMARCA4 alterations (37.5% vs. 11.8%) than SMARCA4-DUS patients. Conclusions Growing understanding of the role SMARCA4 plays in the pathogenesis of these rare cancers may inform recommended genetic testing and counseling in families with these tumor types.

Acute gastroenteritis (AGE) is caused by a wide range of pathogens. Culture methods for the detection of bacterial pathogens is time consuming and labour intensive. This study compared a same-day-to-result commercial molecular method using BD Max™ Enteric Bacterial Panel against conventional culture and laboratory-developed PCR assays (LDTs), and characterised the epidemiology of bacterial AGE in Singapore.PCRs for Campylobacter spp., Salmonella spp., Shigella spp./Enteroinvasive Escherichia coli (EIEC) and Shiga toxin-producing E. coli (STEC)/Shigella dysenteriae were performed on the BD Max™ platform. Concurrent routine bacterial culture ("reference standard") was performed for Campylobacter, Salmonella, Shigella, Vibrio and Aeromonas spp. In the event of a discrepancy, an "expanded reference standard" (bacterial culture with LDT) was used.There were 299 stool specimens in the study, with no bacterial pathogens detected in 190 samples (63.5%). The positive samples (n = 109,36.5%) were detected with Salmonella (n = 57,19.1%), Campylobacter (n = 28,9.4%), Vibrio parahaemolyticus (n = 6,2.0%), Shigella/EIEC (n = 6,2.0%), ETEC (n = 4,1.3%), STEC (n = 2,0.7%), Aeromonas (n = 2,0.7%), Plesiomonas shigelloides (n = 1,0.3%) and 3(1.0%) co-infections. Compared to the "expanded reference standard", conventional culture missed 38/112 (33.9%) pathogens. Conversely, testing by BD Max™ alone failed to detect 17 pathogens. BD Max™ reported seven (2.3%) false-positive results.BD Max™ increased the detection rate of bacterial AGE pathogens in the panel, but was limited by the absence of detection capability for Vibrio and Aeromonas spp.

Introduction: Onychomycosis is a debilitating, difficult-to-treat nail fungal infection with increasing prevalence worldwide. The main etiological agents are dermatophytes, which are common causative pathogens in superficial fungal mycoses. Conventional detection methods such as fungal culture have low sensitivity and specificity and are time-consuming.Aim: The main objective of this study was to design, develop and validate a real-time probe-based multiplex qPCR assay for the detection of dermatophytes and Fusarium species.Methodology: The performance characteristics of the qPCR assays were evaluated. The multiplex qPCR assays targeted four genes (assay 1: pan-dermatophytes/Fusarium spp.; assay 2: Trichophyton rubrum/Microsporum spp.). Analytical validation was accomplished using 150 fungal isolates and clinical validation was done on 204 nail specimens. The performance parameters were compared against the gold standard (fungal culture) and expanded gold standard (culture in conjunction with sequencing).Results: Both the single-plex and multiplex qPCR assays performed well especially when compared against the expanded gold standard. Among the 204 tested nail specimens, the culture method showed that 125 (61.3 %) were infected with at least one organism, of which 40 yielded positive results for dermatophytes and Fusarium spp. These target organisms detected include 20 dermatophytes and 22 Fusarium spp. The developed qPCR assays demonstrated excellent limit of detection, efficiency, coefficient of determination, analytical and clinical sensitivity and specificity.Conclusion: The multiplex qPCR assays were reliable for the diagnosis of onychomycosis, with shorter turn-around time as compared to culture method. This aids in the planning of treatment strategies to achieve optimal therapeutic outcome.

Objectives: Recent studies have identified higher recurrence and lower survival with a minimally-invasive approach (MIS) versus abdominal approach to radical hysterectomy for early-stage cervical cancer. We aim to compare recurrence rate, progression-free survival, and overall survival for cervical cancer after MIS versus abdominal radical hysterectomy. Methods: We searched Medline, Embase, Central, and the Cochrane Library to identify studies from 1990 to 2020 that included women with stage 1 or higher cervical cancer treated with primary radical hysterectomy and compared recurrence and/or progession-free survival (PFS) and overall survival with MIS versus abdominal hysterectomy (PROSPERO 2020 CRD42020173600). Results: 50 studies, including 22,593 women with cervical cancer, met inclusion criteria. 29% of studies had less than 30 months of follow-up and 14% had 60+ months of follow-up. Of the 37 studies reporting PFS, 29 reported no difference and 8 reported decreased PFS with MIS approach. Of the 37 studies reporting OS, 2 studies reported improved OS with MIS approach, 31 reported no difference, and 4 reported decreased overall survival with MIS approach. For progression-free survival, the odds were non-significantly worse for women undergoing MIS radical hysterectomy (OR 1.25, 95% CI 0.98-1.52, 14 studies) when all studies were included. When limited to studies with 30+ months follow-up, the odds of progression-free survival were worse with MIS radical hysterectomy (OR 1.39 for 30+ months, 95% CI 1.09-1.70, 12 studies; OR 1.49 for 48+ months, 95% CI 0.94-2.03, 4 studies). For overall survival, the odds were not significantly different for MIS vs. abdominal hysterectomy (OR 0.82, 95% CI 0.58-1.06, 14 studies). When limited to studies with longer follow-up, the odds of overall survival remained non-significantly different for MIS vs. abdominal (OR 0.90 for 30+ months, 95% CI 0.53-1.26, 11 studies; OR 0.94 for 48+ months, 95% CI 0.42-1.46, 4 studies; OR 1.70 for 60+ months, 95% CI 0.62-2.78, 2 studies). Conclusions: In our meta-analysis of 50 studies, MIS radical hysterectomy was associated with worse progression-free survival compared to open radical hysterectomy for cervical cancer in studies with 30+ months of follow-up. The emergence of this finding with longer follow-up highlights the importance of high-quality studies to guide cancer and surgical treatment.

Objectives: A recent randomized controlled trial of minimally invasive (MIS) radical hysterectomy versus open surgery for early-stage cervical cancer has demonstrated higher recurrence and lower survival with MIS. However, given the lower operative morbidity of MIS, multiple groups have attempted to define subgroups of patients who may be able to undergo laparoscopic or robotic radical hysterectomy safely in retrospective cohort studies. Methods: We conducted a systematic review and meta-analysis of cohort studies and randomized controlled trials from 1990 to 2020 comparing MIS radical hysterectomy to open radical hysterectomy in early-stage cervical cancer (PROSPERO 2020 CRD42020173600). Cancer histology, laparoscopic or robotic approach, and overall survival (OS), progression-free survival (PFS), and recurrence rate were recorded for each study. We conducted a random-effects meta-analysis with inverse-probability weighting. Results: 41 studies including 22,593 unique women (10,686 MIS, 47%) were eligible for analysis; 6 reported outcomes by histology. Squamous cell carcinoma was the most common histology (79%). For adenocarcinoma, meta-analysis of 3 studies no differences in recurrence (RR 0.87, 95% CI 0.41-1.85), PFS (RR 0.98, 95% CI 0.07-1.89), or OS (RR 1.41, 95% CI 0.56-2.27) between open and MIS surgery. For SCC, two studies showed no change in PFS, and one large study reported decreased OS (HR 1.65, 95% CI 1.17-2.33). One small study found no difference in PFS or OS for neuroendocrine tumors of the cervix. One large study showed decreased OS with robotic versus open radical hysterectomy (HR 1.61, 95% CI 1.2-2.2), but no difference with laparoscopic versus open surgery (HR 1.50, 95% CI 0.97-2.3). Download : Download high-res image (132KB) Download : Download full-size image Conclusions: This meta-analysis contributes to examination of current evidence on surgical approach to early-stage cervical cancer. Our results suggest that there is no histological group for which MIS radical hysterectomy is superior to open radical hysterectomy, and equivalent to inferior outcomes with robotic or conventional laparoscopic surgery versus open surgery. Subgroup analysis generally showed equivocal or inferior outcomes with MIS compared to open surgery, though results remain limited by sample size.

The molecular mechanisms leading to resistance to PD-1 blockade are largely unknown. Here, we characterize tumor biopsies from a melanoma patient who displayed heterogeneous responses to anti-PD-1 therapy. We observe that a resistant tumor exhibited a loss-of-function mutation in the tumor suppressor gene FBXW7, while a sensitive tumor from the same patient did not. Consistent with a functional role in immunotherapy response, inactivation of Fbxw7 in murine tumor cell lines caused resistance to anti-PD-1 in immunocompetent animals. Loss of Fbxw7 was associated with altered immune microenvironment, decreased tumor-intrinsic expression of the dsRNA sensors Mda5 and Rig-I, diminished induction of type I interferon and MHC-I expression. In contrast, restoration of dsRNA sensing in Fbxw7-deficient cells was sufficient sensitize them to anti-PD-1. Our results thus establish a new role for the commonly inactivated tumor suppressor FBXW7 in viral sensing and sensitivity to immunotherapy.

Tumor mutational burden correlates with response to immune checkpoint blockade in multiple solid tumors, though in microsatellite stable tumors this association is weak and of limited clinical utility. Here, we uniformly analyzed whole exome sequencing (WES) of 249 tumors and matched normal tissue from patients with clinically annotated outcomes to immune checkpoint therapy, including radiographic response, across multiple cancer types to examine additional tumor genomic features that contribute to selective response. Our analyses identified genomic correlates of response beyond mutational burden, including somatic events in individual driver genes, certain global mutational signatures, and specific HLA-restricted neoantigens. However, these features were often inter-related, highlighting the complexity of identifying genetic driver events that generate an immunoresponsive tumor environment. This study lays a path forward in analyzing large clinical cohorts in an integrated and multifaceted manner to enhance our ability to discover clinically meaningful predictive features of response to immune checkpoint blockade.

SNF'ing out antitumor immunity Immune checkpoint inhibitors induce durable tumor regressions in some, but not all, cancer patients. Understanding the mechanisms that determine tumor sensitivity to these drugs could potentially expand the number of patients who benefit (see the Perspective by Ghorani and Quezada). Pan et al. discovered that tumor cells in which a specific SWI/SNF chromatin remodeling complex had been experimentally inactivated were more sensitive to T cell–mediated killing. The cells were more responsive to interferon-γ, leading to increased secretion of cytokines that promote antitumor immunity. Miao et al. examined the genomic features of tumors from patients with metastatic renal cell carcinoma who had been treated with immune checkpoint inhibitors. Tumors harboring inactivating mutations in PBRM1 , which encodes a subunit of the same SWI/SNF complex, were more likely to respond to the drugs. Science , this issue p. 770 , p. 801 ; see also p. 745

Ex vivo systems that incorporate features of the tumor microenvironment and model the dynamic response to immune checkpoint blockade (ICB) may facilitate efforts in precision immuno-oncology and the development of effective combination therapies. Here, we demonstrate the ability to interrogate ex vivo response to ICB using murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS). MDOTS/PDOTS isolated from mouse and human tumors retain autologous lymphoid and myeloid cell populations and respond to ICB in short-term three-dimensional microfluidic culture. Response and resistance to ICB was recapitulated using MDOTS derived from established immunocompetent mouse tumor models. MDOTS profiling demonstrated that TBK1/IKKϵ inhibition enhanced response to PD-1 blockade, which effectively predicted tumor response in vivo. Systematic profiling of secreted cytokines in PDOTS captured key features associated with response and resistance to PD-1 blockade. Thus, MDOTS/PDOTS profiling represents a novel platform to evaluate ICB using established murine models as well as clinically relevant patient specimens. Significance: Resistance to PD-1 blockade remains a challenge for many patients, and biomarkers to guide treatment are lacking. Here, we demonstrate feasibility of ex vivo profiling of PD-1 blockade to interrogate the tumor immune microenvironment, develop therapeutic combinations, and facilitate precision immuno-oncology efforts. Cancer Discov; 8(2); 196–215. ©2017 AACR. See related commentary by Balko and Sosman, p. 143. See related article by Deng et al., p. 216. This article is highlighted in the In This Issue feature, p. 127

Despite continued widespread use, the genomic effects of cisplatin-based chemotherapy and implications for subsequent treatment are incompletely characterized. Here, we analyze whole exome sequencing of matched pre- and post-neoadjuvant cisplatin-based chemotherapy primary bladder tumor samples from 30 muscle-invasive bladder cancer patients. We observe no overall increase in tumor mutational burden post-chemotherapy, though a significant proportion of subclonal mutations are unique to the matched pre- or post-treatment tumor, suggesting chemotherapy-induced and/or spatial heterogeneity. We subsequently identify and validate a novel mutational signature in post-treatment tumors consistent with known characteristics of cisplatin damage and repair. We find that post-treatment tumor heterogeneity predicts worse overall survival, and further observe alterations in cell-cycle and immune checkpoint regulation genes in post-treatment tumors. These results provide insight into the clinical and genomic dynamics of tumor evolution with cisplatin-based chemotherapy, suggest mechanisms of clinical resistance, and inform development of clinically relevant biomarkers and trials of combination therapies., The impact of cisplatin-based chemotherapy on tumor genomes is complex. Here, the authors study matched pre- and post-chemotherapy primary samples in muscle-invasive bladder cancer, finding a cisplatin-based mutational signature, and highlighting the impact of intratumor heterogeneity on survival.

We present an in silico approach to identify neoepitopes derived from intron retention events in tumor transcriptomes. Using mass spectrometry immunopeptidome analysis, we show that retained intron (RI) neoepitopes are processed and presented on MHC-I on the surface of cancer cell lines. RNA-derived neoepitopes should be considered for prospective personalized cancer vaccine development.

Immune-checkpoint blockade (ICB) has demonstrated efficacy in many tumor types, but predictors of responsiveness to anti-PD1 ICB are incompletely characterized. In this study, we analyzed a clinically annotated cohort of patients with melanoma (n = 144) treated with anti-PD1 ICB, with whole-exome and whole-transcriptome sequencing of pre-treatment tumors. We found that tumor mutational burden as a predictor of response was confounded by melanoma subtype, whereas multiple novel genomic and transcriptomic features predicted selective response, including features associated with MHC-I and MHC-II antigen presentation. Furthermore, previous anti-CTLA4 ICB exposure was associated with different predictors of response compared to tumors that were naive to ICB, suggesting selective immune effects of previous exposure to anti-CTLA4 ICB. Finally, we developed parsimonious models integrating clinical, genomic and transcriptomic features to predict intrinsic resistance to anti-PD1 ICB in individual tumors, with validation in smaller independent cohorts limited by the availability of comprehensive data. Broadly, we present a framework to discover predictive features and build models of ICB therapeutic response., Analysis of fully clinically annotated and sequenced melanoma tumor samples collected before anti-PD1 treatment suggests that determinants of response differ on the basis of previous anti-CTLA4 therapy, and that tumor mutational burden may not be a strong predictor of response across melanoma subtypes.

BACKGROUND. Understanding the integrated immunogenomic landscape of advanced prostate cancer (APC) could impact stratified treatment selection. METHODS. Defective mismatch repair (dMMR) status was determined by either loss of mismatch repair protein expression on IHC or microsatellite instability (MSI) by PCR in 127 APC biopsies from 124 patients (Royal Marsden [RMH] cohort); MSI by targeted panel next-generation sequencing (MSINGS) was then evaluated in the same cohort and in 254 APC samples from the Stand Up To Cancer/Prostate Cancer Foundation (SU2C/PCF). Whole exome sequencing (WES) data from this latter cohort were analyzed for pathogenic MMR gene variants, mutational load, and mutational signatures. Transcriptomic data, available for 168 samples, was also performed. RESULTS. Overall, 8.1% of patients in the RMH cohort had some evidence of dMMR, which associated with decreased overall survival. Higher MSINGS scores associated with dMMR, and these APCs were enriched for higher T cell infiltration and PD-L1 protein expression. Exome MSINGS scores strongly correlated with targeted panel MSINGS scores (r = 0.73, P < 0.0001), and higher MSINGS scores associated with dMMR mutational signatures in APC exomes. dMMR mutational signatures also associated with MMR gene mutations and increased immune cell, immune checkpoint, and T cell–associated transcripts. APC with dMMR mutational signatures overexpressed a variety of immune transcripts, including CD200R1, BTLA, PD-L1, PD-L2, ADORA2A, PIK3CG, and TIGIT. CONCLUSION. These data could impact immune target selection, combination therapeutic strategy selection, and selection of predictive biomarkers for immunotherapy in APC. FUNDING. We acknowledge funding support from Movember, Prostate Cancer UK, The Prostate Cancer Foundation, SU2C, and Cancer Research UK.

Immunotherapies such as anti-PD-1 antibodies can induce durable responses in a subset of cancer patients, but intrinsic or acquired resistance occurs in most cases. Understanding the molecular mechanisms that drive resistance and response to immunotherapies could therefore lead to alternative treatment strategies exhibiting greater efficacy and precision. To identify new tumor cell-intrinsic mechanisms of resistance to PD-1 blockade, we evaluated metastatic melanoma patients who exhibited complete regression of all but one metastatic site following anti-PD-1 therapy. In one patient, we observed a loss-of-function mutation in the tumor suppressor gene F-Box and WD repeat domain containing 7 (FBXW7)—specifically in the resistant tumor. Using an immunocompetent, anti-PD-1 sensitive melanoma mouse model, we demonstrated that Fbxw7 loss of function in tumor cells causes resistance to PD-1 blockade. We showed that the genetic deletion of Fbxw7 in tumor cells alters the tumor immune microenvironment—including decreased effector cells and increased suppressive cells—in addition to diminished responses to viral sensing and interferon signaling pathways in vivo. Mechanistically, we found that Fbxw7 is necessary for the expression of the dsRNA sensors Mda5 and Rig-I. As a consequence, Fbxw7 is required for dsRNA-induced type I interferon production and interferon signaling in tumor cells. Conversely, restoration of the dsRNA signaling pathway in Fbxw7-deficient cells is sufficient to increase MHC-I expression and to suppress growth of Fbxw7-deficient tumors, thereby promoting antitumor immunity. Our findings establish a hitherto unrecognized role of the tumor suppressor gene FBXW7 in tumor immunity and sensitivity to immunotherapy. Collectively, these findings establish a novel tumor-intrinsic pathway of resistance and suggest that therapeutic reactivation of viral sensing pathways could improve clinical responses to checkpoint inhibitors in genomically defined populations. Citation Format: Cécile Gstalder, David Liu, Diana Miao, Alexander Devine, Bart Lutterbach, Priya Pancholi, Megha Shettigar, Elizabeth Buchbinder, Scott Carter, Michael Manos, Vanesa Rojas-Rudilla, Ryan Brennick, Evisa Gjini, Ana Lako, Scott Rodig, Charles Yoon, Gordon Freeman, F. Stephen Hodi, Eliezer M. Van Allen, Rizwan Haq. Inactivation of Fbxw7 impairs dsRNA sensing and confers resistance to PD-1 blockade [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A48.

Mesenchymal tumor subpopulations secrete pro-tumorigenic cytokines and promote treatment resistance1-4. This phenomenon has been implicated in chemorefractory small cell lung cancer and resistance to targeted therapies5-8, but remains incompletely defined. Here, we identify a subclass of endogenous retroviruses (ERVs) that engages innate immune signaling in these cells. Stimulated 3 prime antisense retroviral coding sequences (SPARCS) are oriented inversely in 3' untranslated regions of specific genes enriched for regulation by STAT1 and EZH2. Derepression of these loci results in double-stranded RNA generation following IFN-γ exposure due to bi-directional transcription from the STAT1-activated gene promoter and the 5' long terminal repeat of the antisense ERV. Engagement of MAVS and STING activates downstream TBK1, IRF3, and STAT1 signaling, sustaining a positive feedback loop. SPARCS induction in human tumors is tightly associated with major histocompatibility complex class 1 expression, mesenchymal markers, and downregulation of chromatin modifying enzymes, including EZH2. Analysis of cell lines with high inducible SPARCS expression reveals strong association with an AXL/MET-positive mesenchymal cell state. While SPARCS-high tumors are immune infiltrated, they also exhibit multiple features of an immune-suppressed microenviroment. Together, these data unveil a subclass of ERVs whose derepression triggers pathologic innate immune signaling in cancer, with important implications for cancer immunotherapy.

Personalized cancer vaccine strategies directed at tumor neoantigens derived from somatic mutations in the DNA are currently under prospective evaluation1, 2. Alterations in tumor RNA, rather than DNA, may also represent a previously-unexplored source of neoantigens. Here, we show that intron retention, a widespread feature of cancer transcriptomes3, 4, represents a novel source of tumor neoantigens. We developed an in silico approach to identify retained intron neoantigens from RNA sequencing data and applied this methodology to tumor samples from patients with melanoma treated with immune checkpoint blockade5, 6, discovering that the retained intron neoantigen burden in these samples augments the DNA-derived, somatic neoantigen burden. We validated the existence of retained intron derived neoantigens by implementing this technique on cancer cell lines with mass spectrometry-derived immunopeptidome data7, 8, revealing that retained intron neoantigens were complexed with MHC I experimentally. Unexpectedly, we observed a trend toward lack of clinical benefit from immune checkpoint blockade in high retained intron load-tumors, which harbored transcriptional signatures consistent with cell cycle dysregulation and DNA damage repair. Our results demonstrate the contribution of transcriptional dysregulation to the overall burden of tumor neoantigens, provide a foundation for augmenting personalized cancer vaccine development with a new class of tumor neoantigens, and demonstrate how global transcriptional dysregulation may impact selective response to immune checkpoint blockade.Statement of significanceWe developed and experimentally validated a computational pipeline to identify a novel class of tumor neoantigens derived from RNA-based intron retention, which is prevalent throughout cancer transcriptomes. The discovery of transcriptionally-derived tumor neoantigens expands the tumor immunopeptidome and contributes potential substrates for personalized cancer vaccine development.

3036Background: Immune checkpoint inhibitors have improved survival in multiple malignancies, but genomic biomarkers for efficacy in microsatellite stable tumors are incompletely characterized. Met...

CTLA-4 immune checkpoint blockade is clinically effective in a subset of patients with metastatic melanoma. We identify a subcluster of MAGE-A cancer-germline antigens, located within a narrow 75 kb region of chromosome Xq28, that predicts resistance uniquely to blockade of CTLA-4, but not PD-1. We validate this gene expression signature in an independent anti-CTLA-4-treated cohort and show its specificity to the CTLA-4 pathway with two independent anti-PD-1-treated cohorts. Autophagy, a process critical for optimal anti-cancer immunity, has previously been shown to be suppressed by the MAGE-TRIM28 ubiquitin ligase in vitro. We now show that the expression of the key autophagosome component LC3B and other activators of autophagy are negatively associated with MAGE-A protein levels in human melanomas, including samples from patients with resistance to CTLA-4 blockade. Our findings implicate autophagy suppression in resistance to CTLA-4 blockade in melanoma, suggesting exploitation of autophagy induction for potential therapeutic synergy with CTLA-4 inhibitors.

Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR-Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.

Response to immune checkpoint blockade in mesenchymal tumors is poorly characterized, but immunogenomic dissection of these cancers could inform immunotherapy mediators. We identified a treatment-naive patient who has metastatic uterine leiomyosarcoma and has experienced complete tumor remission for >2 years on anti-PD-1 (pembrolizumab) monotherapy. We analyzed the primary tumor, the sole treatment-resistant metastasis, and germline tissue to explore mechanisms of immunotherapy sensitivity and resistance. Both tumors stained diffusely for PD-L2 and showed sparse PD-L1 staining. PD-1+ cell infiltration significantly decreased in the resistant tumor (p = 0.039). Genomically, the treatment-resistant tumor uniquely harbored biallelic PTEN loss and had reduced expression of two neoantigens that demonstrated strong immunoreactivity with patient T cells in vitro, suggesting long-lasting immunological memory. In this near-complete response to PD-1 blockade in a mesenchymal tumor, we identified PTEN mutations and reduced expression of genes encoding neoantigens as potential mediators of resistance to immune checkpoint therapy.

Objectives Pica is the craving for and consumption of nonfood items, including the ingestion of earth (geophagy), raw starch (amylophagy), and ice (pagophagy). Pica has long been associated with micronutrient deficiencies, but the strength of this relationship is unclear. We aimed to evaluate the association between pica behavior and the risk of being anemic or having low hemoglobin (Hb), hematocrit (Hct), or plasma zinc (Zn) concentrations. Methods We systematically reviewed studies in which micronutrient levels were reported by pica status. We calculated the pooled odds ratio for anemia or weighted mean difference in Hb, Hct, or Zn concentrations between groups practicing or not practicing pica behaviors. Results Forty-three studies including 6,407 individuals with pica behaviors and 10,277 controls were identified. Pica was associated with 2.35 times greater odds of anemia (95% CI: 1.94–2.85, P < 0.001), lower Hb concentration (−0.65 g/dl, 95% CI: −0.83 to −0.48 g/dl, P < 0.001), lower Hct concentration (−1.15%, 95% CI: −1.61 to −0.70%, P < 0.001), and lower Zn concentration (−34.3 μg/dl, 95% CI: −59.58 to −9.02 μg/dl, P = 0.008). Statistical significance persisted after excluding outliers and in subgroup analyses by pica type and life stage. Conclusion Pica is significantly associated with increased risk for anemia and low Hb, Hct, and plasma Zn. Although the direction of the causal relationship between pica and micronutrient deficiency is unknown, the magnitude of these relationships is comparable to other well-recognized causes of micronutrient deficiencies. Pica warrants greater public health attention; specifically the potential physiological mechanisms underpinning the relationship between pica and micronutrient deficiencies merit further study. Am. J. Hum. Biol. 27:84–93, 2015. © 2014 Wiley Periodicals, Inc.

This study evaluated three types of swab transport systems for organism recovery. Swabs with transport media were further assessed for organism viability over 24 hours over a range of different storage temperatures. Test organisms consisted of aerobes, fastidious aerobes and anaerobes. Swabs were tested according to the standardised quantitative elution method published by the Clinical Laboratory Standards Institute (CLSI; M-40A). There were substantial differences in primary organism recovery, with recovery rates from different swabs ranging from0.1% to 78% for Streptococcus pyogenes. Similar differences were noted for other test organisms. In general, the flocked swab (ESwab) demonstrated highest rates of recovery for aerobic organisms, while higher rates of recovery of Fusobacterium nucleatum were demonstrated from a standard swab (Transwab). When considering organism viability, no single swab fulfilled all the criteria stipulated by the M-40A standard for all organism/temperature combinations. Organism viability was marginally better for the gel-based swab transport systems as compared to the liquid-media based ESwab. Significant differences between swab transport systems were demonstrated, including differences for primary organism recovery and viability. The ESwab showed the best recovery of organisms, while gel-based media demonstrated marginally better bacterial viability for most tested retention times and temperatures.