Neoantigen Burden Research Articles

Abstract 3639: Fusion-associated neoantigen burden and predicted immunogenicity of CDK12 biallelic loss-of-function tumors vary substantially across cancer types

Abstract Background: Biallelic inactivation of CDK12 is associated with a distinct genomic signature of focal tandem duplications (FTDs). Gene fusions resulting from CDK12-associated FTDs increase neoantigen load, raising interest in CDK12 as a biomarker of response to immune checkpoint inhibitors (ICIs). Although FTDs have been detected at higher rates in CDK12-altered tumors compared to CDK12 WT tumors for multiple cancer types, fusion-associated neoantigen burden has only been evaluated in metastatic castration-resistant prostate cancer. Methods: By retrospective review of molecular profiles, we identified tumors with CDK12 biallelic loss-of-function (LOF) in a primary cohort of over 9000 patient tumors, representing 39 cancer types, and assessed fusions detected by whole transcriptome sequencing for immune epitopes. Biomarker and fusion results for a second cohort of over 13,000 patient tumors were reviewed for validation of the initial findings. Results: Tumors with CDK12 biallelic LOF (bLOF) (n=29) had significantly higher mean fusion rates (11.7 fusions/tumor) than CDK12 monoallelic LOF (mLOF) and WT tumors (4.1 and 2.6, respectively). Analysis of fusion topologies and breakpoints indicated a predominance of duplication events for CDK12 bLOF tumors and a bimodal distribution of breakpoint distances (modes at ~250-500 kb and ~1.0-2.5 Mb), consistent with prior reports. Fusion rates correlated with the rate of predicted neoantigens with high, intermediate, and low affinity for MHC class I. Fusion-associated neoantigen burden was significantly higher in CDK12 bLOF tumors (145.0 high affinity peptides/tumor) than mLOF (49.7) and WT (26.6) tumors. Among CDK12-altered tumors, fusion rates were significantly higher in prostate (16.4 fusions/tumor, n=11) and ovarian (19.7, n=6) than other cancer types (3.4 overall, n=12), potentially reflecting cancer type-specific roles for CDK12. Co-occurrence of mismatch repair deficiency (dMMR)/high microsatellite instability (MSI-High) with CDK12 bLOF (n=5), most often in colorectal tumors (n=4), correlated with a lower fusion rate (0.2 fusions/tumor); recurrent CDK12 frameshift mutations in these cases (G1461fs, T1463fs, and Q1291fs) coincide with poly-nucleotide tracts, suggesting CDK12 mutations are a secondary effect. In a separate cohort of over 13,000 patient tumors, additional CDK12 bLOF tumors (n=47) were identified, showing a similar distribution and association with high fusion rate (11.0 fusions/tumor). Conclusion: Fusion rates and predicted neoantigen load varied significantly between CDK12 biallelic tumors across cancer types, highlighting the value of biomarkers with a quantitative immunogenic/phenotypic readout. We propose that fusion rates are linked to CDK12 alterations and may serve as useful biomarker to enhance our ability to identify responders of ICI therapy. Citation Format: Andrew Elliott, Phillip Stafford, Jian Zhang, Qing Zhang, Jeff Swensen, Daniel Martin, Joanne Xiu, Zoran Gatalica, Daniel Vaena, Elisabeth Heath, W. Michael Korn. Fusion-associated neoantigen burden and predicted immunogenicity of CDK12 biallelic loss-of-function tumors vary substantially across cancer types [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3639.

Abstract 4278: A composite neoantigen score is more strongly associated with therapeutic response than tumor mutational burden in a cohort of late-stage anti-PD-1-treated melanoma patients

Abstract Checkpoint inhibitor therapy has demonstrated meaningful, if varied antitumor activity, with patient response influenced by a variety of biological factors, including complex interactions between the tumor and immune system. Thus, it is of increasing interest to identify composite biomarkers integrating multiple biological features to better predict immunotherapy response. In this study we use a comprehensive tumor immungenomics profiling platform to examine the effectiveness of our composite neoantigen score for stratifying patient response to checkpoint blockade therapy compared to tumor mutational burden and other biomarkers.Pre-treatment tumor/normal samples from 55 unresectable, stage III/IV melanoma patients who underwent anti-PD-1 therapy were characterized to assess factors influencing response. RECIST criteria were used to evaluate tumor response to therapy, with a median follow-up of 18 months. For each patient, a single paired FFPE tumor and normal blood sample was collected and profiled using Personalis' ImmunoID NeXT platform; an augmented exome/transcriptome platform and analysis pipeline, which produces comprehensive tumor mutation information, gene expression quantification, neoantigen characterization, HLA typing and LOH, TCR repertoire profiling and tumor microenvironment profiling. These data were then analyzed together with clinical outcome, and a composite neoantigen score computed for each patient along with other biomarkers such as tumor mutational burden (TMB).In this cohort, an elevated pretreatment composite neoantigen score combining neoantigen predictions adjusted based on resistance mechanisms that affect neoantigen presentation on the MHC complex was more strongly predictive of response to PD-1 blockade than TMB alone. This was true for both response and non-response via RECIST criteria and progression free survival. We also found that the composite neoantigen score was a stronger predictor of patient response when compared to neoantigen burden alone. Additionally, we observed increased response to anti-PD-1 therapy in patients with elevated pretreatment TCR clonality. Combining the composite neoantigen score and TCR clonality data revealed a significant association with non-response to therapy. Patients with high composite neoantigen score and TCR clonality that failed to achieve complete response revealed potential resistance mechanisms to anti-PD-1 therapy. Specifically, we identified patients with high expression of IDO1 or CTLA4, which may facilitate PD-1-independent immune escape. Additionally, we found patients with mutations within the antigen presentation machinery (APM), likely leading to loss of surface expression of the proteins, and in the case of B2M mutations, improper HLA class I folding and antigen presentation. These APM mutations likely result in reduced neoantigen presentation in these patients, facilitating tumor escape.In summary, our composite neoantigen score which integrates multiple components of MHC class I presentation into a single score, is more significantly associated with response to therapy than individual biomarkers such as tumor mutational burden. These findings highlight the promise of composite biomarkers for the optimization of anti-PD-1 therapy patient selection. Citation Format: Charles W. Abbott, Eric Levy, Rachel Marty Pyke, Rena McClory, Sekwon Jang, Richard Chen, Sean Boyle. A composite neoantigen score is more strongly associated with therapeutic response than tumor mutational burden in a cohort of late-stage anti-PD-1-treated melanoma patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4278.

Predictive Biomarkers of Response to Immunotherapy in Metastatic Renal Cell Cancer.

IntroductionIn the last decades, the therapeutic decision-making approach to metastatic renal cell cancer (mRCC) has dramatically changed thanks to the introduction in the treatment scenario of, first, anti-angiogenic agents and, afterward, immune-checkpoint inhibitors (ICIs). Immunotherapy is now the standard of care in pretreated mRCC patients and has recently entered even the first line setting. Nevertheless, in mRCC as well as in other tumor settings, a durable and clinically meaningful benefit from treatment with ICIs is not obtained for all patients treated. Therefore, the necessity to identify and validate predictive biomarkers of response to immunotherapy has emerged, in order to design the optimal treatment strategy for mRCC patients.DiscussionIn this review, we present and discuss the most promising predictive biomarkers of response to ICIs in mRCC with the recent data available. In details, the first marker that was investigated is the immunohistochemical expression of programmed death receptor ligand 1 (PD-L1), showing a negative prognostic role in mRCC, but the debate about its potential predictive value is still open. Additionally, the high heterogeneity in PD-L1 determination methods adds complexity to this issue. Second, the tumor mutational or neoantigen burden is an emerging biomarker of increased response to immunotherapy, hypothesizing that the higher the TMB, the higher is the production of neoantigens, and thus the stimulation of anti-tumor immune response, even though controversial results have been obtained. Third, the tumor microenvironment, namely the different populations of the immune infiltrate, plays a key role in tumor progression and in the response to immunotherapy. Finally, several studies have collected evidence on the potential association of the occurrence of immune-related adverse events (irAEs) with the benefit from ICIs, first in non-small cell lung cancer (NSCLC) and melanoma, and recently even in mRCC.ConclusionSeveral promising biomarkers of response to immunotherapy with ICIs have been identified, though without conclusive results upon their potential predictive value in mRCC. Therefore, the results of the exploratory analyses of the recently presented first-line trials and hopefully of future prospective, biomarker-driven studies could provide useful tools to be applied in the everyday clinical practice.

High somatic mutation and neoantigen burden do not correlate with decreased progression-free survival in HCC patients

High somatic mutation and neoantigen burden do not correlate with decreased progression-free survival in HCC patients

Abstract A13: Entinostat enhances antitumor immune responses and cooperates with PD-1 blockade in bladder cancer mouse models

Abstract Bladder cancer harbors a high frequency of somatic mutations that occur randomly across the genome. A fraction of these mutations can be expressed and processed into peptides called neoantigens. When presented on the surface of tumor cells by the major histocompatibility complexes (MHC), these neoantigens can invoke an adaptive immune response. In general, a high neoantigen burden is associated with a significant increase in overall survival. However, tumor cells can avoid the detection of immune cells by silencing the expression of the neoantigens. The post-translational acetylation of histone tails is catalyzed by histone acetyltransferases (HATs) and is counterbalanced by histone deacetylases (HDACs), which mediate their removal. Histone acetylation is associated with open chromatin and active transcription. HDAC inhibition would, therefore, promote histone acetylation and restore the expression of suppressed genes. Many of the frequently mutated genes in bladder cancer are involved in chromatin remodeling, such as KMT2D, KDM6A, and KMT2C, suggesting the contribution of epigenetic dysregulation to disease progression. Therefore, therapy that targets epigenetic processes such as HDAC inhibition is a rational treatment approach for bladder cancer. Here, we evaluated the antitumor efficacy and mechanisms of entinostat, a selective HDAC1 and HDAC3 inhibitor, in a preclinical murine model of high-grade muscle-invasive bladder cancer, BBN963. In this study, BBN963 cells were implanted subcutaneously into immunocompetent C57BL/6 (B6) mice or immunodeficient NOD/scid/gamma (NSG) mice. Entinostat exhibited robust in vivo activity in B6 mice implanted with BBN963 cells with a number of complete responses. However, the effect was much weaker in BBN963 cells grown in NSG mice. RNAseq analysis demonstrated increased immune gene signature expression in entinostat treated tumors from B6 (but not NSG) mice. Flow cytometry verified the increased numbers of CD8+ effector memory T cells. Most interestingly, the expressions of predicted neoantigens were almost completely lost in the entinostat-treated tumors in B6 mice but were unchanged in tumors from NSG mice. Additionally, the in vivo antitumor effect of entinostat in the BBN963 cells lacking MHC class 1 was significantly reduced. Finally, the combination of anti-PD1 therapy and entinostat robustly out-performed each agent alone in inhibiting BBN963 tumor growth with 67% incident of complete response. These data support the hypothesis that entinostat mediates antitumor effect through enhanced neoantigen expression and antigen-driven immune response. Citation Format: Andrew S. Truong, Mi Zhou, Takanobu Utsumi, Bhavani Krishnan, Kyle G. Stewart, Ryoichi Saito, Ujjawal Manocha, Sean T. Bailey, William Y. Kim. Entinostat enhances antitumor immune responses and cooperates with PD-1 blockade in bladder cancer mouse models [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A13.

Progress in Neoantigen Targeted Cancer Immunotherapies.

Immunotherapies that harness the immune system to kill cancer cells have showed significant therapeutic efficacy in many human malignancies. A growing number of studies have highlighted the relevance of neoantigens in recognizing cancer cells by intrinsic T cells. Cancer neoantigens are a direct consequence of somatic mutations presenting on the surface of individual cancer cells. Neoantigens are fully cancer-specific and exempt from central tolerance. In addition, neoantigens are important targets for checkpoint blockade therapy. Recently, technological innovations have made neoantigen discovery possible in a variety of malignancies, thus providing an impetus to develop novel immunotherapies that selectively enhance T cell reactivity for the destruction of cancer cells while leaving normal tissues unharmed. In this review, we aim to introduce the methods of the identification of neoantigens, the mutational patterns of human cancers, related clinical trials, neoantigen burden and sensitivity to immune checkpoint blockade. Moreover, we focus on relevant challenges of targeting neoantigens for cancer treatment.

Tumor mutational burden related classifier is predictive of response to PD-L1 blockade in locally advanced and metastatic urothelial carcinoma

BackgroundImmunotherapy has made encouraging progress in the treatment of urothelial carcinoma, but only a small percentage of patients respond effectively to the immune checkpoint blockade (ICB). Our study aims to develop a classifier could effectively predict the response to ICB. MethodsSupport vector machines recursive feature elimination (SVM-RFE) algorithm was used to feature selection, then compared nine common binary classification algorithms through machine learning, we selected the classifier with the highest prediction performance (LASSO logistics classifier). Ten-fold cross-validation was used to avoid the overfitting effect. ResultsWe developed a classifier on a urothelial carcinoma cohort treated with PD-L1 inhibitor Atzolizumab (IMvigor210 cohort, n = 272) and calculated a tumor mutational burden-related LASSO score (TLS) using the LASSO algorithm, which was significantly correlated with Tumor mutational burden (TMB) and neoantigen burden. We validated the efficacy of TLS in predicting prognosis and immunotherapy benefit in internal (IMvigor210) and external validation set (TCGA-BLCA, n = 406), respectively. ConclusionsAfter in-depth analysis, we provide a new idea for stratified treatment of such patients, that is, patients with high TLS should use ICB and also may benefit from hormone therapy, while patients with low TLS respond poorly to ICB and maybe benefit from targeting TGFβ.

Non-Melanoma Skin Cancers: Biological and Clinical Features.

Non-melanoma skin cancers (NMSCs) include basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and Merkel cell carcinoma (MCC). These neoplasms are highly diverse in their clinical presentation, as well as in their biological evolution. While the deregulation of the Hedgehog pathway is commonly observed in BCC, SCC and MCC are characterized by a strikingly elevated mutational and neoantigen burden. As result of our improved understanding of the biology of non-melanoma skin cancers, innovative treatment options including inhibitors of the Hedgehog pathway and immunotherapeutic agents have been recently investigated against these malignancies, leading to their approval by regulatory authorities. Herein, we review the most relevant biological and clinical features of NMSC, focusing on innovative treatment approaches.

Abstract PR17: Immunogenomic landscape of pediatric solid malignancies

Abstract Malignancy remains the leading cause of disease-related death in children. To identify potential tumor-driving molecular targets and immunogenomic profiles in pediatric cancers, we performed RNA-seq analysis on a cohort of 788 pediatric solid malignant tumors across 14 different diagnoses in conjunction with additional 147 normal tissues for comparison. Sequencing data were analyzed for expressed mutations, fusion events, and expressional patterns, providing therapeutic targets and rich cancer biology for these childhood cancers. Furthermore, we describe immunogenomic features of these solid tumors including immune cell infiltrate, neoantigen expression, expression of immunomodulatory molecules, and T-cell receptor repertoire. Across the cohort, we observed a striking correlation between the expressed neoantigen burden in tumors and enrichment of the effector immune signatures. Histology-specific immunogenomic patterns were also apparent. Several of the pediatric cancers such as alveolar soft part sarcoma and osteosarcoma exhibit rich immune cell infiltration and evidence for activated T-cell activities, whereas others such as Wilms’ tumors and synovial sarcoma generally have a very low T-cell infiltration. We demonstrate that RNA-seq is a powerful tool to identify clinically relevant and histology-specific recurrent mutations, novel oncogenic fusions, and translationally relevant immunogenomic patterns for pediatric cancers. This study also represents one of the largest of its type to date and provides a framework for future translational efforts in pediatric cancer. This abstract is also being presented as Poster A69. Citation Format: Jun S. Wei, Andrew S. Brohl, Sivasish Sindiri, Young K. Song, Sushma Najaraj, Vineela Gangalapudi, Xinyu Wen, Marc Ladanyi, Javed Khan. Immunogenomic landscape of pediatric solid malignancies [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr PR17.

Abstract PR05: APOBEC mutagenesis is tightly linked to the immune landscape and immunotherapy biomarkers in head and neck squamous cell carcinoma

Abstract Significant interest is focused on the immune microenvironment of head and neck squamous cell carcinoma (HNSCC) due in part to the recent success of immune modulating therapeutics. HNSCC is known to be an immunologically active tumor with immune cell infiltration levels among the highest of all cancers, high mutational burden, and a subset of patients who respond to immune checkpoint blockade. One of the primary sources of mutations in HNSCC is the cytidine deaminase APOBEC3, which is also a known factor in innate immunity. Why particular HNSCCs have higher rates of APOBEC mutations and how these mutations relate to the immune microenvironment and immune activation remain unknown. We utilized whole-exome and RNA-Seq datasets from 276 HNSCC patients from TCGA to annotate APOBEC mutations, immune cell populations, activating and end effectors of immunity and predict neoantigens. In combination with FFPE tissue sections from matched patients, patient demographic data and germline APOBEC polymorphism status, we interrogated the relationship between APOBEC mutations and the immune landscape. Immune cell populations and composite scores of immune activation and infiltration (IFNy score, CYT score, Estimate Immune score) are tightly associated with APOBEC mutational burden, both univariately and after controlling for HPV status (p = 0.013-0.014). HNSCC has the highest levels of IFNy across cancer types with high APOBEC mutational burden, with the highest IFNy scores in HPV mediated HNSCC (HPVmHNSCC) (p = 0.05). Similarly, PD-L1 expression is positively associated with APOBEC mutational burden (p = 0.04). Mutation-induced tumor-specific neoantigen burden is strongly associated with APOBEC mutational burden while other sources of neoantigens are not associated (p = 8e-12). The presence of a germline APOBEC gene polymorphism is more prevalent in non-white, non-black race patients and within this group, patients with the polymorphism have higher APOBEC mutational burden (p= 0.002). Taken together, these data suggest that multiple mechanisms may exist within HNSCC that lead to APOBEC mutations. Immune upregulation, potentially in response to mutation-induced neoantigens, appears to be the primary driver, with viral infection inducing additional IFNy production in the subset of tumors caused by HPV. These mechanisms may be additive and not mutually exclusive, which could explain the higher levels of APOBEC mutations in HPVmHNSCC. The tight interconnectedness of APOBEC mutations and the immune landscape in HNSCC, including numerous known markers of response to immunotherapy, highlights the importance of the immune microenvironment in mutation acquisition in HNSCC. Citation Format: Yan Lin, Shuyan Zhai, Fengshen Kuo, Timothy A. Chan, Luc G. Morris, Robert L. Ferris, Daniel L. Faden, Fei Ding. APOBEC mutagenesis is tightly linked to the immune landscape and immunotherapy biomarkers in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr PR05.

Geospatial immune variability illuminates differential evolution of lung adenocarcinoma.

Remarkable progress in molecular analyses has improved our understanding of the evolution of cancer cells toward immune escape1-5. However, the spatial configurations of immune and stromal cells, which may shed light on the evolution of immune escape across tumor geographical locations, remain unaddressed. We integrated multiregion exome and RNA-sequencing (RNA-seq) data with spatial histology mapped by deep learning in 100 patients with non-small cell lung cancer from the TRACERx cohort6. Cancer subclones derived from immune cold regions were more closely related in mutation space, diversifying more recently than subclones from immune hot regions. In TRACERx and in an independent multisample cohort of 970 patients with lung adenocarcinoma, tumors with more than one immune cold region had a higher risk of relapse, independently of tumor size, stage and number of samples per patient. In lung adenocarcinoma, but not lung squamous cell carcinoma, geometrical irregularity and complexity of the cancer-stromal cell interface significantly increased in tumor regions without disruption of antigen presentation. Decreased lymphocyte accumulation in adjacent stroma was observed in tumors with low clonal neoantigen burden. Collectively, immune geospatial variability elucidates tumor ecological constraints that may shape the emergence of immune-evading subclones and aggressive clinical phenotypes.

Exome scale liquid biopsy characterization of putative neoantigens and genomic biomarkers pre- and post anti-PD-1 therapy in squamous cell carcinoma of the head and neck.

6557 Background: The reduced scope, and number of genes profiled by typical liquid biopsy panels can result in missed biomarkers including neoantigens, which may change with treatment, as well as potentially undetected resistance mechanisms and pathways beyond the scope of targets typically captured by panels. To address these limitations, we used a whole-exome scale liquid biopsy monitoring platform, NeXT Liquid Biopsy, to analyze head and neck squamous cell carcinoma (HNSCC) patients that have received anti-PD1 therapy. Presently, we sought to (1) monitor neoantigen changes in cfDNA as a complement to tumor biopsy-derived neoantigens, (2) compare the impact of tumor escape mechanisms, including HLA-LOH, on neoantigens identified in tissue and cfDNA and (3) to identify novel biological signatures that combine information from both solid tumor and liquid biopsies. Methods: Pre- and post-intervention matched normal, tumor and plasma samples were collected from a cohort of 12 patients with HNSCC. Following baseline sample collection all patients received a single dose of nivolumab, followed by resection approximately one month later when feasible, or a second biopsy where resection was impractical. Solid tumor and matched normal samples were profiled using ImmunoID NeXT, an augmented exome/transcriptome platform and analysis pipeline. Exome-scale somatic variants were identified in cfDNA from plasma samples using the NeXT Liquid Biopsy platform. Data from these two platforms were compared with corresponding clinical findings. Results: Concordant somatic events were detected between plasma and tumor at pre- and post-treatment timepoints. Neoantigens predicted to arise from these somatic events were reduced in solid tumor post-treatment, but increased in cfDNA, when compared to pre-treatment timepoints. HLA LOH was identified in a number of subjects, likely resulting in reduced neoepitope presentation in those cases. Immune cell infiltration increased in the tumor following treatment, with no changes to the CD8+/Treg cell ratio, suggesting consistent immunoregulation. Conclusions: Exome-wide neoantigen burden was reliably predicted from cfDNA, providing additional insight complementing data from solid tumor. Analyzing HLA LOH, and neoantigen burden from both solid and liquid biopsies together over the course of treatment creates a more comprehensive profile of therapeutic response and resistance mechanisms in HNSCC patients missed with typical liquid biopsy panels.

The role of TGF-β pathway in immune regulation as a potential biomarker of immunotherapy across pan-cancer.

e15187 Background: The transforming growth factor beta (TGF-β) signaling pathway has been reported to be involved in both tumor suppression and tumor promotion. However, the role of TGF-β pathway in immune regulation for cancer patients and its influences on immunotherapy efficacy have not been systematically investigated. Methods: Available data of whole-exome sequencing, mRNA expression, baseline characterization and prognosis information of 10,912 pancancer patients were adopted from The Cancer Genome Altas (TCGA) to explore the role of TGF-β pathway in immune regulation. Formalin-fixed, paraffin-embedded tissue samples from 6,717 Chinese patients with over 17 tumor types were assayed by next-generation sequencing with a panel with 381 cancer related genes as a validation cohort (3DMed cohort). Datasets from the public MSK cohort (N = 1,610) was used to explore the association of TGF-β pathway in patient survival. Results: The highest prevalence of single nucleotide variation (SNVs) in TGF-β pathway fell in digestive system tumors in both TCGA and 3DMed cohorts, including colon adenocarcinoma pancreatic adenocarcinoma, rectum adenocarcinoma, and stomach adenocarcinoma. TGF-β pathway SNVs was significantly correlated with high microsatellite instability, high tumor mutational burden (TMB-H) status and high neoantigen burden (TNB-H) ( p< 0.001) across all tumor types. Notably, the correlation of pathway alternation and TMB or TNB remained significance in microsatellite stable patients ( p< 0.001). Alternations of the pathway genes were associated with significant different expression patterns of immune-related genes such as the up-regulation of CD28, CD40, and CCL5, etc. Consistently, significant higher levels of CD8 ( p< 0.001), dendritic cells ( p< 0.001), and neutrophil cells ( p< 0.001) were observed in the pathway mutated samples. Patients with TGF-β pathway mutations exhibited significant worse prognosis than the wild-type patients regardless of interventions (overall survival, HR 1.20, 95% CI 1.08-1.33; p= 0.001). However, when treated with immune checkpoint inhibitors, superior survival benefit was observed in patients in the mutation group versus the wild-type group (overall survival, HR 0.73, 95% CI 0.61-0.88; p= 0.001). Conclusions: Our study has provided clues for the role of TGF-β pathway in immune regulation in patients with solid tumors and revealed the potential predictive role of TGF-β pathway alternation in cancer immunotherapy.

The prevalence of CDK12 alterations in Chinese cancer patients and the association with tumor mutation burden(TMB) and survival.

e13663 Background: Cyclin-dependent kinase 12 (CDK12) is a cyclin-dependent kinase that regulates transcription and RNA splicing, thereby modulating multiple cellular processes. It has been suggested that CDK12 loss-of-function mutations lead to a higher neoantigen burden and favorable responses to PD-1 inhibitors in advanced prostate cancer. Given this potentially actionable molecular subtype, we sought to determine the prevalence of CDK12 alterations in Chinese cancer patients and the association with TMB and overall survival(OS). Methods: The prevalence of CDK12 alterations were queried in 3D Medicines database with 15,745 Chinese cancer patients involved. Whole-exome sequencing data of 464 patients with prostate adenocarcinoma(PRAD) from the Cancer Genome Altas (TCGA) were downloaded to explore the association between CDK12 gene alteration and OS. And the association with TMB were analyzed in a cohort of 731 patients with various cancer types published by Memorial Sloan Kettering (MSKCC) (Samstein et al., Nature Genetics, 2019). Results: Any CDK12 and known or likely deleterious CDK12 mutations were identified in 598(3.8%) and 98(0.62%) patients, respectively. Across all cancer types, prostate adenocarcinoma(PRAD) was found to have the highest frequency of deleterious mutations(8.75%, 23/263), followed by breast cancer (4.97%, 25/503). Mutations were also detected in multiple cancer types including bladder cancer, ovarian cancer, lung cancer, colorectal cancer and so on with a frequency of less than 1%. CDK12 mutations were associated with shorter OS (HR = 15.25; 95% CI, 2.88-80.73; p < 0.001) in TCGA PRAD and cholangiocarcinoma datasets, which was not seen in other cancer types. Patients harboring CDK12 mutation had a significant higher TMB(p < 0.001) in the pan-cancer study of publicly-available cohort from MSKCC. Conclusions: CDK12 alterations existed across tumor types in Chinese patients with relatively high frequencies detected in PRAD and breast cancer and represent extremely rare events in multiple cancers. CDK12 mutation was a poor prognostic factor in PRAD and cholangiocarcinoma. In a pan cancer analysis patients with CDK12 mutation tended to have a significant higher TMB and may benefit from PD-1/L1 blockade immunotherapy.

CDK12-Mutated Prostate Cancer: Clinical Outcomes With Standard Therapies and Immune Checkpoint Blockade.

Translational studies have shown that CDK12 mutations may delineate an immunoresponsive subgroup of prostate cancer, characterized by high neo-antigen burden. Given that these mutations may define a clinically distinct subgroup, we sought to describe outcomes to standard drugs and checkpoint inhibitors (CPI). Clinical data from consecutive patients with CDK12 mutations were retrospectively collected from 7 centers. Several clinical-grade sequencing assays were used to assess CDK12 status. Descriptive statistics included PSA50 response rate (≥ 50% decline in prostate-specific antigen from baseline) and clinical/radiographic progression-free survival (PFS). Of 52 patients with CDK12-mutated prostate cancer, 27 (52%) had detected biallelic CDK12 alterations. At diagnosis, 44 (88%) had Gleason grade group 4-5, 52% had T3-T4, and 14 (27%) had M1 disease. Median follow-up was 8.2 years (95% CI, 5.6 to 11.1 years), and 49 (94%) developed metastatic disease. Median overall survival from metastasis was 3.9 years (95% CI, 3.2 to 8.1 years). Unconfirmed PSA50 response rates to abiraterone and enzalutamide in the first-line castration-resistant prostate cancer setting were 11 of 17 (65%) and 9 of 12 (75%), respectively. Median PFS on first-line abiraterone and enzalutamide was short, at 8.2 months (95% CI, 6.6 to 12.6 months) and 10.6 months (95% CI, 10.2 months to not reached), respectively. Nineteen patients received CPI therapy. PSA50 responses to CPI were noted in 11%, and PFS was short; however, the estimated 9-month PFS was 23%. PFS was higher in chemotherapy-näıve versus chemotherapypretreated patients (median PFS: not reached v 2.1 months, P = .004). CDK12 mutations define an aggressive prostate cancer subgroup, with a high rate of metastases and short overall survival. CPI may be effective in a minority of these patients, and exploratory analysis supports using anti-programmed cell death protein 1 drugs early. Prospective studies testing CPI in this subset of patients with prostate cancer are warranted.

Immunotherapy in Metastatic Colorectal Cancer: Could the Latest Developments Hold the Key to Improving Patient Survival?

Immunotherapy has considerably increased the number of anticancer agents in many tumor types including metastatic colorectal cancer (mCRC). Anti-PD-1 (programmed death 1) and cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) immune checkpoint inhibitors (ICI) have been shown to benefit the mCRC patients with mismatch repair deficiency (dMMR) or high microsatellite instability (MSI-H). However, ICI is not effective in mismatch repair proficient (pMMR) colorectal tumors, which constitute a large population of patients. Several clinical trials evaluating the efficacy of immunotherapy combined with chemotherapy, radiation therapy, or other agents are currently ongoing to extend the benefit of immunotherapy to pMMR mCRC cases. In dMMR patients, MSI testing through immunohistochemistry and/or polymerase chain reaction can be used to identify patients that will benefit from immunotherapy. Next-generation sequencing has the ability to detect MSI-H using a low amount of nucleic acids and its application in clinical practice is currently being explored. Preliminary data suggest that radiomics is capable of discriminating MSI from microsatellite stable mCRC and may play a role as an imaging biomarker in the future. Tumor mutational burden, neoantigen burden, tumor-infiltrating lymphocytes, immunoscore, and gastrointestinal microbiome are promising biomarkers that require further investigation and validation.

Heterogeneity of neoantigen landscape between primary lesions and their matched metastases in lung cancer.

BackgroundPersonalized cancer vaccines based on tumor-derived neoantigens have shown strong and long-lasting antitumor effect in patients with some solid tumors. However, whether neoantigens identified from primary lesions could represent their metastatic lesions, and consequently the effect of vaccine therapy remained unknown.MethodsTo investigate whether neoantigens identified from primary tumors are similar to their matched metastases in lung cancer, we identified 79 samples from 24 cases. All of samples were collected before any systemic therapy. Major criteria for neoantigen identification included: derived from tumor-specific mutations, fold change >10 comparing to germline expression level, high predicted human leukocyte antigen (HLA) binding affinity and peptide of 9–11 amino acids in length.ResultsWe found a wide range of tumor neoantigen burden in both primaries and metastases. The counts, overall distribution pattern and predicted HLA binding affinity of neoantigens were similar between primaries and metastases. However, only 20% of shared neoantigens (presented in both primaries and metastases) was observed, which were mainly derived from single nucleotide variants (SNVs) and fusions. A variety of corresponding HLA alleles were observed and 50.0% of cases were HLA-C*06:02. Finally, we observed the neoantigen intrametastases homogeneity in patients with sole brain metastases.ConclusionsNeoantigen landscape in terms of the number, type and predicted HLA binding affinity was similar between primaries and metastases, but the percentage of shared neoantigens is only modest, suggesting vaccine development based solely on primary tumor neoantigen may not offer optimal therapeutic outcome, and shared neoantigen needs to be seriously considered.

Abstract B18: Exome scale liquid biopsy monitoring of putative neoantigens and genomic biomarkers in patients on anti-PD-1 therapy in squamous cell carcinoma of the head and neck

Abstract Monitoring treatment response and potential resistance mechanisms to therapy with typical liquid biopsy panels can be limited by the relatively small number of genes profiled. The limited scope can result in missed therapeutic biomarkers such as neoantigens, which may change dynamically with treatment, as well as potentially missed resistance mechanisms and pathways not typically covered by existing panels. To address these shortcomings, we applied an exome scale liquid biopsy monitoring platform, NeXT Liquid Biopsy, to analyze head and neck squamous cell carcinoma (HNSCC) patients who have received anti-PD1 therapy. Using this platform we sought to (1) identify and monitor neoantigen changes in cell free DNA (cfDNA) as a complement to tumor biopsy-derived neoantigens, (2) compare the differences in putative neoantigens identified in tissue and cfDNA, and (3) identify additional biomarker correlations between cfDNA and solid tumor biopsies. Pre- and post-intervention matched tumor, normal, and plasma samples were obtained from a pilot cohort of 9 patients with HNSCC. Following initial sample collection all patients received a single dose of nivolumab, followed by definitive resection of the primary tumor mass approximately one month later when possible, or a second biopsy where resection was impractical (resection, n=5; biopsy, n=4). Solid tumor and matched normal samples were profiled using ImmunoID NeXT, an augmented exome/transcriptome platform and analysis pipeline. For the plasma samples, we performed exome-scale cfDNA profiling with the ImmunoID NeXT Liquid Biopsy platform to detect somatic variants. Data from these two platforms were compared with corresponding clinical findings. We observed a strong concordance of somatic events between the plasma and tumor in both pre- and post-treatment timepoints. Median post-treatment neoantigen burden was reduced in solid tumor, but increased in cfDNA, when compared to pretreatment timepoints. HLA-specific loss of heterozygosity (LOH) was identified in a number of subjects, likely resulting in reduced neoepitope presentation in those cases. These changes were accompanied by increased immune cell infiltration in the tumor following treatment, with no changes to the CD8+/Treg cell ratio, suggesting consistent immunoregulatory influence. Exome-wide somatic events were reliably detected in cfDNA, providing additional potential biomarkers to complement biomarkers identified in solid tumor. As we increase our cohort size we expect that identification of biomarkers from both exome-scale tissue biopsy and cfDNA will provide a more comprehensive view into therapeutic response and resistance mechanisms in HNSCC patients missed with typical liquid biopsy panels. Citation Format: Charles W. Abbott, Nikita Bedi, Simo V. Zhang, Robin Li, Rachel Marty Pyke, Eric Levy, Rebecca Chernock, Mena Mansour, John B. Sunwoo, A. Dimitrios Colevas, Richard Chen, Sean M. Boyle. Exome scale liquid biopsy monitoring of putative neoantigens and genomic biomarkers in patients on anti-PD-1 therapy in squamous cell carcinoma of the head and neck [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 B18.

Genomic landscape and immune microenvironment features of FH-deficient renal cell carcinoma.

717 Background: Fumarate hydratase-deficient renal cell carcinoma (FH-deficient RCC) is a rare but aggressive subtype of RCC, and an understanding of their genomic landscape is lacking. Methods: Thirteen FH-deficient RCC and matched normal samples from our medical center were used for whole-exome sequencing and molecular profiling. Transcriptomic sequencing data were analyzed from The Cancer Genome Atlas (TCGA) RCC samples and two independent datasets of FH-mutated RCC. For each patient in our cohort, clinical outcomes were recorded, with median follow-up until March, 2019. Results: We identified FH-deficient RCC had high mutation burden, frequent somatic mutations in MUC4, MUC16, ANKRD6 and PRDM16 and germline pathologic FANCD2 mutation. In contrast to mainly RCC subtypes, FH-deficient RCC exhibited elevated mutational signatures of homologous recombination deficiency (7/13, 53%) and microsatellite instability (4/13, 30%). Moreover, we revealed that FH-deficient RCC was characteristic of an immunogenic tumor type, with high tumor-specific neoantigen burdens, enhanced immune cells infiltration but immunosuppressive microenvironment. In our cohort, 11 patients received systematic treatments; 63% (5/8) responded poorly to antiangiogenic or anti-mammalian target of rapamycin agents, but all (6/6) achieved benefit from immune checkpoint blockade monotherapy or combination with antiangiogenic agents. Conclusions: Our study provides a better understanding of the mutational landscape of FH-deficient RCC, highlighting the potential of immunotherapy for treating this patient populations.

CDK12-mutated prostate cancer (PC): Clinical outcomes to standard therapies and immune checkpoint blockade.

191 Background: Translational studies have shown that CDK12 mutations may delineate an immunoresponsive subgroup of PC, characterized by high neoantigen burden. Given that these mutations may define a clinically relevant PC subgroup, we sought to describe outcomes to both standard drugs and PD1 inhibitors. Methods: Clinical data from consecutive patients with CDK12 mutations were retrospectively collected from 7 centers. Multiple clinical grade sequencing assays were used to assess for CDK12 alterations. Descriptive statistics included PSA50 (≥50% decline in PSA from baseline) and clinical/radiographic progression-free survival (PFS). Results: Of 56 CDK12-mutated PC patients, 27 (48%) had biallelic CDK12 alterations. At diagnosis, 46 (82%) had Gleason grade group 4-5 disease, 52% had T3-T4 disease and 15 (27%) had M1 disease. Median follow up was 8.2 yrs (95% CI: 5.6-11.1) and 53 (95%) developed metastatic disease. Median overall survival (OS) from metastasis was 3.9 years (95% CI: 3.2-5.8). Nineteen patients received an anti-PD1 therapy. PSA50 responses to PD1 blockade were infrequent and overall PFS was short (Table); however, the estimated 9-month PFS was 23%. PFS was higher in chemotherapy naïve patients who received anti-PD1 therapy (P=0.004). Outcomes were similar in monoallelic vs. biallelic CDK12-mutated patients. Conclusions: CDK12-mutations define an aggressive PC subgroup, with a high rate of metastatic disease and short OS. Immune checkpoint inhibitors may be effective in a minority of these patients and exploratory analysis supports using anti-PD1 drugs early.[Table: see text]