Neoepitope Load Research Articles

Neoepitope load, T cell signatures and PD-L2 as combined biomarker strategy for response to checkpoint inhibition immunotherapy.

Immune checkpoint inhibition for the treatment of cancer has provided a breakthrough in oncology, and several new checkpoint inhibition pathways are currently being investigated regarding their potential to provide additional clinical benefit. However, only a fraction of patients respond to such treatment modalities, and there is an urgent need to identify biomarkers to rationally select patients that will benefit from treatment. In this study, we explore different tumor associated characteristics for their association with favorable clinical outcome in a diverse cohort of cancer patients treated with checkpoint inhibitors. We studied 29 patients in a basket trial comprising 12 different tumor types, treated with 10 different checkpoint inhibition regimens. Our analysis revealed that even across this diverse cohort, patients achieving clinical benefit had significantly higher neoepitope load, higher expression of T cell signatures, and higher PD-L2 expression, which also correlated with improved progression-free and overall survival. Importantly, the combination of biomarkers serves as a better predictor than each of the biomarkers alone. Basket trials are frequently used in modern immunotherapy trial design, and here we identify a set of biomarkers of potential relevance across multiple cancer types, allowing for the selection of patients that most likely will benefit from immune checkpoint inhibition.

Mutational signature profiling classifies subtypes of clinically different mismatch-repair-deficient tumours with a differential immunogenic response potential.

Mismatch repair (MMR) deficiency is the hallmark of tumours from Lynch syndrome (LS), sporadic MLH1 hypermethylated and Lynch-like syndrome (LLS), but there is a lack of understanding of the variability in their mutational profiles based on clinical phenotypes. The aim of this study was to perform a molecular characterisation to identify novel features that can impact tumour behaviour and clinical management. We tested 105 MMR-deficient colorectal cancer tumours (25 LS, 35 LLS and 45 sporadic) for global exome microsatellite instability, cancer mutational signatures, mutational spectrum and neoepitope load. Fifty-three percent of tumours showed high contribution of MMR-deficient mutational signatures, high level of global exome microsatellite instability, loss of MLH1/PMS2 protein expression and included sporadic tumours. Thirty-one percent of tumours showed weaker features of MMR deficiency, 62% lost MSH2/MSH6 expression and included 60% of LS and 44% of LLS tumours. Remarkably, 9% of all tumours lacked global exome microsatellite instability. Lastly, HLA-B07:02 could be triggering the neoantigen presentation in tumours that show the strongest contribution of MMR-deficient tumours. Next-generation sequencing approaches allow for a granular molecular characterisation of MMR-deficient tumours, which can be essential to properly diagnose and treat patients with these tumours in the setting of personalised medicine.

The Prognostic Impact of Circulating Tumour DNA in Melanoma Patients Treated with Systemic Therapies-Beyond BRAF Mutant Detection.

In this study, we evaluated the predictive value of circulating tumour DNA (ctDNA) to inform therapeutic outcomes in metastatic melanoma patients receiving systemic therapies. We analysed 142 plasma samples from metastatic melanoma patients prior to commencement of systemic therapy: 70 were treated with BRAF/MEK inhibitors and 72 with immunotherapies. Patient-specific droplet digital polymerase chain reaction assays were designed for ctDNA detection. Plasma ctDNA was detected in 56% of patients prior to first-line anti-PD1 and/or anti-CTLA-4 treatment. The detection rate in the immunotherapy cohort was comparably lower than those with BRAF inhibitors (76%, p = 0.0149). Decreasing ctDNA levels within 12 weeks of treatment was strongly concordant with treatment response (Cohen's k = 0.798, p < 0.001) and predictive of longer progression free survival. Notably, a slower kinetic of ctDNA decline was observed in patients treated with immunotherapy compared to those on BRAF/MEK inhibitors. Whole exome sequencing of ctDNA was also conducted in 9 patients commencing anti-PD-1 therapy to derive tumour mutational burden (TMB) and neoepitope load measurements. The results showed a trend of high TMB and neoepitope load in responders compared to non-responders. Overall, our data suggest that changes in ctDNA can serve as an early indicator of outcomes in metastatic melanoma patients treated with systemic therapies and therefore may serve as a tool to guide treatment decisions.

Responsiveness to Immune Checkpoint Inhibitors Is Associated With a Peripheral Blood T-Cell Signature in Metastatic Castration-Resistant Prostate Cancer.

Although most patients with microsatellite instable (MSI) metastatic castration-resistant prostate cancer (mCRPC) respond to immune checkpoint blockade (ICB), only a small subset of patients with microsatellite stable (MSS) tumors have similar benefit. Biomarkers defining ICB-susceptible subsets of patients with MSS mCRPC are urgently needed. Using next-generation T-cell repertoire sequencing, we explored immune signatures in 54 patients with MSS and MSI mCRPC who were treated with or without ICB. We defined subset-specific immune metrics as well as T-cell clusters and correlated the signatures with treatment benefit. Consistent overlaps between tumor and peripheral T-cell repertoires suggested that blood was an informative material to identify relevant T-cell signatures. We found considerably higher blood T-cell richness and diversity and more shared T-cell clusters with low generation probability (pGen) in MSI versus MSS mCRPC, potentially reflecting more complex T-cell responses because of a greater neoepitope load in the MSI subset. Interestingly, patients with MSS mCRPC with shared low pGen T-cell clusters showed significantly better outcomes with ICB, but not with other treatments, compared with patients without such clusters. Blood clearance of T-cell clusters on ICB treatment initiation seemed to be compatible with T-cell migration to the primary tumor or metastatic sites during the process of clonal replacement as described for other tumors receiving ICB. The MSI mCRPC subset shows a distinct T-cell signature that can be detected in blood. This signature points to immune parameters that could help identify a subset of patients with MSS mCRPC who may have an increased likelihood of responding to ICB or to combination approaches including ICB.

ATIM-01. NIVOLUMAB AND BEVACIZUMAB FOR RECURRENT GLIOBLASTOMA; A TRANSLATIONAL TRIAL IN PROGRESS

Abstract Glioblastoma multiforme (GBM) is an aggressive brain tumor with a poor prognosis. Standard of care at diagnosis is surgical resection, followed by radiation and temozolomide. Receiving this therapy, the median survival is 14.6 months [1]. We have no standard treatment for relapse and known options have limited effect. There is an urgent need for novel treatment interventions to improve clinical outcomes and quality of life. Recently, improved overall survival has been achieved with immune therapeutics in melanoma and renal cell carcinoma. Accordingly, it has been posited that immunotherapy may offer promise in other difficult cancers such as GBM [2]. We present our translational study; a phase II open label, two-armed translational study of Nivolumab and Bevacizumab for recurrent GBM, who have failed Stupp’s regime [1]. Patients are included in two arms depending on possibly salvage neurosurgical resection. Both arms receive Nivolumab and Bevacizumab administrated every second weekend, but the surgical arm also receive Nivolumab 7 days prior surgery. We expect 40 patients; 20 in each arm. Enrollment period is expected to 20 months, started October 2018. Our primary objective is to make preliminary assessment of immune related biomarkers, including PD-L1; therefore, we perform full genome sequencing on tumor biopsies from the surgical arm and on blood samples from both arms. We evaluate changes in the transcriptomic landscape caused by the check-point inhibition and relation to response as compared with baseline sequencing data, as well as the impact of tumor mutation burden and neoepitope load. We investigate the tumor microenvironment by harvesting tumor infiltrating lymphocytes and study the composition by flow-cytometry. The patients are evaluated by blood samples, FET-PET as wells as clinical examinations to evaluate PFS and OS. Overall the study will provide us with a unique possibility to investigate and thereof predict which patients will profit from the treatment.

T-cell bispecific antibodies in node-positive breast cancer: novel therapeutic avenue for MHC class I loss variants.

Tumor-infiltrating lymphocytes (TILs) represent a prognostic factor for survival in primary breast cancer (BC). Nonetheless, neoepitope load and TILs cytolytic activity are modest in BC, compromising the efficacy of immune-activating antibodies, which do not yet compete against immunogenic chemotherapy. We analyzed by functional flow cytometry the immune dynamics of primary and metastatic axillary nodes [metastatic lymph nodes (mLN)] in early BC (EBC) after exposure to T-cell bispecific antibodies (TCB) bridging CD3ε and human epidermal growth factor receptor 2 (HER2) or Carcinoembryonic Antigen-Related Cell Adhesion Molecule 5 (CEACAM5), before and after chemotherapy. Human leukocyte antigen (HLA) class I loss was assessed by whole exome sequencing and immunohistochemistry. One hundred primary BC, 64 surrounding 'healthy tissue' and 24 mLN-related parameters were analyzed. HLA loss of heterozygosity was observed in EBC, at a clonal and subclonal level and was associated with regulatory T cells and T-cell immunoglobulin and mucin-domain-3 expression restraining the immuno-stimulatory effects of neoadjuvant chemotherapy. TCB bridging CD3ε and HER2 or CEACAM5 could bypass major histocompatibility complex (MHC) class I loss, partially rescuing T-cell functions in mLN. TCB should be developed in BC to circumvent low MHC/peptide complexes.

The neoepitope landscape of breast cancer: implications for immunotherapy

BackgroundCancer immunotherapy with immune checkpoint blockade (CKB) is now standard of care for multiple cancers. The clinical response to CKB is associated with T cell immunity targeting cancer-induced mutations that generate novel HLA-binding epitopes (neoepitopes).MethodsHere, we developed a rapid bioinformatics pipeline and filtering strategy, EpitopeHunter, to identify and prioritize clinically relevant neoepitopes from the landscape of somatic mutations. We used the pipeline to determine the frequency of neoepitopes from the TCGA dataset of invasive breast cancers. We predicted HLA class I-binding neoepitopes for 870 breast cancer samples and filtered the neoepitopes based on tumor transcript abundance.ResultsWe found that the total mutational burden (TMB) was highest for triple-negative breast cancer, TNBC, (median = 63 mutations, range: 2–765); followed by HER-2(+) (median = 39 mutations, range: 1–1206); and lowest for ER/PR(+)HER-2(−) (median = 32 mutations, range: 1–2860). 40% of the nonsynonymous mutations led to the generation of predicted neoepitopes. The neoepitope load (NEL) is highly correlated with the mutational burden (R2 = 0.86).ConclusionsOnly half (51%) of the predicted neoepitopes are expressed at the RNA level (FPKM≥2), indicating the importance of assessing whether neoepitopes are transcribed. However, of all patients, 93% have at least one expressed predicted neoepitope, indicating that most breast cancer patients have the potential for neo-epitope targeted immunotherapy.

Abstract B070: Neoepitope and mutation load as a biomarker in a broad cohort of cancer patients treated with immunotherapy

Abstract Various different treatments of immunotherapy have in recent years made their mark in mainstream oncology, but it is still uncertain which patients will benefit and why. To determine this, different biomarkers have been investigated including tumor specific peptides originating from somatic mutations and recognized by T-cells, neoepitopes. Recent studies show that clinical benefit of immunotherapy correlate with both high mutational burden and high neoantigen load in defined patient groups, melanoma and lung cancer, underlining the possibility to use these measurements as biomarkers, and driving the hypothesis that neoantigen-responsive T-cells are the main drivers to cancer-cell elimination. We propose to investigate if a high mutational load and a high amount of neoepitopes are correlated with a general respond to immunotherapy across different cancer types and immunotherapy treatments. To examine this correlation, we studied 19 patients who have been treated with different kinds of immunotherapy. The data consist of Whole Exome Sequencing (WXS) data from blood and tumor, as well as tumor mRNA sequencing from all patients. Neoepitope load was predicted using Mutant peptide extractor and informer (MuPeXI) to find possible neoepitopes and score the neopeptides by their potential immunogenicity. The analysis of the sequencing data from the 19 patients revealed that even though these patients have cancers of varying origin and treated with different immunotherapies, a significant correlation was observed between a high number of neoepitopes as well as high mutational load with an increased possibility of a clinical benefit to immunotherapy. These findings showed that overall a clinical benefit is correlated with increased mutational load and neoepitopes independent of the cancer type and treatment which if used as a biomarker can possibly stratify patients determining who is likely to respond to cancer immunotherapy. Furthermore, we examined the tumor mRNA for T-cell recpetor sequences, and interestingly patients with high level of predeicted neoepitopes show enhanced oligoclonality of TCRs at the tumor site. Citation Format: Anne-Mette Bjerregaard, Vinicius Araujo Barbosa de Lima, Annie Borch, Olga Araujo Barbosa de Oestrup, Ulrik Lassen, Sine Reker Hadrup. Neoepitope and mutation load as a biomarker in a broad cohort of cancer patients treated with immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B070.

Abstract B083: Defective transcription elongation in a subset of cancers confers immunotherapy resistance

Abstract Immunogenicity of most cancer types is a result of either neoantigenic mutational load, which stokes up an adaptive immune response, or due to oncogenic stress pathways, which elicit an innate antitumor response. There have been exponential gains made in recent times using numerous strategies to reactivate the host antitumor immunity including the development of immune checkpoint inhibitors. Nonetheless, the promise of a cure and durable response in select patients does not refute the low response rates in advanced cases, most of which also relapse. These observations shore up the possibility of other mechanisms beyond the inactivation of local lymphocyte infiltrates that might also play in tumor cell evasion of antitumor immune response. Through comprehensive computational and follow-up experimental validations, we have found that a subset of cancers (~15-20% of all cancers) are characterized by severe defects in almost the entire epigenetic and transcriptional apparatus. These defects result in genome-wide deregulation of histone modifications, mRNA transcription elongation, and mRNA processing and nuclear export, especially in the long genes which we term as Transcriptional Elongation Deficient: TEdef. As such, cancer cells with TEdef suppressed the expression of the pathways enriched for long genes, such as proinflammatory signaling pathways (FasL response, TNF/NF-kB signaling, interferon signaling) at both mRNA and protein levels, and had diminished response to interferon and TNF stimuli. Remarkably, in renal cell carcinoma and metastatic melanoma patients in four cohorts, the TEdef phenotype significantly correlated with poor response and unfavorable outcome to immunotherapy, but not to chemo- or targeted therapy. Importantly, forced induction of TEdef in tumor cells impaired the expression of, and signaling through, the proinflammatory pathways, and imposed a resistance to the innate and adaptive antitumor immune responses and to immune checkpoint inhibitor therapy in vivo. Tumor lymphocyte infiltration (TIL) and somatic neoepitope load (SNL) are some of the best markers of immunotherapy response in the clinic. However, TEdef tumors were characterized by a higher rate of immune cell infiltration, but paradoxically, less immune-mediated local tumor cell lysis, further supporting the notion that TEdef is a tumor cell-autonomous mechanism of immune resistance. As such, combining TIL or SNL with TEdef had superior power in predicting the progression-free and overall survival of melanoma patients treated with the anti-CTLA4 and anti-PD-1 therapy. Overall, TEdef is a novel epigenetic mechanism of resistance to antitumor immune attack, which warrants its assessment in cancer patients undergoing immunotherapy. Citation Format: Vishnu Modur. Defective transcription elongation in a subset of cancers confers immunotherapy resistance [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B083.

Abstract B089: Application of precision cancer immunotherapy design tools to bladder cancer: Non-self-like neoepitopes as a prognostic biomarker

Abstract Precision cancer immunotherapy targeting mutations expressed by cancer cells has proven to effectively control the tumor of patients in multiple clinical trials (Sahin et al., Nature 2017; Ott et al., Nature 2017). However, the selection of immunogenic T-cell neo-epitopes remains challenging and many epitopes selected using traditional methodologies fail to induce effector T-cell responses. Poor performance may partially be due to inclusion of mutated epitopes cross-conserved with self-epitopes recognized by regulatory (Treg), anergic, or deleted T-cells. Vaccination with self-epitopes can lead to weak effector responses, active immune suppression, and toxicity due to immune-mediated adverse effects. In addition, most cancer vaccine studies focus on the selection of CD8 T-cell neo-epitopes due to an apparent lack of robust and accurate CD4 T-cell epitope prediction tools. We have developed Ancer, an integrated and streamlined neo-epitope selection pipeline, that accelerates the selection of both CD4 and CD8 T-cell neo-epitopes from next-generation sequencing (NGS) data. Ancer leverages EpiMatrix and JanusMatrix, predictive algorithms that have been extensively validated in prospective vaccine studies for infectious diseases (Moise et al., Hum Vaccines Immunother 2015; Wada et al., Sci Rep 2017). Distinctive features of Ancer are its ability to accurately predict Class II HLA ligands, or CD4 epitopes, with EpiMatrix, and to identify tolerated or Treg epitopes with JanusMatrix. In addition, screening candidate sequences with JanusMatrix enables to the removal of neo-epitopes that may trigger off-target events, which have in some cases abruptly halted the development of promising cancer therapies. Ancer was applied to NGS data derived from the BLCA bladder cancer cohort from The Cancer Genome Atlas (TCGA) database. On average, 55 out of 204 missense mutations in bladder cancer patients’ tumors met Ancer’s quality control standards, in an initial analysis carried out for a representative set of 11 patients. This subset of high-quality missense variants was then screened using Ancer settings defined by the unique HLA of each patient, to derive the best vaccine candidate sequences encompassing these mutations. A median number of 24 (interquartile range: 15-64) candidate sequences were generated for each patient under study. The time required to select sequences for all of the patients in this study was less than two days. This initial analysis of eleven BLCA bladder cancer cohort patients demonstrates the capacity of Ancer to define a sufficient number of candidate sequences for vaccinating bladder cancer patients in a precision immunotherapy setting. We also assessed Ancer’s ability to predict patient outcomes on a larger subset of 58 individuals. While the disease-free status of BLCA patients could not be explained by their tumor mutational burden (AUC = 0.55, p-value = 0.1328), nor by their load of missense mutations (AUC = 0.54, p-value = 0.1740), the number of neoepitopes highly different from self, as defined by Ancer, significantly segregated disease-free patients from patients who recurred or progressed (AUC = 0.68, p-value = 0.0214). These results suggest that defining the number of true neoepitopes using Ancer may represent a novel biomarker for more robust antitumor immune response and higher likelihood of disease-free survival.Our analysis of the BLCA cohort from the TCGA database showcases the value of Ancer in clinical settings. Ancer can be used to identify high-value candidate sequences for inclusion in personalized therapies while removing potentially tolerated or tolerogenic self-epitopes from consideration. Our next step will be to investigate whether Ancer-defined neoepitope load will serve as a biomarker for prognosis and response to therapy in the full BLCA cohort. Citation Format: Guilhem Richard, Randy F. Sweis, Leonard Moise, Matthew Ardito, William A. Martin, Gad Berdugo, Gary D. Steinberg, Anne S. De Groot. Application of precision cancer immunotherapy design tools to bladder cancer: Non-self-like neoepitopes as a prognostic biomarker [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B089.

Mutational Analysis Identifies Therapeutic Biomarkers in Inflammatory Bowel Disease-Associated Colorectal Cancers.

Purpose: Inflammatory bowel disease-associated colorectal cancers (IBD-CRC) are associated with a higher mortality than sporadic colorectal cancers. The poorly defined molecular pathogenesis of IBD-CRCs limits development of effective prevention, detection, and treatment strategies. We aimed to identify biomarkers using whole-exome sequencing of IBD-CRCs to guide individualized management.Experimental Design: Whole-exome sequencing was performed on 34 formalin-fixed paraffin-embedded primary IBD-CRCs and 31 matched normal lymph nodes. Computational methods were used to identify somatic point mutations, small insertions and deletions, mutational signatures, and somatic copy number alterations. Mismatch repair status was examined.Results: Hypermutation was observed in 27% of IBD-CRCs. All hypermutated cancers were from the proximal colon; all but one of the cancers with hypermutation had defective mismatch repair or somatic mutations in the proofreading domain of DNA POLE Hypermutated IBD-CRCs had increased numbers of predicted neo-epitopes, which could be exploited using immunotherapy. We identified six distinct mutation signatures in IBD-CRCs, three of which corresponded to known mechanisms of mutagenesis. Driver genes were also identified.Conclusions: IBD-CRCs should be evaluated for hypermutation and defective mismatch repair to identify patients with a higher neo-epitope load who may benefit from immunotherapies. Prospective trials are required to determine whether IHC to detect loss of MLH1 expression in dysplastic colonic tissue could identify patients at increased risk of developing IBD-CRC. We identified mutations in genes in IBD-CRCs with hypermutation that might be targeted therapeutically. These approaches would complement and individualize surveillance and treatment programs. Clin Cancer Res; 24(20); 5133-42. ©2018 AACR.

PO-520 POLE gene mutation analysis in colorectal cancer patients with MSS – a pilot study

IntroductionTumours characterised by high-mutation rate and neoepitope load, which are hypothesised to be responsible for their high immunogenicity, are known to respond to the immune checkpoint inhibitor therapy. Microsatellite instability (MSI) in patients with colorectal cancer (CRC) is a biomarker for the high mutation rate is these patients. Besides MSI, there exists a subset of CRC patients with stable microsatellites (MSS) and germline or somatic mutations in functional domains of the DNA polymerase ε encoded by POLE gene who can benefit from the immune checkpoint therapy. The aim of this retrospective pilot study was to detect POLE mutations and identify patients with MSS CRC who could benefit from the immune checkpoint inhibitor therapy. Because the detection of POLE mutations requires sensitive methods, we used single tube nested PCR to improve the sensitivity of the method.Material and methodsIn this pilot study, 34 patients with the diagnosis of CRC aged 52 or less out of 270 CRC patient cohort have been enrolled. The lymphocyte infiltration and MSI were determined. PCR primers were designed for exons 9, 11 and 13 of the POLE gene, one pair of external primers as well as one pair of internal primers for each exon. The amplicons were sequenced according to the Sanger method using internal primers. The sequences were aligned with the reference sequence (ENST0000032057.9).Results and discussionsThe average age of our patients was 43.9 years. The nested PCR was optimised by touch down approach with two different annealing temperatures for external and internal primers. In our pilot study, we were not able to find any of the previously described pathogenic mutations. In one case of colorectal adenocarcinoma with mucinous elements (48y), a silent substitution c.893C>T resulting in p.L283L was detected with forward and reverse primers. This substitution is not described in dSNP. Using MutationTaster, we have found a possible effect of this substitution on RNA splicing, which still has to be confirmed on the RNA level.ConclusionMutations in POLE gene in CRC and endometrial cancer are associated with hypermutated tumours and are promising predictive biomarkers in immune checkpoint inhibitor therapy. It is important to develop sensitive methods that enable the detection of somatic mutations in routine clinical setting. Supported by Biomedical Centre Martin (ITMS 26220220187) and APVV-16–0066.

Abstract P3-05-13: The neo-epitope landscape of breast cancer: Implications for immunotherapy

Abstract Introduction: Immune checkpoint blockade is an effective immunotherapy for multiple cancers, including a subset of TNBCs. The clinical response to checkpoint blockade correlates with high tumor mutational burden. Cancer-induced mutations are predicted to generate new HLA-binding epitopes (neo-epitopes), which are potential targets of T cells. To determine the neo-epitope load in breast cancer, we developed a rapid bioinformatics pipeline and filtering strategy, EpitopeHunter, to identify and prioritize clinically relevant neo-epitopes from the landscape of somatic mutations. Here, we determined the specificity and frequency of neo-epitopes from the TCGA dataset of invasive breast cancers. Methods: We analyzed 842 tumor and normal breast cancer exome sequencing from the TCGA dataset. Subgroup analysis was performed based on the IHC status of ER, PR and HER2: (i) TNBC (n=98); (ii) ER and/or PR(+), Her2(-) (n=604); and (iii) ER and PR(+/-), Her-2(+) (n=140). Cancer specific mutations were called uniformly for the aligned tumor and normal pairs using VarScan2. For each annotated non-synonymous mutation, we generated all possible neo-epitopes (up to 11-mers) including the mutant amino acid. HLA typing of A, B and C MHC class I genes of each patient was determined using the POLYSOLVER algorithm. We selected high affinity binding epitopes using the IEDB prediction algorithm based on the known epitopes to identify likely binding of each neo-epitope within the suite of patient-specific HLA alleles (IEDB score &amp;lt; 500). We then filtered the epitopes based on tumor transcript abundance using RNAseq expression for 837/842 patients. An expression value of FPKM &amp;gt; 2 was used as a cut-off for the expressed neo-epitopes. Results: Total mutational burden was highest for TNBC (median=53, range: 2-1162); followed by ER and PR(+/-), Her-2(+) (median=40, range: 6-4983); and lowest for ER and/or PR (+), Her-2(-) (median=30, range: 0-5515). About 15% of the nonsynonymous mutations led to the generation of neo-epitopes. The neo-epitope load (high affinity neo-epitopes with IEDB score &amp;lt;500) is highly correlated with the mutational burden (R2= 0.8-0.9). Number of patients with at least one neo-epitope expressed in each category was: TNBC (n= 79/97, 81%); ER and/or PR(+), Her2(-) (n=478/600, 79%); and ER and PR(+/-), Her-2(+) (n=115/140, 82%). Overall, 672/837 patients have at least one predicted high affinity neo-epitope expressed, with an average of 14.7 (1-525) neo-epitopes expressed per patient. Conclusions: We have developed a rapid bioinformatics pipeline, EpitopeHunter, for the identification of neo-epitopes from tumor sequencing data. Eighty percent of breast cancer patients have at least one expressed neo-epitope, which may serve as candidates for targeted immunotherapy. Citation Format: Narang P, Chen M, Sharma AA, Anderson KS, Wilson Sayres MA. The neo-epitope landscape of breast cancer: Implications for immunotherapy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-13.

Abstract 5923: Developing a novel system for studying tumor microenvironment heterogeneity in pancreatic adenocarcinoma

Abstract Pancreatic cancer (PDA) has a dismal prognosis and responds poorly to all existing therapies. This is largely attributed to the existence of dense stroma and lack of T cell infiltration. PDA exhibits heterogeneous tumor microenvironments that are composed of various stromal cells. Human PDA has been shown to have various levels of T cell infiltration. The mechanisms underlying the inter-tumor heterogeneity of immune microenvironment in PDA and its effects on distinct responses of PDA to therapies, especially immune-modulating therapies, remain largely unknown. Our lab has demonstrated that there is no difference in somatic mutation burden or neo-epitope load between human PDA tumors with high or low amount of cytolytic T cells. We hypothesize that tumor-cell-intrinsic determinants, transcriptional and/or epigenetic factors, shape the heterogenous tumor immune microenvironment, and thereby result in different responses to therapies. For the reason that tumor cellularity of PDA is very low, the profiling of bulk tumor samples cannot provide enough information for understanding tumor-cell-intrinsic molecular events associated with differential immune microenvironment. Therefore, we developed a panel of ~60 cell lines (diversity-cell-panel) from PDA tumors derived from inbred C57BL/6 KPC mouse model, which recapitulates the major histopathological and molecular features of human PDA. Our preliminary results have demonstrated that the diversity-cell-panel recapitulates the immune microenvironments heterogeneity in PDA. Tumor cells mainly fell into two subgroups: T-cell-high and T-cell-low. Tumors resulted from T-cell-high tumor cells also possess less amount of immune-suppressive myeloid cells. To study whether this difference is caused by various amount of mutations, we performed exome-sequencing for 8 cell lines within this cell-panel, and showed that T cell-high and T-cell-low tumor cells have similar amount of mutations and neo-epitopes. To further study the distinct molecular features of T-cell-high and T-cell-low tumor cells, we will perform RNA-seq and ATAC-seq on tumor cells sorted from resulting tumors. To understand whether T cell infiltration affects the response of PDA tumors to immune-modulating therapies, I have been testing the response of T-cell-high and T-cell-low tumors to a group of therapies, including chemotherapy, CD40 therapy and immune checkpoint blockade therapy. Utilizing this novel system, the cell-panel, this study will delineate in unprecedented detail the novel molecular determinants of immune heterogeneity in PDA and the implication of the heterogeneity to therapies. This diversity-cell-panel will also be useful for understanding other aspects of tumor heterogeneity, including angiogenesis, drug resistance and metastatic disease. Citation Format: Jinyang Li. Developing a novel system for studying tumor microenvironment heterogeneity in pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5923. doi:10.1158/1538-7445.AM2017-5923

Immune Cytolytic Activity Stratifies Molecular Subsets of Human Pancreatic Cancer.

Purpose: Immunotherapy has the potential to improve the dismal prognosis in pancreatic ductal adenocarcinoma (PDA), but clinical trials, including those with single-agent PD-1 or PD-L1 inhibition, have been disappointing. Our aim was to examine the immune landscape of PDA as it relates to aspects of tumor biology, including neoepitope burden.Experimental Design: We used publicly available expression data from 134 primary resection PDA samples from The Cancer Genome Atlas to stratify patients according to a cytolytic T-cell activity expression index. We correlated cytolytic immune activity with mutational, structural, and neoepitope features of the tumor.Results: Human PDA displays a range of intratumoral cytolytic T-cell activity. PDA tumors with low cytolytic activity exhibited significantly increased copy number alterations, including recurrent amplifications of MYC and NOTCH2 and recurrent deletions and mutations of CDKN2A/B In sharp contrast to other tumor types, high cytolytic activity in PDA did not correlate with increased mutational burden or neoepitope load (MHC class I and class II). Cytolytic-high tumors exhibited increased expression of multiple immune checkpoint genes compared to cytolytic-low tumors, except for PD-L1 expression, which was uniformly low.Conclusions: These data identify a subset of human PDA with high cytolytic T-cell activity. Rather than being linked to mutation burden or neoepitope load, immune activation indices in PDA were inversely linked to genomic alterations, suggesting that intrinsic oncogenic processes drive immune inactivity in human PDA. Furthermore, these data highlight the potential importance of immune checkpoints other than PD-L1/PD-1 as therapeutic targets in this lethal disease. Clin Cancer Res; 23(12); 3129-38. ©2016 AACR.

Abstract A12: Analysis of tumor immunogenicity in germline BRCA1/2 mutation associated breast and ovarian cancers

Abstract Germline mutations in BRCA1 or BRCA 2 can lead to increased genomic instability and mutational burden in mutation-associated breast and ovarian cancer. Given the association of tumor neoepitope load and clinical response to immune checkpoint therapy, we used whole exome sequencing (WES) to predict missense mutational burden and potential neoepitope expression in 34 breast and 71 ovarian tumors with germline BRCA1/2 mutations (combining Penn and TCGA cohorts). Analysis of WES data found that tumors from patients with BRCA1/2 mutations had significantly higher mutational burden than non-BRCA1/2 tumors (p&amp;lt;0.01). Based on a novel bioinformatics pipeline for human leukocyte antigen (HLA) genotyping and MHC class I neoantigen prediction, we found that neoantigen load (predicted IC50 &amp;lt;500 nM) varied across patients (breast: mean - 147, std. dev. – 127; ovarian: mean – 126, std. dev. – 96). While higher neoantigen load promotes cytotoxic CD8+ T-cell responses in a number of cancers, we found among the BRCA1/2 subset that neoantigen load did not correlate with cytolytic signature, an RNA-based metric of anti-tumor immune response. From TCGA RNA-seq data, immune pathways (q&amp;lt;0.01) including PD-1 signaling, reflective of counter-regulatory responses often observed in response to immune pressure, positively correlated with cytolytic signature. Neoantigen load, however, correlated only with GPCR signaling and extracellular matrix (ECM) proteins (q&amp;lt;0.01). By contrast, the expression of genes involved in ECM and angiogenesis, which are involved in tumor immune escape and suppression of immune cell infiltration, negatively correlated with cytolytic signature (q&amp;lt;0.01). Increased PDL1 and PDL2 (p&amp;lt;0.05) expression associated with copy number loss in PTEN. These data were confirmed by tissue microarray analysis, which found that levels of T cell infiltration did not associate with neoantigen load, whereas PTEN loss was associated with decreased T cell infiltration (p&amp;lt;0.05). Our work demonstrates that neoantigen load alone is not sufficient to induce T-cell infiltration and cytotoxicity in tumors from patients with BRCA1/2 mutations, but rather points to tumor intrinsic pathways involving PTEN copy alterations and a dense extracellular matrix as determinants of tumor immunity, both of which may lead to novel opportunities for combination immune therapy in these cancers. Citation Format: Adam Kraya, Kara N. Maxwell, Brandon M. Wenz, Bradley Wubbenhorst, Daniel De Sloover, Nicole Lunceford, Amanda Barrett, Jennifer D. Morrissette, Michael Feldman, Robert H. Vonderheide, Susan M. Domcheck, Katherine L. Nathanson. Analysis of tumor immunogenicity in germline BRCA1/2 mutation associated breast and ovarian cancers. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A12.

Abstract IA23: Immune landscape of human pancreatic cancer

Abstract Survival rates for pancreatic ductal adenocarcinoma (PDA) remain dismal. Immunotherapy has the potential to improve outcomes, but clinical trial results – particularly those employing single-agent CTLA4 or PD1/PD-L1 inhibition – have thus far been disappointing. We stratified human PDA based on a signature of cytolytic T-cell activity. Differences in cytolytic index correlated with defined PDA subtypes and distinct mutational events. Specifically, PDA tumors with low cytolytic activity exhibited increased genomic structural variation, including reproducible copy number alterations in MYC, FGFR1, NOTCH2, and CDKN2. In contrast to other tumors such as kidney cancer or melanoma, PDA tumors with a high cytolytic index lacked an increased mutation or neo-epitope load. Tumors with high cytolytic activity exhibited increased expression of several immune checkpoint genes, but PD-L1 expression was uniformly low. These data establish a link between genomic alterations and immune activation in PDA and suggest that immune checkpoints other than PD-L1/PD-1 may be attractive therapeutic targets in this lethal disease. Citation Format: David Balli, Andrew J. Rech, Ben Z. Stanger, Robert H. Vonderheide.{Authors}. Immune landscape of human pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr IA23.