Spencer D. Martin

Neo-antigens predicted by tumor genome meta-analysis correlate with increased patient survival

Somatic missense mutations can initiate tumorogenesis and, conversely, anti-tumor cytotoxic T cell (CTL) responses. Tumor genome analysis has revealed extreme heterogeneity among tumor missense mutation profiles, but their relevance to tumor immunology and patient outcomes has awaited comprehensive evaluation. Here, for 515 patients from six tumor sites, we used RNA-seq data from The Cancer Genome Atlas to identify mutations that are predicted to be immunogenic in that they yielded mutational epitopes presented by the MHC proteins encoded by each patients autologous HLA-A alleles. Mutational epitopes were associated with increased patient survival. Moreover, the corresponding tumors had higher CTL content, inferred from CD8A gene expression, and elevated expression of the CTL exhaustion markers PDCD1 and CTLA4. Mutational epitopes were very scarce in tumors without evidence of CTL infiltration. These findings suggest that the abundance of predicted immunogenic mutations may be useful for identifying patients likely to benefit from checkpoint blockade and related immunotherapies.

Surveillance of the Tumor Mutanome by T Cells during Progression from Primary to Recurrent Ovarian Cancer

Purpose: Cancers accumulate mutations over time, each of which brings the potential for recognition by the immune system. We evaluated T-cell recognition of the tumor mutanome in patients with ovarian cancer undergoing standard treatment. Experimental Design: Tumor-associated T cells from 3 patients with ovarian cancer were assessed by ELISPOT for recognition of nonsynonymous mutations identified by whole exome sequencing of autologous tumor. The relative levels of mutations and responding T cells were monitored in serial tumor samples collected at primary surgery and first and second recurrence. Results: The vast majority of mutations (78/79) were not recognized by tumor-associated T cells; however, a highly specific CD8+ T-cell response to the mutation hydroxysteroid dehydrogenase–like protein 1 (HSDL1)L25V was detected in one patient. In the primary tumor, the HSDL1L25V mutation had low prevalence and expression, and a corresponding T-cell response was undetectable. At first recurrence, there was a striking increase in the abundance of the mutation and corresponding MHC class I epitope, and this was accompanied by the emergence of the HSDL1L25V-specific CD8+ T-cell response. At second recurrence, the HSDL1L25V mutation and epitope continued to be expressed; however, the corresponding T-cell response was no longer detectable. Conclusion: The immune system can respond to the evolving ovarian cancer genome. However, the T-cell response detected here was rare, was transient, and ultimately failed to prevent disease progression. These findings reveal the limitations of spontaneous tumor immunity in the setting of standard treatments and suggest a high degree of ignorance of tumor mutations that could potentially be reversed by immunotherapy. Clin Cancer Res; 20(5); 1125–34. ©2013 AACR.

Low Mutation Burden in Ovarian Cancer May Limit the Utility of Neoantigen-Targeted Vaccines

Due to advances in sequencing technology, somatically mutated cancer antigens, or neoantigens, are now readily identifiable and have become compelling targets for immunotherapy. In particular, neoantigen-targeted vaccines have shown promise in several pre-clinical and clinical studies. However, to date, neoantigen-targeted vaccine studies have involved tumors with exceptionally high mutation burdens. It remains unclear whether neoantigen-targeted vaccines will be broadly applicable to cancers with intermediate to low mutation burdens, such as ovarian cancer. To address this, we assessed whether a derivative of the murine ovarian tumor model ID8 could be targeted with neoantigen vaccines. We performed whole exome and transcriptome sequencing on ID8-G7 cells. We identified 92 somatic mutations, 39 of which were transcribed, missense mutations. For the 17 top predicted MHC class I binding mutations, we immunized mice subcutaneously with synthetic long peptide vaccines encoding the relevant mutation. Seven of 17 vaccines induced robust mutation-specific CD4 and/or CD8 T cell responses. However, none of the vaccines prolonged survival of tumor-bearing mice in either the prophylactic or therapeutic setting. Moreover, none of the neoantigen-specific T cell lines recognized ID8-G7 tumor cells in vitro, indicating that the corresponding mutations did not give rise to bonafide MHC-presented epitopes. Additionally, bioinformatic analysis of The Cancer Genome Atlas data revealed that only 12% (26/220) of HGSC cases had a ≥90% likelihood of harboring at least one authentic, naturally processed and presented neoantigen versus 51% (80/158) of lung cancers. Our findings highlight the limitations of applying neoantigen-targeted vaccines to tumor types with intermediate/low mutation burdens.

Targeting the undruggable: immunotherapy meets personalized oncology in the genomic era

Owing to recent advances in genomic technologies, personalized oncology is poised to fundamentally alter cancer therapy. In this paradigm, the mutational and transcriptional profiles of tumors are assessed, and personalized treatments are designed based on the specific molecular abnormalities relevant to each patients cancer. To date, such approaches have yielded impressive clinical responses in some patients. However, a major limitation of this strategy has also been revealed: the vast majority of tumor mutations are not targetable by current pharmacological approaches. Immunotherapy offers a promising alternative to exploit tumor mutations as targets for clinical intervention. Mutated proteins can give rise to novel antigens (called neoantigens) that are recognized with high specificity by patient T cells. Indeed, neoantigen-specific T cells have been shown to underlie clinical responses to many standard treatments and immunotherapeutic interventions. Moreover, studies in mouse models targeting neoantigens, and early results from clinical trials, have established proof of concept for personalized immunotherapies targeting next-generation sequencing identified neoantigens. Here, we review basic immunological principles related to T-cell recognition of neoantigens, and we examine recent studies that use genomic data to design personalized immunotherapies. We discuss the opportunities and challenges that lie ahead on the road to improving patient outcomes by incorporating immunotherapy into the paradigm of personalized oncology.

A library-based screening method identifies neoantigen-reactive T cells in peripheral blood prior to relapse of ovarian cancer

ABSTRACT Mutated cancer antigens, or neoantigens, represent compelling immunological targets and appear to underlie the success of several forms of immunotherapy. While there are anecdotal reports of neoantigen-specific T cells being present in the peripheral blood and/or tumors of cancer patients, effective adoptive cell therapy (ACT) against neoantigens will require reliable methods to isolate and expand rare, neoantigen-specific T cells from clinically available biospecimens, ideally prior to clinical relapse. Here, we addressed this need using “mini-lines”, large libraries of parallel T cell cultures, each originating from only 2,000 T cells. Using small quantities of peripheral blood from multiple time points in an ovarian cancer patient, we screened over 3.3 × 106 CD8+ T cells by ELISPOT for recognition of peptides corresponding to the full complement of somatic mutations (n = 37) from the patients tumor. We identified ten T cell lines which collectively recognized peptides encoding five distinct mutations. Six of the ten T cell lines recognized a previously described neoantigen from this patient (HSDL1L25V), whereas the remaining four lines recognized peptides corresponding to four other mutations. Only the HSDL1L25V-specific T cell lines recognized autologous tumor. HSDL1L25V-specific T cells comprised at least three distinct clonotypes and could be identified and expanded from peripheral blood 3–9 months prior to the first tumor recurrence. These T cells became undetectable at later time points, underscoring the dynamic nature of the response. Thus, neoantigen-specific T cells can be expanded from small volumes of blood during tumor remission, making pre-emptive ACT a plausible clinical strategy.

Mapping the human T cell repertoire to recurrent driver mutations in MYD88 and EZH2 in lymphoma

ABSTRACT Oncogenic “driver” mutations are theoretically attractive targets for the immunotherapy of lymphoid cancers, yet the proportion that can be recognized by T cells remains poorly defined. To address this issue without any confounding effects of the patients immune system, we assessed T cells from 19 healthy donors for recognition of three common driver mutations in lymphoma: MYD88L265P, EZH2Y641F, and EZH2Y641N. Donors collectively expressed the 10 most prevalent HLA class I alleles, including HLA-A*02:01. Peripheral blood T cells were primed with peptide-loaded dendritic cells (DC), and reactive T cells were assessed for recognition of naturally processed mutant versus wild type full-length proteins. After screening three driver mutations across 17–26 HLA class I alleles and 3 × 106−3 × 107 T cells per donor, we identified CD4+ T cells against EFISENCGEII from EZH2Y641N (presented by HLA-DRB1*13:02) and CD8+ T cells against RPIPIKYKA from MYD88L265P (presented by HLA-B*07:02). We failed to detect RPIPIKYKA-specific T cells in seven other HLA-B*07:02-positive donors, including two lymphoma patients. Thus, healthy donors harbor T cells specific for common driver mutations in lymphoma. However, such responses appear to be rare due to the combined limitations of antigen processing, HLA restriction, and T cell repertoire size, highlighting the need for highly individualized approaches for selecting targets.

Personalized Immunotherapy Targeting the Cancer Mutanome

Recent advances in DNA sequencing have revealed substantial mutational heterogeneity across tumors, which presents an enormous challenge for designing effective treatments. Although the majority of mutations have proven difficult to target pharmacologically, the exquisite sensitivity and specificity of the adaptive immune system offers a promising therapeutic alternative. Mutant proteins can give rise to novel T cell epitopes (called neoantigens) that are presented on the surface of tumor cells by the major histocompatibility complex. While the existence of neoantigens has been appreciated for decades, next-generation sequencing (NGS) has enabled rapid, systematic identification of neoantigens and corresponding T cell responses in patients treated with conventional and immune-based therapies. Herein, we review the characteristics of tumor neoantigens and the methods and challenges related to their identification. We discuss several recent clinical studies in which NGS data have been used to identify mutation-reactive T cells, culminating in vaccine and cell therapy trials in which neoantigens have been deliberately targeted. Improvements in DNA sequencing have revealed the complexity of human tumors, but these advances also provide a promising new pathway toward personalized immunotherapy.

Somatic mutation-associated T follicular helper cell elevation in lung adenocarcinoma

ABSTRACT T follicular helper cells (Tfh) play crucial roles in the development of humoral immunity. In the B cell-rich germinal center of lymphoid organs, they select for high-affinity B cells and aid in their maturation. While Tfh have known roles in B cell malignancies and have prognostic value in some epithelial cancers, their role in lung tumour initiation and development is unknown. Through immune cell deconvolution, we observed significantly increased Tfh in tumours from two independent cohorts of lung adenocarcinomas and found that this upregulation occurs early in tumour development. A subset of tumours were stained for T and B cells using multicolour immunohistochemistry, which revealed the presence of tumour-adjacent tertiary lymphoid organs in 17/20 cases each with an average of 16 Tfh observed in the germinal center. Importantly, Tfh levels were correlated with tumour mutational load and immunogenic cancer testis antigens, suggesting their involvement in mounting an active immune response against tumour neoantigens.

Abstract B15: Increased presence of T follicular helper cells in lung adenocarcinoma is associated with mutational load

Tertiary lymphoid organs are ectopic lymphoid formations found in inflamed tissues such as tumors, and their presence has been associated with improved patient outcome. T follicular helper cells (Tfh) reside in the germinal centre of tertiary lymphoid organs, and are required for the maturation of B cells and subsequent antibody response. Whereas the prognostic value of Tfh has been described in breast and colon tumors, they remain uncharacterized in lung tumors. We hypothesize that Tfh cells reside in lung adenocarcinomas and are associated with an increased immune response due to neoantigen exposure. Gene expression profiles were obtained from 83 paired lung adenocarcinomas and nonmalignant lung tissues from the BC Cancer Agency, and 571 unpaired samples from The Cancer Genome Atlas. Relative proportions of 22 immune cell subsets were inferred from gene expression data using CIBERSORT, a deconvolution algorithm. Identification of tertiary lymphoid organs was achieved through multicolor immunohistochemistry (IHC) staining for T- and B-cell lineage markers (CD3 and CD79a) using whole tissue sections. Proportions of Tfh cells were correlated with tumor mutation load defined as non-silent mutations per megabase (Mann Whitney U test) and patient outcome (Cox proportional hazard model). The proportion of Tfh cells was significantly increased in tumor tissue compared to nonmalignant lung in both cohorts. We also observed concomitant upregulation of Tfh markers PD1 and CXCR5 . Multicolor IHC validated the presence of tertiary lymphoid organs in 19 out of 20 cases assessed. Intriguingly, the increase in the proportion of Tfh cells revealed by CIBERSORT was observed across all disease stages and was validated in an additional cohort of Stage I lung adenocarcinomas. The relative proportion of Tfh cells did, however, increase with increasing tumor mutation burden, suggesting their involvement in an active immune response against tumor neoantigens. Tfh recruitment appears to be an early event in lung tumor progression and a function of neoantigen exposure, suggesting involvement in an active antitumor response rather than a passive result of chronic inflammation. Further investigation into Tfh in lung adenocarcinomas may lead to prognostic applications. Citation Format: Katey S.S. Enfield, Kevin W. Ng, Erin A. Marshall, Spencer D. Martin, Wan L. Lam. Increased presence of T follicular helper cells in lung adenocarcinoma is associated with mutational load [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B15.

Abstract PR11: Hyperspectral imaging tools capture the spatial organization of cell subsets within the tumor microenvironment

Immune cells are a major component of the tumor microenvironment (TME). The spatial organization of immune cell subpopulations within the TME is recognized to have biologic significance and clinical relevance. For example, spatial organization of immune cell subsets within the TME is critical for the inhibition of cytotoxic T-cell activity through direct interaction of ligand (PD-L1) with receptor (PD-1)). However, precise spatial deconvolution is limited by the lack of imaging algorithms for in situ multiplex single cell analyses as flow cytometry does not preserve data in the spatial dimension. To this end, we have developed a hyperspectral imaging platform designed for analyzing multichannel immunohistochemical-stained tissue sections for generating cell density data and reconstructing spatial architecture for tumor biology as well as clinical association studies. Whole-tissue sections from 20 lung adenocarcinomas with at least 5 years’ follow-up were stained for CD3 (pan-T cell), CD8 (cytotoxic T cell), and CD79a (B cell and plasma cell) and counterstained with hematoxylin. Multispectral images were acquired for five fields of view and analyzed to quantify cell types. Regions of Interest (ROIs) were then identified and analyzed in order to quantify cell-cell spatial relationships. Nonrandom patterns of immune cell distributions were identified using the Monte Carlo resampling method (500 iterations). Cell counts, densities, spatial relationships, and significant immune cell distributions were associated with clinical features (Kruskal - Wallis p Our analysis generated 234 image files for analysis, with an average of 16,400 cells per image. The densities of intratumoral CD8+ cytotoxic T cells were significantly higher in nonrecurrent cases, agreeing with literature reports. Similarly, cell sociology deductions identified relationships associated with metastasis: tumor cells in nonmetastatic cases had increased numbers of CD8+ cytotoxic T-cell neighbors. Following Monte Carlo analysis, nonrandom cell~cell spatial proximities emerged that were not identified at a cell density level. We have developed a hyperspectral imaging platform capable of quantifying cell-cell spatial relationships within tissue sections. This technology can be applied to larger clinical cohorts for the study of therapeutically targetable immune cell subsets with the goal of identifying patterns that correlate with clinical response and patient outcome. This abstract is also being presented as Poster B16. Citation Format: Katey S.S. Enfield, Spencer D. Martin, Sonia H.Y. Kung, Paul Gallagher, Calum E. MacAulay, Martial Guillaud, Wan L. Lam. Hyperspectral imaging tools capture the spatial organization of cell subsets within the tumor microenvironment [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR11.