Clonal Neoantigens Research Articles

Abstract PR13: Inactivation of DNA repair triggers dynamic neoantigen evolution and impairs cancer growth

Abstract Colorectal, ovarian, endometrial and other tumors carrying defects in DNA mismatch repair often show favorable prognosis and indolent progression. The genomes of these tumors -also known as microsatellite unstable (MSI) cancers- bear hundreds of thousands of somatic mutations, a feature which fosters cancer progression and rapid evolution of resistance to targeted therapies. The molecular bases for the favorable outcome of MMR deficient cancers have long remained a mystery. Recent evidences that a subset of MSI tumors respond prominently to immune checkpoint blockade led to the seminal hypothesis that the presence of high number of somatic mutations may be responsible for effective immune-surveillance. However, several reports indicate that a relevant fraction of hyper-mutated tumors have unfavorable prognosis and do not respond to immune-modulators. Intrigued by these contradictory findings, we used the CRISPR system to genetically inactivated MutL homolog 1 (MLH1) in colorectal, breast and pancreatic mouse cancer cells. The growth of MMR deficient cells was comparable to their proficient counterparts in vitro and upon transplantation in immune-compromised mice. Strikingly however, isogenic MMR deficient colorectal, breast and pancreatic cancer cells were largely unable to form tumors when injected subcutaneously or orthotopically in syngeneic mouse models. MMR deficient tumors initially established in immune-deficient mice continued to grow exponentially when transplanted in syngeneic animals but regressed promptly when immune checkpoint inhibitors (anti PD-1 and anti CTLA-4) were administered. Exome sequencing of MMR proficient cells revealed mutational loads and neo-antigen profiles that were stable over time. MMR inactivation further increased the mutation burden and led to highly dynamic mutational profiles, resulting in persistent renewal of neoantigens. These results led us hypothesize that enforced increase of the number of mutations in cancer cells could be paradoxically- beneficial for therapeutic purposes. We therefore performed a pharmacological screen to identify agents capable of permanent inactivation of MMR in colorectal, breast and PDAC cancer cells. We found that temozolomide triggers MLH1 inactivation and leads to rapid clonal evolution and dynamic neoantigen profiles. Temozolomide-treated cells were unable to form tumors in syngeneic animals, while cells treated with other alkylating agents did. Genomic analysis of these tumor models revealed that fluctuating levels of neoantigens, rather than the absolute number of mutations is critical to provoke immune surveillance. These results provide the rationale for developing innovative anticancer therapies that target DNA repair proteins. This abstract is also being presented as Poster B11. Citation Format: Giovanni Germano, Simona Lamba, Giuseppe Rospo, Alessandro Magrì, Federica Maione, Mariangela Russo, Nabil Amirouchene Angelozzi, Ludovuc Barault, Monica Montone, Federica Di Nicolantonio, Enrico Giraudo, Alberto Bardelli. Inactivation of DNA repair triggers dynamic neoantigen evolution and impairs cancer growth. [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 PR13.

Abstract IA19: Novel insights into T cell recognition and regulation at the tumor site

Abstract Regulation of T cell function represents a key obstacle to the successful clinical application of tumor immunotherapy. Antibody-mediated blockade of co-inhibitory immune checkpoint molecules seeks to address such regulation. Durable remissions and even cure has been observed in patients with advanced cancer, however such responses are limited to a small fraction of treated patients. The identification of biomarkers predictive of response and resistance to therapy therefore remains an area of high scientific priority. Recent studies highlight the impact of the genomic landscape on anti-tumor immunity. Tumor-specific mutations may serve as neoantigens, eliciting T cell responses, and appear to be enriched in patients with advanced melanoma and non-small cell lung cancer (NSCLC) deriving benefit from CTLA-4 and PD-1 blockade respectively. Genetic heterogeneity within single tumors is well described, however the impact of intra-tumor heterogeneity (ITH) upon the neoantigen landscape and anti-tumor immunity has remained unclear. Through integrated analysis of ITH and neoantigen burden, we demonstrate the impact of clonal neoantigens, present in every cancer cell, on survival in primary lung adenocarcinoma. We identify neoantigen-reactive CD8+ T cells recognizing clonal neoantigens in patients with early stage NSCLC and demonstrate expression of high levels of co-inhibitory immune checkpoint molecules including PD-1. In keeping with these observations, sensitivity to CTLA-4 and PD-1 blockade in patients with advanced melanoma and NSCLC was enhanced in tumors enriched for clonal neoantigens. Heterogeneity in the neoantigen landscape may therefore influence immune surveillance. Clonal neoantigens, shared by all tumor cells, represent a key substrate for T cell recognition and an attractive target for adoptive cell-based and vaccination strategies which may hold promise to address the challenges of ITH. Citation Format: Andrew J. S. Furness. Novel insights into T cell recognition and regulation at the tumor site. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA19.

ED01.03 Insights from TCGA

ED01.03 Insights from TCGA

Predictors of Chemosensitivity in Triple Negative Breast Cancer: An Integrated Genomic Analysis.

BackgroundTriple negative breast cancer (TNBC) is a highly heterogeneous and aggressive disease, and although no effective targeted therapies are available to date, about one-third of patients with TNBC achieve pathologic complete response (pCR) from standard-of-care anthracycline/taxane (ACT) chemotherapy. The heterogeneity of these tumors, however, has hindered the discovery of effective biomarkers to identify such patients.Methods and FindingsWe performed whole exome sequencing on 29 TNBC cases from the MD Anderson Cancer Center (MDACC) selected because they had either pCR (n = 18) or extensive residual disease (n = 11) after neoadjuvant chemotherapy, with cases from The Cancer Genome Atlas (TCGA; n = 144) and METABRIC (n = 278) cohorts serving as validation cohorts. Our analysis revealed that mutations in the AR- and FOXA1-regulated networks, in which BRCA1 plays a key role, are associated with significantly higher sensitivity to ACT chemotherapy in the MDACC cohort (pCR rate of 94.1% compared to 16.6% in tumors without mutations in AR/FOXA1 pathway, adjusted p = 0.02) and significantly better survival outcome in the TCGA TNBC cohort (log-rank test, p = 0.05). Combined analysis of DNA sequencing, DNA methylation, and RNA sequencing identified tumors of a distinct BRCA-deficient (BRCA-D) TNBC subtype characterized by low levels of wild-type BRCA1/2 expression. Patients with functionally BRCA-D tumors had significantly better survival with standard-of-care chemotherapy than patients whose tumors were not BRCA-D (log-rank test, p = 0.021), and they had significantly higher mutation burden (p < 0.001) and presented clonal neoantigens that were associated with increased immune cell activity. A transcriptional signature of BRCA-D TNBC tumors was independently validated to be significantly associated with improved survival in the METABRIC dataset (log-rank test, p = 0.009). As a retrospective study, limitations include the small size and potential selection bias in the discovery cohort.ConclusionsThe comprehensive molecular analysis presented in this study directly links BRCA deficiency with increased clonal mutation burden and significantly enhanced chemosensitivity in TNBC and suggests that functional RNA-based BRCA deficiency needs to be further examined in TNBC.

Abstract A023: Inducing neoantigens in disseminated tumor lesions to enhance their susceptibility to PD-1 blockade therapy

Abstract Correlation between “clonal” neoantigen burden (neoantigens generated early during cancer development and therefore represented at high frequency in all tumor lesions) and responsiveness to checkpoint blockade therapy has underscored the importance of neoantigens in promoting tumor immunogenicity, and has spurred a broad effort to develop methods to identify neoantigens for use in vaccination protocols. Yet, the majority of patients do not express, or express low numbers of, clonal neoantigens, and consequently will be less likely to benefit from checkpoint blockade and other forms of immune potentiating therapies. Here we describe several strategies to generate de novo neoantigens in the patient' disseminated tumor lesions and show that in murine tumor models they potentiate checkpoint blockade therapy. We have previously shown that oligonucleotide aptamer-targeted siRNA inhibition of the Nonsense-mediated mRNA (NMD) process in tumor cells in situ results in the induction of neoatigens and inhibition of tumor growth (Pastor et al., Nature, 2010, 465:227). We now demonstrate that tumor-targeted NMD inhibition potentiates PD-1 blockade therapy. A general concern and potential limitation of the NMD approach is that a significant proportion of NMD inhibition-induced neoantigens are generated as a result of random mutations or aberrant splicing and hence will not be represented at high frequency in all the tumor lesions of the patient. To that end, we are exploring alternative strategies to generate clonal neoantigens by targeting key components of antigen presentation pathways, specifically the TAP transporter, ERAAP peptidase, and Invariant chain, the latter to generate class II-restricted neoantigens. Studies have shown that downregulation of these products, using genetic means or antisense RNA, not only inhibits the canonical antigen presentation pathway but also upregulates alternative pathways that present new, otherwise silent or subdominant, epitopes. Since such epitopes are not generated as a result of random mutations they are more likely to represent “clonal” neoepitopes generated in every cell in which the target was downregulated. Here we are developing clinically feasible broadly applicable approaches to inhibit the aforementioned mediators of antigen presentation using corresponding siRNAs targeted to tumor cells in situ by conjugation to a nucleolin-binding aptamer. Nucleolin, a nucleolar product, is translocated to the surface the majority of tumor cells of both human and murine origin and thereby serves as a broad, if not universal, target to deliver therapeutic cargo to the disseminated tumor cells of the patient. We show that nucleolin aptamer-targeted downregulation of ERAAP or Invariant chain in tumor-bearing mice inhibits tumor growth and potentiates PD-1 blockade therapy. Ongoing studies explore the combinatorial use of neoantigen induction methods and combination of neoantigen induction with immune potentiating strategies including but not limited to checkpoint blockade. Citation Format: Greta Garrido Hidalgo, Agata Levay, Alexey Berezhnoy, Brett Schrand, Eli Gilboa. Inducing neoantigens in disseminated tumor lesions to enhance their susceptibility to PD-1 blockade therapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A023.

Immunoscore and Immunoprofiling in cancer: an update from the melanoma and immunotherapy bridge 2015.

The fifth “Melanoma Bridge Meeting” took place in Naples, December 1–5th, 2015. The main topics discussed at this meeting were: Molecular and Immuno advances, Immunotherapies and Combination Therapies, Tumor Microenvironment and Biomarkers and Immunoscore. The natural history of cancer involves interactions between the tumor and the immune system of the host. The immune infiltration at the tumor site may be indicative of host response. Significant correlations were shown between the levels of immune cell infiltration in tumors and patient’s clinical outcome. Moreover, incredible progress comes from the discovery of mutation-encoded tumor neoantigens. In fact, as tumors grow, they acquire mutations that are able to influence the response of patients to immune checkpoint inhibitors. It has been demonstrated that sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. The road ahead is still very long, but the knowledge of the mechanisms of immune escape, the study of tumor neo-antigens as well as of tumor microenvironment and the development of new immunotherapy strategies, will make cancer a more and more treatable disease.

Immunotherapy: Clonal neoantigens and immune response: a balancing act.

Immunotherapy: Clonal neoantigens and immune response: a balancing act.

The cellular ancestry of tumor antigens

Tumor Immunology![Figure][1] Tumors contain cells expressing a variety of novel antigens CREDIT: MCGRANAHAN ET AL. . One contributing factor in antitumor immunity is the repertoire of neoantigens created by genetic mutations within tumor cells. Like the corresponding mutations, these neoantigens show intratumoral heterogeneity. Some are present in all tumor cells (clonal), and others are present in only a fraction of cells (subclonal). In a study of lung cancer and melanoma, McGranahan et al. found that a high burden of clonal tumor neoantigens correlated with improved patient survival, an increased presence of tumor-infiltrating lymphocytes, and a durable response to immunotherapy. Science , this issue p. [1463][2] [1]: pending:yes [2]: /lookup/doi/10.1126/science.aaf1490

Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade.

As tumors grow, they acquire mutations, some of which create neoantigens that influence the response of patients to immune checkpoint inhibitors. We explored the impact of neoantigen intratumor heterogeneity (ITH) on antitumor immunity. Through integrated analysis of ITH and neoantigen burden, we demonstrate a relationship between clonal neoantigen burden and overall survival in primary lung adenocarcinomas. CD8(+)tumor-infiltrating lymphocytes reactive to clonal neoantigens were identified in early-stage non-small cell lung cancer and expressed high levels of PD-1. Sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. T cells recognizing clonal neoantigens were detectable in patients with durable clinical benefit. Cytotoxic chemotherapy-induced subclonal neoantigens, contributing to an increased mutational load, were enriched in certain poor responders. These data suggest that neoantigen heterogeneity may influence immune surveillance and support therapeutic developments targeting clonal neoantigens.