Abstract NG01: Evolution of neoantigen landscape during immune checkpoint blockade in non-small cell lung cancer
Authors: Anagnostou Valsamo , Smith Kellie N. , Forde Patrick M. , Niknafs Noushin , Bhattacharya Rohit , White James , Adleff Vilmos , Phallen Jillian , Wali Neha , Hruban Carolyn , Guthrie Violeta B. , Rodgers Kristen , Naidoo Jarushka , Kang Hyunseok , Sharfman William , Georgiades Christos , Verde Franco , Illei Peter , Li Qing Ka , Gabrielson Edward , Brock Malcolm , Zahnow Cynthia , Baylin Stephen B. , Scharpf Rob , Brahmer Julie R. , Karchin Rachel , Pardoll Drew M. , Velculescu Victor E. 
Published: July 2017

Abstract
Tumor cells contain nonsynonymous somatic mutations that alter the amino acid sequences of the proteins encoded by the affected genes. Those alterations are foreign to the immune system and may therefore represent tumor-specific neoantigens capable of inducing antitumor immune responses. Somatic mutational and neoantigen density has recently been shown to correlate with long-term benefit from immune checkpoint blockade in non-small cell lung cancer (NSCLC) and melanoma, suggesting that a high density of neoepitopes stemming from somatic mutations may enhance clinical benefit from blockade of immune checkpoints that unleash endogenous responses to these mutation-associated neoantigens (MANAs). Expression of the programmed cell death ligand 1 (PD-L1) in tumors or tumor-infiltrating immune cells has been associated with responses to PD-1 blockade; however, PD-L1 expression or other immune biomarkers have not been sufficient to fully explain therapeutic outcomes. Among the patients that initially respond to PD-1 blockade, some become resistant to the therapy. Up-regulation of alternate immune checkpoints, loss of HLA haplotypes, or somatic mutations in HLA or JAK1/JAK2 genes have been proposed as mechanisms of evasion to immune recognition in some patients, but the mechanisms underlying response and acquired resistance to immune checkpoint blockade have remained elusive. To examine mechanisms of resistance to immunotherapy, we performed genome-wide sequence analysis of protein coding genes and T-cell receptor (TCR) clonotype analysis, followed by functional assays of autologous T-cell activation of patients who demonstrated initial response to immune checkpoint blockade but ultimately developed progressive disease.Of a cohort of 42 NSCLC patients treated with single-agent PD-1 or combined PD-1 and CTLA4 blockade, we identified all consecutive cases that at the time of the analysis developed acquired resistance (two subjects treated with nivolumab and two with ipilimumab and nivolumab) and where paired tumor specimens were available both before and after therapy. To examine the landscape of genomic alterations and associated neoantigens, we performed whole exome sequencing of tumors from these patients. Pretreatment and postprogression specimens were obtained from the same anatomic location or from sites in close anatomic proximity. We examined multiple immune-related parameters of peptides stemming from somatic alterations using a computational multidimensional neoantigen prediction platform. This approach allowed for identification of peptides within mutated genes that were predicted to be processed and presented by MHC class I proteins and therefore had the potential to elicit an immune response. The algorithm evaluated the binding of mutant peptides (8-11mers) to patient-specific HLA class I alleles and ranked the neoantigens according to MHC binding affinity, antigen processing, and self-similarity.
Analyses of matched pretreatment and resistant tumors identified genomic changes resulting in loss of 7 to 18 putative mutation-associated neoantigens in resistant clones. While algorithm-based predictions of antigenicity are valuable in narrowing down the large number of peptides capable of being generated by a mutation to a set potential antigenic peptides presented by self-MHC alleles, functional T cell recognition is critical to evaluate immune responsiveness. To this end, we developed a sensitive approach for assessing T-cell response to candidate MANAs (cMANAs) that utilized next-generation sequencing of TCR-Vb CDR3 regions as a measure of T-cell clonality. To evaluate T-cell recognition of eliminated neoantigens, purified peripheral blood T cells from the patients described above were stimulated with autologous peripheral blood mononuclear cells (PBMCs) loaded with cMANA peptides in a ten-day culture system. We subsequently used TCR next-generation sequencing to assess the differential abundance of neoantigen-specific T cell clonotypes in these expanded T cell populations. In order to further investigate the importance of eliminated MANAs, we generated peptides from retained and gained MANAs and assessed their potential to elicit a MANA-specific T-cell expansion. Peptides generated from the eliminated neoantigens elicited clonal T-cell expansion in autologous T-cell cultures, suggesting that they generated functional immune responses. These findings indicate that patient-derived T cells recognized the eliminated neoantigens and suggest that these neoantigens were relevant targets for the achievement of initial therapeutic response to checkpoint blockade. Conceptually, neoantigen loss occurs through elimination of tumor subclones or through deletion of chromosomal regions containing truncal alterations. To evaluate the contribution of these mechanisms to the loss of neoantigens, we analyzed the tumors both before and after therapy using the SCHISM pipeline and incorporating mutation frequency, tumor purity, and copy number variation to infer the fraction of cells containing a specific mutation (mutation cellularity). Consistent with our hypothesis, we observed both mechanisms of neoantigen elimination: loss of truncal changes through genetic events involving chromosomal deletions and loss of heterozygosity (LOH) and loss of subclonal neoantigens either by LOH or through elimination of tumor subclones. Both truncal and subclonal changes were among the eliminated neoantigens that were functionally validated.
To evaluate the impact of changes in neoantigen landscape on cytotoxic T-cell receptor repertoire, we analyzed serially collected PBMCs, prior to immunotherapy initiation, at clinical response, and at resistance. We hypothesized that loss of neoantigens would lead to a decrease in clonality of cytotoxic TCR clonotypes, thus reflecting tumor immune evasion at the time of emergence of resistance. We observed peripheral T-cell expansion of a subset of the top 100 most frequent intratumoral clones, with the most frequent clones reaching up to a 44-fold increase in abundance in the blood at the time of response, followed by a decrease to pretreatment levels at the time of resistance. As a comparison, such decreases in TCR frequencies were not observed in a NSCLC patient with durable response to PD-1 blockade and no change in intratumoral TCR frequencies was seen in a NSCLC patient with primary resistance to PD-1 blockade. Taken together, these observations suggest that TCR expansion may be both a useful measure of response to checkpoint blockade and an indicator of acquired therapeutic resistance through neoantigen loss.
As immune checkpoint therapy has become standard of care for many cancer types, the development of acquired resistance is being recognized more commonly. Through our comprehensive genomic analyses, we have identified changes in the genomic landscape of tumors during immune checkpoint blockade. These analyses show that emergence of acquired resistance is associated with loss of mutations encoding for putative tumor-specific neoantigens, through both elimination of tumor subclones and chromosomal loss of truncal alterations. Using a new approach to assess neoantigen reactivity by T cells, we found that some of these eliminated mutations indeed encoded peptides recognized by T cells in the peripheral circulation of the respective patients. Given that the antitumor efficacy of checkpoint blockade likely involves release of endogenous T-cell responses to tumor antigens generated by coding mutations, our findings are consistent with a mechanism of acquired resistance to immune checkpoint blockade that involves therapy-induced immune editing of MANAs.
Acquisition of somatic resistance mutations is a common mechanism of therapeutic resistance to targeted therapies. However, elimination of genomic alterations and more specifically loss of somatic mutations through subsequent genetic events is uncommon in the context of natural tumor evolution or therapeutic resistance. Elimination of mutation associated antigens by a T-cell-dependent immunoselection process has been proposed as a mechanism of cancer immunoediting in melanoma after adoptive T cell transfer; however, the evolution of neoantigen loss as an escape mechanism under the selective pressure of immune checkpoint blockade in lung cancer has not been previously studied. In addition, we examined a variety of other mechanisms that have been proposed in the development of resistance to immunotherapies. We did not observe any differences in PD-L1 expression in tumor cells between responsive and resistant tumor samples. Likewise, we evaluated known potential genomic mechanisms of immunotherapy resistance; however, there were no new genomic alterations in the CD274 gene encoding for PD-L1, PDCD1 encoding for PD-1, CTLA4, JAK1 or JAK2 genes, in HLA genes, beta 2 microglobulin, or other antigen presentation-associated genes.
This work demonstrates for the first time that acquired resistance to anti-PD-1 or anti-PD1/anti-CTLA4 therapy in lung cancer can arise in association with the evolving landscape of mutations, some of which encode tumor neoantigens recognizable by T cells. These observations imply that widening the breadth of tumor neoantigen reactivity may mitigate the development of acquired resistance.
Citation Format: Valsamo Anagnostou, Kellie N. Smith, Patrick M. Forde, Noushin Niknafs, Rohit Bhattacharya, James White, Vilmos Adleff, Jillian Phallen, Neha Wali, Carolyn Hruban, Violeta B. Guthrie, Kristen Rodgers, Jarushka Naidoo, Hyunseok Kang, William Sharfman, Christos Georgiades, Franco Verde, Peter Illei, Qing Ka Li, Edward Gabrielson, Malcolm Brock, Cynthia Zahnow, Stephen B. Baylin, Rob Scharpf, Julie R. Brahmer, Rachel Karchin, Drew M. Pardoll, Victor E. Velculescu. Evolution of neoantigen landscape during immune checkpoint blockade in non-small cell lung cancer [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 NG01. doi:10.1158/1538-7445.AM2017-NG01