Abstract: We conducted the first trial of neoadjuvant PD-1 blockade in resectable non-small cell lung cancer (NSCLC), finding nivolumab monotherapy to be safe and feasible with an encouraging rate of pathologic response. Building on these results, and promising data for nivolumab plus ipilimumab (anti-CTLA-4) in advanced NSCLC, we expanded our study to include an arm investigating neoadjuvant nivolumab plus ipilimumab. Methods Patients with resectable stage IB (≥4 cm)–IIIA (American Joint Committee on Cancer Tumor Node Metastases seventh edition), histologically confirmed, treatment-naïve NSCLC received nivolumab 3 mg/kg intravenously plus ipilimumab 1 mg/kg intravenously 6 weeks prior to planned resection. Nivolumab 3 mg/kg was given again approximately 4 and 2 weeks preoperatively. Primary endpoints were safety and feasibility with a planned enrollment of 15 patients. Pathologic response was a key secondary endpoint. Results While the treatment regimen was feasible per protocol, due to toxicity, the study arm was terminated early by investigator consensus after 9 of 15 patients were enrolled. All patients received every scheduled dose of therapy and were fit for planned surgery; however, 6 of 9 (67%) experienced treatment-related adverse events (TRAEs) and 3 (33%) experienced grade ≥3 TRAEs. Three of 9 patients (33%) had biopsy-confirmed tumor progression precluding definitive surgery. Of the 6 patients who underwent resection, 3 are alive and disease-free, 2 experienced recurrence and are actively receiving systemic treatment, and one died postoperatively due to acute respiratory distress syndrome. Two patients who underwent resection had tumor pathologic complete responses (pCRs) and continue to remain disease-free over 24 months since surgery. Pathologic response correlated with pre-treatment tumor PD-L1 expression, but not tumor mutation burden. Tumor KRAS/STK11 co-mutations were identified in 5 of 9 patients (59%), of whom two with disease progression precluding surgery had tumor KRAS/STK11/KEAP1 co-mutations. Conclusions Though treatment was feasible, due to toxicity the study arm was terminated early by investigator consensus. In light of this, and while the long-term disease-free status of patients who achieved pCR is encouraging, further investigation of neoadjuvant nivolumab plus ipilimumab in patients with resectable NSCLC requires the identification of predictive biomarkers that enrich for response.

Abstract: Background: Programmed death-1 (PD-1) blocking antibodies improve survival in patients with advanced non-small-cell lung cancer (NSCLC) but have not been tested in resectable NSCLC, where little progress has been made over the last decade. Methods: Adults with untreated surgically resectable stage I-IIIA NSCLC received two doses of nivolumab (anti-PD-1) preoperatively. The primary endpoints of the study were safety and feasibility. Tumor pathologic response, PD-L1 expression, mutation burden and mutation-associated neoantigen-specific T-cell responses were evaluated. Results: Neoadjuvant nivolumab was had an acceptable side effect profile without surgical delays, and 20 of 21 tumors were completely resected. Major pathologic response occurred in 45% (9/20) of resected tumors. Responses occurred in both PD-L1 positive and negative tumors. Pathologic response significantly correlated with pre-treatment tumor somatic mutation burden. T-cell clones shared between the tumor and peripheral blood increased systemically upon anti-PD-1 treatment in 8 of 9 patients analyzed. Mutation-associated neoantigen-specific T-cell clones, from a primary tumor that underwent pathologic complete response, rapidly expanded in peripheral blood at 2-4 weeks post-treatment, some of these clones were not detected before anti-PD-1. Conclusions: Neoadjuvant nivolumab was associated with few side effects, did not delay surgery, and induced major pathologic responses in 45% of resected tumors. Tumor mutation burden is predictive of pathologic response to anti-PD-1. Anti-PD-1 can induce expansion of mutation-associated neoantigen-specific T-cell clones in peripheral blood. [P.M.F., J.E.C., and K.N.S. contributed equally to this work.] Citation Format: Patrick M. Forde, Jamie E. Chaft, Kellie N. Smith, Valsamo Anagnostou, Tricia R. Cottrell, Matthew D. Hellmann, Marianna Zahurak, Stephen C. Yang, David R. Jones, Stephen Broderick, Richard J. Battafarano, Moises J. Velez, Natasha Rekhtman, Zachary Olah, Jarushka Naidoo, Kristen A. Marrone, Franco Verde, Haidan Guo, Jiajia Zhang, Justina X. Caushi, Hok Yee Chan, John-William Sidhom, Robert B. Scharpf, James White, Edward Gabrielson, Hao Wang, Gary L. Rosner, Valerie Rusch, Jedd D. Wolchok, Taha Merghoub, Janis M. Taube, Victor E. Velculescu, Suzanne L. Topalian, Julie R. Brahmer, Drew M. Pardoll. Neoadjuvant PD-1 blockade in resectable lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT079.

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

Abstract: Immuno-oncology (I-O) has required a shift in the established paradigm of toxicity and response assessment in clinical research. The design and interpretation of cancer clinical trials has been primarily driven by conventional toxicity and efficacy patterns observed with chemotherapy and targeted agents, which are insufficient to fully inform clinical trial design and guide therapeutic decisions in I-O. Responses to immune-targeted agents follow nonlinear dose–response and dose–toxicity kinetics mandating the development of novel response evaluation criteria. Biomarker-driven surrogate endpoints may better capture the mechanism of action and biological response to I-O agents and could be incorporated prospectively in early-phase I-O clinical trials. While overall survival remains the gold standard for evaluation of clinical efficacy of I-O agents in late-phase clinical trials, exploration of potential novel surrogate endpoints such as objective response rate and milestone survival is to be encouraged. Patient-reported outcomes should also be assessed to help redefine endpoints for I-O clinical trials and drive more efficient drug development. This paper discusses endpoints used in I-O trials to date and potential optimal endpoints for future early- and late-phase clinical development of I-O therapies. Clin Cancer Res; 23(17); 4959–69. ©2017 AACR. See all articles in this CCR Focus section, “Clinical Trial Design Considerations in the Immuno-oncology Era.”

Abstract: The dynamic nature of the cancer-immune system crosstalk has rendered single biomarker approaches insufficient to predict outcome from immune-targeted agents. These therapies also pose a challenge to radiographic response assessments that may underestimate the therapeutic benefit. There is therefore an urgent clinical need to develop molecular assays of response and resistance.In order to capture the dynamic nature of the anti-tumor immune response under checkpoint blockade, we devised an integrative non-invasive approach that captures the evolving neoantigen landscape and the associated T-cell receptor (TCR) repertoire. We employed whole exome sequencing to determine the genomic landscape of tumors and identify tumor-derived alterations in subsequent analyses of circulating cell-free tumor DNA (ctDNA), for 14 patients with metastatic NSCLC treated with immune checkpoint blockade. Liquid biopsies were obtained prior to treatment, at week 4-6 and at additional timepoints until disease progression. We used the ultrasensitive targeted error correction sequencing approach to analyze 58 cancer driver genes in the circulation of these patients and assessed the value of longitudinal ctDNA monitoring as a surrogate for response. In parallel, we evaluated dynamics changes in the TCR repertoire by TCR next generation sequencing of serially collected peripheral T cells and tumor infiltrating lymphocytes for each patient. Radiographic response assessments were performed using the RECIST 1.1 criteria. These analyses revealed that ctDNA dynamics predict outcome significantly earlier than imaging. Patients that responded to therapy had a significant drop in ctDNA early during the treatment course whereas non-responders had either limited changes or significant increase in ctDNA. For patients with acquired resistance, ctDNA kinetics revealed clearing of ctDNA at the time of response followed by a new peak at the time of progression. ctDNA detection early during the treatment course was a significant prognostic factor for progression-free and overall survival (log rank p=.004 and .002 respectively) and ctDNA elimination was superior to tumor mutation burden as a predictor of response to therapy. In parallel, we identified changes in the TCR repertoire that are predictive of outcome: peripheral T cell expansion of a subset of intra-tumoral clones was noted at the time of response whereas there was no evidence of T cell expansion in non-responders. Peripheral T cell expansion of a subset of intra-tumoral clones was noted to peak at the time of response and decrease to baseline levels at the time of resistance for patients with acquired resistance. Our findings suggest that a dynamic assay able to capture the tumor-immune system equilibrium would be superior to conventional analyses of static time points. Such an integrated approach would be highly relevant to tailored cancer immunotherapy strategies. Citation Format: Valsamo Anagnostou, Patrick Forde, Jarushka Naidoo, Kristen Marrone, Vilmos Adleff, James White, Jillian Phallen, Alessandro Leal, Carolyn Hruban, Ashok Sivakumar, Franco Verde, Rachel Karchin, Julie Brahmer, Victor Velculescu. ctDNA and TCR dynamics predict response toimmune checkpoint blockade in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3668.

Abstract: Despite the initial successes of immunotherapy, there is an urgent clinical need for molecular assays that identify patients more likely to respond. Here, we report that ultrasensitive measures of circulating tumor DNA (ctDNA) and T-cell expansion can be used to assess responses to immune checkpoint blockade in metastatic lung cancer patients (N = 24). Patients with clinical response to therapy had a complete reduction in ctDNA levels after initiation of therapy, whereas nonresponders had no significant changes or an increase in ctDNA levels. Patients with initial response followed by acquired resistance to therapy had an initial drop followed by recrudescence in ctDNA levels. Patients without a molecular response had shorter progression-free and overall survival compared with molecular responders [5.2 vs. 14.5 and 8.4 vs. 18.7 months; HR 5.36; 95% confidence interval (CI), 1.57–18.35; P = 0.007 and HR 6.91; 95% CI, 1.37–34.97; P = 0.02, respectively], which was detected on average 8.7 weeks earlier and was more predictive of clinical benefit than CT imaging. Expansion of T cells, measured through increases of T-cell receptor productive frequencies, mirrored ctDNA reduction in response to therapy. We validated this approach in an independent cohort of patients with early-stage non–small cell lung cancer (N = 14), where the therapeutic effect was measured by pathologic assessment of residual tumor after anti-PD1 therapy. Consistent with our initial findings, early ctDNA dynamics predicted pathologic response to immune checkpoint blockade. These analyses provide an approach for rapid determination of therapeutic outcomes for patients treated with immune checkpoint inhibitors and have important implications for the development of personalized immune targeted strategies. Significance: Rapid and sensitive detection of circulating tumor DNA dynamic changes and T-cell expansion can be used to guide immune targeted therapy for patients with lung cancer. See related commentary by Zou and Meyerson, p. 1038

Abstract: Immune checkpoint inhibitors have shown significant therapeutic responses against tumors containing increased mutation-associated neoantigen load. We have examined the evolving landscape of tumor neoantigens during the emergence of acquired resistance in patients with non–small cell lung cancer after initial response to immune checkpoint blockade with anti–PD-1 or anti–PD-1/anti–CTLA-4 antibodies. Analyses of matched pretreatment and resistant tumors identified genomic changes resulting in loss of 7 to 18 putative mutation-associated neoantigens in resistant clones. Peptides generated from the eliminated neoantigens elicited clonal T-cell expansion in autologous T-cell cultures, suggesting that they generated functional immune responses. Neoantigen loss occurred through elimination of tumor subclones or through deletion of chromosomal regions containing truncal alterations, and was associated with changes in T-cell receptor clonality. These analyses provide insight into the dynamics of mutational landscapes during immune checkpoint blockade and have implications for the development of immune therapies that target tumor neoantigens. Significance: Acquired resistance to immune checkpoint therapy is being recognized more commonly. This work demonstrates for the first time that acquired resistance to immune checkpoint blockade 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 neoantigen reactivity may mitigate the development of acquired resistance. Cancer Discov; 7(3); 264–76. ©2017 AACR. See related commentary by Yang, p. 250. This article is highlighted in the In This Issue feature, p. 235