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Predicting in vivo response to antineoplastics remains an elusive challenge. We performed a first-of-kind evaluation of two transcriptome-based precision cancer medicine methodologies to predict tumor sensitivity to a comprehensive repertoire of clinically relevant oncology drugs, whose mechanism of action we experimentally assessed in cognate cell lines. We enrolled patients with histologically distinct, poor-prognosis malignancies who had progressed on multiple therapies, and developed low-passage, patient-derived xenograft models that were used to validate 35 patient-specific drug predictions. Both OncoTarget, which identifies high-affinity inhibitors of individual master regulator (MR) proteins, and OncoTreat, which identifies drugs that invert the transcriptional activity of hyperconnected MR modules, produced highly significant 30-day disease control rates (68% and 91%, respectively). Moreover, of 18 OncoTreat-predicted drugs, 15 induced the predicted MR-module activity inversion in vivo. Predicted drugs significantly outperformed antineoplastic drugs selected as unpredicted controls, suggesting these methods may substantively complement existing precision cancer medicine approaches, as also illustrated by a case study. Significance: Complementary precision cancer medicine paradigms are needed to broaden the clinical benefit realized through genetic profiling and immunotherapy. In this first-in-class application, we introduce two transcriptome-based tumor-agnostic systems biology tools to predict drug response in vivo. OncoTarget and OncoTreat are scalable for the design of basket and umbrella clinical trials.

Anti-PD-1/PD-L1 agents have transformed the treatment landscape of advanced non-small cell lung cancer (NSCLC). To expand our understanding of the molecular features underlying response to checkpoint inhibitors in NSCLC, we describe here the first joint analysis of the Stand Up To Cancer-Mark Foundation cohort, a resource of whole exome and/or RNA sequencing from 393 patients with NSCLC treated with anti-PD-(L)1 therapy, along with matched clinical response annotation. We identify a number of associations between molecular features and outcome, including (1) favorable (for example, ATM altered) and unfavorable (for example, TERT amplified) genomic subgroups, (2) a prominent association between expression of inducible components of the immunoproteasome and response and (3) a dedifferentiated tumor-intrinsic subtype with enhanced response to checkpoint blockade. Taken together, results from this cohort demonstrate the complexity of biological determinants underlying immunotherapy outcomes and reinforce the discovery potential of integrative analysis within large, well-curated, cancer-specific cohorts.

Treatment with immune checkpoint blockade (ICB) often fails to elicit durable antitumor immunity. Recent studies suggest that ICB does not restore potency to terminally dysfunctional T cells, but instead drives proliferation and differentiation of self-renewing progenitor T cells into fresh, effector-like T cells. Antitumor immunity catalyzed by ICB is characterized by mobilization of antitumor T cells in systemic circulation and tumor. To address whether abundance of self-renewing T cells in blood is associated with immunotherapy response, we used flow cytometry of peripheral blood from a cohort of patients with metastatic non–small cell lung cancer (NSCLC) treated with ICB. At baseline, expression of T-cell factor 1 (TCF1), a marker of self-renewing T cells, was detected at higher frequency in effector-memory (CCR7–) CD8+ T cells from patients who experienced durable clinical benefit compared to those with primary resistance to ICB. On-treatment blood samples from patients benefiting from ICB also exhibited a greater frequency of TCF1+CCR7–CD8+ T cells and higher proportions of TCF1 expression in treatment-expanded PD-1+CCR7–CD8+ T cells. The observed correlation of TCF1 frequency in CCR7–CD8+ T cells and response to ICB suggests that broader examination of self-renewing T-cell abundance in blood will determine its potential as a noninvasive, predictive biomarker of response and resistance to immunotherapy.

Immune checkpoint blockade is limited by resistance to treatment, with many patients not achieving durable antitumor responses. Self-renewing (T cell factor 1+ [TCF1+]) CD8+ T cells have recently been implicated in efficacy of anti–programmed cell death protein 1 (anti–PD-1). Mice challenged with syngeneic tumors were treated with anti–PD-1 and/or a reversible inhibitor of PI3K δ, designed to promote T cell self-renewal. Growth of tumors in untreated mice was characterized by waning proportions of TCF1+ T cells, suggesting self-renewing T cells become limiting for successful immunotherapy. Higher proportions of TCF1+ T cells in tumor and blood correlated with better control of tumor growth. Combining anti–PD-1 and inhibitor of PI3K δ conferred superior protection compared with either monotherapy and was associated with higher frequency of TCF1+ T cells in tumor and blood compared with anti–PD-1 alone. These findings reveal predictive importance of self-renewing T cells in anti-tumor immunity and suggest that resistance-directed strategies to enhance T cell self-renewal could potentiate the efficacy of PD-1 blockade.

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Treatment with immune checkpoint blockade (ICB) often fails to elicit durable antitumor immunity. Recent studies suggest that ICB does not restore potency to terminally dysfunctional T cells, but instead drives proliferation and differentiation of self-renewing progenitor T cells into fresh, effector-like T cells. Antitumor immunity catalyzed by ICB is characterized by mobilization of antitumor T cells in systemic circulation and tumor. To address whether abundance of self-renewing T cells in blood is associated with immunotherapy response, we used flow cytometry of peripheral blood from a cohort of patients with metastatic non–small cell lung cancer (NSCLC) treated with ICB. At baseline, expression of T-cell factor 1 (TCF1), a marker of self-renewing T cells, was detected at higher frequency in effector-memory (CCR7–) CD8+ T cells from patients who experienced durable clinical benefit compared to those with primary resistance to ICB. On-treatment blood samples from patients benefiting from ICB also exhibited a greater frequency of TCF1+CCR7–CD8+ T cells and higher proportions of TCF1 expression in treatment-expanded PD-1+CCR7–CD8+ T cells. The observed correlation of TCF1 frequency in CCR7–CD8+ T cells and response to ICB suggests that broader examination of self-renewing T-cell abundance in blood will determine its potential as a noninvasive, predictive biomarker of response and resistance to immunotherapy.

Purpose:Programmed death ligand 1 (PD-L1) is expressed in tumor cells and immune cells, and both have been associated with response to anti-PD-1 axis immunotherapy. Here, we examine the expression of PD-L1 to determine which cell type carries the predictive value of the test.Experimental Design:We measured the expression of PD-L1 in multiple immune cells with two platforms and confocal microscopy on three retrospective Yale NSCLC cohorts (425 nonimmunotherapy-treated cases and 62 pembrolizumab/nivolumab/atezolizumab-treated cases). The PD-L1 level was selectively measured in different immune cell subsets using two multiplexed quantitative immunofluorescence panels, including CD56 for natural killer cells, CD68 for macrophages, and CD8 for cytotoxic T cells.Results:PD-L1 was significantly higher in macrophages in both tumor and stromal compartment compared with other immune cells. Elevated PD-L1 in macrophages was correlated with high PD-L1 level in tumor as well as CD8 and CD68 level (P < 0.0001). High PD-L1 expression in macrophages was correlated with better overall survival (OS; P = 0.036 by cell count/P = 0.019 by molecular colocalization), while high PD-L1 expression in tumor cells was not.Conclusions:In nearly 500 non–small cell lung cancer (NSCLC) cases, the predominant immune cell type that expresses PD-L1 is CD68+ macrophages. The level of PD-L1 in macrophages is significantly associated with the level of PD-L1 in tumor cells and infiltration by CD8+ T cells, suggesting a connection between high PD-L1 and “hot” tumors. In anti-PD-1 axis therapy–treated patients, high levels of PD-L1 expression in macrophages are associated with longer OS and may be responsible for the predictive effect of the marker.

Purpose:To determine the tumor tissue/cell distribution, functional associations, and clinical significance of PD-1, LAG-3, and TIM-3 protein expression in human non–small cell lung cancer (NSCLC).Experimental Design:Using multiplexed quantitative immunofluorescence, we performed localized measurements of CD3, PD-1, LAG-3, and TIM-3 protein in >800 clinically annotated NSCLCs from three independent cohorts represented in tissue microarrays. Associations between the marker's expression and major genomic alterations were studied in The Cancer Genome Atlas NSCLC dataset. Using mass cytometry (CyTOF) analysis of leukocytes collected from 20 resected NSCLCs, we determined the levels, coexpression, and functional profile of PD-1, LAG-3, and TIM-3 expressing immune cells. Finally, we measured the markers in baseline samples from 90 patients with advanced NSCLC treated with PD-1 axis blockers and known response to treatment.Results:PD-1, LAG-3, and TIM-3 were detected in tumor-infiltrating lymphocytes (TIL) from 55%, 41.5%, and 25.3% of NSCLC cases, respectively. These markers showed a prominent association with each other and limited association with major clinicopathologic variables and survival in patients not receiving immunotherapy. Expression of the markers was lower in EGFR-mutated adenocarcinomas and displayed limited association with tumor mutational burden. In single-cell CyTOF analysis, PD-1 and LAG-3 were predominantly localized on T-cell subsets/NKT cells, whereas TIM-3 expression was higher in NK cells and macrophages. Coexpression of PD-1, LAG-3, and TIM-3 was associated with prominent T-cell activation (CD69/CD137), effector function (Granzyme-B), and proliferation (Ki-67), but also with elevated levels of proapoptotic markers (FAS/BIM). LAG-3 and TIM-3 were present in TIL subsets lacking PD-1 expression and showed a distinct functional profile. In baseline samples from 90 patients with advanced NSCLC treated with PD-1 axis blockers, elevated LAG-3 was significantly associated with shorter progression-free survival.Conclusions:PD-1, LAG-3, and TIM-3 have distinct tissue/cell distribution, functional implications, and genomic correlates in human NSCLC. Expression of these immune inhibitory receptors in TILs is associated with prominent activation, but also with a proapoptotic T-cell phenotype. Elevated LAG-3 expression is associated with insensitivity to PD-1 axis blockade, suggesting independence of these immune evasion pathways.

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Single-cell genomics enables dissection of tumor heterogeneity and molecular underpinnings of drug response at an unprecedented resolution1-11. However, broad clinical application of these methods remains challenging, due to several practical and preanalytical challenges that are incompatible with typical clinical care workflows, namely the need for relatively large, fresh tissue inputs. In the present study, we show that multimodal, single-nucleus (sn)RNA/T cell receptor (TCR) sequencing, spatial transcriptomics and whole-genome sequencing (WGS) are feasible from small, frozen tissues that approximate routinely collected clinical specimens (for example, core needle biopsies). Compared with data from sample-matched fresh tissue, we find a similar quality in the biological outputs of snRNA/TCR-seq data, while reducing artifactual signals and compositional biases introduced by fresh tissue processing. Profiling sequentially collected melanoma samples from a patient treated in the KEYNOTE-001 trial12, we resolved cellular, genomic, spatial and clonotype dynamics that represent molecular patterns of heterogeneous intralesional evolution during anti-programmed cell death protein 1 therapy. To demonstrate applicability to banked biospecimens of rare diseases13, we generated a single-cell atlas of uveal melanoma liver metastasis with matched WGS data. These results show that single-cell genomics from archival, clinical specimens is feasible and provides a framework for translating these methods more broadly to the clinical arena.

BackgroundDespite the prominent role of innate immunity in the antitumor response, little is known about the myeloid composition of human non-small cell lung cancer (NSCLC) with respect to histology and molecular subtype. We used multiplexed quantitative immunofluorescence (QIF) to measure the distribution and clinical significance of major myeloid cell subsets in large retrospective NSCLC collections.MethodsWe established a QIF panel to map major myeloid cell subsets in fixed human NSCLC including 4’,6-Diamidino-2-Phenylindole for all cells, pancytokeratin for tumor-epithelial cells, CD68 for M1-like macrophages; and CD11b plus HLA-DR to interrogate mature and immature myeloid cell populations such as myeloid derived suppressor cells (MDSCs). We interrogated 793 NSCLCs represented in four tissue microarray-based cohorts: #1 (Yale, n=379) and #2 (Greece, n=230) with diverse NSCLC subtypes; #3 (Yale, n=138) with molecularly annotated lung adenocarcinomas (ADC); and #4 (Yale, n=46) with patient-matched NSCLC and morphologically-normal lung tissue. We examined associations between marker levels, myeloid cell profiles, clinicopathologic/molecular variables and survival.ResultsThe levels of CD68+ M1 like macrophages were significantly lower and the fraction of CD11b+/HLA-DR− MDSC-like cells was prominently higher in tumor than in matched non-tumor lung tissues. HLA-DR was consistently higher in myeloid cells from tumors with elevated CD68 expression. Stromal CD11b was significantly higher in squamous cell carcinomas (SCC) than in ADC across the cohorts and EGFR-mutated lung ADCs displayed lower CD11b levels than KRAS-mutant tumors. Increased stromal CD68- and HLA-DR-expressing cells was associated with better survival in ADCs from two independent NSCLC cohorts. In SCC, increased stromal CD11b or HLA-DR expression was associated with a trend towards shorter 5-year survival.ConclusionsNSCLCs display an unfavorable myeloid immune contexture relative to non-tumor lung and exhibit distinct myeloid-cell profiles across histologies and presence of major oncogenic driver-mutations. Elevated M1-like stromal proinflammatory myeloid cells are prognostic in lung ADC, but not in SCC.

PURPOSE The routine use of large next-generation sequencing (NGS) pan-cancer panels is required to identify the increasing number of, but often uncommon, actionable alterations to guide therapy. Inconsistent coverage and variable payment is hindering NGS adoption into clinical practice. A review of test utilization, clinical utility, coverage, and reimbursement was conducted in a cohort of patients diagnosed with high-risk cancer who received pan-cancer panel testing as part of their clinical care. MATERIALS AND METHODS The Columbia Combined Cancer Panel (CCCP), a 467-gene panel designed to detect DNA variations in solid and liquid tumors, was performed in the Laboratory of Personalized Genomic Medicine at Columbia University Irving Medical Center. Utilization was characterized at test order. Results were reviewed by a molecular pathologist, followed by a multidisciplinary molecular tumor board where clinical utility was classified by consensus. Reimbursement was reviewed after payers provided final coverage decisions. RESULTS NGS was performed on 359 high-risk tumors from 349 patients. Reimbursement data were available for 246 cases. The most common reason providers ordered CCCP testing was for patients diagnosed with a treatment-resistant or recurrent tumor (n = 214; 61%). Findings were clinically impactful for 229 cases (64%). Molecular alterations that may inform future therapy in the event of progression or relapse were found in 42% of cases, and a targeted therapy was initiated in 23 cases (6.6%). The majority of tests were denied coverage by payers (n = 190; 77%). On average, insurers reimbursed 10.75% of the total NGS service charge. CONCLUSION CCCP testing identified clinically impactful alterations in 64% of cases. Limited coverage and low reimbursement remain barriers, and broader reimbursement policies are needed to adopt pan-cancer NGS testing that benefits patients into clinical practice.

SUMMARYAnti-PD-1/PD-L1 agents have transformed the treatment landscape of advanced non-small cell lung cancer (NSCLC). While our understanding of the biology underlying immune checkpoint blockade in NSCLC is still incomplete, studies to date have established predictive roles for PD-L1 tumor expression and tumor mutational burden (TMB). To expand our understanding of the molecular features underlying response to checkpoint inhibitors in NSCLC, we describe here the first joint analysis of the Stand Up 2 Cancer - Mark Foundation (SU2C-MARK) Cohort, a resource of whole exome and/or RNA sequencing from 393 patients with NSCLC treated with anti-PD-(L)1 therapy, along with matched clinical response annotation. We identify a number of associations between molecular features and outcome, including: 1) favorable (e.g., ATM altered), and unfavorable (e.g., TERT amplified) genomic subgroups, 2) distinct immune infiltration signatures associated with wound healing (unfavorable) and immune activation (favorable), and 3) a novel de-differentiated tumor-intrinsic subtype characterized by expression of endodermal lineage genes, immune activation, and enhanced response rate. Taken together, results from this cohort extend our understanding of NSCLC-specific predictors, providing a rich set of molecular and immunologic hypotheses with which to further our understanding of the biology of checkpoint blockade in NSCLC.

PD-1 is an inhibitory receptor in T cells, and antibodies that block its interaction with ligands augment anti-tumor immune responses. The clinical potential of these agents is limited by the fact that half of all patients develop immune-related adverse events (irAEs). To generate insights into the cellular changes that occur during anti-PD-1 treatment, we performed single-cell RNA sequencing of circulating T cells collected from patients with cancer. Using the K-nearest-neighbor-based network graph-drawing layout, we show the involvement of distinctive genes and subpopulations of T cells. We identify that at baseline, patients with arthritis have fewer CD8 T

Checkpoint inhibitor (CPI) therapies provide limited benefit to patients with tumors of low immune reactivity. T cell-inducing vaccines hold promise to exert long-lasting disease control in combination with CPI therapy. Safety, tolerability and recommended phase 2 dose (RP2D) of an individualized, heterologous chimpanzee adenovirus (ChAd68) and self-amplifying mRNA (samRNA)-based neoantigen vaccine in combination with nivolumab and ipilimumab were assessed as primary endpoints in an ongoing phase 1/2 study in patients with advanced metastatic solid tumors (NCT03639714). The individualized vaccine regimen was safe and well tolerated, with no dose-limiting toxicities. Treatment-related adverse events (TRAEs)10% included pyrexia, fatigue, musculoskeletal and injection site pain and diarrhea. Serious TRAEs included one count each of pyrexia, duodenitis, increased transaminases and hyperthyroidism. The RP2D was 10

Single-cell genomics are enabling technologies, but their broad clinical application remains challenging. We report an easily adaptable approach for single-cell transcriptome and T cell receptor (TCR)-sequencing, and matched whole-genome sequencing from tiny, frozen clinical specimens. We achieve similar quality and biological outputs while reducing artifactual signals compared to data from matched fresh tissue samples. Profiling sequentially collected melanoma samples from the KEYNOTE-001 trial, we resolve cellular, genomic, and clonotype dynamics that encapsulate molecular patterns of tumor evolution during anti-PD-1 therapy. To demonstrate applicability to banked biospecimens of rare diseases, we generate a large uveal melanoma liver metastasis single-cell and matched WGS atlas, which revealed niche-specific impairment of clonal T cell expansion. This study provides a foundational framework for propelling single-cell genomics to the clinical arena.

Background: Hypersialylation (excessive sialoglycans) on tumor cells has been known to be associated with poorer cancer outcomes for more than 40 years. Sialoglycans are immune suppressive and promote tumor immune evasion by binding to sialoglycan receptors (e.g. Siglecs) expressed on immune cells. However, redundant immune cell expression among Siglecs has posed a challenge for receptor-targeting therapeutic approaches. E-602 is a first-in-class fusion protein of engineered human sialidase (Neu2) and a human IgG1 Fc region. The dimeric sialidase moieties of E-602 circumvent the redundancy of this biology by directly cleaving terminal sialic acid residues from sialoglycans on immune and tumor cells. In preclinical studies, E-602 enhanced immune function by augmenting antigen-specific priming and activation of T cells and restoring function of exhausted-like T cells, and E-602 demonstrated antitumor activity as monotherapy in multiple mouse tumor models. Methods: A Phase 1/2 first-in-human dose escalation study is evaluating the safety, pharmacokinetics (PK), pharmacodynamics (PD), and antitumor activity of E-602 in patients with advanced cancers. Eligible patients with select advanced solid tumors were treated with E-602 IV once weekly at dose levels between 1 and 30 mg/kg. Circulating immune cells were analyzed for changes in sialylation and immunophenotyping by flow cytometry. Changes in circulating cytokines were measured by immunoassays. Results: As of January 6, 2023, 32 patients were treated with at least one dose of E-602. The most common tumors treated were colorectal (n=18) and pancreatic (n=6). Doses up to 30 mg/kg were tolerated with no dose-limiting toxicities. The most frequent adverse event was infusion-related reactions which were primarily grade 1-2 and clinically manageable. PK was linear across the evaluated dose range with an estimated T1/2 of 9-24 hours. Dose dependent PD observations included (1) desialylation of peripheral CD8+ T, CD4+ T, and NK cells, remaining detectable in some patients at 7 days post-dose; (2) increases in the immune activation marker, CD69, on peripheral CD8+ T, CD4+ T, and NK cells; and (3) increases in the IFNγ-dependent chemokine CXCL10, TNF-α, and MIP1-β. Thus far, response-evaluable patients have had stable or progressive disease. Additional safety, PK, PD, and clinical outcomes for other patients treated as part of backfill at multiple dose levels will be reported at the meeting. Conclusion: E-602 is tolerated at doses up to 30 mg/kg. Consistent with preclinical findings, dose-dependent desialylation and immune system activation were observed. Based on the observed tolerability and PD effects, the Phase 2 portion of the study to evaluate clinical activity of E-602 monotherapy in patients with checkpoint-inhibitor resistant NSCLC and melanoma will proceed. Citation Format: Jason J. Luke, Melissa Johnson, Anthony Tolcher, Christopher T. Chen, Tong Dai, Brendan D. Curti, Anthony El-Khoueiry, Mario Sznol, Brian S. Henick, Christine Horak, Pushpa Jayaraman, Christopher B. Cole, Dawn Wilson, Lizhi Cao, Li Peng, David Feltquate, Deanne Lathers, Manish R. Sharma. GLIMMER-01: initial results from a phase 1 dose escalation trial of a first-in-class bi-sialidase (E-602) in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT034.

Introduction: Neoadjuvant chemoimmunotherapy is promising in improving outcomes for patients with resectable lung cancer in the phase III setting, though data are immature at this time for overall survival (OS). Here, we present outcomes after 3 years of follow-up from the first reported study of neoadjuvant immunotherapy + chemotherapy in this population. Methods: This open-label, multi-center single-arm investigator-initiated phase II study was conducted at three hospitals in the USA. Neoadjuvant atezolizumab, carboplatin, and nab-paclitaxel were administered for up to 4 cycles prior to surgical resection. The primary endpoint of major pathologic response (MPR) has been previously reported; here we report mature disease-free survival (DFS) and OS as well as clinical characteristics of patients with recurrent brain metastases (BM) with integrated data from tumor genomics, gene expression, and quantitative immunofluorescent (QIF) measurement of immune markers. Results: Of 30 enrolled patients, 29 were taken to the operating room with 26 completing successful R0 resection. 17 patients experienced MPR, of whom 10 had pathological complete response (pCR). The median follow-up time was 39.5 months (95% CI: 30.9-49.4). Median OS was 55.8 months (95% CI: 43.6-NA); median DFS was 34.5 months (95% CI: 18.4-NA). Landmark OS at 12, 24, and 36 months were 97%, 80%, and 77%, respectively; DFS: 80%, 60%, and 49%. Radiographic and pathologic measures of response, as well as baseline PD-L1 levels, did not statistically significantly associate with DFS or OS. The strongest trends were observed with pCR (OS: HR 0.22, 95% CI 0.03-1.83; DFS: HR 0.45, 95% CI 0.12-1.65). Percent decrease of radiographic response correlated inversely with percent pathological regression (Pearson correlation -0.44, 95% CI: -0.71—-0.08). Of 14 patients evaluable for site of recurrence, 6 developed BM. 5/6 patients with BM died, with median time to recurrence of 12.4 months (95% CI: 9.9-32.6) and time to death of 45.3 months (95% CI: 22.7-55.8). 3/6 patients who developed BM experienced MPR (2 achieved pCR). Radiographic response and PD-L1 level did not significantly associate with development of BM. Patients whose tumors had mutations in STK11 and KEAP1 did not experience statistically worse DFS/OS, though incidence of BM trended higher. Reduced copy number of STK11 and KEAP1, which both reside on chromosome 19p, was observed in about 1/3 of tumors. Deletion of either gene was significantly associated with worse pathologic response, lower levels of PD-L1+ cells by QIF, and BM. Conclusions: Mature OS data reveal sustained clinical benefit of neoadjuvant chemoimmunotherapy with atezolizumab. Predictivity of radiographic and pathologic surrogates for survival was limited. Further study is warranted to establish the associations between STK11 and KEAP1 genomic alterations and key clinical outcomes in early-stage NSCLC. Citation Format: Brian S. Henick, Justin F. Gainor, Mark M. Awad, Codruta Chiuzan, Stephanie Izard, Yohanna Georgis, Peter D. Koch, Robert F. Garofano, Cheryl V. Wong, Anjali Saqi, Jessica M. Grindheim, Katja Schulze, Naiyer A. Rizvi, Benjamin Izar, Alison M. Taylor, Catherine A. Shu. 3-year update of neoadjuvant atezolizumab + chemotherapy in patients with resectable non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT217.

Therapeutic vaccines hold promise to broaden the potency of immune checkpoint blockade (ICB) therapy in tumors lacking immune reactivity. A heterologous chimpanzee adenovirus (ChAd68) and self-amplifying mRNA (samRNA)-based individualized neoantigen vaccine regimen in combination with nivolumab 480 mg IV and ipilimumab 30mg SC (NCT03639714) has previously demonstrated safety, durable immunogenicity, and clinical benefit in patients with previously treated metastatic disease. Genomic correlates of response were studied over time in 29 patients (13 MSS-CRC, 13 GEA, 3 NSCLC) to understand novel mechanisms of action. Exome and transcriptome sequencing from archival biopsies was used for neoantigen selection. Monthly circulating tumor DNA (ctDNA) samples were collected for monitoring using a comprehensive tumor-naïve and tumor-informed hybrid-capture based ctDNA assay. Paired pre- and post-vaccine tumor transcriptomes were analyzed for 10 patients with 6 having accompanying DNA T cell receptor Β CDR3 repertoire sequencing (TCRseq) in biopsies and longitudinal PBMCs. Prior to vaccination patient tumors were not enriched for immune infiltration or tumor mutation burden (TMB), median 4.3 mut/Mb (range: 2-17 mut/Mb). Minimal neoantigen and mutation drift was observed with a median of 92.5% of neoantigens (range: 45-100%) and a median of 84% (range: 24-99%) of individual mutations detected in biopsies and ctDNA. Notably, paired pre- and post-vaccine biopsy gene expression analyses show upregulation in gene signatures associated with immune infiltration aligning with evidence of T cell expansion measured by significantly expanding CDR3 clonotypes (p &lt0.01). Longitudinal TCRseq in PBMCs demonstrate vaccine induced TCR repertoire dynamics and expanding and contracting clones observed in tumor biopsies could be monitored throughout treatment. In 4 patients the most drastic TCR repertoire changes were observed at time points measured after a 2nd dose of ChAd68. Lastly, we observe evidence of acquired immune evasion through ctDNA monitoring in two patients each following a year of study treatment. One GEA patient acquired HLA-LOH after remaining stable on treatment and one MSS-CRC with a molecular response (MR) for &gt7 months acquired novel biallelic loss-of-function mutations in TAP1 following 1 year of study treatment. We demonstrate that our neoantigen-directed immunotherapy regimen drives durable immune pressure on the tumor in patients with advanced disease where CPI alone has provided minimal benefit. Further, the evidence of acquired resistance supports the induction of immune pressure on tumors following individualized neoantigen vaccination. Comprehensive ctDNA longitudinal monitoring enables real-time assessment of clinical response and acquired resistance. Citation Format: Desiree Schenk, Rita Zhou, Olivia Petrillo, Alexis Mantilla, Italo Faria do Valle, Steven Maron, Brian S. Henick, Chih-Yi Liao, Daniel V.T. Catenacci, Sameek Roychowdhury, Benjamin Solomon, Alexander I. Spira, Ankur Dhanik, Andrew R. Fergusson, Karin Jooss, Matthew Davis. Disease monitoring with comprehensive genomics provides evidence of mechanism of action and immune evasion in patients receiving an individualized neoantigen cancer vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1126.

PURPOSE: Neoadjuvant PD-1 blockade is a promising treatment for resectable non-small cell lung cancer (NSCLC), yet immunological mechanisms contributing to tumor regression and biomarkers of response are unknown. Using paired tumor/blood samples from a phase 2 clinical trial (NCT02259621), we explored whether the peripheral T cell clonotypic dynamics can serve as a biomarker for response to neoadjuvant PD-1 blockade. EXPERIMENTAL DESIGN: T cell receptor (TCR) sequencing was performed on serial peripheral blood, tumor and normal lung samples from resectable NSCLC patients treated with neoadjuvant PD-1 blockade. We explored the temporal dynamics of the T cell repertoire in the peripheral and tumoral compartments in response to neoadjuvant PD-1 blockade by using the TCR as a molecular barcode. RESULTS: Higher intratumoral TCR clonality was associated with reduced percent residual tumor at the time of surgery, and the TCR repertoire of tumors with major pathologic response (MPR

Neoantigen directed immunotherapy holds promise to increase the likelihood of patients with solid tumor devoid of immune infiltration benefiting from immune checkpoint immunotherapy (CPI). A heterologous prime-boost vaccination approach consisting of Chimpanzee Adenovirus (ChAd) prime and multiple self-amplifying mRNA (SAM) boosts, delivering 20 neoantigens, has been evaluated in a Phase 1/2 clinical trial in late-stage solid tumor patients in combination with nivolumab and ipilimumab (NCT03639714). Neoantigen dynamics, tumor burden and genomic correlates of response were studied over time in 20 patients (8 GEA, 2 NSCLC, 10 MSS-CRC). Exome sequences from archival (sample used for neoantigen selection), baseline (start of immunization) and on-treatment biopsies were analyzed for 20, 16 and 10 patients respectively. Paired pre- and post-vaccine tumor transcriptomes were analyzed for 6 patients. Personalized capture baits were designed for all non-synonymous mutations detected in archival biopsies (mean 146; range: 67-402) for ctDNA monitoring. Longitudinal ctDNA samples were collected monthly on treatment (mean 7; range: 1-18). ctDNA duplex UMI libraries were captured and sequenced to a target mean raw depth >80,000x and reduced to 3x per strand consensus duplex reads. The majority of vaccine neoantigens were detected in ctDNA (87%; range 45%-100%) and mean neoantigen variant allele frequency (VAF) strongly correlated with all monitored mutations VAF (R2 = 0.90, p < 0.0001) through treatment. The percentage of vaccine neoantigens detected was higher than that for all monitored mutations in the same samples with a median of 80% (21%-98%) in ctDNA and 70% (44%-100%) in biopsies. Five of 9 MSS-CRC patients with measurable baseline ctDNA achieved molecular responses (MR, >50% reduction in ctDNA from baseline) that correlated with OS and PFS, and in some patients, was accompanied by radiologic tumor shrinkage. One MSS-CRC patient with MR for >7 months acquired novel biallelic loss-of-function mutations in TAP1 following 1 year of study treatment. Differential gene expression analysis from paired pre- and post-vaccine biopsies (including 2 MSS-CRC pairs with MR) demonstrated significant upregulation in gene signatures associated with immune-inflamed tumor microenvironments including interferon alpha and gamma responses. We demonstrate that tumor-informed neoantigen selection and vaccine manufacturing while patients receive chemotherapy is feasible, since the majority of neoantigens are retained in the tumor post-chemotherapy. Further, neoantigen-directed immunotherapy appears to drive clinical benefit in patients with advanced MSS-CRC tumors, where CPI alone has provided minimal benefit. Comprehensive ctDNA longitudinal monitoring enables real time assessment of clinical response and acquired resistance. Citation Format: Desiree Schenck, Rita Zhou, Alexis Mantilla, Oliver Spiro, Taylor Patch, Adrienne Johnson, Daniel Navarro Gomez, Brian S. Henick, Chih-Yi Liao, Sameek Roychowdhury, Steve Maron, Benjamin Solomon, Alexander I. Spira, Daniel V. Catenacci, Andrew R. Fergusson, Raphael F. Rousseau, Karin Jooss, Matthew J. Davis. Comprehensive ctDNA monitoring provides early signal of clinical benefit with a novel personalized neoantigen directed immunotherapy for late-stage cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1238.

Recent studies have suggested that ICB does not primarily restore function to terminally exhausted T cells, but instead drives proliferation and differentiation of progenitor T cells into fresh effector cells. T cell factor 1 (TCF1) marks and maintains T cell self-renewal while repressing differentiation and has been associated with increased antitumor activity. These findings suggest ICB may depend on the continued availability of self-renewing TCF1+ progenitor T cells as the source of fresh effector cells. We hypothesized that characterizing the immunophenotype of T cells in the peripheral blood, with a focus on markers of self-renewal, might be predictive of ICB response. Utilizing an institution-wide biospecimen repository, we evaluated peripheral blood samples using multi-parametric flow cytometry from patients with metastatic NSCLC treated with single-agent ICB who experienced durable clinical benefit (DCB; >1 year of treatment and/or clinical response >1 year) or primary resistance (RES; Citation Format: Rohan Maniar, Peter H. Wang, Robert S. Washburn, Naiyer A. Rizvi, Steven L. Reiner, Brian S. Henick. Peripherally measured T cell self-renewal capacity associates with response to immune checkpoint blockade (ICB) in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1240.

e20593 Background: Epidermal growth factor receptor-mutated [ EGFR(+)] NSCLC has historically been associated with young, non-smoking patients, especially those of Asian descent. Our medical center serves a diverse population within New York City and includes patients from neighborhoods that are predominantly Hispanic/Latino. We therefore sought to describe the population of patients with EGFR(+) NSCLC treated at our cancer center. Methods: We used our single institution lung cancer database to identify 2092 patients diagnosed with NSCLC between 2013-2019; of these, 375 had EGFR(+) disease. We collected retrospective data on patient demographics and disease outcomes. The chi-square test and t-tests were performed to compare between subgroups of patients. Results: Between 2013-2019, 17.9% of patients diagnosed with NSCLC at our center had an identified EGFR mutation. Incidence of EGFR mutations was higher in Asian NSCLC patients (46.7%) compared to Black (13.1%), white (16.8%), and Hispanic (16.0%) NSCLC patients. Within the 375 patients diagnosed with EGFR(+) NSCLC, however, the distribution was 62.1% white (233 patients), 13.6% Asian (51 patients), 11.2% Hispanic (42 patients), 8.3% Black (31 patients), and 4.8% unknown/other race (18 patients). Over half (57.7%) of patients with EGFR(+) NSCLC had a history of smoking, and most (68.9%) were female. Median age at diagnosis was 71 years. Frequency of tyrosine kinase inhibitor (TKI)-sensitive mutations (L858R and exon 19 deletion) was 85.7% in Hispanic patients, 91.7% in Asian patients, 76.4% in white patients, and 71.0% in Black patients. Significantly more Hispanic EGFR(+) patients were diagnosed at Stage IV (65.4%) compared to 40.4% of Asian patients, 36.7% of Black patients, and 25.7% of white patients (p < 0.02 for all comparisons). Among patients diagnosed with Stage IV disease, Hispanic patients had worse average survival compared to non-Hispanic patients (19.4 months vs. 27.9 months, p = 0.01). Conclusions: EGFR-mutant NSCLC is thought to be especially common among patients who are younger, Asian, and/or never smokers. Our population of EGFR(+) NSCLC, however, encompasses a racially diverse group of patients, most of whom were older at the time of diagnosis and many of whom had a history of smoking. This population of patients, most of whom harbor a TKI-sensitive mutation, supports the use of routine mutational testing that is agnostic to patient demographics. Our data also suggest that Hispanic patients in particular are diagnosed with more advanced disease and have shorter survival; the reasons for such disparities within the EGFR(+) NSCLC population warrant further study.

2545 Background: Pneumonitis is one of the most morbid complications from immune checkpoint inhibitor (ICI) treatment, but pathogenic mechanisms are unclear and no biomarkers permit pre-treatment risk assessment. We sought to characterize peripheral T cell subsets of pneumonitis patients on the single cell level. Methods: Blood was collected before and during ICI treatment in 24 patients. Cells were processed for single cell RNA sequencing (scRNAseq) employing CITEseq methodology using multiplexed cell surface markers labelled with a cocktail of oligonucleotide-tagged Total-Seq anti-human antibodies against CD4, CD8, CD45RA and CD27 followed by Chromium 10X sequencing. Principal Component Analysis was performed with iCellR, K-nearest-neighbor-based Network graph drawing Layout, and PhenoGraph clustering to assign cell types. CT scans were performed per standard of care and were reviewed by an experienced thoracic radiologist. Results: Seven of 24 patients developed pneumonitis; 9 did not experience an immune-related adverse event, and the remainder experienced arthritis (4), thyroiditis (3), or neurotoxicity (1). Pneumonitis patients had expanded proportions of TH2 TCF7+ T cells at baseline as compared to the other patients. Radiographically, two patients’ pneumonitis manifested as Chronic Hypersensitivity Pneumonitis (CHP), and four had Organized Pneumonia (OP). At baseline, CHP patients had significantly lower levels of CD8+ TCM cells (CXCR3+), double-positive T cells, gamma-delta T cells, and higher levels of naïve-like CD4+ TN TCF7+LEF1+ and CD4+ TH1/2 CXCR3+GATA3+ cells compared to OP. Gene expression levels also distinguished between these radiographic phenotypes. Conclusions: The peripheral T cell composition of patients who developed pneumonitis was distinct from those who did not in our cohort and unique by radiographic manifestation, suggesting potential pathogenic mechanisms and a prelude to circulating predictive markers of ICI toxicity.

e21178 Background: ICIs are cornerstones of therapy for advanced NSCLC. Despite dramatic and sometimes durable responses to therapy, most patients (pts) either (i) do not respond to therapy (intrinsic resistance), or (ii) subsequently progress after initial clinical benefit (acquired resistance). Currently, insights into the molecular mechanisms of resistance to ICIs in NSCLC are lacking. Methods: To investigate clinical and molecular features of pts progressing on ICIs, we identified pts who underwent repeat tumor biopsies on and/or after disease progression on ICIs and were included in the Stand Up 2 Cancer (SU2C)/Mark Foundation multi-institutional cohort. Biopsy specimens underwent whole-exome sequencing (WES) and/or whole transcriptome sequencing (RNAseq). Results: We identified 37 pts who underwent a total of 47 repeat biopsies on or after ICIs. Six pts underwent multiple post-ICI biopsies (range 2-4). Twenty-five pts (68%) received PD-(L)1 inhibitor monotherapy, 6 (16%) received PD-(L)1 plus CTLA-4 inhibitors, and 6 (16%) received other PD-1 inhibitor-based combinations. Overall, the objective response rate was 46% among pts undergoing repeat biopsies (complete response 2 [5%], partial response 15 [41%], stable disease 14 [38%], progressive disease 5 [14%] and not evaluable 1 [3%]). Median progression-free survival (PFS) was 8.1 months. In total, pre-ICI biopsy specimens were available in 20 pts. WES and RNAseq were performed on 67 and 44 specimens, respectively. Median tumor mutation burden (TMB) in pre-ICI specimens was 5.0 mutations/Mb versus 4.9 mutations/Mb in post-ICI specimens ( p= 0.3, Mann-Whitney U test). Among 20 paired pre/post-ICI specimens, there was no significant difference in TMB (pre-treatment median 3.9 mutations/Mb; post-treatment median 4.3 mutations/Mb; p= 0.7, Wilcoxon signed-rank test). One pt with a complete response acquired a nonsense mutation in B2M, and one pt with a partial response acquired a nonsense mutation in JAK1. Among 10 paired pre/post-ICI specimens that underwent RNAseq, we observed significant decreases in granzyme B and perforin in post-ICI specimens ( p= 4×10-5 and p= 2×10-3, respectively, limma-voom analysis). Conclusions: Genomic alterations impairing antigen presentation (e.g., B2M) or immune activation (e.g., JAK1) may enable resistance to ICIs in a small subset of cases. However, the majority of repeat biopsies obtained from pts progressing on ICIs lacked clear genetic mediators of resistance, suggesting the presence of additional tumor-intrinsic and/or tumor-extrinsic factors underlying resistance to ICIs in NSCLC.

Introduction: The development of immune-related adverse events (irAEs) has been associated with improved efficacy of immune checkpoint inhibitors in patients with urothelial cancer, melanoma, and NSCLC. Whether this association exists in patients with SCLC is currently unknown. Methods: We conducted a multicenter retrospective study to evaluate the relationship between irAEs and immunotherapy efficacy in SCLC. To account for the lead-time bias resulting from the time-dependent nature of irAEs, the development of irAEs was considered as a time-varying covariate in univariate and multivariate Cox proportional hazard models. Results: Of the 183 patients treated with immunotherapy, 73 (39.9%) experienced at least one irAE. A total of 42 patients (22.9%) had grade 1 to 2 irAEs, whereas 31 patients (16.9%) had grade 3 to 4 irAEs. The median time of onset to the first irAE was 24 days (interquartile range: 14–55). The baseline clinicopathologic features were well-balanced between patients with and without irAEs. At a median follow-up of 24 months (95% confidence interval [CI]: 17.0–31.6), the median progression-free survival was significantly longer in the irAE group than the non-irAE group (3.8 versus 1.3 mo, p < 0.0001). The median overall survival was also significantly longer among patients with irAEs than patients without irAEs (13.8 versus 2.9 mo, p < 0.0001). When analyzed as a time-varying covariate, the development of irAEs was associated with a significant improvement in progression-free survival (hazard ratio: 0.44 [95% CI: 0.29–0.66], p < 0.001) and overall survival (hazard ratio: 0.47 [95% CI: 0.32–0.71], p < 0.001) in multivariate models. Conclusions: The development of irAEs is associated with improved clinical outcomes for immunotherapy in patients with advanced SCLC.

Purpose: To analyze the distribution, associated immune contexture, and clinical significance of human leukocyte antigen (HLA) class-I and HLA class-II subunits in non–small cell lung cancer (NSCLC). Experimental Design: Using spatially resolved and quantitative multiplexed immunofluorescence we studied the tumor/stromal tissue distribution, cancer cell–specific defects, and clinicopathologic/survival associations of β2 microglobulin (β2M), HLA-A, and HLA-B,-C heavy chains, as well as HLA class-II β chain in >700 immunotherapy-naïve NSCLCs from four independent cohorts. Genomic analysis of HLA genes in NSCLC was performed using two publicly available cohorts. Results: Cancer cell–specific downregulation of HLA markers was identified in 30.4% of cases. β2M was downregulated in 9.8% (70/714), HLA-A in 9% (65/722), HLA-B,-C in 12.1% (87/719), and HLA class-II in 17.7% (127/717) of evaluable samples. Concurrent downregulation of β2M, HLA-B,-C, and HLA class-II was commonly identified. Deleterious mutations in HLA genes were detected in Conclusions: Our results reveal frequent and differential defects in HLA class-I and HLA class-II protein subunit expression in immunotherapy-naïve NSCLCs associated with distinct tumor microenvironment composition and patient survival.

INTRODUCTION: CMTM6 has been described as a PD-L1 regulator at the protein level by modulating stability via ubiquitination. In this study, we describe the patterns of CMTM6 expression and assess its association with response to PD-1 pathway blockade in non-small-cell lung cancer (NSCLC). METHODS: We used multiplexed quantitative immunofluorescence to determine the expression of CMTM6 and PD-L1 in 438 NSCLCs represented in tissue microarrays, including two independent retrospective cohorts of immunotherapy treated (n = 69) and untreated (n = 258) patients, and a third collection of EGFR and KRAS genotyped tumors (n = 111). RESULTS: Tumor and stromal CMTM6 expression was detected in approximately 70 % of NSCLCs. CMTM6 expression was not associated with clinical features or EGFR/KRAS mutational status and showed a modest correlation with T-cell infiltration (R(2) < 0.40). We found a significant correlation between CMTM6 and PD-L1, higher in the stroma (R(2) = 0.51) than tumor cells (R(2) = 0.35). In our retrospective NSCLC cohort, neither CMTM6 nor PD-L1 expression alone significantly predicted immunotherapy outcomes. However, high CMTM6 and PD-L1 co-expression in the stroma and CD68 compartments (adjusted HR 0.38, p = 0.03), but not in tumor cells (p = 0.15), was significantly associated with longer OS in treated patients, but not observed in the absence of immunotherapy. CONCLUSION: This study supports the mechanistic role for CMTM6 in stabilization of PD-L1 in patient tumors and suggests that high co-expression of CMTM6 and PD-L1, particularly in stromal immune-cells (macrophages), might identify the greatest benefit from PD-1 axis blockade in NSCLC.

Purpose: Programmed death ligand 1 (PD-L1) is expressed in tumor cells and immune cells, and both have been associated with response to anti-PD-1 axis immunotherapy. Here, we examine the expression of PD-L1 to determine which cell type carries the predictive value of the test. Experimental Design: We measured the expression of PD-L1 in multiple immune cells with two platforms and confocal microscopy on three retrospective Yale NSCLC cohorts (425 nonimmunotherapy-treated cases and 62 pembrolizumab/nivolumab/atezolizumab-treated cases). The PD-L1 level was selectively measured in different immune cell subsets using two multiplexed quantitative immunofluorescence panels, including CD56 for natural killer cells, CD68 for macrophages, and CD8 for cytotoxic T cells. Results: PD-L1 was significantly higher in macrophages in both tumor and stromal compartment compared with other immune cells. Elevated PD-L1 in macrophages was correlated with high PD-L1 level in tumor as well as CD8 and CD68 level (P < 0.0001). High PD-L1 expression in macrophages was correlated with better overall survival (OS; P = 0.036 by cell count/P = 0.019 by molecular colocalization), while high PD-L1 expression in tumor cells was not. Conclusions: In nearly 500 non–small cell lung cancer (NSCLC) cases, the predominant immune cell type that expresses PD-L1 is CD68+ macrophages. The level of PD-L1 in macrophages is significantly associated with the level of PD-L1 in tumor cells and infiltration by CD8+ T cells, suggesting a connection between high PD-L1 and “hot” tumors. In anti-PD-1 axis therapy–treated patients, high levels of PD-L1 expression in macrophages are associated with longer OS and may be responsible for the predictive effect of the marker.

The advent of PD-1/PD-L1 agents has transformed the therapeutic landscape of many advanced cancers, including non-small cell lung cancer (NSCLC). However, our understanding of the genomic biomarkers underlying effective treatment response remain limited. Here we provide updated results from our ongoing effort, the Stand Up To Cancer Lung (SU2C-Lung)/Mark Foundation EXTOLConsortium, a multi-institution collaboration to expand our understanding of the molecular determinants of immunotherapy response in NSCLC. Comprising a set of nearly 400 patients, this cohort enables evaluation of both genomic and transcriptomic factors associated with checkpoint blockade response. In addition to validating previously known associations including TMB and neoantigen burden, we examined transcriptional predictors associated with response. We generated a list of differentially expressed genes with respect to best overall response (BOR), and performed dimensionality reduction using semi-supervised Bayesian Non-Negative Factorization (ssBNMF). We identified 3 distinct clusters with strong sample membership. Characterization of these subtypes revealed varying levels of immune infiltrate, histologic composition, and response rates to checkpoint blockade. Of these three subtypes, two were associated with low response rates to PD-1/PD-L1 blockade, suggesting the existence of distinct avenues toward resistance. To further characterize these transcriptional subtypes, we used ssBNMF marker genes to classify publicly available NSCLC samples from The Cancer Genome Atlas (TCGA), along with gene expression from a smaller cohort of large-cell neuroendocrine (LCNE) samples. Histologic composition showed good concordance with our SU2C samples, and redemonstrated observations within our smaller SU2C cohort of distinct immuno-suppressive and immuno-depleted milieus associated with resistance. Citation Format: Monica B. Arniella, Arvind Ravi, Justin Gainor, Chip Stewart, Sam Freeman, Mark Awad, Patrick Forde, Valsamo Anagnostou, Brian Henick, Jonathan W. Riess, Don Gibbons, Nathan Pennell, Vamsidhar Velcheti, Ignaty Leshchiner, Jaegil Kim, Subba Digumarthy, Mari Mino-Kenudson, John Heymach, Nir Hacohen, Naiyer Rizvi, Roy Herbst, Victor E. Velculescu, Julie Brahmer, Kurt Schalper, Pasi Jänne, Jedd Wolchok, Alice Shaw, Gad Getz, Matthew D. Hellman. An SU2C-Mark Foundation Lung collaborative update: integrative genomics identifies distinct transcriptional states associated with checkpoint blockade resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB197.

Introduction EGFR is a therapeutic target in NSCLC for EGFR-mutant patients. Proximity ligation assay (PLA) is a method to detect functional signaling associated protein complexes. Growth factor receptor bound protein 2 (GRB2) is an adaptor protein that binds to the phosphorylated residues of active EGFR. Interaction of EGFR and GRB2 correlates with active EGFR signaling and leads to activation of the MAPK/ERK pathway. Methods A PLA developed to detect EGFR-GRB2 interaction was measured by quantitative immunofluorescence using Automated Quantitative Analysis technology. EGFR pathway activation was assessed in patients with NSCLC with different mutation status along with overall EGFR expression. Additionally, the PLA to detect EGFR-GRB2 interaction was evaluated as a prognostic marker in two cohorts of patients with lung adenocarcinoma. Results The PLA to detect EGFR-GRB2 interaction was unrelated to overall EGFR expression or mutation in a series of patients with NSCLC with known mutation status. EGFR-mutant ( p = 0.04) and EGFR/KRAS wild-type tumors ( p = 0.0049) had significantly higher EGFR pathway activation compared with KRAS-mutant cases, with no significant difference shown between mutation sites. In two series of patients with lung adenocarcinoma, the PLA to detect EGFR-GRB2 interaction was independently associated with longer survival (hazard ratio = 0.46, 95% confidence interval: 0.2–0.78, p = 0.0085 and hazard ratio = 0.48, 95% confidence interval: 0.2–0.85, p = 0.017). Total EGFR protein expression alone was not correlated with outcome. Conclusions EGFR colocalization with GRB2 as assessed by PLA is not correlated with EGFR expression levels or mutation status, defining a patient group that may show EGFR pathway activation, as illustrated by its prognostic value. Future studies may determine whether this group is more likely to respond to EGFR-targeted therapies.

PD-1/PD-L1 checkpoint blockade has been a landmark advance for many patients suffering from advanced non-small cell lung cancer (NSCLC). However, detailed biomarkers of response beyond tumor mutational burden (TMB) are still poorly understood. As part of the effort to elucidate these additional signatures, we describe our progress on the Stand Up to Cancer Lung (SU2C-Lung) cohort. Initial characterization of exomes recapitulates mutational and copy number profiles seen in The Cancer Genome Atlas (TCGA) project. To better define expression subtypes using RNA sequencing, we performed non-negative matrix factorization (NMF) across an aggregated set of publicly available NSCLC expression data (including adenocarcinoma, squamous cell carcinoma, and large cell neuroendocrine histologies), and demonstrate good concordance in the SU2C-Lung cohort between this expression-based classifier and clinically annotated histology. To gain further insight into how immune cell infiltrates vary across our cohort, we additionally tested two common deconvolution algorithms, EPIC and CIBERSORT. While these two methods agree for some prominent cell types, such as B cells and CD4 T cells, discrepancies in minor infiltrating components such as NK cells may suggest a limit to the inference of rare subpopulations from bulk sequencing data.Finally, we describe a novel approach for determining single-gene predictors of response. Using the method, which is based on comparison of top single transcriptional features identified from random bootstraps of the full cohort as compared to a set of background shuffles, we are able to show that we remain powered for discovery of RNA response biomarkers despite the typically burdensome toll of multiple hypothesis correction at genome wide scale. Acknowledgment: Supported by Stand Up To Cancer-American Cancer Society Lung Cancer Dream Team Translational Research Grant SU2C-AACR-DT17-15. Citation Format: Monica Arniella, Arvind Ravi, Chip Stewart, Sam Freeman, Mark Awad, Patrick Forde, Valsamo Anagnostou, Brian Henick, Jonathan Riess, Don Gibbons, Nathan Pennell, Vamsidhar Velcheti, Ignaty Leshchiner, Jaegil Kim, Subba Digumarthy, Mari Mino-Kenudson, John Heymach, Nir Hacohen, Naiyer Rizvi, Roy Herbst, Victor Velculescu, Julie Brahmer, Kurt Schalper, Pasi Jänne, Jedd Wolchok, Alice Shaw, Justin Gainor, Matthew Hellmann, Gad Getz. Integrative genomic analysis of checkpoint blockade in lung cancer: A multi-institution SU2C collaborative [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5902.

Background: Neoantigens arising from somatic mutations are attractive targets for cancer immunotherapy as they may be recognized as foreign by the immune system. RO7198457, a systemically administered RNA-Lipoplex iNeST was designed to stimulate T cell responses against neoantigens. A first-in-human Phase Ib study of RO7198457, in combination with the aPD-L1 antibody atezolizumab is being conducted in patients with locally advanced or metastatic solid tumors. Methods: RO7198457 is manufactured on a per-patient basis and contains up to 20 tumor-specific neoepitopes. Nine doses of RO7198457 were administered i.v. in weekly and bi-weekly intervals during the 12-week induction stage and every 24 weeks during the maintenance stage. Atezolizumab 1200 mg was administered on Day 1 of each 21-day cycle. Results: In total, 132 patients enrolled in cohorts with doses ranging from 15-50 μg RO7198457 in combination with atezolizumab. Most common tumor types were NSCLC, TNBC, melanoma and CRC. The median number of prior therapies was 3 (range 1-11). 39% of patients received prior immunotherapy. Most patients had low levels of PD-L1 expression (93% patients with Citation Format: Juanita S. Lopez, Ross Camidge, Marco Iafolla, Sylvie Rottey, Martin Schuler, Matthew Hellmann, Ani Balmanoukian, Luc Dirix, Michael Gordon, Ryan Sullivan, Brian S. Henick, Charles Drake, Kit Wong, Patricia LoRusso, Patrick Ott, Lawrence Fong, Aglaia Schiza, Jeffery Yachnin, Christian Ottensmeier, Fadi Braiteh, Johanna Bendell, Rom Leidner, George Fisher, Guy Jerusalem, Laura Molenaar-Kuijsten, Marcus Schmidt, Scott A. Laurie, Raid Aljumaily, Achim Rittmeyer, Eelke Gort, Ignacio Melero, Lars Mueller, Rachel Sabado, Patrick Twomey, Jack Huang, Manesh Yadav, Jingbin Zhang, Felicitas Mueller, Evelyna Derhovanessian, Ugur Sahin, Özlem Türeci, Thomas Powles. A phase Ib study to evaluate RO7198457, an individualized Neoantigen Specific immunoTherapy (iNeST), in combination with atezolizumab in patients with locally advanced or metastatic solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT301.

Introduction. VISTA/PD-1H is an Ig domain-containing type I transmembrane protein able to suppress T-cell activation and is being evaluated as a candidate anti-cancer immunotherapy target. VISTA can bind P-selectin glycoprotein ligand-1 (PSGL1) and V-set and immunoglobulin domain containing 3 (VSIG3) suggesting these interactions could mediate VISTA's immunomodulatory effect. We studied the tissue distribution of VISTA, PSGL1 and VSIG3 in human non-small cell lung cancer (NSCLC). Methods We used multiplexed quantitative immunofluorescence (QIF) to simultaneously measure DAPI, cytokeratin (CK), VISTA, PSGL1 and VSIG3 in 48 paired tumor/normal lung samples and in 850 stage I-IV NSCLCs from four independent cohorts represented in tissue microarray format. The first 2 NSCLC cohorts (Cohorts #1, n=382 and #2, n=282) included cases treated with standard of care non-immunotherapy. Cohort #3 included 137 lung adenocarcinomas with analysis of mutant oncogenic drivers; and Cohort #4 included 49 NSCLC cases treated with PD-1 axis blockers. The targets were selectively measured in CK+ tumor cells and CK-negative stromal cells. We also determined the spatial association between VISTA and its binding partners by fluorescence signal co-localization. The association between target levels, clinicopathologic/molecular variables, tumor infiltrating lymphocytes (TILs), PD-L1 expression and survival was established. Results PSGL1 was located predominantly in stromal/immune cells and VSIG3 was detected in both tumor and stromal compartments. Both targets were expressed at higher levels in NSCLC than in non-tumor lung tissue and showed a positive association with VISTA in cancer tissues. Using the visual detection threshold PSGL1 and VSIG3 expression were detected in >90% of cases from cohorts #1 and #2 and showed positive association with CD3+ TILs and PD-L1 levels. PSGL1 was higher in lung adenocarcinomas harboring EGFR mutations than in tumors with KRAS variants or cases lacking mutations in both oncogenes. Elevated VISTA/PSGL1 co-localization was significantly associated with longer 5-year overall survival in cases not treated with immunostimulatory therapy (Cohorts #1 and #2). However, an opposite association was seen in cases treated with PD-1 axis blockers, where elevated VISTA/PSGL1 co-expression was associated with shorter survival. Conclusions PSGL1 and VSIG3 are frequently expressed in human NSCLC. Expression of PSGL1 is associated with increased tumor immune infiltration and activating EGFR mutations. High baseline VISTA/PSGL1 co-expression is associated with adverse outcome after PD-1 axis blockers. The latter suggest VISTA/PSGL1 as a dominant immune evasion pathway independent from PD-1/PD-L1 axis in a subset of human NSCLC. Validation of these findings is ongoing. Citation Format: Alicia Ding, Franz Villarroel-Espindola, Adam Ducler, Brian S. Henick, Shruti Desai, Nicole Gianino, Jon Zugazagoitia, David L. Rimm, Alain Robert, Francisco Cruzalegui, Pierre Ferré, Roy Herbst, Miguel Sanmamed, Lieping Chen, Kurt A. Schalper. VISTA/PSGL1 axis as a dominant immunomodulatory pathway in human non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5525.

Background The development of immune-related adverse events (irAEs) has been associated with improved efficacy of immune checkpoint inhibitors in patients with urothelial cancer, melanoma, and non-small cell lung cancer. Whether this association exists in patients with small cell lung cancer (SCLC) is currently unknown. Methods We conducted a multicenter retrospective study to evaluate the relationship between irAEs and immunotherapy efficacy in SCLC. To account for the lead-time bias resulting from the time-dependent nature of irAEs, the development of irAEs was considered as a time-varying covariate in univariate and multivariate Cox proportional hazard models. Results Among 183 patients treated with immunotherapy, 73 (39.9%) experienced at least one irAE. Forty-two (22.9%) patients had grade 1-2 irAEs, while 31 patients (16.9%) had grade 3-4 irAEs. The median time of onset to the first irAE was 24 days (IQR: 14-55). Baseline clinicopathologic features were well balanced between patients with and without irAEs. At a median follow-up of 24 months (95%CI:17.0-31.6), the median progression-free survival (mPFS) was significantly longer in the irAE group compared to the non-irAE group (3.8 versus 1.3 months, P Conclusions The development of irAEs is associated with improved clinical outcomes to immunotherapy in patients with advanced SCLC.

3137 Background: Neoantigens are key targets of a tumor-specific immune response and CD8 T cells targeting neoantigens drive clinical benefit in patients (pts) treated with checkpoint inhibitors. Methods: Two Phase I studies are being conducted to assess the safety, immunogenicity, and early clinical activity of a viral-based neoantigen-targeting prime/boost immunotherapy aimed at maximizing the CD8 T cell response. Both studies use a chimpanzee adenovirus prime followed by increasing doses of repeat boosts with a self-amplifying mRNA in combination with IV nivolumab +/- SC ipilimumab. In the first study, GO-004, patient-specific neoantigens are predicted using Gritstone's EDGE model and incorporated into both prime/boost vectors. In GO-005, shared neoantigens derived from common driver mutations (including several from KRAS) are encoded in off-the-shelf prime/boost vectors. Results: To date, 12 pts have been treated: 6 pts with GEA, NSCLC, or MSS-CRC (GO-004) and 6 pts with NSCLC, MSS-CRC, or PDA (GO-005) with all pts receiving IV nivolumab and 5 pts also receiving SC ipilimumab. Nine pts continue to receive study treatment. No DLTs have been observed. Treatment-related AEs are reversible and include Grade 1/2 fever (7/12), injection site reactions (4/12), fatigue (3/12), diarrhea (2/12), hypotension (2/12), pruritus (2/12), skin reactions (2/12), anorexia (1/12), dyspnea (1/12), hyponatremia (1/12), infusion-related reactions (1/12), myalgia (1/12), and asymptomatic Grade 3 CK elevation (1/12). At the time of analysis, 8 of 12 pts with ≥ 1 radiographic assessment have a best response of stable disease (SD) (3) and progressive disease (PD) (4), and one pt with no evaluable disease at baseline continues on study > 8 months. In GO-005, 1 pt with SD has a 20% reduction in tumor dimensions that correlates with a decrease in ctDNA. In 4 pts in GO-004 analyzed to date, all pts showed substantial neoantigen-specific CD8 T cell responses to multiple neoantigens after priming which increase further in 2 of 3 pts analyzed after subsequent boosts. In GO-005, 1 of 3 pts showed a robust KRAS G12C-specific CD8 T cell response. Induced T cells express IFNg and granzyme B, consistent with an effector response. Conclusions: Taken together, these early data support the tolerability of a viral-based prime/boost immunotherapy, demonstrate marked immunogenicity, and are consistent with potential clinical activity. Additional pts and data at higher dose levels will be presented. Clinical trial information: NCT03639714, NCT03953235 .

Purpose: To determine the tumor tissue/cell distribution, functional associations, and clinical significance of PD-1, LAG-3, and TIM-3 protein expression in human non–small cell lung cancer (NSCLC). Experimental Design: Using multiplexed quantitative immunofluorescence, we performed localized measurements of CD3, PD-1, LAG-3, and TIM-3 protein in >800 clinically annotated NSCLCs from three independent cohorts represented in tissue microarrays. Associations between the marker's expression and major genomic alterations were studied in The Cancer Genome Atlas NSCLC dataset. Using mass cytometry (CyTOF) analysis of leukocytes collected from 20 resected NSCLCs, we determined the levels, coexpression, and functional profile of PD-1, LAG-3, and TIM-3 expressing immune cells. Finally, we measured the markers in baseline samples from 90 patients with advanced NSCLC treated with PD-1 axis blockers and known response to treatment. Results: PD-1, LAG-3, and TIM-3 were detected in tumor-infiltrating lymphocytes (TIL) from 55%, 41.5%, and 25.3% of NSCLC cases, respectively. These markers showed a prominent association with each other and limited association with major clinicopathologic variables and survival in patients not receiving immunotherapy. Expression of the markers was lower in EGFR-mutated adenocarcinomas and displayed limited association with tumor mutational burden. In single-cell CyTOF analysis, PD-1 and LAG-3 were predominantly localized on T-cell subsets/NKT cells, whereas TIM-3 expression was higher in NK cells and macrophages. Coexpression of PD-1, LAG-3, and TIM-3 was associated with prominent T-cell activation (CD69/CD137), effector function (Granzyme-B), and proliferation (Ki-67), but also with elevated levels of proapoptotic markers (FAS/BIM). LAG-3 and TIM-3 were present in TIL subsets lacking PD-1 expression and showed a distinct functional profile. In baseline samples from 90 patients with advanced NSCLC treated with PD-1 axis blockers, elevated LAG-3 was significantly associated with shorter progression-free survival. Conclusions: PD-1, LAG-3, and TIM-3 have distinct tissue/cell distribution, functional implications, and genomic correlates in human NSCLC. Expression of these immune inhibitory receptors in TILs is associated with prominent activation, but also with a proapoptotic T-cell phenotype. Elevated LAG-3 expression is associated with insensitivity to PD-1 axis blockade, suggesting independence of these immune evasion pathways.

Immunotherapy is emerging as a powerful approach in cancer treatment. Preclinical data predicted the antineoplastic effects seen in clinical trials of programmed death-1 (PD-1) pathway inhibitors, as well as their observed toxicities. The results of early clinical trials are extraordinarily promising in several cancer types and have shaped the direction of ongoing and future studies.This review describes the biological rationale for targeting the PD-1 pathway with monoclonal antibodies for the treatment of cancer as a context for examining the results of early clinical trials. It also surveys the landscape of ongoing clinical trials and discusses their anticipated strengths and limitations.PD-1 pathway inhibition represents a new frontier in cancer immunotherapy, which shows clear evidence of activity in various tumor types including NSCLC and melanoma. Ongoing and upcoming trials will examine optimal combinations of these agents, which should further define their role across tumor types. Current limitations include the absence of a reliable companion diagnostic to predict likely responders, as well as lack of data in early-stage cancer when treatment has the potential to increase cure rates.

Background: The immune checkpoint target and companion diagnostic, PD-L1 is expressed in both tumor cell and immune cells and both are read by the pathologist and associated with response to therapy in different organ systems. Here, we examine the expression of PD-L1 using molecular assessment of cellular localization to determine which cell type carries the predictive value of the companion diagnostic test. Methods: We used the quantitative immunofluorescence (QIF) methods (AQUA analysis of images from the PM2000 and inForm analysis of images from the PerkinElmer Vectra and Polaris Automated Quantitative Pathology Imaging System) and measured the expression of PD-L1 in multiple immune cell types by phenotyping each cell. We also acquired high resolution images from the multiplex for precise identification of co-localization using confocal microscopy. The PD-L1 localization was measured using two multiplex QIF panels including CD56 for natural killer (NK) cells, CD68 for macrophages and CD8 for cytotoxic T cells. This multiplex was performed on three retrospective Yale NSCLC cohorts (457 non-immunotherapy-treated cases and 62 PD-1 axis immunotherapy treated cases) represented in tissue microarrays. Results: PD-L1 localization was significantly higher in macrophages compared to NK cells and CD8 T cells in both tumor and stroma compartment. In stroma, 80% of PD-L1 is expressed by macrophages, while 5% and 3% by NK cells and CD8 T cells, respectively. High CD8 level (top tertile) was associated with better overall survival (P Conclusion: In over 500 lung cancer cases the predominant immune cell type that expresses PD-L1 CD68 positive macrophages, as compared to NK cells and CD8 T cells. The level of PD-L1 in macrophages is significantly associated with the level of PD-L1 in tumor and with high levels of CD8+ T cells, suggesting a connection between high PD-L1 and “hot” tumors. Similar to our previous findings in melanoma, we find that high levels of PD-L1 in macrophages is a positive predictive biomarker for PD-1 pathway blockade therapy. Due to the small size of the treated cohort in the study, further work is required to confirm this observation. Citation Format: Yuting Liu, Jon Zugazagoitia, Brian Henick, Kurt A. Schalper, David L. Rimm. Immune profiling of in NSCLC shows expression of PD-L1 in macrophages is better associated with outcomes than PD-L1 in tumor cells in PD-1 axis immunotherapy treated patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2674.

12002Background: Despite innate immunity’s prominent role in the anti-tumor response, little is known about the MC composition of human NSCLC. We used multiplexed quantitative immunofluorescence (Q...

12015Background: Defective antigen presenting machinery (APM) limits tumor recognition by cognate T cells mediating resistance to immune checkpoint inhibitors (ICIs). The expression frequency, biol...

e14611 Background: PD-1 axis blockade induces lasting clinical responses in ~20% of patients with advanced NSCLC. However, most patients eventually develop resistance. Acquired resistance is poorly understood, but may be mediated by alternative immune suppressive pathways. Methods: Using multiplex immunofluorescence we simultaneously measured levels of DAPI, CD3 (D7AE6), PD-1 (EH33), TIM-3 (D5D5R) and LAG-3 (17B4) in 11 whole tissue sections obtained from patients with NSCLC before PD-1 axis blockade and after acquired resistance (8 cases with progression on-treatment and 3 with progression off-therapy). Markers were measured in the whole tumor area or in CD3+ T-cells using fluorescence co-localization. The association between markers and changes upon acquired resistance were studied. Results: Expression of PD-1, TIM-3 and LAG-3 was seen in all cases with membranous staining pattern and signal predominantly located in CD3+ T-cells. Levels of TIM-3 and LAG-3 in T-cells were significantly correlated (Spearman’s R = 0.65, P = 0.001), but were not associated with PD-1 (R = -0.03, P = 0.86 for TIM-3 and PD-1; and R = 0.24, P = 0.28 for LAG-3 and PD-1). Compared to pre-treatment samples, 6 cases (55%) showed significantly higher levels of PD-1 or LAG-3 on acquired resistance and 5 cases (45%) had higher TIM-3. Of these, 4 cases had higher levels of the 3 markers and were on-therapy at progression. Lower levels of PD-1, TIM-3, and LAG-3 were found on acquired resistance in 5 (45%), 6 (55%), and 4 (36%) cases, respectively. Four of these cases showed lower levels of all inhibitory receptors, 3 of which were off-therapy at progression. Only one case had no change in LAG-3 levels. Conclusions: PD-1, TIM-3 and LAG-3 were expressed in the majority of NSCLCs with signal predominantly located in T-lymphocytes. Among acquired resistance cases, higher levels of PD-1, TIM-3 and LAG-3 were associated with progression on-treatment. Lower levels of the markers were associated with progression off-therapy. Although multiple mechanisms may exist, up-regulation of alternative immune inhibitory receptors such as TIM-3 and LAG-3 could mediate acquired resistance to PD-1 axis blockers in a proportion of NSCLCs.

e20652 Background: Detection of EGFR mutations in ctDNA can help determine appropriateness of TKI therapy for patients with NSCLC. We investigated whether longitudinal monitoring of ctDNA levels can be used to assess response to therapy and disease progression, with a focus on EGFR mutation-positive patients treated with immunotherapy. Methods: Serially collected blood from patients with EGFR mutation-positive NSCLC treated with TKIs and/or immunotherapy was analyzed using an ultrasensitive 24-gene next-generation sequencing assay. Clinical characteristics and outcomes were analyzed retrospectively by chart review. Results: We studied quantitative changes in ctDNA levels during treatment by analyzing somatic mutations in 91 plasma samples from 8 patients with EGFR-mutant NSCLC, including samples collected around the time of disease progression for a subset of patients. Two patients treated with PD-1 inhibitor monotherapy experienced a rise in ctDNA harboring EGFR-sensitizing mutations prior to radiographic progression. A third patient was started on anti-PD-1 monotherapy following disease progression on erlotinib. Plasma levels of L858R, T790M, and TP53 mutations were detectable on treatment initiation and decreased with radiographic response. The levels of these mutations rose at progression,fell with response to EGFR-directed therapy, and increased again before disease progression. Another patient was found to have mutations in EGFR, T790M, and TP53 that fell upon treatment with combination TKI therapy. The remaining four patients studied were treated with concurrent TKI and immunotherapy. In all of these cases, sensitizing EGFR mutations were present in plasma at low levels during response to treatment. Two of the four patients had a rise in ctDNA level at the time of radiographic progression; the other two patients had durable responses with persistently low ctDNA levels. Analysis of additional cases is ongoing. Conclusions: Monitoring quantitative changes in ctDNA may enable assessment of response or disease progression in immunotherapy- and TKI-treated EGFR-mutant NSCLC patients.