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Small-cell lung cancer (SCLC) is speculated to harbor complex genomic intratumor heterogeneity (ITH) associated with high recurrence rate and suboptimal response to immunotherapy. Here, using multi-region whole exome/T cell receptor (TCR) sequencing as well as immunohistochemistry, we reveal a rather homogeneous mutational landscape but extremely cold and heterogeneous TCR repertoire in limited-stage SCLC tumors (LS-SCLCs). Compared to localized non-small cell lung cancers, LS-SCLCs have similar predicted neoantigen burden and genomic ITH, but significantly colder and more heterogeneous TCR repertoire associated with higher chromosomal copy number aberration (CNA) burden. Furthermore, copy number loss of IFN-γ pathway genes is frequently observed and positively correlates with CNA burden. Higher mutational burden, higher T cell infiltration and positive PD-L1 expression are associated with longer overall survival (OS), while higher CNA burden is associated with shorter OS in patients with LS-SCLC., Small-cell lung cancer (SCLC) is an aggressive disease with limited therapeutic options. Here the authors perform an immunogenomic analysis of limited-stage SCLC, revealing a homogeneous mutational landscape, but limited T-cell infiltration and a cold and heterogeneous T cell repertoire.

PURPOSE To compare clinical outcomes in a cohort of patients with advanced non–small-cell lung cancer (NSCLC) with targetable genomic alterations detected using plasma-based circulating tumor DNA (ctDNA) or tumor-based next-generation sequencing (NGS) assays treated with US Food and Drug Administration–approved therapies at a large academic research cancer center. METHODS A retrospective review from our MD Anderson GEMINI database identified 2,224 blood samples sent for ctDNA NGS testing from 1971 consecutive patients with a diagnosis of advanced NSCLC. Clinical, treatment, and outcome information were collected, reviewed, and analyzed. RESULTS Overall, 27% of the ctDNA tests identified at least one targetable mutation and 73% of targetable mutations were EGFR-sensitizing mutations. Among patients treated with first-line epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapies, there were no significant differences in progression-free survival of 379 days and 352 days ( P value = .41) with treatment based on tissue (n = 40) or ctDNA (n = 40), respectively. Additionally, there were no differences in progression-free survival or objective response rate among those with low (n = 8, 0.01%-0.99%) versus high (n = 16, ≥ 1%) levels of ctDNA of the targetable mutation as measured by variant allele frequency (VAF). Overall, there was excellent testing concordance (n = 217 tests) of > 97%, sensitivity of 91.7%, and specificity of 99.7% between blood-based ctDNA NGS and tissue-based NGS assays. CONCLUSION There were no significant differences in clinical outcomes among patients treated with approved EGFR-TKIs whose mutations were identified using either tumor- or plasma-based comprehensive profiling and those with very low VAF as compared with high VAF, supporting the use of plasma-based profiling to guide initial TKI use in patients with metastatic EGFR-mutant NSCLC.

Introduction Immune infiltration in lung adenocarcinomas (LUADs) has been associated with response to immune checkpoint inhibitors. Clinical features underlying differential responses of patients with LUADs to immunotherapy are not well understood. Here, we analyzed the association between LUAD immune infiltration and clinicopathologic variables. Materials and Methods Intratumoral CD3, CD8, and CD68 cell densities (tumor-associated immune cells [TAICs]) were immunohistochemically assessed in 146 surgically resected LUADs. LUADs were classified into 2 groups, low and high TAICs, based on the median values of cell densities for CD3, CD8, and CD68. Somatic mutation burden and driver gene mutation status were analyzed in a subset of the cases (n = 92). We statistically analyzed the association between the TAIC groups and various clinicopathologic and molecular variables by using the χ2/Fisher and Wilcoxon sum tests and multivariable logistic regression models. Results Patient gender, tumor size, and STK11 mutations were significantly associated with TAIC levels in LUAD. Female patients exhibited significantly elevated TAIC levels (P = .005) compared with male patients. Tumor size was inversely associated with TAIC levels (P = .012). STK11 mutated tumors were associated with lower TAICs (P = .008). Higher TAICs were consistently observed in female patients with LUADs after adjusting for stage, tumor size, and age. Multivariable regression models confirmed female gender as an independent variable associated with TAIC levels in LUAD (P = .0141). Conclusion Immune infiltration in LUADs was significantly higher in female patients, warranting further exploration into the association between this clinical variable and immunotherapeutic response in LUAD.

Introduction Lung adenocarcinomas harboring EGFR mutations do not respond to immune checkpoint blockade therapy and their EGFR wildtype counterpart. The mechanisms underlying this lack of clinical response have been investigated but remain incompletely understood. Methods We analyzed three cohorts of resected lung adenocarcinomas (Profiling of Resistance Patterns of Oncogenic Signaling Pathways in Evaluation of Cancer of Thorax, Immune Genomic Profiling of NSCLC, and The Cancer Genome Atlas) and compared tumor immune microenvironment of EGFR-mutant tumors to EGFR wildtype tumors, to identify actionable regulators to target and potentially enhance the treatment response. Results EGFR-mutant NSCLC exhibited low programmed death-ligand 1, low tumor mutational burden, decreased number of cytotoxic T cells, and low T cell receptor clonality, consistent with an immune-inert phenotype, though T cell expansion ex vivo was preserved. In an analysis of 75 immune checkpoint genes, the top up-regulated genes in the EGFR-mutant tumors (NT5E and ADORA1) belonged to the CD73/adenosine pathway. Single-cell analysis revealed that the tumor cell population expressed CD73, both in the treatment-naive and resistant tumors. Using coculture systems with EGFR-mutant NSCLC cells, T regulatory cell proportion was decreased with CD73 knockdown. In an immune-competent mouse model of EGFR-mutant lung cancer, the CD73/adenosine pathway was markedly up-regulated and CD73 blockade significantly inhibited tumor growth. Conclusions Our work revealed that EGFR-mutant NSCLC has an immune-inert phenotype. We identified the CD73/adenosine pathway as a potential therapeutic target for EGFR-mutant NSCLC.

Ipilimumab improves clinical outcomes when combined with nivolumab in metastatic non-small cell lung cancer (NSCLC), but its efficacy and impact on the immune microenvironment in operable NSCLC remain unclear. We report the results of the phase 2 randomized NEOSTAR trial (NCT03158129) of neoadjuvant nivolumab or nivolumab + ipilimumab followed by surgery in 44 patients with operable NSCLC, using major pathologic response (MPR) as the primary endpoint. The MPR rate for each treatment arm was tested against historical controls of neoadjuvant chemotherapy. The nivolumab + ipilimumab arm met the prespecified primary endpoint threshold of 6 MPRs in 21 patients, achieving a 38% MPR rate (8/21). We observed a 22% MPR rate (5/23) in the nivolumab arm. In 37 patients resected on trial, nivolumab and nivolumab + ipilimumab produced MPR rates of 24% (5/21) and 50% (8/16), respectively. Compared with nivolumab, nivolumab + ipilimumab resulted in higher pathologic complete response rates (10% versus 38%), less viable tumor (median 50% versus 9%), and greater frequencies of effector, tissue-resident memory and effector memory T cells. Increased abundance of gut Ruminococcus and Akkermansia spp. was associated with MPR to dual therapy. Our data indicate that neoadjuvant nivolumab + ipilimumab-based therapy enhances pathologic responses, tumor immune infiltrates and immunologic memory, and merits further investigation in operable NSCLC. Neoadjuvant treatment with nivolumab plus ipilimumab is well tolerated and demonstrates clinical efficacy in patients with early stage lung cancer.

The advent of immune checkpoint inhibitors (ICIs) has dramatically changed the treatment of patients with locally advanced unresectable and metastatic non-small cell lung cancer (NSCLC). Now, ICIs are undergoing evaluation as neoadjuvant therapy in patients with early-stage, resectable NSCLC using candidate surrogate endpoints of clinical efficacy, i.e., major pathologic response (MPR, ≤10% viable tumor cells in resected tumors). The initial results from early, small-scale trials are encouraging; however, they also reveal that a substantial number of patients with operable disease may not benefit from neoadjuvant ICIs. Consequently, much investigative effort is currently directed toward identifying mechanisms of resistance to ICI therapy in resectable NSCLC. There is also an urgent need for biomarkers that could be used to guide the clinical decision-making process and maximize the clinical benefit of ICIs in patients with early-stage, resectable NSCLC. Here, we summarize the initial results from the trials of neoadjuvant ICIs in patients with early-stage and locally advanced operable NSCLC and review the findings of studies investigating emerging biomarkers associated with those trials.

Background Metastasis is the primary cause of cancer mortality accounting for 90% of cancer deaths. Our understanding of the molecular mechanisms driving metastasis is rudimentary. Results We perform whole exome sequencing (WES), RNA sequencing, methylation microarray, and immunohistochemistry (IHC) on 8 pairs of non-small cell lung cancer (NSCLC) primary tumors and matched distant metastases. Furthermore, we analyze published WES data from 35 primary NSCLC and metastasis pairs, and transcriptomic data from 4 autopsy cases with metastatic NSCLC and one metastatic lung cancer mouse model. The majority of somatic mutations are shared between primary tumors and paired distant metastases although mutational signatures suggest different mutagenesis processes in play before and after metastatic spread. Subclonal analysis reveals evidence of monoclonal seeding in 41 of 42 patients. Pathway analysis of transcriptomic data reveals that downregulated pathways in metastases are mainly immune-related. Further deconvolution analysis reveals significantly lower infiltration of various immune cell types in metastases with the exception of CD4+ T cells and M2 macrophages. These results are in line with lower densities of immune cells and higher CD4/CD8 ratios in metastases shown by IHC. Analysis of transcriptomic data from autopsy cases and animal models confirms that immunosuppression is also present in extracranial metastases. Significantly higher somatic copy number aberration and allelic imbalance burdens are identified in metastases. Conclusions Metastasis is a molecularly late event, and immunosuppression driven by different molecular events, including somatic copy number aberration, may be a common characteristic of tumors with metastatic plasticity.

BackgroundEGFR tyrosine kinase inhibitors (TKIs) induce cytolysis and release of tumour proteins, which can stimulate antigen-specific T cells. The safety and efficacy of durvalumab and gefitinib in combination for TKI-naive patients with advancedEGFRm NSCLC was evaluated.MethodsThis Phase 1 open-label, multicentre trial (NCT02088112) was conducted in 56 patients with NSCLC. Dose expansion permitted TKI-naive patients, primarily with activating L858R or Ex19delEGFRm. Arms 1 + 1a received concurrent therapy; Arm 2 received 4 weeks of gefitinib induction followed by concurrent therapy.ResultsFrom dose escalation, the recommended dose of durvalumab was 10 mg/kg Q2W with 250 mg QD gefitinib. Pharmacokinetics were as expected, consistent with inhibition of soluble PD-L1 and no treatment-emergent immunogenicity. In dose expansion, 35% of patients had elevated liver enzymes leading to drug discontinuation. In Arms 1 + 1a, objective response rate was 63.3% (95% CI: 43.9–80.1), median progression-free survival (PFS) was 10.1 months (95% CI: 5.5–15.2) and median response duration was 9.2 months (95% CI: 3.7–14.0).ConclusionsDurvalumab and gefitinib in combination had higher toxicity than either agent alone. No significant increase in PFS was detected compared with historical controls. Therefore, concurrent PD-L1 inhibitors with gefitinib should be generally avoided in TKI-naive patients withEGFRm NSCLC.

Summary Non-small cell lung cancer (NSCLC) is the deadliest form of cancer worldwide, due in part to its proclivity to metastasize. Identifying novel drivers of invasion and metastasis holds therapeutic potential for the disease. We conducted a gain-of-function invasion screen, which identified two separate hits, IMPAD1 and KDELR2, as robust, independent drivers of lung cancer invasion and metastasis. Given that IMPAD1 and KDELR2 are known to be localized to the ER-Golgi pathway, we studied their common mechanism of driving in vitro invasion and in vivo metastasis and demonstrated that they enhance Golgi-mediated function and secretion. Therapeutically inhibiting matrix metalloproteases (MMPs) suppressed both IMPAD1- and KDELR2-mediated invasion. The hits from this unbiased screen and the mechanistic validation highlight Golgi function as one of the key cellular features altered during invasion and metastasis.

Purpose: Nintedanib enhances the activity of chemotherapy in metastatic non–small cell lung cancer (NSCLC). In this phase I/II study, we assessed safety and efficacy of nintedanib plus neoadjuvant chemotherapy, using major pathologic response (MPR) as primary endpoint. Patients and Methods: Eligible patients had stage IB (≥4 cm)–IIIA resectable NSCLC. A safety run-in phase was followed by an expansion phase with nintedanib 200 mg orally twice a day (28 days), followed by three cycles of cisplatin (75 mg/m2), docetaxel (75 mg/m2) every 21 days plus nintedanib, followed by surgery. With 33 planned patients, the study had 90% power to detect an MPR increase from 15% to 35%. Results: Twenty-one patients (stages I/II/III, N = 1/8/12) were treated. One of 15 patients treated with nintedanib 200 mg achieved MPR [7%, 95% confidence interval (CI), 0.2%–32%]. Best ORR in 20 evaluable patients was 30% (6/20, 95% CI, 12%–54%). Twelve-month recurrence-free survival and overall survival were 66% (95% CI, 47%–93%) and 91% (95% CI, 79%–100%), respectively. Most frequent treatment-related grade 3–4 toxicities were transaminitis and electrolyte abnormalities. On the basis of an interim analysis the study was discontinued for futility. Higher levels of CD3+ and cytotoxic CD3+CD8+ T cells were found in treated tumors of patients who were alive than in those who died (652.8 vs. 213.4 cells/mm2, P = 0.048; 142.3 vs. 35.6 cells/mm2, P = 0.018). Conclusions: Although tolerated, neoadjuvant nintedanib plus chemotherapy did not increase MPR rate compared with chemotherapy historical controls. Additional studies of the combination in this setting are not recommended. Posttreatment levels of tumor-infiltrating T cells were associated with patient survival. Use of MPR facilitates the rapid evaluation of neoadjuvant therapies. See related commentary by Blakely and McCoach, p. 3499

Current strategies to improve clinical outcomes in KRAS-mutant non-small cell lung cancer (NSCLC) patients include MEK inhibitor and PD-1 / PD-L1 immune checkpoint blockade (ICB) combinations. Experience from melanoma suggests that anti-CTLA-4 and anti-PD-1 / PD-L1 combinations improve outcomes, but similar benefits remain to be seen in NSCLC. This report describes a single center, investigator-initiated Phase I/II clinical trial comparing two combination schedules of intermittent or continuous selumetinib (AZD6244, ARRY-142886), tremelimumab (anti-CTLA-4) and durvalumab (anti-PD-L1) with historical controls in patients with previously treated, unresectable NSCLC. Forty patients will be accrued at the University of Texas MD Anderson Cancer Center. Primary objectives include maximum tolerated dose (MTD; dose-escalation phase) and Progression-Free Survival (PFS; dose expansion phase). Secondary objectives include response rate by RECIST 1.1, disease control rate, overall survival, safety and duration of response. Exploratory objectives will assess biomarkers of response and resistance based on pre-and on-treatment biopsies and peripheral blood using immune profiling, transcriptome and protein readouts. MICRO ABSTRACT: This investigator-initiated trial was designed to provide novel data regarding: 1) the safety of durvalumab, tremelimumab and selumetinib combination therapy; 2) whether this combination can improve PFS in metastatic NSCLC; 3) potential biomarkers of response and resistance based on pre-and on-treatment biopsies and peripheral blood.

Enhanced tumor glycolytic activity is a mechanism by which tumors induce an immunosuppressive environment to resist adoptive T cell therapy; therefore, methods of assessing intratumoral glycolytic activity are of considerable clinical interest. In this study, we characterized the relationships among tumor (18)F-fluorodeoxyglucose (FDG) retention, tumor metabolic and immune phenotypes, and survival in patients with resected non-small cell lung cancer (NSCLC). We retrospectively analyzed tumor preoperative positron emission tomography (PET) (18)F-FDG uptake in 59 resected NSCLCs and investigated correlations between PET parameters (SUV(Max), SUV(Total), SUV(Mean), TLG), tumor expression of glycolysis- and immune-related genes, and tumor-associated immune cell densities that were quantified by immunohistochemistry. Tumor glycolysis-associated immune gene signatures were analyzed for associations with survival outcomes. We found that each (18)F-FDG PET parameter was positively correlated with tumor expression of glycolysis-related genes. Elevated (18)F-FDG SUV(Max) was more discriminatory of glycolysis-associated changes in tumor immune phenotypes than other (18)F-FDG PET parameters. Increased SUV(Max) was associated with multiple immune factors characteristic of an immunosuppressive and poorly immune infiltrated tumor microenvironment, including elevated PD-L1 expression, reduced CD57(+) cell density, and increased T cell exhaustion gene signature. Elevated SUV(Max) identified immune-related transcriptomic signatures that were associated with enhanced tumor glycolytic gene expression and poor clinical outcomes. Our results suggest that (18)F-FDG SUV(Max) has potential value as a noninvasive, clinical indicator of tumor immunometabolic phenotypes in patients with resectable NSCLC and warrants investigation as a potential predictor of therapeutic response to immune-based treatment strategies.

BackgroundWhile the clinical outcomes of immune checkpoint inhibitor (ICI) use in older adults with advanced-stage non-small cell lung cancer (NSCLC) have been described, the role of ICI use continued beyond disease progression (BDP) remains to be well defined for this population. This retrospective single-center study explored the clinical outcomes of continuing ICIs BDP among older adult patients with advanced NSCLC.MethodsUsing MD Anderson’s Gemini Lung Cancer database, we retrospectively reviewed the clinical outcomes of older adults (≥70 years) diagnosed with advanced-stage NSCLC treated with anti-PD-(L)1 monotherapy from March 2015 through April 2019 to correlate clinicopathologic features with clinical outcomes. Clinical therapy responses were evaluated by Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST v1.1). Toxicities were assessed using Common Terminology Criteria for Adverse Events (CTCAE), version 5. Patients treated BDP were defined as individuals receiving ICIs for ≥8 weeks prior to documentation of progression who subsequently remained on ICIs for ≥6 weeks.ResultsOf the 159 older adults meeting the inclusion criteria, 33 (21%) received ICIs BDP (64% male, median age 74.9 years (70.1–82.0) at the start of ICI treatment, 3 received first-line ICI therapy). Most patients were former (85%) or current (6%) smokers. 79% had adenocarcinoma histology. The median duration of immunotherapy continued BDP was 7.1 months (95% CI: 3.0–8.2). After a follow-up of 30.1 months, the median overall survival (mOS) was 31.5 months (95% CI: 16.5-not reached). Eight (24%) received local consolidative radiotherapy with a median duration of ICI BDP of 8.2 months (95% CI: 1.9–13.3). Twenty-five (76%) did not receive local consolidative therapy and achieved a median duration of ICI BDP of 4.1 months (95% CI: 2.3–7.8). Six (18%) exhibited pseudo-progression (i.e. delayed response to immunotherapy with decreased tumor burden on subsequent radiologic studies), with 4 achieving ”stable disease” as best response and 2 achieving a partial response. The median duration of immunotherapy continued beyond pseudo-progression was 11.7 months (95% CI: 7.1–35.7), and the mOS was 26.2 months (95% CI: 16.5–40.0). Patients treated with ICI BDP most commonly experienced fatigue (18%), pneumonitis (12%), rash (9%), and hypothyroidism (9%). Three patients (9%) experienced grade 3 or higher toxicities (one grade 3 arthralgias and two grade 3 pneumonitis).ConclusionsICI-use BDP in older adults with advanced NSCLC may benefit a subset of patients. Additionally, local consolidative therapy with radiation may offer prolonged duration of ICI treatment.AcknowledgementsSupported by the generous philanthropic contributions to The University of Texas MD Anderson Lung Moon Shot Program and the MD Anderson Cancer Center Support Grant P30 CA01667. Special acknowledgment to the GEMINI team.Ethics ApprovalThis study was approved and conducted in accordance with the institutional review board at the University of Texas MD Anderson Cancer Center; approval number (PA13-0589).

BackgroundHow neoadjuvant chemo-immunotherapy modulates tumor immune composition and response is not completely understood. We interrogate immunomodulation of neoadjuvant platinum-based chemotherapy (C), nivolumab (N), and N-plus-C (NC) and their connections to therapeutic efficacy in resected non-small cell lung cancer (NSCLC) by integrating immunomic data from the ImmunogenomiC PrOfiling of NSCLC (ICON) study and NEOSTAR trial cohorts.MethodsIn NEOSTAR (NCT03158129), patients with stage I-IIIA (single N2) resectable NSCLC (AJCC7th) received N (3 mg/kg IV, D1,15,29); patients with stage IB(≥4cm)-IIIA (single N2) resectable NSCLC received NC (N 360 mg IV plus C, D1,22,43 for 3 cycles, every 3 weeks) before surgery; major pathologic response (MPR) was the primary endpoint. In ICON, patients with stage IB(≥4cm)-IIIA resectable NSCLC received C before surgery. Surgically resected tumor samples underwent immune profiling via flow cytometry (n=16,13,9 for C,N,NC), immunohistochemistry (IHC;n=0,18,14), and multiplexed immunofluorescence (mIF;n=28,16,10). Treatment-associated immunomodulation and associations with therapeutic efficacy were analyzed using: 1) a shared nearest neighbors-based network we developed linking measurements across datasets; 2) MetaCyto, a specialized cytometry analysis method for identifying cell subsets by clustering.ResultsWe holistically explored the immunomic data by integration across cohorts. Through hierarchical regression of the integrated data, we determined the overall effect of a given treatment controlling for the presence or absence of the other treatment.We examined C’s effects across all cohorts controlling for N. Across all patients, regardless of MPR, C is associated with immunosuppression, increasing PD1+ T cell (CD45+CD3+) populations: regulatory (CD4+CD25+FOXP3+), helper (CD4+), and effector (CD8+) (effect size(ES):1.48,1.61,1.26;qConversely, we examined N’s effects across all cohorts controlling for C. Across all patients, regardless of MPR, N is associated with immune activation, increasing ICOS+ T cell populations: regulatory, helper, and effector (ES:1.29,1.29,1.47;qConclusionsWe report the first integrated examination of the immunomodulatory effect of neoadjuvant C and N. C is associated with immunosuppression while N with immune activation; together, N appears to lessen C’s suppressive effects. Incorporation of transcriptomics into this integrated network of flow cytometry, mIF, and IHC immune profiling data is ongoing to augment translational insights for neoadjuvant chemo/immunotherapies.

BackgroundLung cancer is the leading cause of cancer death, partially owing to its extensive heterogeneity. The analysis of intertumor heterogeneity has been limited by an inability to concurrently obtain tissue from synchronous metastases unaltered by multiple prior lines of therapy.MethodsIn order to study the relationship between genomic, epigenomic and T cell repertoire heterogeneity in a rare autopsy case from a young female never-smoker with late-stage lung adenocarcinoma (LUAD), we did whole-exome sequencing (WES), DNA methylation and T-cell receptor (TCR) sequencing to characterize the immunogenomic landscape of one primary and 19 synchronous metastatic tumors.ResultsWe observed heterogeneous mutation, methylation, and T cell patterns across distinct metastases including a set of prevalent T cell clonotypes which were completely excluded from left-side thoracic tumors. Though a limited number of predicted neoantigens were shared, these were associated with homology of the T cell repertoire across metastases. Lastly, ratio of methylated neoantigen coding mutations was negatively associated with T-cell density, richness and clonality, suggesting neoantigen methylation may partially drive immunosuppression.ConclusionsOur study demonstrates heterogeneous genomic and T cell profiles across synchronous metastases and how restriction of unique T cell clonotypes within an individual may differentially shape the genomic and epigenomic landscapes of synchronous lung metastases.

Introduction Immune checkpoint blockade (ICB) has revolutionized the treatment of NSCLC, but only approximately 15% of patients achieve durable benefit. Understanding mechanisms of resistance to ICB is pivotal in developing more effective treatment strategies. Recent studies showed that human leukocyte antigen (HLA) class I heterozygosity might be important in mediating benefit from ICB. We aimed to investigate the impact of HLA class I genotype on outcomes of patients with NSCLC treated with ICB. Methods We collected HLA typing, genomic, and clinical data from patients with advanced NSCLC treated with ICB at M. D. Anderson Cancer Center. We compared HLA class I–heterozygous and HLA class I–homozygous patients for progression-free survival (PFS) and overall survival (OS). HLA I supertype/alleles were also analyzed. To validate our findings, we also analyzed two previously published independent cohorts of patients with NSCLC (the CheckMate-012 and Chowell cohorts). Results No significant correlations were observed for HLA class I zygosity and PFS or OS in the M. D. Anderson Cancer Center (n = 200), CheckMate-012 (n = 75), or Chowell (n = 371) cohorts. No HLA class I supertype/allele was consistently shown to be correlated with PFS or OS. Predictors of worse outcome across the three cohorts included presence of targetable driver mutation, serine/threonine kinase 11 gene (STK11) mutation, negative programmed death ligand 1 expression, and low tumor mutational burden. Conclusions HLA class I genotype is not correlated with survival in advanced NSCLC treated with ICB. This suggests that the impact of HLA class I diversity may be disease specific and that tumor genomic and immune markers are more impactful in predicting benefit from ICB in NSCLC.

PURPOSE Targeted therapies against non–small-cell lung cancer (NSCLC) harboring HER2 mutations remain an unmet need. In this study, we assessed the efficacy and safety of poziotinib in patients with HER2 exon 20 mutant advanced NSCLC in a single-arm, open-label, phase II study. PATIENTS AND METHODS Patients with advanced HER2 exon 20 mutant NSCLC were enrolled to receive poziotinib at a dose of 16 mg/d for 28-day cycles. The primary end point was objective response rate per RECIST version 1.1. Confirmatory scans were performed at least 28 days from initial radiologic response. RESULTS Thirty patients received poziotinib treatment. At baseline, 90% of patients received prior platinum-based chemotherapy and 53% had two lines or more prior systemic therapies. As of data cutoff on March 1, 2021, the confirmed objective response rate was 27% (95% CI, 12 to 46). Responses were observed across HER2 exon 20 mutation subtypes. The median duration of response was 5.0 months (95% CI, 4.0 to not estimable). The median progression-free survival was 5.5 months (95% CI, 4.0 to 7.0). The median overall survival was 15 months (95% CI, 9.0 to not estimable). The most common grade 3 treatment-related adverse events were skin rash (47%) and diarrhea (20%). There was one possible treatment-related death because of pneumonitis. CONCLUSION Poziotinib showed promising antitumor activity in patients with HER2 exon 20 mutant NSCLC including patients who had previously received platinum-based chemotherapy.

BackgroundNon-small cell lung cancer (NSCLC) patients bearing targetable oncogene alterations typically derive limited benefit from immune checkpoint blockade (ICB), which has been attributed to low tumor mutation burden (TMB) and/or PD-L1 levels. We investigated oncogene-specific differences in these markers and clinical outcome.MethodsThree cohorts of NSCLC patients with oncogene alterations (n=4189 total) were analyzed. Two clinical cohorts of advanced NSCLC patients treated with ICB monotherapy [MD Anderson (MDACC; n=172) and Flatiron Health-Foundation Medicine Clinico-Genomic Database (CGDB; n=894 patients)] were analyzed for clinical outcome. The FMI biomarker cohort (n=4017) was used to assess the association of oncogene alterations with TMB and PD-L1 expression.ResultsHigh PD-L1 expression (PD-L1 ≥50%) rate was 19%–20% in classic EGFR, EGFR exon 20 and HER2-mutant tumors, and 34%–55% in tumors with ALK, BRAF V600E, ROS1, RET, or MET alterations. Compared with KRAS-mutant tumors, BRAF non-V600E group had higher TMB (9.6 vs KRAS 7.8 mutations/Mb, p=0.003), while all other oncogene groups had lower TMB (pEGFR, HER2, ALK, ROS1, RET, or MET alterations had short progression-free survival (PFS; 1.8–3.7 months), while BRAF V600E group was associated with greater clinical benefit from ICB (CGDB cohort: PFS 9.8 months vs KRAS 3.7 months, HR 0.66, p=0.099; MDACC cohort: response rate 62% vs KRAS 24%; PFS 7.4 vs KRAS 2.8 months, HR 0.36, p=0.026). KRAS G12C and non-G12C subgroups had similar clinical benefit from ICB in both cohorts. In a multivariable analysis, BRAF V600E mutation (HR 0.58, p=0.041), PD-L1 expression (HR 0.57, p=0.022), and high TMB (HR 0.66, pConclusionsHigh TMB and PD-L1 expression are predictive for benefit from ICB treatment in oncogene-driven NSCLCs. NSCLC harboring BRAF mutations demonstrated superior benefit from ICB that may be attributed to higher TMB and higher PD-L1 expression in these tumors. Meanwhile EGFR and HER2 mutations and ALK, ROS1, RET, and MET fusions define NSCLC subsets with minimal benefit from ICB despite high PD-L1 expression in NSCLC harboring oncogene fusions. These findings indicate a TMB/PD-L1-independent impact on sensitivity to ICB for certain oncogene alterations.

Background: SARS-CoV-2 infection is the cause of the respiratory illness COVID-19, which presents most frequently with respiratory symptoms SARS-CoV-2 cell entry requires interactions with ACE2 and TMPRSS2 on the surface of the host cell Cancer patients and, specifically, those with thoracic malignancies seem to experience poorer clinical outcomes Methods: We utilized bulk and single-cell transcriptional data from a combination of normal and malignant tissues and cells from aerodigestive and respiratory tracts to explore mechanisms governing the expression of ACE2 and TMPRSS2 Additionally, we determined the effect of EMT induction, ZEB1 modulation, and SARS-CoV-2 infection on ACE2 expression Results: Our bulk data suggests that aerodigestive and lung cancer models express a broad range of ACE2 and TMRPSS2, particularly in epithelial cells, and would serve as good models for studying SARS-CoV-2 infection We assessed the relationship between ACE2 and epithelial differentiation in numerous datasets, and found consistent positive correlations with transcriptional and microRNA signifiers of epithelial differentiation The miR-200 family – zinc finger E-box-binding homeobox 1 (ZEB1) pathway, which is an established regulator of EMT, also directly regulates ACE2 expression, likely via putative ZEB1 repressor sites located in the ACE2 promoter Furthermore, SARS-CoV-2 infection reduces ACE2 expression and shifts cells to a more mesenchymal phenotype with loss of EPCAM and upregulation of ZEB1 and other EMT-associated genes Conclusions: ACE2-positive cells are almost exclusively epithelial and unexpectedly rare, considering the devastating impact of this infection Following viral entry, SARS-CoV-2 infection induces molecular changes within the cells that are reminiscent of EMT, including increased ZEB1 ZEB1, in turn, appears to directly repress the expression of ACE2 This SARS-CoV-2-induced ACE2 deficiency, compounded by the downregulation of genes, including claudins, which play a critical role in restricting epithelial and endothelial permeability, exposes respiratory cells to increased risk of edema and acute respiratory distress syndrome (ARDS) Legal entity responsible for the study: The authors Funding: NIH/NCI R01-CA207295 (L A B ), NIH/NCI U01-CA213273 (L A B , J V H ), CCSG P30-CA01667 (L A B ), University of Texas SPORE in Lung Cancer P5-CA070907 (L A B , D L G , J V H , C M G ), the Department of Defense (LC170171;L A B ), Khalifa Bin Zayed Al Nahyan Foundation (C M G ), RP170067 (EMP), through generous philanthropic contributions to The University of Texas MD Anderson Lung Cancer Moon Shot Program and Andrew Sabin Family Fellowship, and The Rexanna Foundation for Fighting Lung Cancer Disclosure: C Gay: Research grant/Funding (self): Astra Zeneca J V Heymach: Advisory/Consultancy: AstraZeneca;Advisory/Consultancy: Boehringer Ingelheim;Advisory/Consultancy: Exelixis;Advisory/Consultancy: Genentech;Advisory/Consultancy: GlaxoSmithKline;Advisory/Consultancy: Guardant Health;Advisory/Consultancy: Hengrui;Advisory/Consultancy: Spectrum L A Byers: Advisory/Consultancy, Research grant/Funding (self): AstraZeneca;Advisory/Consultancy, Research grant/Funding (self): AbbVie;Advisory/Consultancy, Research grant/Funding (self): GenMab;Advisory/Consultancy: BergenBio;Advisory/Consultancy: Pharma Mar SA;Advisory/Consultancy, Research grant/Funding (self): Sierra Oncology;Advisory/Consultancy: Merck;Advisory/Consultancy: Bristol Myers Squibb;Advisory/Consultancy: Genentech;Advisory/Consultancy: Pfizer;Research grant/Funding (self): Tolero Pharmaceuticals All other authors have declared no conflicts of interest

Lack of sustained response to therapeutic agents in patients with KRAS-mutant lung cancer poses a major challenge and arises partly due to intratumor heterogeneity that defines phenotypically distinct tumor subpopulations. To attain better therapeutic outcomes, it is important to understand the differential therapeutic sensitivities of tumor cell subsets. Epithelial-mesenchymal transition is a biological phenomenon that can alter the state of cells along a phenotypic spectrum and cause transcriptional rewiring to produce distinct tumor cell subpopulations. We utilized functional shRNA screens, in in vitro and in vivo models, to identify and validate an increased dependence of mesenchymal tumor cells on cyclin-dependent kinase 4 (CDK4) for survival, as well as a mechanism of resistance to MEK inhibitors. High zinc finger E-box binding homeobox 1 levels in mesenchymal tumor cells repressed p21, leading to perturbed CDK4 pathway activity. Increased dependence on CDK4 rendered mesenchymal cancer cells particularly vulnerable to selective CDK4 inhibitors. Coadministration of CDK4 and MEK inhibitors in heterogeneous tumors effectively targeted different tumor subpopulations, subverting the resistance to either single-agent treatment.

Background Tumor and nodal downstaging following neoadjuvant therapy in resectable non-small cell lung cancer (NSCLC) are important markers of therapeutic response associated with favorable prognosis. We studied the impact of four different systemic neoadjuvant therapies on tumor, nodal and overall pathological downstaging of surgically resectable I-IIIA NSCLC (AJCC 7th edition). Methods Our study cohorts consisted of NSCLC patients treated with three cycles of neoadjuvant platinum doublet chemotherapy from 2001–2012 (N=302, 84%), and patients treated on the NEOSTAR study (NCT03158129) who received neoadjuvant nivolumab (N=21,6%), nivolumab plus ipilimumab (N=16, 4%), or platinum doublet chemotherapy plus nivolumab (N=22, 6%). Clinical and pathological (yp) T and N staging were evaluated for downstaging and upstaging; differences were assessed using Fisher’s exact test. Results Following neoadjuvant platinum doublet chemotherapy, nivolumab, nivolumab plus ipilimumab and platinum doublet chemotherapy plus nivolumab, the rates of clinical-to-pathological ypT downstaging were 26% (N=79), 29% (N=6), 38% (N=6) and 59% (N=13), respectively, p =0.012 (table 1). The rates of clinical-to-pathological ypN downstaging in patients with clinical N1 or N2 disease with each therapy were 55% (N=96), 50% (N=3), 50% (N=2), and 42% (N=5) respectively, p =0.862. Overall clinical-to-pathological (ypT and/or ypN) downstaging rates were 38% (N=114), 38% (N=8), 38% (N=6), and 68% (N=15) respectively, p=0.048. The proportions of patients being overall upstaged following each therapy were 28% (N=85), 38% (N=8), 38% (N=6) and 14% (N=3), respectively, p=0.251. These results suggest superior downstaging effect and clinically meaningful lower upstaging probability of combined platinum doublet chemotherapy plus nivolumab as compared to other neoadjuvant regimens. Conclusions The combination of neoadjuvant platinum doublet chemotherapy with nivolumab achieves the most robust tumor and overall pathological downstaging and decreases the probability of upstaging at surgery. Whether the overall downstaging effect results in improved survival will be determined with longer follow-up, in conjunction with results from ongoing phase III neoadjuvant chemo-immunotherapy trials. Trial Registration NCT03158129 Ethics Approval This study was approved by the University of Texas MD Anderson Institutional Review Board with a waiver of informed consent, protocol 2020-0337.

Introduction Plasma-based circulating tumor DNA (ctDNA) is an established biomarker for molecular profiling with emerging applications in disease monitoring in multiple tumor types, including, NSCLC. However, determinants of ctDNA shedding and correlation with tumor burden are incompletely understood, particularly in advanced-stage disease. Methods We retrospectively analyzed ctDNA-based and tissue-based genomic data and imaging from 144 patients with NSCLC. Tumor burden was quantified with computed tomography (CT) and brain magnetic resonance imaging for the overall cohort and 18F-fludeoxyglucose positron emission tomography-CT in a subset of patients. Results There was a moderate but statistically significant correlation between ctDNA variant allele frequency and multiple imaging measures of tumor burden such as CT volume (rho = 0.34, p ≤ 0.0001) and metabolic tumor volume (rho = 0.36, p = 0.003). This correlation was strongest in KRAS-mutant tumors (rho = 0.56, p ≤ 0.001), followed by TP53 mutants (rho = 0.43, p ≤ 0.0001), and weakest in EGFR-mutated (EGFR+) tumors (rho = 0.24, p = 0.077). EGFR+ tumors with EGFR copy number gain had significantly higher variant allele frequency than EGFR+ without copy number gain (p ≤ 0.00001). In multivariable analysis, TP53 and EGFR mutations, visceral metastasis, and tumor burden were independent predictors of increased ctDNA shedding. Conclusions Levels of detectable ctDNA were affected not only by tumor burden but also by tumor genotype. The genotype-specific differences observed may be due to variations in DNA shedding and cellular turnover. These findings have implications for the emerging use of ctDNA in NSCLC disease monitoring and early detection.

Tumor extracellular matrix has been associated with drug resistance and immune suppression. Here, proteomic and RNA profiling reveal increased collagen levels in lung tumors resistant to PD-1/PD-L1 blockade. Additionally, elevated collagen correlates with decreased total CD8+ T cells and increased exhausted CD8+ T cell subpopulations in murine and human lung tumors. Collagen-induced T cell exhaustion occurs through the receptor LAIR1, which is upregulated following CD18 interaction with collagen, and induces T cell exhaustion through SHP-1. Reduction in tumor collagen deposition through LOXL2 suppression increases T cell infiltration, diminishes exhausted T cells, and abrogates resistance to anti-PD-L1. Abrogating LAIR1 immunosuppression through LAIR2 overexpression or SHP-1 inhibition sensitizes resistant lung tumors to anti-PD-1. Clinically, increased collagen, LAIR1, and TIM-3 expression in melanoma patients treated with PD-1 blockade predict poorer survival and response. Our study identifies collagen and LAIR1 as potential markers for immunotherapy resistance and validates multiple promising therapeutic combinations., Tumor extracellular matrix has been associated with cancer progression, therapy resistance and immune suppression. Here, the authors show that collagen generates resistance to PD-1/PD-L1 immunotherapy by upregulating LAIR1 expression and downstream signaling, leading to increased CD8+ T cell exhaustion.

The epithelial-to-mesenchymal transition (EMT) is a dynamic epigenetic reprogramming event that occurs in a subset of tumor cells and is an initiating step toward invasion and distant metastasis. The process is reversible and gives plasticity to cancer cells to survive under variable conditions, with the acquisition of cancer stem cell–like characteristics and features such as drug resistance. Therefore, understanding survival dependencies of cells along the phenotypic spectrum of EMT will provide better strategies to target the spatial and temporal heterogeneity of tumors and prevent their ability to bypass single-inhibitor treatment strategies. To address this, we integrated the data from a selective drug screen in epithelial and mesenchymal KRAS/p53 (KP)-mutant lung tumor cells with separate datasets including reverse-phase protein array and an in vivo shRNA dropout screen. These orthogonal approaches identified AXL and MEK as potential mesenchymal and epithelial cell survival dependencies, respectively. To capture the dynamicity of EMT, incorporation of a dual fluorescence EMT sensor system into murine KP lung cancer models enabled real-time analysis of the epigenetic state of tumor cells and assessment of the efficacy of single agent or combination treatment with AXL and MEK inhibitors. Both two- and three-dimensional culture systems and in vivo models revealed that this combination treatment strategy of MEK plus AXL inhibition synergistically killed lung cancer cells by specifically targeting each phenotypic subpopulation. In conclusion, these results indicate that cotargeting the specific vulnerabilities of EMT subpopulations can prevent EMT-mediated drug resistance, effectively controlling tumor cell growth and metastasis. Significance: This study shows that a novel combination of MEK and AXL inhibitors effectively bypasses EMT-mediated drug resistance in KRAS/p53-mutant non–small cell lung cancer by targeting EMT subpopulations, thereby preventing tumor cell survival.

BackgroundThe biological underpinnings of the prognostic and predictive significance of a relative neutrophilia in patients with non-small lung cancer (NSCLC) are undefined. We sought to comprehensively examine the relationships between circulating and intratumoral neutrophil populations and features of the immune contexture in patients undergoing NSCLC resection.MethodsPreoperative soluble cytokine and angiogenic factors; tumor multiplex immunofluorescence; RNA, whole exome, and T-cell receptor sequencing; and flow cytometry were analyzed for relationships with populations of circulating (from complete blood counts) and intratumoral neutrophils (transcriptional signatures) in a prospectively enrolled resected NSCLC cohort (n=66). In a historical cohort (n=1524), preoperative circulating neutrophil and lymphocyte counts were analyzed for associations with overall survival (OS).ResultsCirculating neutrophil populations were positively correlated with increased tumor burden, and surgical tumor resection was followed by a subsequent reduction in peripheral neutrophil counts. Expansion of the circulating neutrophil compartment was associated with increased levels of pro-granulopoietic (IL-1β, IL-17A, TNFα, IL-6) and TH2-associated (IL-5, IL-13) cytokines. Tumors with high intratumoral neutrophil burden were marked by a blunted T-cell response characterized by reduced expression of cytotoxic T-cell genes (CD8A,CD8B,GZMA,GZMB), decreased CD3+CD8+cell infiltration, and diminished expression of IFNγ-related genes. The associations between increased intratumoral neutrophil burden and reduced CD3+CD8+infiltration persisted after adjustment for tumor size, histology, mutational burden, and PD-L1 expression. In 1524 patients, elevated preoperative circulating neutrophil count was independently associated with worse OS (main effect HR 1.82, 95% CI 1.24 to 2.68, p=0.002).ConclusionsOur findings demonstrate that neutrophil expansion reflects protumorigenic and immunosuppressive processes that manifest as worse OS in patients undergoing NSCLC resection. These results justify further investigation of therapeutic strategies targeting neutrophil-associated immune evasion.

Understanding resistance mechanisms to targeted therapies and immune checkpoint blockade in mutant KRAS lung cancers is critical to developing novel combination therapies and improving patient survival. Here, we show that MEK inhibition enhanced PD-L1 expression while PD-L1 blockade upregulated MAPK signaling in mutant KRAS lung tumors. Combined MEK inhibition with anti-PD-L1 synergistically reduced lung tumor growth and metastasis, but tumors eventually developed resistance to sustained combinatorial therapy. Multi-platform profiling revealed that resistant lung tumors have increased infiltration of Th17 cells, which secrete IL-17 and IL-22 cytokines to promote lung cancer cell invasiveness and MEK inhibitor resistance. Antibody depletion of IL-17A in combination with MEK inhibition and PD-L1 blockade markedly reduced therapy-resistance in vivo. Clinically, increased expression of Th17-associated genes in patients treated with PD-1 blockade predicted poorer overall survival and response in melanoma and predicated poorer response to anti-PD1 in NSCLC patients. Here we show a triple combinatorial therapeutic strategy to overcome resistance to combined MEK inhibitor and PD-L1 blockade. Recent clinical trials combining MEK inhibitors with anti-PD-L1 in solid tumours show moderate responses. Here, the authors demonstrate that the combination of MEK inhibition and PD-L1 blockade in KRAS mutant lung cancer models leads to a transient tumour regressions and resistance due to increased infiltration of Th17 cells and that the triple therapy targeting MEK, PD-L1 and IL-17 produced better in vivo responses.

Objectives The influence of surgical approach on systemic inflammatory response and the subsequent oncologic impact for non-small cell lung cancer is debated. We aimed to measure the effects of thoracic surgical approach on peripheral cytokine milieu over time. Methods Patients undergoing primary lung resection without neoadjuvant therapy (2016–2018) were evaluated. A panel of 43 cytokines, angiogenic factors, and inflammatory molecules (CAFs) were evaluated in peripheral blood preoperatively, at 24 -hs and 4-weeks postoperatively. Differences between CAFs in patients undergoing thoracotomy versus video-assisted thoracoscopic surgery (VATS) at all timepoints were assessed using Student’s t-test. Results 76 patients with available peripheral CAF panels met inclusion criteria. Thoracotomy was performed in 53 (70 %) patients while VATS was undertaken in 23 (30 %). Upon examination of known inflammatory CAFs, including IL-1β, IL-6, IL-8, IL-10, IFN-γ, and soluble (s) CD27, no differences were detected at 24 h or 4 weeks postoperatively between surgical groups. Examination of trends over time did not demonstrate any temporal derangements for these CAFs, with return to baseline levels by 4 weeks postoperatively for both groups. Evaluation of soluble (s) checkpoint molecules, including sPD-1, sPD-L1, sTIM-3, and sCTLA-4, did not reveal any differences in the immediate postoperative or long-term recovery period. Conclusions Peripheral immune profiles following pulmonary resection do not appear to differ between VATS and thoracotomy postoperatively. CAF fluctuations are transient and recover rapidly. These results, at the peripheral cytokine level, suggest that the surgical approach for lung cancer is unlikely to alter the effectiveness of novel immune-modulating systemic therapies, although more studies are needed to validate these findings.

Genetic aberrations driving pro-oncogenic and pro-metastatic activity remain an elusive target in the quest of precision oncology. To identify such drivers, we use an animal model of KRAS-mutant lung adenocarcinoma to perform an in vivo functional screen of 217 genetic aberrations selected from lung cancer genomics datasets. We identify 28 genes whose expression promoted tumor metastasis to the lung in mice. We employ two tools for examining the KRAS-dependence of genes identified from our screen: 1) a human lung cell model containing a regulatable mutant KRAS allele and 2) a lentiviral system permitting co-expression of DNA-barcoded cDNAs with Cre recombinase to activate a mutant KRAS allele in the lungs of mice. Mechanistic evaluation of one gene, GATAD2B, illuminates its role as a dual activity gene, promoting both pro-tumorigenic and pro-metastatic activities in KRAS-mutant lung cancer through interaction with c-MYC and hyperactivation of the c-MYC pathway., KRAS-driven lung cancers represent an aggressive form of NSCLC. In this study the authors perform an in vivo functional screening and identify GATAD2B as a driver of tumor growth and metastasis in KRAS-driven lung cancer.

BackgroundWhile immune checkpoint blockade is regarded as standard of care for treatment of non-small cell lung cancer (NSCLC), up to 50% of patients with metastatic NSCLC do not achieve an optimal response.1–3 Previous work by our group and others in adoptive cell therapy (ACT) of metastatic melanoma (MM) has shown that infusion of a CD8+-rich TIL product significantly improved clinical outcomes, yet traditional IL-2 expansion methods have resulted in a predominantly CD4+ NSCLC TIL expansion product.7–12 This preclinical study explores the feasibility of producing a tumor-specific, CD8+-enriched NSCLC TIL product for ACT with an improved culture method.MethodsTIL from resected NSCLC tumors were cultured using 1) the traditional method using IL-2 alone in 24-well plates (TIL 1.0) or 2) IL-2 in combination with agonistic antibodies against CD3 and 4-1BB (Urelumab) in a G-Rex flask (TIL 3.0). Expanded TIL were phenotyped using flow cytometry for CD4 and CD8 subset assessment and the CDR3-beta variable region of the T-cell receptor (TCR) involved in antigen binding was sequenced to assess the T-cell repertoire.ResultsIn a shorter manufacturing time (median of 14 days vs 27.5 days), TIL 3.0 expanded on average 5.3-times more NSCLC TIL (95% CI= 4.3–6.2, pConclusionsThis study reports the feasibility of using the TIL 3.0 methodology to robustly expand a CD8+ T-cell repertoire which maintains the respective clonal hierarchy in NSCLC tumors and enriches for putative tumor-specific TIL clones. The robustness and speed of the new process may facilitate testing and implementing effective TIL ACT in NSCLC.ReferencesGaron EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, et al. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med 2015;372(21):2018–28.Borghaei H, Paz-Ares L, Horn L, Spigel DR, Steins M, Ready NE, et al. Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. N Engl J Med 2015;373(17):1627–39.Gettinger S, Horn L, Jackman D, Spigel D, Antonia S, Hellmann M, et al. Five-Year Follow-Up of Nivolumab in Previously Treated Advanced Non-Small-Cell Lung Cancer: Results from the CA209–003 Study. J Clin Oncol 2018;36(17):1675–84.Melioli G, Ratto G, Guastella M, Meta M, Biassoni R, Semino C, et al. Isolation and in vitro expansion of lymphocytes infiltrating non-small cell lung carcinoma: functional and molecular characterisation for their use in adoptive immunotherapy. Eur J Cancer 1994;30A(1):97–102.McGranahan N, Furness AJ, Rosenthal R, Ramskov S, Lyngaa R, Saini SK, et al. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science 2016;351(6280):1463–9.Rosenberg SA, Yang JC, Sherry RM, Kammula US, Hughes MS, Phan GQ, et al. Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy. Clin Cancer Res 2011;17(13):4550–7.Besser MJ, Shapira-Frommer R, Treves AJ, Zippel D, Itzhaki O, Hershkovitz L, et al. Clinical responses in a phase II study using adoptive transfer of short-term cultured tumor infiltration lymphocytes in metastatic melanoma patients. Clin Cancer Res 2010;16(9):2646–55.Pilon-Thomas S, Kuhn L, Ellwanger S, Janssen W, Royster E, Marzban S, et al. Efficacy of adoptive cell transfer of tumor-infiltrating lymphocytes after lymphopenia induction for metastatic melanoma. J Immunother 2012;35(8):615–20.Radvanyi LG, Bernatchez C, Zhang M, Fox PS, Miller P, Chacon J, et al. Specific Lymphocyte Subsets Predict Response to Adoptive Cell Therapy Using Expanded Autologous Tumor-Infiltrating Lymphocytes in Metastatic Melanoma Patients. Clinical Cancer Research 2012;18(24):6758–70.Forget MA, Haymaker C, Hess KR, Meng YJ, Creasy C, Karpinets T, et al. Prospective Analysis of Adoptive TIL Therapy in Patients with Metastatic Melanoma: Response, Impact of Anti-CTLA4, and Biomarkers to Predict Clinical Outcome. Clin Cancer Res 2018;24(18):4416–28.Ben-Avi R, Farhi R, Ben-Nun A, Gorodner M, Greenberg E, Markel G, et al. Establishment of adoptive cell therapy with tumor infiltrating lymphocytes for non-small cell lung cancer patients. Cancer Immunol Immunother 2018;67(8):1221–30.Ma Y, Ou J, Lin T, Chen L, Wang J, Qiao D, et al. Phenotypic analysis of tumor-infiltrating lymphocytes from non-small cell lung cancer and their potential application for adoptive cell therapy. Immunopharmacol Immunotoxicol 2020;42(4):319–29Ethics ApprovalThis study was performed on NSCLC tumor tissue resected from 16 patients enrolled, following informed consent, in the ImmunogenomiC prOfiling of early-stage NSCLC (ICON) project. This study was approved by the University of Texas MD Anderson Cancer Center‘s Institutional Review Board (protocol number PA15-1112_MODCR001).

Immunotherapies targeting immune checkpoints have proven efficacious in reducing the burden of lung cancer in patients; however, the antigenic targets of these reinvigorated T cells remain poorly defined. Lung cancer tumors contain tumor-associated macrophages (TAM) and neutrophils, which release the serine proteases neutrophil elastase (NE) and proteinase 3 (P3) into the tumor microenvironment. NE and P3 shape the antitumor adaptive immune response in breast cancer and melanoma. In this report, we demonstrate that lung cancer cells cross-presented the tumor-associated antigen PR1, derived from NE and P3. Additionally, NE and P3 enhanced the expression of human leukocyte antigen (HLA) class I molecules on lung cancer cells and induced unique, endogenous peptides in the immunopeptidome, as detected with mass spectrometry sequencing. Lung cancer patient tissues with high intratumoral TAMs were enriched for MHC class I genes and T-cell markers, and patients with high TAM and cytotoxic T lymphocyte (CTL) infiltration had improved overall survival. We confirmed the immunogenicity of unique, endogenous peptides with cytotoxicity assays against lung cancer cell lines, using CTLs from healthy donors that had been expanded against select peptides. Finally, CTLs specific for serine proteases–induced endogenous peptides were detected in lung cancer patients using peptide/HLA-A2 tetramers and were elevated in tumor-infiltrating lymphocytes. Thus, serine proteases in the tumor microenvironment of lung cancers promote the presentation of HLA class I immunogenic peptides that are expressed by lung cancer cells, thereby increasing the antigen repertoire that can be targeted in lung cancer. Cancer Immunol Res; 5(4); 319–29. ©2017 AACR.

Radiographic imaging is the standard approach for evaluating the disease involvement of lymph nodes in patients with operable NSCLC although the impact of neoadjuvant immune checkpoint inhibitors (ICIs) on lymph nodes has not yet been characterized. Herein, we present an ad hoc analysis of the NEOSTAR trial (NCT03158129) where we observed a phenomenon we refer to as “nodal immune flare” (NIF) in which patients treated with neoadjuvant ICIs demonstrate radiologically abnormal nodes post-therapy that upon pathological evaluation are devoid of cancer and demonstrate de novo non-caseating granulomas. Abnormal lymph nodes are analyzed by computed tomography and 18F-fluorodeoxyglucose positron emission tomography/computer tomography to evaluate the size and the maximum standard uptake value post- and pre-therapy in NEOSTAR and an independent neoadjuvant chemotherapy cohort. NIF occurs in 16% (7/44) of patients treated with ICIs but in 0% (0/28) of patients after neoadjuvant chemotherapy. NIF is associated with an inflamed nodal immune microenvironment and with fecal abundance of genera belonging to the family Coriobacteriaceae of phylum Actinobacteria, but not with tumor responses or treatment-related toxicity. Our findings suggest that this apparent radiological cancer progression in lymph nodes may occur due to an inflammatory response after neoadjuvant immunotherapy, and such cases should be evaluated by pathological examination to distinguish NIF from true nodal progression and to ensure appropriate clinical treatment planning., Granulomatous/sarcoid-like lesions have been reported in patients treated with immune checkpoint inhibitors (ICIs). Here the authors report the occurrence of “nodal immune flare”, an apparent radiological cancer progression in the nodes characterized by the absence of cancer and the presence of non-caseating granulomas, in patients with non-small cell lung cancer following neoadjuvant ICI treatment.

Heightened secretion of protumorigenic effector proteins is a feature of malignant cells. Yet, the molecular underpinnings and therapeutic implications of this feature remain unclear. Here, we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIβ (PI4KIIIβ). Molecular, biochemical, and cell biological studies show that PI4KIIIβ-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIβ-dependent secreted factors maintain 1q-amplified cancer cell survival and influence prometastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIβ antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIβ-dependent secretion for cancer cell survival and tumor progression.

BACKGROUND: The tumor immune microenvironment (TIME) of lung cancer brain metastasis is largely unexplored. We carried out immune profiling and sequencing analysis of paired resected primary tumors and brain metastases of non-small-cell lung carcinoma (NSCLC). PATIENTS AND METHODS: TIME profiling of archival formalin-fixed and paraffin-embedded specimens of paired primary tumors and brain metastases from 39 patients with surgically resected NSCLCs was carried out using a 770 immune gene expression panel and by T-cell receptor beta repertoire (TCRβ) sequencing. Immunohistochemistry was carried out for validation. Targeted sequencing was carried out to catalog hot spot mutations in cancer genes. RESULTS: Somatic hot spot mutations were mostly shared between both tumor sites (28/39 patients; 71%). We identified 161 differentially expressed genes, indicating inhibition of dendritic cell maturation, Th1, and leukocyte extravasation signaling pathways, in brain metastases compared with primary tumors (P

Introduction Although the combination of anti–programmed cell death-1 or anti–programmed cell death ligand-1 (PD-L1) with platinum chemotherapy is a standard of care for NSCLC, clinical responses vary. Even though predictive biomarkers (which include PD-L1 expression, tumor mutational burden, and inflamed immune microenvironment) are validated for immunotherapy, their relevance to chemoimmunotherapy combinations is less clear. We have recently reported that activation of the stimulator of interferon genes (STING) innate immune pathway enhances immunotherapy response in SCLC. Here, we hypothesize that STING pathway activation may predict and underlie predictive correlates of antitumor immunity in NSCLC. Methods We analyzed transcriptomic and proteomic profiles in two NSCLC cohorts from our institution (treatment-naive patients in the Profiling of Resistance Patterns and Oncogenic Signaling Pathways in Evaluation of Cancers of the Thorax study and relapsed patients in the Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination study) and The Cancer Genome Atlas (N = 1320). Tumors were stratified by STING activation on the basis of protein or mRNA expression of cyclic GMP-AMP synthase, phospho-STING, and STING-mediated chemokines (chemokine ligand 5 [CCL5] and C-X-C motif chemokine 10 [CXCL10]). STING activation in patient tumors and in platinum-treated preclinical NSCLC models was correlated with biomarkers of immunotherapy response. Results STING activation is associated with higher levels of intrinsic DNA damage, targetable immune checkpoints, and chemokines in treatment-naive and relapsed lung adenocarcinoma. We observed that tumors with lower STING and immune gene expression show higher frequency of serine-threonine kinase 11 (STK11) mutations; however, we identified a subset of these tumors that are TP53 comutated and display high immune- and STING-related gene expression. Treatment with cisplatin increases STING pathway activation and PD-L1 expression in multiple NSCLC preclinical models, including adeno- and squamous cell carcinoma. Conclusions STING pathway activation in NSCLC predicts features of immunotherapy response and is enhanced by cisplatin treatment. This suggests a possible predictive biomarker and mechanism for improved response to chemoimmunotherapy combinations.

PURPOSE Our previously published findings reported that local consolidative therapy (LCT) with radiotherapy or surgery improved progression-free survival (PFS) and delayed new disease in patients with oligometastatic non–small-cell lung cancer (NSCLC) that did not progress after front-line systemic therapy. Herein, we present the longer-term overall survival (OS) results accompanied by additional secondary end points. PATIENTS AND METHODS This multicenter, randomized, phase II trial enrolled patients with stage IV NSCLC, three or fewer metastases, and no progression at 3 or more months after front-line systemic therapy. Patients were randomly assigned (1:1) to maintenance therapy or observation (MT/O) or to LCT to all active disease sites. The primary end point was PFS; secondary end points were OS, toxicity, and the appearance of new lesions. All analyses were two sided, and P values less than .10 were deemed significant. RESULTS The Data Safety and Monitoring Board recommended early trial closure after 49 patients were randomly assigned because of a significant PFS benefit in the LCT arm. With an updated median follow-up time of 38.8 months (range, 28.3 to 61.4 months), the PFS benefit was durable (median, 14.2 months [95% CI, 7.4 to 23.1 months] with LCT v 4.4 months [95% CI, 2.2 to 8.3 months] with MT/O; P = .022). We also found an OS benefit in the LCT arm (median, 41.2 months [95% CI, 18.9 months to not reached] with LCT v 17.0 months [95% CI, 10.1 to 39.8 months] with MT/O; P = .017). No additional grade 3 or greater toxicities were observed. Survival after progression was longer in the LCT group (37.6 months with LCT v 9.4 months with MT/O; P = .034). Of the 20 patients who experienced progression in the MT/O arm, nine received LCT to all lesions after progression, and the median OS was 17 months (95% CI, 7.8 months to not reached). CONCLUSION In patients with oligometastatic NSCLC that did not progress after front-line systemic therapy, LCT prolonged PFS and OS relative to MT/O.

Immunotherapy targeting T cells is increasingly utilized to treat solid tumors including non-small cell lung cancer (NSCLC). This requires a better understanding of the T cells in the lungs of patients with NSCLC. Here, we report T cell repertoire analysis in a cohort of 236 early-stage NSCLC patients. T cell repertoire attributes are associated with clinicopathologic features, mutational and immune landscape. A considerable proportion of the most prevalent T cells in tumors are also prevalent in the uninvolved tumor-adjacent lungs and appear specific to shared background mutations or viral infections. Patients with higher T cell repertoire homology between the tumor and uninvolved tumor-adjacent lung, suggesting a less tumor-focused T cell response, exhibit inferior survival. These findings indicate that a concise understanding of antigens and T cells in NSCLC is needed to improve therapeutic efficacy and reduce toxicity with immunotherapy, particularly adoptive T cell therapy., Relevant features of T cell repertoire in human cancer remain to be delineated. Here the authors show, by TCR sequencing in a large cohort of lung cancer patients, that while a majority of T cell clones are shared between tumor and adjacent lung tissue, less frequent tumor-unique T cell clones correlate with worse prognosis.

The implementation of cancer immunotherapeutics for solid tumors including lung cancers has improved clinical outcomes in a small percentage of patients. However, the majority of patients show little to no response or acquire resistance during treatment with checkpoint inhibitors delivered as a monotherapy. Therefore, identifying resistance mechanisms and novel combination therapy approaches is imperative to improve responses to immune checkpoint inhibitors. To address this, we performed an in vivo shRNA dropout screen that focused on genes encoding for FDA-approved drug targets (FDAome). We implanted epithelial and mesenchymal Kras/p53 (KP) mutant murine lung cancer cells expressing the FDAome shRNA library into syngeneic mice treated with an anti-PD-1 antibody. Sequencing for the barcoded shRNAs revealed Ntrk1 was significantly depleted from mesenchymal tumors challenged with PD-1 blockade, suggesting it provides a survival advantage to tumor cells when under immune system pressure. Our data confirmed Ntrk1 transcript levels are upregulated in tumors treated with PD-1 inhibitors. Additionally, analysis of tumor-infiltrating T cell populations revealed that Ntrk1 can promote CD8+ T cell exhaustion. Lastly, we found that Ntrk1 regulates Jak/Stat signaling to promote expression of PD-L1 on tumor cells. Together, these data suggest that Ntrk1 activates Jak/Stat signaling to regulate expression of immunosuppressive molecules including PD-L1, promoting exhaustion within the tumor microenvironment.

Background: We have previously demonstrated that the presence of a proinflammatory peripheral cytokine milieu correlates with high levels of circulating neutrophils at the time of surgery and reduced overall survival and recurrence-free survival. In this study, we hypothesized that functional immune features or states in circulation may indicate early tumor recurrence when assessed longitudinally. We also investigated whether potential deficiencies in peripheral immune functionality at the time of lung cancer resection could identify correlates with subsequent outcome. Methods: We performed flow cytometry and luminex profiling of blood samples collected from patients with stage I-IIIA resected NSCLC (n=150) and enrolled on the Immunogenomic profiling of NSCLC (ICON) prospective protocol. Patient characteristics include 75 adenocarcinomas, 30 squamous and 12 mixed or other histologies. Only patients who underwent primary cancer resection without neoadjuvant therapy were included. At a median follow up of 18.2 months, 37 patients had disease recurrence. Blood was collected at the time of primary lung cancer resection, at 4 weeks, and 4 months thereafter with PBMCs utilized for flow cytometry and plasma for cytokine assessment. Changes in cytokines were assessed by normalizing to baseline levels. Results: Larger tumors as well as advanced clinical and pathological stages were associated with higher frequencies of proliferating Ki67+CD4+ and Ki67+CD8+ T cells in circulation at time of resection, suggesting an activated circulating immune response. We identified novel strong correlations in the plasma between soluble BTLA and Tim3 (r= 0.87, p=1.74e-140), PD1 and CD80 (r=0.72, p=6.41e-74) and moderate correlations between soluble PD1 and PDL1 (r=0.39, p=8.41e-18). Efforts are ongoing to determine the association of specific circulating immune states with the presence of these soluble receptors. CD8+Tim3+ T cells (FC=5.2%, p=0.028) and CTLA4+NK cells (FC=47.7%, p=0.002) as well as CTLA4+Tregs (FC=25.2%, p=0.05) were found to be increased in circulation at pre-recurrence time points (either 4 weeks or 4 months) relative to resection. This suggests the emergence of a suppressive cell type as well as induction of specific checkpoint receptors on effector cells that correlate with tumor recurrence. Finally, we identified a cytokine signature associated with recurrence by testing three sets within the ICON cohort with a training set AUC = 0.76 and the test set AUC=0.72, which was validated in a third set of patients yielding an AUC = 0.76. Conclusions: We identified circulating immune features associated with initial tumor size and overall stage as well as unique associations among soluble proteins. Increased presence of potentially inhibited or suppressed CD8+ T cells and NK cells as well as Tregs are suggestive of mechanisms of immune suppression relative to tumor recurrence. Citation Format: Younghee Lee, Daniel McGrail, Hai Tran, Mayra E. Vasquez, Carlos Ramos, Alexandre Reuben, Ara A. Vaporciyan, Annikka Weissferdt, Chantale Bernatchez, Tina Cascone, Ignacio I. Wistuba, Jianjun Zhang, John Heymach, Marcelo V. Negrao, Don L. Gibbons, Boris Sepesi, Cara L. Haymaker. Circulating biomarkers are associated with recurrence following complete resection of non-small cell lung cancer [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 1670.

Introduction: Differences in the host immune environment are thought to mediate heterogeneous treatment responses in non-small cell lung cancer (NSCLC). Unlike individual platform analyses, integrative analysis of multi-platform profiling allows for the discovery of novel interactions that expand our understanding of the disease. Utilizing the ImmunogenomiC prOfiling of NSCLC patient cohort (ICON), a prospective multi-omics protocol of operable early-stage NSCLC tumors with integrated immune, genomic, and clinical data, we hypothesized that multi-platform analyses would identify differences in the immune-genomic landscape that are associated with disease recurrence. Methods: Tumor and tumor-adjacent uninvolved lung was collected at resection; blood was collected before and after surgery. Tissue samples underwent WES, RNAseq, TCR sequencing (TCRseq), multiplex immunofluorescence (mIF), and RPPA profiling; tissue and blood (PBMC) samples were analyzed by flow cytometry. An integrated, inter-modality network was built using Spearman correlations between measurement pairs from different data modalities. Multivariate analysis was performed to adjust for stage and histology. Results: A total of 89 treatment-naïve patients with Stage 1-3 resected NSCLC (Squamous: 19; Non-squamous: 70) and 24 months of follow-up were analyzed (recurrence N = 24; no recurrence N = 65). The data network includes over 4,000 measurements linked by over 50,000 correlations. InfoMap, a community detection approach, was used to extract sub-network modules, which were used to contextualize the results of multivariate analysis. Tumors from patients with recurrence demonstrated decreased immune cell infiltration and activation including decreased cytotoxic CD8 T-cells (CD8+PD1+; fold-change (FC) = 0.898, p = 0.018; flow cytometry), decreased T-cell clonality (FC = 0.954, p = 0.017; TCRseq), and decreased tumor-associated macrophages (CD68+PD-L1+; FC = 0.426, p = 0.011; mIF). Furthermore, circulating CD8+ICOS+ activated T cells were decreased in patients with recurrence suggesting an impaired systemic anti-tumor immune response (FC = 0.552, p = 0.042; PBMC Flow). Finally, tumor-adjacent uninvolved lungs showed distinct T-cell phenotypes with accumulation of inactive CD8 T-cells (CD8+PD1-TIM3-) in patients with recurrence and increased populations of activated CD8 T-cells (CD8+PD1+) in patients without recurrence. Conclusion: Integrative multi-omic analysis suggests preserved anti-tumor immune surveillance in patients who are disease-free after 2 years from surgical resection with curative intent for treatment of NSCLC relative to patients with disease recurrence. Further analysis is ongoing to interrogate genomic and immune variables that are associated with disease recurrence. Citation Format: Neal Akhave, Stephanie Schmidt, Alexandre Reuben, Tina Cascone, Jianhua Zhang, Jun Li, Junya Fujimoto, Lauren A. Byers, Beatriz Sanchez-Espiridion, Lixia Diao, Jing Wang, Lorenzo Federico, Marie-Andree Forget, Daniel J. McGrail, Annikka Weissferdt, Shiaw-Yih Lin, Younghee Lee, Carmen Behrens, Ignacio I. Wistuba, Andrew Futreal, Ara Vaporciyan, Boris Sepesi, John V. Heymach, Chantale Bernatchez, Cara Haymaker, Jianjun Zhang, Christopher A. Bristow, Marcelo V. Negrao, Don L. Gibbons. Integrated multi-platform profiling of early-stage non-small cell lung cancer identifies relationship between disease recurrence and decreased native immune response in treatment-naïve resected NSCLC [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 619.

Lung adenocarcinoma (LUAD) is the most commonly diagnosed histological subtype of lung cancer. While earlier work has underscored genomic and immune alterations in LUAD, the roles of individual cell populations in early-stage human LUAD evolution in space remain unknown. Here, we provide a detailed cellular atlas of early-stage LUAD and its spatial ecosystem along the peripheral lung. We performed single-cell RNA sequencing of 186,916 cells including enriched epithelial fractions from five early-stage LUADs with fourteen multi-region normal lung tissues of defined spatial proximities from the primary LUADs. We show that major epithelial and immune cellular lineages, states, and transcriptomic features geospatially and progressively evolve across normal regions and with increasing LUAD proximity. Analysis of 70,030 lung epithelial cells unraveled diverse lineage trajectories, transcriptional lineage plasticity programs underlying KRAS-mutant cells, and intratumoral heterogeneity within single sites. T regulatory cell programs including multiple immune checkpoints increased in tissues with closer proximity to LUADs, in sharp contrast to signatures of CD8+ cytotoxic T cells, antigen presentation by macrophages, and inflammatory dendritic cells. We found that some spatial signatures (e.g. a B cell signature score) were increased along the pathologic spectrum of normal lung, preneoplastic lesions, and matched invasive LUADs. LUAD cell-cell communication networks were enriched with ligand-receptor interactions involving CD24, LGALS9 and TIM3 immune checkpoints, including crosstalk between CD24 antigen in LUAD epithelial cells and SIGLEC10 in myeloid subsets. CD24 was markedly increased in preneoplasias relative to normal lung and further in LUAD, and its expression was highly positively correlated with immunosuppressive phenotypes. These data provide an atlas of cellular states and phenotypes underlying early-stage LUAD evolution in space, and a scalable resource for identification of targets for early treatment. Citation Format: Ansam Sinjab, Guangchun Han, Warapen Treekitkarnmongkol, Kieko Hara, Patrick Brennan, Minghao Dang, Dapeng Hao, Ruiping Wang, Enyu Dai, Hitoshi Dejima, Jiexin Zhang, Elena Bogatenkova, Beatriz Sanchez-Espiridion, Kyle Chang, Danielle R. Little, Samer Bazzi, Linh Tran, Kostyantyn Krysan, Carmen Behrens, Dzifa Duose, Edwin R. Parra, Maria Gabriela Raso, Luisa M. Solis, Junya Fukuoka, Jianjun Zhang, Boris Sepesi, Tina Cascone, Lauren A. Byers, Don L. Gibbons, Jichao Chen, Seyed Javad Moghaddam, Edwin J. Ostrin, Daniel G. Rosen, John V. Heymach, Paul Scheet, Steven Dubinett, Ignacio I. Wistuba, Junya Fujimoto, Christopher S. Stevenson, Avrum E. Spira, Linghua Wang, Humam Kadara. Resolving the spatial and cellular architecture of lung adenocarcinoma by multi-region single-cell sequencing [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 130.

The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Severely symptomatic COVID-19 is associated with lung inflammation, pneumonia, and respiratory failure, thereby raising concerns of elevated risk of COVID-19-asociated mortality among lung cancer patients. Angiotensin converting enzyme 2 (ACE2) is the major receptor for SARS-CoV-2 entry into lung cells. Yet, the single-cell expression landscape of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung cancer patients remains unknown. To fill these voids, we leveraged our ongoing efforts in single-cell transcriptomic analysis of 186,916 cells including a large number of epithelial cells (n=70,030) derived from 5 LUADs and 14 matching uninvolved normal lung tissues, to delineate expression levels and cellular distribution of ACE2 and SARS-CoV-2 priming proteases TMPRSS2 and TMPRSS4. Single-cell RNA sequencing of 186,916 cells revealed epithelial-specific expression of ACE2, TMPRSS2 and TMPRSS4. Analysis of 70,030 LUAD- and normal-derived epithelial cells showed that ACE2 levels were highest in normal alveolar type 2 (AT2) cells and that TMPRSS2 was expressed in 65% of normal AT2 cells. Conversely, expression of TMPRSS4 was highest and most frequently detected (75%) in malignant lung cells. ACE2-positive cells co-expressed genes implicated in lung pathobiology, including COPD-associated HHIP, and the scavengers CD36 and DMBT1. Notably, the viral scavenger DMBT1 was significantly positively correlated with ACE2 expression in AT2 cells. In conclusion, we describe normal and tumor lung epithelial populations that express SARS-CoV-2 receptor and proteases, as well as major host defense genes, and that thus comprise potential treatment targets for COVID-19 particularly among lung cancer patients. Citation Format: Guangchun Han, Ansam Sinjab, Kieko Hara, Warapen Treekitkarnmongkol, Patrick Brennan, Kyle Chang, Elena Bokatenkova, Beatriz Sanchez-Espiridion, Carmen Behrens, Luisa M. Solis, Boning Gao, Luc Girard, Jianjun Zhang, Boris Sepesi, Tina Cascone, Lauren A. Byers, Don L. Gibbons, Jichao Chen, Seyed Javad Moghaddam, Edwin J. Ostrin, Paul Scheet, Junya Fujimoto, Jerry Shay, John V. Heymach, John D. Minna, Steven Dubinett, Ignacio I. Wistuba, Christopher S. Stevenson, Avrum E. Spira, Linghua Wang, Humam Kadara. Single-cell expression landscape of SARS-CoV-2 receptor ACE2 and host proteases in human lung adenocarcinoma [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 702.

Non-small-cell lung cancer (NSCLC) demonstrates remarkable molecular diversity. With the completion of The Cancer Genome Atlas (TCGA), there is opportunity for systematic analyses of the entire TCGA NSCLC cohort, including comparisons and contrasts between different disease subsets. On the basis of multidimensional and comprehensive molecular characterization (including DNA methylation and copy, and RNA and protein expression), 1023 NSCLC cases-519 from TCGA adenocarcinoma (AD) project and 504 from TCGA squamous cell carcinoma (SQCC) project-were classified using a 'cluster-of-clusters' analytic approach. Patterns from TCGA NSCLC subsets were examined in independent external databases, including the PROSPECT (Profiling of Resistance patterns and Oncogenic Signaling Pathways in Evaluation of Cancers of the Thorax) NSCLC data set. Nine genomic subtypes of NSCLC were identified, three within SQCC and six within AD. SQCC subtypes were associated with transcriptional targets of SOX2 or p63. One predominately AD subtype (with a large proportion of SQCC) shared molecular features with neuroendocrine tumors. Two AD subtypes manifested a CpG island methylator phenotype. Three AD subtypes showed high p38 and mTOR pathway activation. AD subtypes associated with low differentiation showed relatively worse prognosis. SQCC subtypes and two of the AD subtypes expressed cancer testis antigen genes, whereas three AD subtypes expressed several immune checkpoint genes including PDL1 and PDL2, corresponding with patterns of greater immune cell infiltration. Subtype associations for several immune-related markers-including PD1, PDL1, CD3 and CD8-were confirmed in the PROSPECT cohort using immunohistochemistry. NSCLC molecular subtypes have therapeutic implications and lend support to a personalized approach to NSCLC management based on molecular characterization.

Purpose By applying the principles of real-time biopsy, biomarker-based, adaptively randomized studies in non–small-cell lung cancer (NSCLC) established by the Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) trial, we conducted BATTLE-2 (BATTLE-2 Program: A Biomarker-Integrated Targeted Therapy Study in Previously Treated Patients With Advanced Non-Small Cell Lung Cancer), an umbrella study to evaluate the effects of targeted therapies focusing on KRAS-mutated cancers. Patients and Methods Patients with advanced NSCLC (excluding sensitizing EGFR mutations and ALK gene fusions) refractory to more than one prior therapy were randomly assigned, stratified by KRAS status, to four arms: (1) erlotinib, (2) erlotinib plus MK-2206, (3) MK-2206 plus AZD6244, or (4) sorafenib. Tumor gene expression profiling–targeted next-generation sequencing was performed to evaluate predictive and prognostic biomarkers. Results Two hundred patients, 27% with KRAS-mutated (KRAS mut+) tumors, were adaptively randomly assigned to erlotinib (n = 22), erlotinib plus MK-2206 (n = 42), MK-2206 plus AZD6244 (n = 75), or sorafenib (n = 61). In all, 186 patients were evaluable, and the primary end point of an 8-week disease control rate (DCR) was 48% (arm 1, 32%; arm 2, 50%; arm 3, 53%; and arm 4, 46%). For KRAS mut+ patients, DCR was 20%, 25%, 62%, and 44% whereas for KRAS wild-type patients, DCR was 36%, 57%, 49%, and 47% for arms 1, 2, 3, and 4, respectively. Median progression-free survival was 2.0 months, not different by KRAS status, 1.8 months for arm 1, and 2.5 months for arms 2 versus arms 3 and 4 in KRAS mut+ patients (P = .04). Median overall survival was 6.5 months, 9.0 and 5.1 months for arms 1 and 2 versus arms 3 and 4 in KRAS wild-type patients (P = .03). Median overall survival was 7.5 months in mesenchymal versus 5 months in epithelial tumors (P = .02). Conclusion Despite improved progression-free survival on therapy that did not contain erlotinib for KRAS mut+ patients and improved prognosis for mesenchymal tumors, better biomarker-driven treatment strategies are still needed.

Purpose: Promising results in the treatment of non–small cell lung cancer (NSCLC) have been seen with agents targeting immune checkpoints, such as programmed cell death 1 (PD-1) or programmed death ligand-1 (PD-L1). However, only a select group of patients respond to these interventions. The identification of biomarkers that predict clinical benefit to immune checkpoint blockade is critical to successful clinical translation of these agents. Methods: We conducted an integrated analysis of three independent large datasets, including The Cancer Genome Atlas of lung adenocarcinoma and two datasets from MD Anderson Cancer Center (Houston, TX), Profiling of Resistance Patterns and Oncogenic Signaling Pathways in Evaluation of Cancers of the Thorax (named PROSPECT) and Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (named BATTLE-1). Comprehensive analysis of mRNA gene expression, reverse-phase protein array, IHC, and correlation with clinical data were performed. Results: Epithelial–mesenchymal transition (EMT) is highly associated with an inflammatory tumor microenvironment in lung adenocarcinoma, independent of tumor mutational burden. We found immune activation coexistent with elevation of multiple targetable immune checkpoint molecules, including PD-L1, PD-L2, PD-1, TIM-3, B7-H3, BTLA, and CTLA-4, along with increases in tumor infiltration by CD4+Foxp3+ regulatory T cells in lung adenocarcinomas that displayed an EMT phenotype. Furthermore, we identify B7-H3 as a prognostic marker for NSCLC. Conclusions: The strong association between EMT status and an inflammatory tumor microenvironment with elevation of multiple targetable immune checkpoint molecules warrants further investigation of using EMT as a predictive biomarker for immune checkpoint blockade agents and other immunotherapies in NSCLC and possibly a broad range of other cancers. Clin Cancer Res; 22(14); 3630–42. ©2016 AACR. See related commentary by Datar and Schalper, p. 3422

The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Severely symptomatic COVID-19 is associated with lung inflammation, pneumonia, and respiratory failure, thereby raising concerns of elevated risk of COVID-19-associated mortality among lung cancer patients. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for SARS-CoV-2 entry into lung cells. The single-cell expression landscape of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung cancer patients remains unknown. We sought to delineate single-cell expression profiles of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung adenocarcinoma (LUAD) patients. We examined the expression levels and cellular distribution of ACE2 and SARS-CoV-2-priming proteases TMPRSS2 and TMPRSS4 in 5 LUADs and 14 matched normal tissues by single-cell RNA-sequencing (scRNA-seq) analysis. scRNA-seq of 186,916 cells revealed epithelial-specific expression of ACE2, TMPRSS2, and TMPRSS4. Analysis of 70,030 LUAD- and normal-derived epithelial cells showed that ACE2 levels were highest in normal alveolar type 2 (AT2) cells and that TMPRSS2 was expressed in 65% of normal AT2 cells. Conversely, the expression of TMPRSS4 was highest and most frequently detected (75%) in lung cells with malignant features. ACE2-positive cells co-expressed genes implicated in lung pathobiology, including COPD-associated HHIP, and the scavengers CD36 and DMBT1. Notably, the viral scavenger DMBT1 was significantly positively correlated with ACE2 expression in AT2 cells. We describe normal and tumor lung epithelial populations that express SARS-CoV-2 receptor and proteases, as well as major host defense genes, thus comprising potential treatment targets for COVID-19 particularly among lung cancer patients.

SUMMARY Cancer cells function as primary architects of the tumor microenvironment. However, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblast (CAF) heterogeneity is driven by lung adenocarcinoma (LUAD) cells at either end of the epithelial-to-mesenchymal transition (EMT) spectrum. LUAD cells that have high expression of the EMT-activating transcription factor ZEB1 reprogram CAFs through a ZEB1-dependent secretory program and direct CAFs to the tips of invasive projections through a ZEB1-driven CAF repulsion process. The EMT, in turn, sensitizes LUAD cells to pro-metastatic signals from CAFs. Thus, CAFs respond to contextual cues from LUAD cells to promote metastasis., In brief Bota-Rabassedas et al. show that EMT in lung adenocarcinoma cells activates a secretory process that governs CAF heterogeneity and, in turn, sensitizes lung adenocarcinoma cells to pro-metastatic signals from CAFs. Thus, EMT positions lung adenocarcinoma cells at the apex of a signaling hierarchy in the tumor microenvironment., Graphical Abstract

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.

Lung adenocarcinoma (LUAD) is the most frequently diagnosed histological subtype of lung cancer and accounts for most smoking-related cancer deaths, warranting strategies for early intervention. Earlier work revealed genome-wide aberrations in LUADs and the adjacent premalignant field, known as “field carcinogenesis”, that are pertinent to LUAD pathogenesis. Yet, we still poorly understand the cellular and molecular architecture of LUAD and its nearby “field”. To fill this void, we performed, using the 10X Genomics system and NovaSeq 6000 platform, single-cell RNA sequencing (scRNA-seq) analysis of 4 early-stage resected LUADs as well as 11 matched normal lung tissues with differing spatial proximity from the tumors. We first analyzed 15,739 cells from a LUAD and matched tumor-adjacent and -distant normal tissues from patient 1. The resulting fraction of EPCAM+ cells from this analysis was approximately 4%. For deeper single-cell resolution of LUAD, we thus performed scRNA-seq of separately sorted/enriched epithelial cells (EPCAM+; n = 50,883) and non-epithelial cells (EPCAM-; n= 91,093) from patients 2, 3 and 4, each with an early-stage LUAD and three matched spatially-distributed normal-appearing tissues. Overall, we achieved on average approximately 150,000 reads and 1,956 genes detected per cell. Divergent populations of malignant and non-malignant cells and multiple epithelial and immune subsets clustered in an overall spatially modulated pattern according to proximity to the tumor. Hierarchical clustering revealed multiple distinct populations of airway lineage cells, including alveolar type 1 (AT1), alveolar type 2 (AT2), AT1/AT2 bipotential cells, club, goblet, basal and ciliated cells, with cells from the resected LUAD present in some but not all clusters. By analysis of epithelial-enriched fractions, we were also able to identify and interrogate rare cell subpopulations including CFTR-expressing ionocytes. By inference of copy number variation along with analysis of key oncogene expression patterns, we also identified epithelial cells that represent potential tumor cells-of-origin. Spatial reprogramming of field carcinogenesis encompassed immune cell populations including tumor-exclusive FOXP3+ regulatory T cells with marked over-expression of the major immune checkpoints CTLA4 and TIGIT. In contrast, for at least some of the cases, tumors were nearly devoid of natural killer cells, and abundance of CD8+ T cells dampened with increasing proximity to the tumor. Our single-cell surveys offer insights into novel cues in LUAD pathogenesis. Efforts are underway to interrogate the single-cell epithelial and immune landscape of additional LUADs and matched nearby normal-appearing lung, including those from non-smokers, to better understand the evolution of the disease and, thus, identify a low-hanging fruit of targets for early management of this fatal malignancy. Citation Format: Ansam Sinjab, Guangchun Han, Warapen Treekitkarnmongkol, Patrick Brennan, Kieko Hara, Kyle Chang, Elena Bogatenkova, Beatriz Sanchez-Espiridion, Carmen Behrens, Jianjun Zhang, Boris Sepesi, Tina Cascone, Don L. Gibbons, Jichao Chen, George Eapen, Edwin J. Ostrin, Junya Fujimoto, Avrum E. Spira, Paul A. Scheet, Ignacio I. Wistuba, Linghua Wang, Humam Kadara. A single-cell transcriptomic atlas of lung adenocarcinoma and adjacent normal-appearing tissue [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 1518.

Mitogen-activated protein kinase kinase (MEK) inhibitors have failed to show clinical benefit in Kirsten rat sarcoma (KRAS)-mutant lung cancer due to various resistance mechanisms. To identify differential therapeutic sensitivities between epithelial and mesenchymal lung tumors, we performed in vivo small hairpin RNA (shRNA) screens, proteomic profiling, and analysis of patient tumor datasets, which revealed an inverse correlation between mitogen-activated protein kinase (MAPK) signaling dependency and a zinc finger E-box-binding homeobox 1 (ZEB1)-regulated epithelial-to-mesenchymal transition (EMT). Mechanistic studies determined that MAPK signaling dependency in epithelial lung cancer cells is due to the scaffold protein interleukin 17 receptor D (IL17RD), which is directly repressed by ZEB1. Lung tumors in multiple Kras mutant murine models with increased ZEB1 displayed low IL17RD expression, accompanied by MAPK-independent tumor growth and therapeutic resistance to MEK inhibition. Suppression of ZEB1 function with miR-200 expression or the histone deacetylase (HDAC) inhibitor mocetinostat sensitized resistant cancer cells to MEK inhibition and markedly reduced in vivo tumor growth, showing a promising combinatorial treatment strategy for KRAS-mutant cancers. In human lung tumor samples, high ZEB1 and low IL17RD expression correlated with low MAPK signaling, presenting potential markers that predict patient response to MEK inhibitors.

Lung cancer is the foremost cause of cancer related deaths in the U.S. It is a heterogeneous disease composed of genetically and phenotypically distinct tumor cells surrounded by heterotypic cells and extracellular matrix dynamically interacting with the tumor cells. Research in lung cancer is often restricted to patient-derived tumor specimens, in vitro cell cultures and limited animal models, which fail to capture the cellular or microenvironment heterogeneity of the tumor. Therefore, our knowledge is primarily focused on cancer-cell autonomous aberrations. For a fundamental understanding of lung cancer progression and an exploration of therapeutic options, we focused our efforts to develop an Ex Vivo Tumor platform to culture tumors in 3D matrices, which retains tumor cell heterogeneity arising due to in vivo selection pressure and environmental influences and recapitulate responses of tumor cells to external manipulations. To establish this model, implanted syngeneic murine tumors from a mutant KRAS/p53 model were harvested to yield multicellular tumor aggregates followed by culture in 3D extracellular matrices. Using this system, we identified Src signaling as an important driver of invasion and metastasis in lung cancer and demonstrate that EVTs are a robust experimental tool bridging the gap between conventional in vitro and in vivo models.