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Recently, low human epidermal growth factor receptor 2 (HER2) protein expression has been proposed as a predictive biomarker for response to the antibody-drug conjugate trastuzumab deruxtecan (T-DXd) in metastatic breast cancer. HER2 expression in non-small cell lung cancer (NSCLC) patients has never been carefully measured, and little is known about the frequency of cases with unamplified but detectable levels of the protein. Although some HER2-targeted therapies have been studied in NSCLC patients, they have been restricted to those with genomic ERBB2 gene alterations, which only represent relatively rare cases of NSCLC. Still, emerging investigations of T-DXd in NSCLC have shown promise in patients with unamplified HER2. Taken together, we hypothesize that there may be many cases of NSCLC with levels of HER2 protein expression comparable with levels seen in breast cancer that benefit from T-DXd. Here, we used a previously validated, analytic, quantitative immunofluorescence (QIF) assay that is more sensitive than legacy clinical HER2 immunohistochemistry assays. We measured HER2 protein levels in NSCLC cases to determine the proportion of cases with detectable HER2 expression. Using cell line calibration microarrays alongside our QIF method enabled us to convert HER2 signal into units of attomoles per mm 2 . We found that over 63% of the 741 analyzed NSCLC cases exhibited HER2 expression above the limit of detection, with more than 17% of them exceeding the lower limit of quantification. Although the threshold for response to T-DXd in breast cancer is still unknown, many cases of NSCLC have expression in a range comparable to breast cancer cases with immunohistochemistry scores of 1+ or 2+. Our assay could potentially select NSCLC cases with a detectable target (ie, HER2) that might benefit from HER2 antibody-drug conjugates, irrespective of ERBB2 genomic alterations., (Copyright © 2024 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

Basal Cell Adhesion Molecule (BCAM), a receptor for laminin subunit α5, plays a crucial role in the pathogenesis of various malignancies. Notably, evidence of hypermethylation at multiple immune checkpoints in patients with low BCAM expression suggests these individuals may respond favorably to immunotherapy using ICIs (immune checkpoint inhibitors). This finding lays the foundation for the hypothesis that BCAM may serve as an important biomarker in cancer patients. To investigate this potential, we evaluated BCAM expression patterns in 3114 patients from both discovery and validation cohorts, spanning seven cancer types, using quantitative immunofluorescence (QIF). We also explored the correlation between BCAM and PD-L1 expressions within these cohorts, aiming to establish its potential predictive value for immunotherapy response. Our findings indicate that BCAM was highly expressed in ovarian (79.2%) and lung (78.5%) tumors, with lower yet significant expression in breast (37.7%), head and neck (31.3%), and bladder-urothelial tumors (27.6%). Notably, high BCAM expression was associated with better OS in NSCLC. More importantly, BCAM expression did not correlate with PD-L1 protein expression in any of these tumors, highlighting its independent predictive potential. The widespread expression of BCAM across multiple tumor types, coupled with its lack of correlation with PD-L1 expression, highlights its potential as a predictive novel biomarker across various cancer types., (© 2024. The Author(s).)

Therapies targeting the programmed cell death protein-1/programmed death-ligand 1 (PD-L1) (abbreviated as PD-(L)1) axis are a significant advancement in the treatment of many tumor types. However, many patients receiving these agents fail to respond or have an initial response followed by cancer progression. For these patients, while subsequent immunotherapies that either target a different axis of immune biology or non-immune combination therapies are reasonable treatment options, the lack of predictive biomarkers to follow-on agents is impeding progress in the field. This review summarizes the current knowledge of mechanisms driving resistance to PD-(L)1 therapies, the state of biomarker development along this axis, and inherent challenges in future biomarker development for these immunotherapies. Innovation in the development and application of novel biomarkers and patient selection strategies for PD-(L)1 agents is required to accelerate the delivery of effective treatments to the patients most likely to respond., Competing Interests: Competing interests: SW—Salary and employment: Kite Pharma, Gilead; Consulting fees: SciSpot; Stock ownership: Kite Pharma, Gilead. JCB—Salary and employment: Precede Biosciences; Ownership: JCarl Consulting, LLC; Stock Ownership: AstraZeneca. CG-B—Salary and employment: Bristol Myers Squibb. Stock ownership: Bristol Myers Squibb. KAS—Consulting fees: Shattuck Labs, EMD Serono, Clinica Alemana de Santiago, Genmab, Takeda, Merck Sharpe & Dohme, Bristol Myers Squibb, AstraZeneca, Agenus, Repertoire; Fees for non-CE services: Bristol Myers Squibb, Fluidigm Corporation, Genmab, Merck, Takeda; Contracted research: Navigate Biopharma, Tesaro/GSK, Moderna Inc., Pierre-Fabre, Takeda, Surface Oncology, Merck Sharpe & Dohme, Bristol Myers Squibb, AstraZeneca, Ribon Therapeutics, Akoya Biosciences, Boehringer-Ingelheim, Eli Lilly. JMT—Research funding: Bristol Myers Squibb, Akoya Biosciences; Consulting fees: Bristol Myers Squibb, Roche/Genentech, Merck & Co., AstraZeneca, Regeneron; Stock ownership: Akoya Biosciences. BT—Salary and employment: Merck & Co.; Stock ownership: Merck & Co. AS—Salary and employment: Generate Biomedicines; Stock ownership, Generate Biomedicines, Merck & Co., Allogene. AM—Nothing to disclose. SITC Staff: FD, PJI—Nothing to disclose., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Despite the impressive outcomes with immune checkpoint inhibitor (ICI) in non-small cell lung cancer (NSCLC), only a minority of the patients show long-term benefits from ICI. In this study, we used retrospective cohorts of ICI treated patients with NSCLC to discover and validate spatially resolved protein markers associated with resistance to programmed cell death protein-1 (PD-1) axis inhibition., Methods: Pretreatment samples from 56 patients with NSCLC treated with ICI were collected and analyzed in a tissue microarray (TMA) format in including four different tumor regions per patient using the GeoMx platform for spatially informed transcriptomics. 34 patients had assessable tissue with tumor compartment in all 4 TMA spots, 22 with leukocyte compartment and 12 with CD68 compartment. The patients' tissue that was not assessable in fourfold redundancy in each compartment was designated as the validation cohort; cytokeratin (CK) (N=22), leukocytes CD45 (N=31), macrophages, CD68 (N=43). The human whole transcriptome, represented by~18,000 individual genes assessed by oligonucleotide-tagged in situ hybridization, was sequenced on the NovaSeq platform to quantify the RNAs present in each region of interest., Results: 54,000 gene variables were generated per case, from them 25,740 were analyzed after removing targets with expression lower than a prespecified frequency. Cox proportional-hazards model analysis was performed for overall and progression-free survival (OS, PFS, respectively). After identifying genes significantly associated with limited survival benefit (HR>1)/progression per spot per patient, we used the intersection of them across the four TMA spots per patient. This resulted in a list of 12 genes in the tumor-cell compartment ( RPL13A, GNL3, FAM83A, CYBA, ACSL4, SLC25A6, EPAS1, RPL5, APOL1, HSPD1, RPS4Y1, ADI1). RPL13A, GNL3 in tumor-cell compartment were also significantly associated with OS and PFS, respectively, in the validation cohort (CK: HR, 2.48; p=0.02 and HR, 5.33; p=0.04). In CD45 compartment, secreted frizzled-related protein 2, was associated with OS in the discovery cohort but not in the validation cohort. Similarly, in the CD68 compartment ARHGAP and PNN interacting serine and arginine rich protein were significantly associated with PFS and OS, respectively, in the majority but not all four spots per patient., Conclusion: This work highlights RPL13A and GNL3 as potential indicative biomarkers of resistance to PD-1 axis blockade that might help to improve precision immunotherapy strategies for lung cancer., Competing Interests: Competing interests: DLR has served as an advisor for AstraZeneca, Agendia, Amgen, BMS, Cell Signaling Technology, Cepheid, Danaher, Daiichi Sankyo, Genoptix/Novartis, GSK, Konica Minolta, Merck, NanoString, PAIGE.AI, Roche, and Sanofi. Amgen, Cepheid, NavigateBP, NextCure, and Konica Minolta fund or have funded research in DLR’s laboratory. KAS has served as a Consultant or advisor: AbbVie Inc.; Agenus Inc.; AstraZeneca; Bristol Myers Squibb; CDR-Life Inc.; Clinica Alemana de Santiago; EMD Serono Inc.; F.Hoffmann-La Roche; Genmab A/S; Indaptus Therapeutics; Janssen Pharmaceuticals, Inc.; Merck Sharpe & Dohme; Moderna, Inc.; Molecular Templates, Inc.; OnCusp Therapeutics; Parthenon/Incendia Therapeutics; Repertoire Therapeutics; Sanofi; Sensei Biotherapeutics; Shattuck Labs, Inc.; and Takeda Pharmaceutical Company Limited., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: The tissue immune microenvironment is associated with key aspects of tumor biology. The interaction between the immune system and cancer cells has predictive and prognostic potential across different tumor types. Spatially resolved tissue-based technologies allowed researchers to simultaneously quantify different immune populations in tumor samples. However, bare quantification fails to harness the spatial nature of tissue-based technologies. Tumor-immune interactions are associated with specific spatial patterns that can be measured. In recent years, several computational tools have been developed to increase our understanding of these spatial patterns., Topics Covered: In this review, we cover standard techniques as well as new advances in the field of spatial analysis of the immune microenvironment. We focused on marker quantification, spatial intratumor heterogeneity analysis, cell‒cell spatial interaction studies and neighborhood analyses., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

T cells are often absent from human cancer tissues during both spontaneously induced immunity and therapeutic immunotherapy, even in the presence of a functional T cell-recruiting chemokine system, suggesting the existence of T cell exclusion mechanisms that impair infiltration. Using a genome-wide in vitro screening platform, we identified a role for phospholipase A2 group 10 (PLA2G10) protein in T cell exclusion. PLA2G10 up-regulation is widespread in human cancers and is associated with poor T cell infiltration in tumor tissues. PLA2G10 overexpression in immunogenic mouse tumors excluded T cells from infiltration, resulting in resistance to anti-PD-1 immunotherapy. PLA2G10 can hydrolyze phospholipids into small lipid metabolites, thus inhibiting chemokine-mediated T cell mobility. Ablation of PLA2G10's enzymatic activity enhanced T cell infiltration and sensitized PLA2G10-overexpressing tumors to immunotherapies. Our study implicates a role for PLA2G10 in T cell exclusion from tumors and suggests a potential target for cancer immunotherapy.

Melanoma has the highest propensity among solid tumors to metastasize to the brain. Melanoma brain metastases (MBM) are a leading cause of death in melanoma and affect 40-60% of patients with late-stage disease. Therefore, uncovering the molecular mechanisms behind MBM is necessary to enhance therapeutic interventions. Vascular mimicry (VM) is a form of neovascularization linked to invasion, increased risk of metastasis, and poor prognosis in many tumor types, but its significance in MBM remains poorly understood. We found that VM density is elevated in MBM compared to paired extracranial specimens and is associated with tumor volume and CNS edema. In addition, our studies indicate a relevant role of YAP and TAZ, two transcriptional co-factors scarcely studied in melanoma, in tumor cell-vasculogenesis and in brain metastasis. We recently demonstrated activation of the Hippo tumor suppressor pathway and increased degradation of its downstream targets YAP and TAZ in a metastasis impaired cell line model. In the current study we establish the utility of anti-YAP/TAZ therapy in mouse models of metastatic melanoma whereby treatment effectively inhibits VM and prolongs survival of mice with MBM. The data presented herein suggest that VM may be an important and targetable mechanism in melanoma and that VM inhibition might be useful for treating MBM, an area of high unmet clinical need, thus having important implications for future treatment regimens for these patients., (© 2024. The Author(s).)

Purpose: The spatial arrangement of lymphocytes in the tumor bed (e.g., immune infiltrated, immune excluded, immune desert) is expected to reflect distinct immune evasion mechanisms and to associate with immunotherapy outcomes. However, data supporting these associations are scant and limited by the lack of a clear definition for lymphocyte infiltration patterns and the subjective nature of pathology-based approaches., Experimental Design: We used multiplexed immunofluorescence to study major tumor-infiltrating lymphocyte (TIL) subsets with single-cell resolution in baseline whole-tissue section samples from NSCLC patients treated with immune checkpoint inhibitors (ICI). The spatial TIL patterns were analyzed using a qualitative pathologist-based approach, and an objective analysis of TIL density ratios in tumor/stromal tissues. The association of spatial patterns with outcomes was studied for different TIL markers., Results: The analysis of CD8+ TIL patterns using qualitative assessment identified prominent limitations including the presence of a broad spectrum of phenotypes within most tumors and limited association with outcomes. The utilization of an objective method to classify NSCLCs showed the existence of at least three subgroups with partial overlap with those defined using visual patterns. Using this strategy, a subset of cases with "immune excluded-like" tumors showed prominently worse outcomes, suggesting reduced sensitivity to ICI; however, these results need to be validated. The analysis for other TIL subsets showed different results, underscoring the relevance of the marker selected for spatial TIL pattern evaluation and opportunities for market integration., Conclusions: Our results identified major challenges associated with the qualitative spatial TIL pattern evaluation. We devised a novel objective strategy to overcome some of these limitations that has strong biomarker potential., (©2023 American Association for Cancer Research.)

Introduction: Pathologic response has been proposed as an early clinical trial end point of survival after neoadjuvant treatment in clinical trials of NSCLC. The International Association for the Study of Lung Cancer (IASLC) published recommendations for pathologic evaluation of resected lung cancers after neoadjuvant therapy. The aim of this study was to assess pathologic response interobserver reproducibility using IASLC criteria., Methods: An international panel of 11 pulmonary pathologists reviewed hematoxylin and eosin-stained slides from the lung tumors of resected NSCLC from 84 patients who received neoadjuvant immune checkpoint inhibitors in six clinical trials. Pathologic response was assessed for percent viable tumor, necrosis, and stroma. For each slide, tumor bed area was measured microscopically, and pre-embedded formulas calculated unweighted and weighted major pathologic response (MPR) averages to reflect variable tumor bed proportion., Results: Unanimous agreement among pathologists for MPR was observed in 68 patients (81%), and inter-rater agreement (IRA) was 0.84 (95% confidence interval [CI]: 0.76-0.92) and 0.86 (95% CI: 0.79-0.93) for unweighted and weighted averages, respectively. Overall, unweighted and weighted methods did not reveal significant differences in the classification of MPR. The highest concordance by both methods was observed for cases with more than 95% viable tumor (IRA = 0.98, 95% CI: 0.96-1) and 0% viable tumor (IRA = 0.94, 95% CI: 0.89-0.98). The most common reasons for discrepancies included interpretations of tumor bed, presence of prominent stromal inflammation, distinction between reactive and neoplastic pneumocytes, and assessment of invasive mucinous adenocarcinoma., Conclusions: Our study revealed excellent reliability in cases with no residual viable tumor and good reliability for MPR with the IASLC recommended less than or equal to 10% cutoff for viable tumor after neoadjuvant therapy., (Copyright © 2023. Published by Elsevier Inc.)

CD8 + T cells are essential components of the immune response against viral infections and tumours, and are capable of eliminating infected and cancerous cells. However, when the antigen cannot be cleared, T cells enter a state known as exhaustion 1 . Although it is clear that chronic antigen contributes to CD8 + T cell exhaustion, less is known about how stress responses in tissues regulate T cell function. Here we show a new link between the stress-associated catecholamines and the progression of T cell exhaustion through the β 1 -adrenergic receptor ADRB1. We identify that exhausted CD8 + T cells increase ADRB1 expression and that exposure of ADRB1 + T cells to catecholamines suppresses their cytokine production and proliferation. Exhausted CD8 + T cells cluster around sympathetic nerves in an ADRB1-dependent manner. Ablation of β 1 -adrenergic signalling limits the progression of T cells towards the exhausted state in chronic infection and improves effector functions when combined with immune checkpoint blockade (ICB) in melanoma. In a pancreatic cancer model resistant to ICB, β-blockers and ICB synergize to boost CD8 + T cell responses and induce the development of tissue-resident memory-like T cells. Malignant disease is associated with increased catecholamine levels in patients 2,3 , and our results establish a connection between the sympathetic stress response, tissue innervation and T cell exhaustion. Here, we uncover a new mechanism by which blocking β-adrenergic signalling in CD8 + T cells rejuvenates anti-tumour functions., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Although immune checkpoint inhibitor (ICI) therapy has dramatically improved outcome for metastatic melanoma patients, many patients do not benefit. Since adverse events may be severe, biomarkers for resistance would be valuable, especially in the adjuvant setting. We performed high-plex digital spatial profiling (DSP) using the NanoString GeoMx® on 53 pre-treatment specimens from ICI-treated metastatic melanoma cases. We interrogated 77 targets simultaneously in four molecular compartments defined by S100B for tumor, CD68 for macrophages, CD45 for leukocytes, and nonimmune stromal cells defined as regions negative for all three compartment markers but positive for SYTO 13. For DSP validation, we confirmed the results obtained for some immune markers, such as CD8, CD4, CD20, CD68, CD45, and PD-L1, by quantitative immunofluorescence (QIF). In the univariable analysis, 38 variables were associated with outcome, 14 of which remained significant after multivariable adjustment. Among them, CD95 was further validated using multiplex immunofluorescence in the Discovery immunotherapy (ITX) Cohort and an independent validation cohort with similar characteristics, showing an association between high levels of CD95 and shorter progression-free survival. We found that CD95 in stroma was associated with resistance to ICI. With further validation, this biomarker could have value to select patients that will not benefit from immunotherapy., Competing Interests: SMM is a current employee at Boehringer Ingelheim Inc. PD is a current employee and stockholder at NanoString Technologies; PFW currently works at Verily Life Sciences, Alphabet Inc. (CA, USA); H.M. Kluger reports receiving commercial research grants from Merck, Bristol-Myers Squibb, and Apexigen, and is a consultant/advisory board member for Alexion, Corvus, Nektar, Biodesix, Genentech, Merck, Celldex, Pfizer, Iovance, Array Biopharma, Clinigen, Bristol-Myers Squibb, Instil Bio and Immunocore. D.L. Rimm declares that in the last two years he has served as a consultant to Astra Zeneca, Amgen, BMS, Cell Signaling Technology, Cepheid, Daiichi Sankyo, Danaher, GSK, Konica/Minolta, Merck, NanoString, Novartis, PAIGE.AI, Perkin Elmer/Akoya, and Roche/Ventana. No potential conflict of interest was reported by the author(s)., (© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Stereotactic ablative radiotherapy (SABR) is a standard-of-care for medically-inoperable-early-stage non-small cell lung cancer (NSCLC). One third of patients progress and chemotherapy is rarely used in this population. We questioned if addition of the immune-checkpoint-inhibitor (ICI) atezolizumab to standard-of-care SABR can improve outcomes. We initiated a multi-institutional single-arm phase I study (NCT02599454) enrolling twenty patients with the primary endpoint of maximum tolerated dose (MTD); secondary endpoints of safety and efficacy; and exploratory mechanistic correlatives. Treatment is well tolerated and full dose atezolizumab (1200 mg) is the MTD. Efficacy signals include early responses (after 2 cycles of ICI, before initiation of SABR) in 17% of patients. Biomarkers of functional adaptive immunity, including T cell activation in the tumor and response to ex-vivo stimulation by circulating T cells, are highly predictive of benefit. These results require validation and are being tested in a phase III randomized trial., (© 2023. Springer Nature Limited.)

Purpose: O 6 -methylguanine DNA methyltransferase ( MGMT )-silenced tumors reveal sensitivity to temozolomide (TMZ), which may be enhanced by PARP inhibitors. Approximately 40% of colorectal cancer has MGMT silencing and we aimed to measure antitumoral and immunomodulatory effects from TMZ and olaparib in colorectal cancer., Experimental Design: Patients with advanced colorectal cancer were screened for MGMT promoter hypermethylation using methylation-specific PCR of archival tumor. Eligible patients received TMZ 75 mg/m 2 days 1-7 with olaparib 150 mg twice daily every 21 days. Pretreatment tumor biopsies were collected for whole-exome sequencing (WES), and multiplex quantitative immunofluorescence (QIF) of MGMT protein expression and immune markers., Results: MGMT promoter hypermethylation was detected in 18/51 (35%) patients, 9 received study treatment with no objective responses, 5/9 had stable disease (SD) and 4/9 had progressive disease as best response. Three patients had clinical benefit: carcinoembryonic antigen reduction, radiographic tumor regression, and prolonged SD. MGMT expression by multiplex QIF revealed prominent tumor MGMT protein from 6/9 patients without benefit, while MGMT protein was lower in 3/9 with benefit. Moreover, benefitting patients had higher baseline CD8 + tumor-infiltrating lymphocytes. WES revealed 8/9 patients with MAP kinase variants (7 KRAS and 1 ERBB2 ). Flow cytometry identified peripheral expansion of effector T cells., Conclusions: Our results indicate discordance between MGMT promoter hypermethylation and MGMT protein expression. Antitumor activity seen in patients with low MGMT protein expression, supports MGMT protein as a predictor of alkylator sensitivity. Increased CD8 + TILs and peripheral activated T cells, suggest a role for immunostimulatory combinations., Significance: TMZ and PARP inhibitors synergize in vitro and in vivo in tumors with MGMT silencing. Up to 40% of colorectal cancer is MGMT promoter hypermethylated, and we investigated whether TMZ and olaparib are effective in this population. We also measured MGMT by QIF and observed efficacy only in patients with low MGMT, suggesting quantitative MGMT biomarkers more accurately predict benefit to alkylator combinations., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Purpose: Veliparib is a PARP inhibitor (PARPi) with activity in BRCA 1/2/ PALB2 -deficient tumors. Preclinical observations reveal topoisomerase inhibitors like irinotecan are synergistic with PARPi irrespective of homologous recombination deficiency (HRD), potentially expanding the role for PARPi., Experimental Design: NCI 7977 was a multicohort phase I clinical trial evaluating the safety and efficacy of multiple dose schedules of veliparib with irinotecan for solid tumors. In the intermittent veliparib cohort, escalating doses of veliparib were given twice daily at dose level (DL) 1 (50 mg) and DL 2 (100 mg) days 1-4 and 8-11 with irinotecan 100 mg/m 2 days 3 and 10 in 21-day cycles., Results: Fifteen patients enrolled, 8 of 15 (53%) received ≥4 prior systemic treatments. At DL1, 1 of 6 patients experienced a dose-limiting toxicity (DLT) of diarrhea. At DL2, 9 patients were treated, with 3 unevaluable for DLT, and 2 of 6 evaluable patients experienced a DLT of grade 3 neutropenia. Irinotecan 100 mg/m 2 and veliparib 50 mg twice daily was the MTD. No objective responses were observed, although 4 patients had progression-free survival >6 months., Conclusions: The MTD of intermittent veliparib is 50 mg twice daily days 1-4 and 8-11 with weekly irinotecan 100 mg/m 2 days 3 and 10 every 21 days. Multiple patients experienced prolonged stable disease irrespective of HRD and prior irinotecan. However, due to the toxicities with higher dose intermittent veliparib and irinotecan, this schedule was determined too toxic for further development and the arm was closed prematurely., Significance: The combination of intermittent veliparib with weekly irinotecan was deemed too toxic for further development. Future PARPi combinations should focus on agents with nonoverlapping toxicities to improve tolerability. The treatment combination showed limited efficacy with prolonged stable disease observed in multiple heavily pretreated patients, but no objective responses were seen., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Checkpoint inhibitors represent an effective treatment approach for a variety of cancers through their inhibition of immune regulatory pathways within the tumor microenvironment (TME). Unfortunately only a minority of patients with cancer achieve clinical benefit from immunotherapy, with the TME emerging as an important predictor of outcomes and sensitivity to therapy. The extent and pattern of T-cell infiltration can vary prominently within/across tumors and represents a biological continuum. Three immune profiles have been identified along this continuum: 'immune-desert' or 'T-cell cold' phenotype, 'immune-active', 'inflamed', or 'T-cell hot' phenotype, and 'immune excluded' phenotype. Of the three profiles, immune excluded remains the most ill-defined with no clear, universally accepted definition even though it is commonly associated with lack of response to immune checkpoint inhibitors and poor clinical outcomes. To address this, 16 multidisciplinary cancer experts from around the world were invited to participate in a symposium using a three-round modified Delphi approach. The first round was an open-ended questionnaire distributed via email and the second was an in-person discussion of the first round results that allowed for statements to be revised as necessary to achieve a maximum consensus (75% agreement) among the rating committee (RC). The final round questionnaire was distributed to the RC via email and had a 100% completion rate. The Delphi process resulted in moving us closer to a consensus definition for immune exclusion that is practical, clinically pertinent, and applicable across a wide range of cancer histologies. A general consensus of the role of immune exclusion in resistance to checkpoint therapy and five research priorities emerged from this process. Together, these tools could help efforts designed to address the underlying mechanisms of immune exclusion that span cancer types and, ultimately, aid in the development of treatments to target these mechanisms to improve patient outcomes., Competing Interests: Competing interests: GTC is an employee of Parthenon Therapeutics. MR is a former Ingram Professor of Cancer Research at the Vanderbilt-Ingram Cancer Center and former Chief Medical Officer of Pfizer; he is also a paid member of the Scientific Advisory Board of Parthenon Therapeutics. PAA has/had a consultant/advisory role for Bristol-Myers Squibb, Roche-Genentech, Merck Sharp & Dohme, Novartis, Merck Serono, Pierre-Fabre, AstraZeneca, Sun Pharma, Sanofi, Idera, Sandoz, Immunocore, 4SC, Italfarmaco, Nektar, Boehringer-Ingelheim, Eisai, Regeneron, Daiichi Sankyo, Pfizer, Oncosec, Nouscom, Lunaphore, Seagen, iTeos, Medicenna, Bio-Al Health, ValoTX, Replimmune, Bayer. He also received research funding from Bristol-Myers Squibb, Roche-Genentech, Pfizer, Sanofi. Travel support by Pfizer, Bio-Al Health, Replimmune. GB is a paid consultant of Parthenon Therapeutics. MC has received honoraria from Eisai, Agios Pharmaceuticals, DAVA Oncology, BAYER, Seattle Genetics, Daiichi Sankyo and AstraZeneca, travel/accommodation/expenses from Genentech/Roche and previously received research support as part of the Yale Cancer Center. JPE is an employee of Parthenon Therapeutics. FG received fees for oral communication from Lilly, Sanofi, BMS, AstraZeneca and Amgen, received funding for clinical trials by AstraZeneca, received travel grants from Roche France, Amgen and Servier; and is an advisory board member for Merck Serano, Amgen, Roche France and Sanofi, all outside of the submitted work. AI is on the advisory board: AstraZeneca, Bayer, BMS, Brenus Pharma, Domain Therapeutics, Epizyme, Ipsen, Lilly, Merck, MSD, Novartis, Parthenon, Roche; Research Grant: AstraZeneca, Bayer, BMS, Domain Therapeutics, Ipsen, Merck, MSD, Novartis, Parthenon, Pharmamar, and Roche. MK has none. RL is a paid member of Scientific Advisory Board of Parthenon Therapeutics. FM-G research grants from Innate Pharma, Roche, BMS. SIP has none. PP paid member of Scientific Advisory Board of Parthenon Therapeutics. EP has received research support from Boehringer-Ingelheim, Capstan Therapeutics, Incyte and TMUNITY; co-founder and holds equity in Capstan Therapeutics. AR has received honoraria from consulting with Amgen, Bristol-Myers Squibb and Merck, is or has been a member of the scientific advisory board and holds stock in Advaxis, Appia, Apricity, Arcus, Compugen, CytomX, Highlight, ImaginAb, ImmPact, ImmuneSensor, Inspirna, Isoplexis, Kite-Gilead, Lutris, MapKure, Merus, PACT, Pluto, RAPT, Synthekine and Tango, has received research funding from Agilent and from Bristol-Myers Squibb through Stand Up to Cancer (SU2C), and patent royalties from Arsenal Bio. KAS received research support (last 24 months) from Navigate Biopharma, Tesaro/GSK, Moderna, Takeda, Surface Oncology, Merck, Bristol-Myers Squibb, AstraZeneca, Ribon Therapeutics, Eli Lilly, Boehringer-Ingelheim, Akoya Biosciences and Roche; consulting or advisory board: Clinica Alemana Santiago, Shattuck Labs, AstraZeneca, EMD Serono, Takeda, Agenus, Genmab, OnCusp, Bristol-Myers Squibb, Roche, CDR life, Sensei Therapeutics, Molecular Templates and Merck; Speaking engagements and other: Merck, Parthenon Therapeutics, PeerView, Forefront collaborative. WHF is Professor Emeritus at University Paris Cité working in Immuno-Oncology for more than 50 years. Paid member of the Scienfic Advisory Board and consultant for Parthenon, Ichnos, Oxford Biotherapeutics, Anaveon, Catalym, Genenta and holds patents for Immuoscore., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Anti-PD-1/PD-L1 agents have transformed the treatment landscape of advanced non-small cell lung cancer (NSCLC). To expand our understanding of the molecular features underlying response to checkpoint inhibitors in NSCLC, we describe here the first joint analysis of the Stand Up To Cancer-Mark Foundation cohort, a resource of whole exome and/or RNA sequencing from 393 patients with NSCLC treated with anti-PD-(L)1 therapy, along with matched clinical response annotation. We identify a number of associations between molecular features and outcome, including (1) favorable (for example, ATM altered) and unfavorable (for example, TERT amplified) genomic subgroups, (2) a prominent association between expression of inducible components of the immunoproteasome and response and (3) a dedifferentiated tumor-intrinsic subtype with enhanced response to checkpoint blockade. Taken together, results from this cohort demonstrate the complexity of biological determinants underlying immunotherapy outcomes and reinforce the discovery potential of integrative analysis within large, well-curated, cancer-specific cohorts., (© 2023. The Author(s).)

Background: Head and neck squamous cell cancer (HNSCC) occurs at higher rates among persons with HIV (PWH). This study compares the impact of sociodemographic and clinicopathologic characteristics on outcomes among PWH-HNSCC compared with HNSCC patients without HIV., Methods: Patient data from HNSCC individuals were collected at a single academic hospital center between 2002 and 2018. Forty-eight patients with HIV (HIV-HNSCC) and 2894 HNSCC patients without HIV were included. Multivariate analysis determined predictors of survival using Cox proportional hazards regression model. HIV-positive and -negative tumors were analyzed by quantitative immunofluorescence for expression of CD4, CD8, CD20 and PD-L1., Results: HIV-HNSCC patients had a lower median overall survival than HNSCC patients without HIV (34 [18-84] vs 94 [86-103] months; P < .001). In multivariate analysis that included age, sex, race/ethnicity, stage, site, tobacco use, time to treatment initiation, and insurance status, HIV was an independent predictor of poorer survival, with a hazard ratio of 1.98 (95% CI: 1.32-2.97; P < .001). PWH with human papillomavirus (HPV)-positive oropharyngeal tumors also had worse prognosis than HPV-positive oropharyngeal tumors in the population without HIV (P < .001). The tumor microenvironment among HIV-HNSCC patients revealed lower intratumoral CD8 infiltration among HIV+ HPV+ tumors compared with HIV- HPV+ tumors (P = .04)., Conclusions: HIV-HNSCC patients had worse prognosis than the non-HIV population, with HIV being an independent predictor of poor clinical outcomes when accounting for important sociodemographic and clinicopathologic factors. Our findings highlight differences in tumor biology that require further detailed characterization in large cohorts and increased inclusion of PWH in immunotherapy trials., Competing Interests: Potential conflicts of interest. B. E. reports consulting fees from Theratechnologies and Genentech/Roche unrelated to this work; participation on an Advisory Board for Theratechnologies unrelated to this work; and stock or stock options with Bausch Health Companies and Bristol Myers Squibb. G. G. reports support unrelated to this work from the Yale Transplant Group (author works as a statistician working at the Yale Center for Analytical Sciences; part of the author's effort is covered by Yale Transplant for providing statistical support for their research studies). K. A. S. reports grants or contracts unrelated to this work from Navigate Biopharma, Tesaro/GSK, Moderna, Inc, Takeda, Surface Oncology, Pierre-Fabre Research Institute, Merck, Bristol-Myers Squibb, AstraZeneca, Ribon Therapeutics, Eli Lilly, Boehringer-Ingelheim, and Akoya Biosciences (all contracted to Yale University); consulting fees paid to the author from Clinica Alemana Santiago, Shattuck Labs, Genmab, AstraZeneca, EMD Serono, Takeda, Torque/Repertoire Therapeutics, Takeda, Agenus, Genmab, OnCusp, Parthenon Therapeutics, Bristol-Myers Squibb, Roche, CDR Life, Sensei Therapeutics, Molecular Templates, and Merck; payment or honoraria for lectures, presentations, speaker’s bureaus, manuscript writing, or educational events from PeerView, Forefront Collaborative, Merck; payment for expert testimony from AstraZeneca; and support for attending meetings and/or travel from Fluidigm. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Despite the clinical efficacy of immune checkpoint inhibitors (ICIs) in NSCLC, only approximately 20% of patients remain disease-free at 5 years. Here, we use digital spatial profiling to find candidate biomarker proteins associated with ICI resistance., Methods: Pretreatment samples from 56 patients with NSCLC treated with ICI were analyzed using the NanoString GeoMx digital spatial profiling method. A panel of 71 photocleavable oligonucleotide-labeled primary antibodies was used for protein detection in four molecular compartments (tumor, leukocytes, macrophages, and immune stroma). Promising candidates were orthogonally validated with quantitative immunofluorescence. Available pretreatment samples from 39 additional patients with NSCLC who received ICI and 236 non-ICI-treated patients with operable NSCLC were analyzed to provide independent cohort validation., Results: Biomarker discovery using the protein-based molecular compartmentalization strategy allows 284 protein variables to be assessed for association with ICI resistance by univariate analysis using continuous log-scaled data. Of the 71 candidate protein biomarkers, CD66b in the CD45+CD68 molecular compartment (immune stroma) predicted significantly shorter overall survival (OS) (hazard ratio [HR] 1.31, p = 0.016) and was chosen for validation. Orthogonal validation by quantitative immunofluorescence illustrated that CD66b was associated with resistance to ICI therapy but not prognostic for poor outcomes in untreated NSCLC (discovery cohort [OS HR 2.49, p = 0.026], validation cohort [OS HR 2.05, p = 0.046], non-ICI-treated cohort [OS HR 1.67, p = 0.06])., Conclusions: Using the technique, we have discovered that CD66b expression is indicative of resistance to ICI therapy in NSCLC. Given that CD66b identifies neutrophils, further studies are warranted to characterize the role of neutrophils in ICI resistance., (Copyright © 2022 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Importance: Although tumor mutation burden (TMB) has been explored as a potential biomarker of immunotherapy efficacy in solid tumors, there still is a lack of consensus about the optimal TMB threshold that best discriminates improved outcomes of immune checkpoint inhibitor therapy among patients with non-small cell lung cancer (NSCLC)., Objectives: To determine the association between increasing TMB levels and immunotherapy efficacy across clinically relevant programmed death ligand-1 (PD-L1) levels in patients with NSCLC., Design, Setting, and Participants: This multicenter cohort study included patients with advanced NSCLC treated with immunotherapy who received programmed cell death-1 (PD-1) or PD-L1 inhibition in the Dana-Farber Cancer Institute (DFCI), Memorial Sloan Kettering Cancer Center (MSKCC), and in the Stand Up To Cancer (SU2C)/Mark Foundation data sets. Clinicopathological and genomic data were collected from patients between September 2013 and September 2020. Data analysis was performed from November 2021 to February 2022., Exposures: Treatment with PD-1/PD-L1 inhibition without chemotherapy., Main Outcomes and Measures: Association of TMB levels with objective response rate (ORR), progression-free survival (PFS), and overall survival (OS)., Results: In the entire cohort of 1552 patients with advanced NSCLC who received PD-1/PD-L1 blockade, the median (range) age was 66 (22-92) years, 830 (53.5%) were women, and 1347 (86.8%) had cancer with nonsquamous histologic profile. A regression tree modeling ORR as a function of TMB identified 2 TMB groupings in the discovery cohort (MSKCC), defined as low TMB (≤19.0 mutations per megabase) and high TMB (>19.0 mutations per megabase), which were associated with increasing improvements in ORR, PFS, and OS in the discovery cohort and in 2 independent cohorts (DFCI and SU2C/Mark Foundation). These TMB levels also were associated with significant improvements in outcomes of immunotherapy in each PD-L1 tumor proportion score subgroup of less than 1%, 1% to 49%, and 50% or higher. The ORR to PD-1/PD-L1 inhibition was as high as 57% in patients with high TMB and PD-L1 expression 50% or higher and as low as 8.7% in patients with low TMB and PD-L1 expression less than 1%. Multiplexed immunofluorescence and transcriptomic profiling revealed that high TMB levels were associated with increased CD8-positive, PD-L1-positive T-cell infiltration, increased PD-L1 expression on tumor and immune cells, and upregulation of innate and adaptive immune response signatures., Conclusions and Relevance: These findings suggest that increasing TMB levels are associated with immune cell infiltration and an inflammatory T-cell-mediated response, resulting in increased sensitivity to PD-1/PD-L1 blockade in NSCLC across PD-L1 expression subgroups.

Background: Most patients with advanced non-small-cell lung cancer (NSCLC) fail to derive significant benefit from programmed cell death protein-1 (PD-1) axis blockade, and new biomarkers of response are needed. In this study, we aimed to discover and validate spatially resolved protein markers associated with sensitivity to PD-1 axis inhibition in NSCLC., Methods: We initially assessed a discovery cohort of 56 patients with NSCLC treated with PD-1 axis inhibitors at Yale Cancer Center. Using the GeoMx Digital Spatial Profiling (DSP) system, 71 proteins were measured in spatial context on each spot in a tissue microarray. We used the AQUA method of quantitative immunofluorescence (QIF) to orthogonally validate candidate biomarkers. For external independent validation, we assessed whole tissue sections derived from 128 patients with NSCLC treated with single-agent PD-1 axis inhibitors at the 12 de Octubre Hospital (Madrid) using DSP. We further analyzed two immunotherapy untreated cohorts to address prognostic significance (n=252 from Yale Cancer Center; n=124 from University Clinic of Navarra) using QIF and DSP, respectively., Results: Using continuous log-scaled data, we identified CD44 expression in the tumor compartment (pan-cytokeratin (CK)+) as a novel predictor of prolonged progression-free survival (PFS) (multivariate HR=0.68, p=0.043) in the discovery set. We validated by QIF that tumor CD44 levels assessed as continuous QIF scores were associated with longer PFS (multivariate HR=0.31, p=0.022) and overall survival (multivariate HR=0.29, p=0.038). Using DSP in an independent immunotherapy treated cohort, we validated that CD44 levels in the tumor compartment, but not in the immune compartment (panCK-/CD45+), were associated with clinical benefit (OR=1.22, p=0.018) and extended PFS under PD-1 axis inhibition using the highest tertile cutpoint (multivariate HR=0.62, p=0.03). The effect of tumor cell CD44 in predicting PFS remained significant after correcting for programmed death-ligand 1 (PD-L1) Tumor Proportion Score (TPS) in both cohorts. High tumor cell CD44 was not prognostic in the absence of immunotherapy. Using DSP data, intratumoral regions with elevated tumor cell CD44 expression showed prominent (fold change>1.5, adjusted p<0.05) upregulation of PD-L1, TIM-3, ICOS, and CD40 in two independent cohorts., Conclusions: This work highlights CD44 as a novel indicative biomarker of sensitivity to PD-1 axis blockade that might help to improve immunotherapy strategies for NSCLC., Competing Interests: Competing interests: SMM is currently a Boehringer Ingelheim employee. LMM reports speakers honoraria from AstraZeneca and research grants from AstraZeneca and BMS. KAS reports consulting or advisory roles for Shattuck Labs, Pierre Fabre, EMD Serono, Clinica Alemana de Santiago, Genmab, Takeda, Merck Sharpe & Dohme, Bristol Myers Squibb, AstraZeneca, Agenus, Repertoire Therapeutics, OnCusp and Ariagen. Reports grants or research funding from Navigate Biopharma, Tesaro/GSK, Moderna, Takeda, Surface Oncology, Pierre Fabre Research Institute, Merck Sharpe & Dohme, Bristol Myers Squibb, AstraZeneca, Ribon Therapeutics, Akoya Biosciences, Boehringer Ingelheim and Eli Lilly. RSH has served as Non-Executive Director for Immunocore and is a member of the Board of Directors (non-executive/independent) for Junshi Biosciences; is a consultant for AbbVie, Armo Biosciences, AstraZeneca, Bristol Myers Squibb, Bayer HealthCare Pharmaceuticals, Bolt Biotherapeutics, Candel Therapeutics, Checkpoint Therapeutics, Cybrexa Therapeutics, DynamiCure Biotechnology, Eli Lilly and Company, eFFECTOR Therapeutics, EMD Serono, Foundation Medicine, Genentech/Roche, Genmab, Gilead, HiberCell, I‐Mab Biopharma, Immune‐Onc Therapeutics, Immunocore, Johnson & Johnson, Loxo Oncology, Merck and Company, Mirati Therapeutics, NextCure, Novartis, Ocean Biomedical, Oncocyte, Oncternal Therapeutics, Pfizer, Refactor Health, Ribbon Therapeutics, Sanofi, STCube Pharmaceuticals, Takeda, WindMIL Therapeutics, Xencor; has received research support from AstraZeneca, Eli Lilly and Company, Genentech/Roche, and Merck and Company; is a committee chair in American Association for Cancer Research, International Association for the Study of Lung Cancer, Society for Immunotherapy of Cancer, Southwest Oncology Group LP-A reports receiving honoraria from Amgen, AstraZeneca, Bayer, Blueprint Medicines, Bristol Myers Squibb, Celgene, Ipsen, Eli Lilly, Merck Serono, Mirati Therapeutics, Merck Sharp & Dohme, Novartis, Pfizer, PharmaMar, Roche/Genentech, Sanofi, Servier, and Takeda; leadership fees from Genomica and ALTUM Sequencing; research funding from AstraZeneca, Bristol Myers Squibb, Kura Oncology, PharmaMar, and Merck Sharp & Dohme; speaker fees from Bristol Myers Squibb, Eli Lilly, Merck Serono, Merck Sharp & Dohme Oncology, Pfizer, and Roche/Genentech; and travel, accommodation, and expenses from AstraZeneca, Bristol Myers Squibb, Merck Sharp & Dohme, Pfizer, Roche, and Takeda. DLR reports grants from Navigate Biopharma and Konica/Minolta/Invicro during the conduct of the study. Activities outside this work include honoraria and/or grants and/or instrument support from Akoya, Amgen, AstraZeneca, BMS, Cell Signaling Technology, Cepheid, Danaher, Konica/Minolta, Lilly, Merck, NanoString, NextCure, Odonate, Paige.AI, Roche, Sanofi, and Ventana. JZ has served as a consultant for AstraZeneca, BMS, Roche, Pfizer, Novartis, and Guardant Health. Reports speakers honoraria from BMS, Pfizer, Roche, AstraZeneca, NanoString and Guardant Health. Reports travel honoraria from BMS, Pfizer, Roche, AstraZeneca, and NanoString. Receives research support/funds from BMS, AstraZeneca, and Roche. The rest of the authors declare no conflicts of interest., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: The identification of actionable oncogenic alterations has enabled targeted therapeutic strategies for subsets of patients with advanced malignancies, including lung adenocarcinoma (LUAD). We sought to assess the frequency of known drivers and identify new candidate drivers in a cohort of LUAD from patients with minimal smoking history., Experimental Design: We performed genomic characterization of 103 LUADs from patients with ≤10 pack-year smoking history. Tumors were subjected to targeted molecular profiling and/or whole-exome sequencing and RNA sequencing in search of established and previously uncharacterized candidate drivers., Results: We identified an established oncogenic driver in 98 of 103 tumors (95%). From one tumor lacking a known driver, we identified a novel gene rearrangement between OCLN and RASGRF1. The encoded OCLN-RASGRF1 chimera fuses the membrane-spanning portion of the tight junction protein occludin with the catalytic RAS-GEF domain of the RAS activator RASGRF1. We identified a similar SLC4A4-RASGRF1 fusion in a pancreatic ductal adenocarcinoma cell line lacking an activating KRAS mutation and an IQGAP1-RASGRF1 fusion from a sarcoma in The Cancer Genome Atlas. We demonstrate these fusions increase cellular levels of active GTP-RAS, induce cellular transformation, and promote in vivo tumorigenesis. Cells driven by RASGRF1 fusions are sensitive to targeting of the RAF-MEK-ERK pathway in vitro and in vivo., Conclusions: Our findings credential RASGRF1 fusions as a therapeutic target in multiple malignancies and implicate RAF-MEK-ERK inhibition as a potential treatment strategy for advanced tumors harboring these alterations. See related commentary by Moorthi and Berger, p. 2983., (©2022 American Association for Cancer Research.)

Background: The expression of SYK in cancer cells has been associated with both tumor promoting and tumor suppressive effects. Despite being proposed as anticancer therapeutic target, the possible role of SYK in modulating local adaptive antitumor immune responses remains uncertain. Using detailed analysis of primary human tumors and in vitro models, we reveal the immunomodulatory effect of SYK protein in human solid cancer., Methods: We spatially mapped SYK kinase in tumor cells, stromal cells and tumor-infiltrating leukocytes (TILs) in 808 primary non-small cell lung carcinomas (NSCLCs) from two cohorts and in 374 breast carcinomas (BCs) from two independent cohorts. We established the associations of localized SYK with clinicopathologic variables and outcomes. The immunomodulatory role of SYK on tumor cells was assessed using in vitro cytokine stimulation, transcriptomic analysis and selective SYK blockade using a small molecule inhibitor. Functional responses were assessed using cocultures of tumor cells with peripheral blood lymphocytes. T cell responses in baseline and post-treatment biopsies from patients with BC treated with a SYK inhibitor in a phase I clinical trial were also studied., Results: Elevated tumor cell or leukocyte SYK expression was associated with high CD4 + and CD8 + TILs and better outcome in both NSCLC and BC. Tumor cell SYK was associated with oncogenic driver mutations in EGFR or KRAS in lung adenocarcinomas and with triple negative phenotype in BC. In cultured tumor cells, SYK was upregulated by TNFα and required for the TNFα-induced proinflammatory responses and T cell activation. SYK blockade after nivolumab in a phase I clinical trial including three patients with advanced triple negative BC reduced TILs and T cell proliferation. Our work establishes the proinflammatory function of tumor cell SYK in lung and breast cancer. SYK signaling in cultured tumor cells is required for T cell activation and SYK blockade limits adaptive antitumor immune responses and tumor rejection in patients with cancer., Conclusions: Together, our results establish the immunomodulatory role of SYK expression in human solid tumors. This information could be used to develop novel biomarkers and/or therapeutic strategies., Competing Interests: Competing interests: KAS reports research funding from Navigate Biopharma, Tesaro/GlaxoSmithKline, Moderna Inc, Takeda, Surface Oncology, Pierre-Fabre Research Institute, Merck-Sharp & Dohme, Bristol-Myers Squibb, AstraZeneca, Ribon Therapeutics, Akoya Biosciences, Eli Lilly and Boehringer-Ingelheim. KAS has received honoraria for consultant/advisory/speaker roles from Moderna Inc, Shattuck Labs, Pierre-Fabre, AstraZeneca, EMD Serono, Ono Pharmaceuticals, Clinica Alemana de Santiago, Dynamo Therapeutics, PeerView, Abbvie, Fluidigm, Takeda/Millenium Pharmaceuticals, Merck-Sharp & Dohme, Bristol Myers-Squibb, Agenus, Parthenon Therapeutics, Molecular Templates, and Torque Therapeutics., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Despite the prominent role of innate immunity in the antitumor response, little is known about the myeloid composition of human non-small cell lung cancer (NSCLC) with respect to histology and molecular subtype. We used multiplexed quantitative immunofluorescence (QIF) to measure the distribution and clinical significance of major myeloid cell subsets in large retrospective NSCLC collections., Methods: We established a QIF panel to map major myeloid cell subsets in fixed human NSCLC including 4',6-Diamidino-2-Phenylindole for all cells, pancytokeratin for tumor-epithelial cells, CD68 for M1-like macrophages; and CD11b plus HLA-DR to interrogate mature and immature myeloid cell populations such as myeloid derived suppressor cells (MDSCs). We interrogated 793 NSCLCs represented in four tissue microarray-based cohorts: #1 (Yale, n=379) and #2 (Greece, n=230) with diverse NSCLC subtypes; #3 (Yale, n=138) with molecularly annotated lung adenocarcinomas (ADC); and #4 (Yale, n=46) with patient-matched NSCLC and morphologically-normal lung tissue. We examined associations between marker levels, myeloid cell profiles, clinicopathologic/molecular variables and survival., Results: The levels of CD68+ M1 like macrophages were significantly lower and the fraction of CD11b+/HLA-DR- MDSC-like cells was prominently higher in tumor than in matched non-tumor lung tissues. HLA-DR was consistently higher in myeloid cells from tumors with elevated CD68 expression. Stromal CD11b was significantly higher in squamous cell carcinomas (SCC) than in ADC across the cohorts and EGFR-mutated lung ADCs displayed lower CD11b levels than KRAS-mutant tumors. Increased stromal CD68- and HLA-DR-expressing cells was associated with better survival in ADCs from two independent NSCLC cohorts. In SCC, increased stromal CD11b or HLA-DR expression was associated with a trend towards shorter 5-year survival., Conclusions: NSCLCs display an unfavorable myeloid immune contexture relative to non-tumor lung and exhibit distinct myeloid-cell profiles across histologies and presence of major oncogenic driver-mutations. Elevated M1-like stromal proinflammatory myeloid cells are prognostic in lung ADC, but not in SCC., Competing Interests: Competing interests: BSH reports research funding from Neximmune and has served as a consultant for AstraZeneca and Ideaya. FVE has received a research grant from Chilean National Fund for Scientific and Technological Development (FONDECYT grant nº 1221415). MFS took part in an advisory board for Numab and BMS; reports speaker honorarium from MSD and Replimune; reports a research grant from Roche. JY is employed by Abbvie. DLR reports research support from Amgen, Cepheid, Navigate BioPharma, NextCure, Konica/Minota, instrument support from Akoya, royalty from Rarecyte, major honoraria from consultative activities with Cell Signaling Technology, Cepheid, Danaher, and Konica/Minolta, and minor honororia from consultative activities with Amgen, AstraZeneca, Fluidigm, GSK, Immunogen, Lilly, Merck, Monopteros, Nanostring, NextCure, Odonate, PAIGE.AI, Regeneron, Roche, Sanofi, Ventana, and Verily. In the past year, L.C. has been a scientific founder, consultant, and board member for NextCure, Junshi, Normunity, Tayu, Zai Lab, Tpioneer, Vcanbio, and GenomiCare and has sponsored research funds from NextCure, Normunity, and DynamiCure. RSH is on the Board of Directors of Immunocore and Junshi Pharmaceuticals and reports consultative activities with AstraZeneca, Bolt Biotherapeutics, Bristol-Myers Squibb, Candel Therapeutics, Inc., Checkpoint Therapeutics, Cybrexa Therapeutics, DynamiCure Biotechnology, LLC, eFFECTOR Therapeutics, Inc, Eli Lilly and Company, EMD Serono, Genentech, Gilead, HiberCell, Inc, I-Mab Biopharma, Immune-Onc Therapeutics, Inc., Immunocore, Janssen, Johnson and Johnson, Loxo Oncology, Merck and Company, Mirati, NextCure, Novartis, Ocean, Biomedical, Inc, Oncocyte Corp, Oncternal Therapeutics, Pfizer, Regeneron Pharmaceuticals, Revelar Biotherapeuitcs, Inc, Ribbon Therapeutics, Roche, Sanofi, Xencor, Inc. RSH reports research support from AstraZeneca, Eli Lilly and Company, Genentech/Roche, Merck and Company. RSH serves a leadership role for the American Association for Cancer Research, International Association for the Study of Lung Cancer, Society for Immunotherapy of Cancer, and Southwest Oncology Group. KAS reports research funding from Navigate Biopharma, Tesaro/GlaxoSmithKline, Moderna, Takeda, Surface Oncology, Pierre-Fabre Research Institute, Merck Sharp & Dohme, Bristol Myers Squibb, AstraZeneca, Ribon Therapeutics, Boehringer-Ingelheimer, Akoya Biosciences and Eli Lilly. KAS has received honoraria for consultant/advisory/speaker roles from Moderna, Shattuck Labs, Pierre-Fabre, AstraZeneca, EMD Serono, Ono Pharmaceuticals, Clinica Alemana de Santiago, Dynamo Therapeutics, PeerView, AbbVie, Fluidigm, Takeda Pharmaceuticals, Merck Sharp & Dohme, Bristol Myers Squibb, Agenus, Parthenon Therapeutics, OnCusp and Torque Therapeutics., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Neoadjuvant chemotherapy and radiation followed by surgical resection of the rectum is a standard treatment for locally advanced rectal cancer. A subset of rectal cancer is caused by a deficiency in mismatch repair. Because mismatch repair-deficient colorectal cancer is responsive to programmed death 1 (PD-1) blockade in the context of metastatic disease, it was hypothesized that checkpoint blockade could be effective in patients with mismatch repair-deficient, locally advanced rectal cancer., Methods: We initiated a prospective phase 2 study in which single-agent dostarlimab, an anti-PD-1 monoclonal antibody, was administered every 3 weeks for 6 months in patients with mismatch repair-deficient stage II or III rectal adenocarcinoma. This treatment was to be followed by standard chemoradiotherapy and surgery. Patients who had a clinical complete response after completion of dostarlimab therapy would proceed without chemoradiotherapy and surgery. The primary end points are sustained clinical complete response 12 months after completion of dostarlimab therapy or pathological complete response after completion of dostarlimab therapy with or without chemoradiotherapy and overall response to neoadjuvant dostarlimab therapy with or without chemoradiotherapy., Results: A total of 12 patients have completed treatment with dostarlimab and have undergone at least 6 months of follow-up. All 12 patients (100%; 95% confidence interval, 74 to 100) had a clinical complete response, with no evidence of tumor on magnetic resonance imaging, 18 F-fluorodeoxyglucose-positron-emission tomography, endoscopic evaluation, digital rectal examination, or biopsy. At the time of this report, no patients had received chemoradiotherapy or undergone surgery, and no cases of progression or recurrence had been reported during follow-up (range, 6 to 25 months). No adverse events of grade 3 or higher have been reported., Conclusions: Mismatch repair-deficient, locally advanced rectal cancer was highly sensitive to single-agent PD-1 blockade. Longer follow-up is needed to assess the duration of response. (Funded by the Simon and Eve Colin Foundation and others; ClinicalTrials.gov number, NCT04165772.)., (Copyright © 2022 Massachusetts Medical Society.)

Immune checkpoint inhibitors (ICIs) show prominent clinical activity across multiple advanced tumors. However, less than half of patients respond even after molecule-based selection. Thus, improved biomarkers are required. In this study, we use an image analysis to capture morphologic attributes relating to the spatial interaction and architecture of tumor cells and tumor-infiltrating lymphocytes (TILs) from digitized H&E images. We evaluate the association of image features with progression-free (PFS) and overall survival in non-small cell lung cancer (NSCLC) ( N = 187) and gynecological cancer ( N = 39) patients treated with ICIs. We demonstrated that the classifier trained with NSCLC alone was associated with PFS in independent NSCLC cohorts and also in gynecological cancer. The classifier was also associated with clinical outcome independent of clinical factors. Moreover, the classifier was associated with PFS even with low PD-L1 expression. These findings suggest that image analysis can be used to predict clinical end points in patients receiving ICI.

Background: Tumor infiltrating lymphocytes (TILs) reflect adaptive antitumor immune responses in cancer and are generally associated with favorable prognosis. However, the relationships between TILs subsets and their spatial arrangement with clinical benefit from immune checkpoint inhibitors (ICI) in non-small cell lung cancer (NSCLC) remains less explored., Methods: We used multiplexed quantitative immunofluorescence panels to determine the association of major TILs subpopulations, CD8 + cytotoxic T cells, CD4 + helper T cells and CD20 + B cells, and T cell exhaustion markers, programmed cell death protein-1 (PD-1),lymphocyte-activation gene 3 (LAG-3) and T cell immunoglobulin mucin-3 (TIM-3) with outcomes in a multi-institutional cohort of baseline tumor samples from 179 patients with NSCLC treated with ICI. The analysis of full-face tumor biopsies including numerous fields of view allowed a detailed spatial analysis and assessment of tumor immune heterogeneity using a multiparametric quadratic entropy metric (Rao's Q Index (RQI))., Results: TILs were preferentially located in the stromal tissue areas surrounding tumor-cell nests and CD8 + T cells were the most abundant subset. Higher density of stromal CD8 + cytotoxic T cells was significantly associated with longer survival, and this effect was more prominent in programmed death ligand-1 (PD-L1) positive cases. The role of baseline T cell infiltration to stratify PD-L1 expressing cases was confirmed measuring the T cell receptor-burden in an independent NSCLC cohort studied with whole-exome DNA sequencing. High levels of LAG-3 on T cells or elevated RQI heterogeneity index were associated with worse survival in the cohort., Conclusion: Baseline T cell density and T cell exhaustion marker expression can stratify outcomes in PD-L1 positive patients with NSCLC treated with ICI. Spatial immune heterogeneity can be measured using the RQI and is associated with survival in NSCLC., Competing Interests: Competing interests: JG has served as a compensated consultant or received honoraria from Bristol Myers Squibb, Takeda, Loxo/Lilly, Blueprint, Oncorus, Regeneron, Gilead, Moderna, AstraZeneca, EMD Serono, Pfizer, Novartis, Merck, and GlydeBio; research support from Novartis; institutional research support from Bristol Myers Squibb, Tesaro, Moderna, Blueprint, Jounce, Array Biopharma, Merck, Adaptimmune, Novartis, and Alexo; and has an immediate family member who is an employee with equity at Ironwood Pharmaceuticals. AR has served as a consultant for Halo and Tyra. AM is an equity holder in Elucid Bioimaging and in Inspirata. In addition, he has served as a scientific advisory board member for Inspirata, AstraZeneca, Bristol Meyers Squibb and Merck. Currently he serves on the advisory board of Aiforia and currently consults for Caris, Roche, Cernostics and Aiforia. He also has sponsored research agreements with Philips, AstraZeneca, Boehringer-Ingelheim and Bristol Meyers Squibb. His technology has been licensed to Elucid Bioimaging. He is also involved in three different R01 grants with Inspirata. MM-K has served as a compensated consultant for H3 Biomedicine and AstraZeneca and has received research (institutional) funding from Novartis and royalty from Elsevier, all of which are not related to this work. JWR reports institutional research support from Spectrum, AstraZeneca, Novartis, Merck, Revolution Medicines. JWR has received honoraria for consultant/advisory roles from EMD Serono, Daiichi Sankyo, BMS, Beigene, Turning Point, Janssen, Novartis, Boehringer Ingelheim, Blueprint, Regeneron. KAS reports research funding from Navigate Biopharma, Tesaro/GlaxoSmithKline, Moderna, Takeda, Surface Oncology, Pierre-Fabre Research Institute, Merck Sharp & Dohme, Bristol Myers Squibb, AstraZeneca, Ribon Therapeutics, Eli Lilly, Boehringer Ingelheim and Akoya Biosciences. KAS has received honoraria for consultant/advisory/speaker roles from Moderna, Shattuck Labs, Pierre-Fabre, AstraZeneca, EMD Serono, Ono Pharmaceuticals, Clinica Alemana de Santiago, Dynamo Therapeutics, PeerView, AbbVie, Fluidigm, Takeda/Millenium Pharmaceuticals, Merck Sharp & Dohme, Bristol Myers Squibb, Agenus, Torque Therapeutics, Genmab, Parthenon Therapeutics and OnCusp. AM’s research efforts are supported by grants from the National Cancer Institute under award numbers R01CA249992-01A1, R01CA202752-01A1, R01CA208236-01A1, R01CA216579-01A1, R01CA220581-01A1, R01CA257612-01A1, 1U01CA239055-01, 1U01CA248226-01, 1U54CA254566-01, National Heart, Lung and Blood Institute 1R01HL15127701A1, R01HL15807101A1, National Institute of Biomedical Imaging and Bioengineering 1R43EB028736-01, National Center for Research Resources under award number 1 C06 RR12463-01, VA Merit Review Award IBX004121A from the US Department of Veterans Affairs Biomedical Laboratory Research and Development Service the Office of the Assistant Secretary of Defense for Health Affairs, through the Breast Cancer Research Program (W81XWH-19-1-0668), the Prostate Cancer Research Program (W81XWH-15-1-0558, W81XWH-20-1-0851), the Lung Cancer Research Program (W81XWH-18-1-0440, W81XWH-20-1-0595), the Peer Reviewed Cancer Research Program (W81XWH-18-1-0404, W81XWH-21-1-0345), the Kidney Precision Medicine Project (KPMP) Glue Grant and sponsored research agreements from Bristol Myers Squibb, Boehringer-Ingelheim, and AstraZeneca., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: People living with HIV (PLWH) have increased risk of developing cancers after controlling traditional risk factors and viral suppression. This study explores whether T cells can serve as a marker of risk for cancer among HIV-infected virally suppressed patients., Methods: A nested case control study design was pursued with 17 cancer cases and 73 controls (PLWH without cancer)ouidentified among the US Military HIV Natural History Study cohort, and were matched for CD4 + count, duration of HIV infection, and viral suppression. Cells were obtained from PLWH on an average of 12 months prior to clinical cancer diagnosis. Expression of inhibitory receptors (PD-1, CD160, CD244, Lag-3, and TIGIT), and transcription factors (T-bet, Eomesodermin, TCF-1, and (TOX) was measured on CD8 +T cells from that early time point., Results: We found that cases have increased expression of PD-1 +CD160+CD244+ ('triple positive') on total and effector CD8 + compared with controls (p=0.02). Furthermore, CD8 +T cells that were both PD-1 +CD160+CD244+ and T-bet dim Eomes hi were significantly elevated in cases at time point before cancer detection, compared with controls without cancer (p=0.008). This was driven by the finding that transcriptional factor profile of cells was altered in cancers compared with controls. Triple-positive cells were noted to retain the ability for cytotoxicity and cytokine secretion mediated by expression of CD160 and PD-1, respectively. However, triple-positive cells demonstrated high expression of TOX-1, a transcription factor associated with T cell exhaustion., Conclusion: In conclusion, we have found a subset of dysfunctional CD8 +T cells, PD-1 +CD160+CD244+T-bet dim Eomes hi , that is elevated 12 months before cancer diagnosis, suggesting that peripheral T cell alterations may serve as a biomarker of increased cancer risk among PLWH., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: To evaluate the tissue distribution and clinical significance of OX40 and OX40L in human non-small cell lung cancer (NSCLC)., Experimental Design: Using multiplexed quantitative immunofluorescence, we conducted simultaneous and localized measurements of OX40 and OX40L proteins, major T-cell subsets, and conventional type 1 dendritic cells (cDC1) in 614 primary NSCLCs from three independent cohorts represented in tissue microarrays. We also measured OX40L protein in samples from a phase I clinical trial of intratumor administration of a lipid nanoparticle encapsulated mRNA encoding OX40L (mRNA-2416) in human solid tumors. Finally, we studied the OX40 pathway in 212 uterine/ovarian serous carcinomas., Results: OX40 protein was expressed in approximately 90% of NSCLCs, and OX40L was detected in approximately 10% of cases. Increased expression of OX40 was associated with higher CD4 + and CD8 + T lymphocytes, as well as cDC1s. Elevated expression of OX40L was consistently associated with increased CD4 + tumor-infiltrating lymphocytes and longer overall survival. No association was found between OX40 or OX40L levels and oncogenic driver mutations in EGFR and KRAS in lung adenocarcinomas. Delivering OX40L mRNA using intratumor mRNA-2416 injection mediated increased local OX40L protein levels that was most prominent in a patient with ovarian serous carcinoma. Detectable OX40L protein levels were observed in 15% of primary uterine/ovarian serous malignancies and associated with longer survival., Conclusions: The OX40 pathway is expressed in a fraction of NSCLCs and is associated with a favorable immune contexture. Although OX40L is uncommonly expressed in NSCLC and serous malignancies, it is associated with better prognosis and can be introduced using exogenous mRNA., (©2021 American Association for Cancer Research.)

Substantial progress has been made in understanding how tumors escape immune surveillance. However, few measures to counteract tumor immune evasion have been developed. Suppression of tumor antigen expression is a common adaptive mechanism that cancers use to evade detection and destruction by the immune system. Epigenetic modifications play a critical role in various aspects of immune invasion, including the regulation of tumor antigen expression. To identify epigenetic regulators of tumor antigen expression, we established a transplantable syngeneic tumor model of immune escape with silenced antigen expression and used this system as a platform for a CRISPR-Cas9 suppressor screen for genes encoding epigenetic modifiers. We found that disruption of the genes encoding either of the chromatin modifiers activating transcription factor 7-interacting protein (Atf7ip) or its interacting partner SET domain bifurcated histone lysine methyltransferase 1 (Setdb1) in tumor cells restored tumor antigen expression. This resulted in augmented tumor immunogenicity concomitant with elevated endogenous retroviral (ERV) antigens and mRNA intron retention. ERV disinhibition was associated with a robust type I interferon response and increased T-cell infiltration, leading to rejection of cells lacking intact Atf7ip or Setdb1 . ATF7IP or SETDB1 expression inversely correlated with antigen processing and presentation pathways, interferon signaling, and T-cell infiltration and cytotoxicity in human cancers. Our results provide a rationale for targeting Atf7ip or Setdb1 in cancer immunotherapy., (©2021 The Authors; Published by the American Association for Cancer Research.)

Despite unique genetic alterations within brain metastases (BrMs) and an immunologically distinct surrounding microenvironment, the composition and functional properties of tumor-infiltrating lymphocytes within BrM remain largely unexplored. In particular, the expression of coinhibitory receptors, such as programmed cell death 1 (PD-1), T cell immunoglobulin mucin receptor 3 (TIM-3), and lymphocyte activation gene 3 (LAG-3), within BrMs is unknown. Using multiplexed quantitative immunofluorescence (QIF), this study evaluates the localized expression of PD-L1, level and functional profile of major T cell subsets, and coinhibitory receptors within lung cancer-associated BrMs and primary lung tumors. Clinicopathologically annotated samples from 95 patients with lung cancer between 2002 and 2015 were represented in a tissue microarray format. Spatially resolved and multiplexed QIF was used to evaluate PD-L1 protein, phenotype markers for major T cell subsets (CD3, CD4, CD8, and FOXP3), cell-localized activation and proliferation markers (granzyme B and Ki67), and coinhibitory receptors (PD-1, LAG-3, and TIM-3). The signal for each marker was measured in marker-selected tissue compartments, and associations between marker levels, tumor location, and major clinicopathological variables were studied. In total, 41 primary lung tumors and 65 BrMs were analyzed, including paired samples from 11 patients. Levels of tumor PD-L1 expression were comparable between BrMs and primary lung tumors. BrMs had significantly lower levels of all T cell subsets relative to primary lung tumors, and T cells in BrMs displayed lower levels of granzyme B than primary lesions. PD-1, TIM-3, and LAG-3 levels in CD3 + T-cells were also significantly lower in BrMs. Marker expression in patients with paired samples from BrMs and primary lung tumors showed comparable results. High CD3 + T-cells, as well as high levels of TIM-3 and LAG-3 in CD3 + T-cells, were associated with longer overall survival in BrMs but not primary lung tumors. Lung cancer-associated BrMs display lower T cell infiltration, markers of cytolytic function, and immune regulatory signals than primary lung tumors. Despite these differences, high TIM-3 and high LAG-3 expressions in CD3 + T-cells were associated with longer survival. These features are accompanied by comparable levels of PD-L1 protein expression compared with primary lung tumors. These results highlight unique aspects of the tumor immune microenvironment within the brain and provide further support for intracranially focused therapies., Competing Interests: Competing interests: BYL, RG, AA, HW, MR, VLC, JNC, AJA, and LBJ have nothing to disclose. HMK reports grants and personal fees from Merck and Bristol-Myers Squibb, grants from Apexigen, personal fees from Iovance, Immunocore, Celldex, Array BioPharma, Elevate Bio, Instil Bio, Clinigen, Shionogi, and Chemocentryx, outside the submitted work. KAS reports research funding from Navigate Biopharma, Tesaro/GlaxoSmithKline, Moderna Inc, Takeda, Surface Oncology, Pierre-Fabre Research Institute, Merck-Sharp & Dohme, Bristol-Myers Squibb, AstraZeneca, Ribon Therapeutics, and Eli Lilly. He has received honoraria for consultant/advisory roles from Moderna Therapeutics, Shattuck Labs, Pierre-Fabre, AstraZeneca, EMD Serono, Ono Pharmaceuticals, Clinica Alemana de Santiago, Dynamo Therapeutics, PeerView, Abbvie, Fluidigm, Takeda/Millenium Pharmaceuticals, Merck-Sharp & Dohme, Bristol Myers-Squibb, Agenus and Torque Therapeutics. SBG reports grants and personal fees from AstraZeneca and Boehringer Ingelheim, and personal fees from Bristol-Myers Squibb, Amgen, Spectrum, Blueprint Medicine, Sanofi Genzyme, Daiichi-Sankyo, Regeneron, Janssen, and Takeda., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Neutrophil extracellular traps (NETs) are webs of extracellular nuclear DNA extruded by dying neutrophils infiltrating tissue. NETs constitute a defence mechanism to entrap and kill fungi and bacteria. Tumours induce the formation of NETs to the advantage of the malignancy via a variety of mechanisms shown in mouse models. Here, we investigated the presence of NETs in a variety of human solid tumours and their association with IL-8 (CXCL8) protein expression and CD8 + T-cell density in the tumour microenvironment. Multiplex immunofluorescence panels were developed to identify NETs in human cancer tissues by co-staining with the granulocyte marker CD15, the neutrophil marker myeloperoxidase and citrullinated histone H3 (H3Cit), as well as IL-8 protein and CD8 + T cells. Three ELISA methods to detect and quantify circulating NETs in serum were optimised and utilised. Whole tumour sections and tissue microarrays from patients with non-small cell lung cancer (NSCLC; n = 14), bladder cancer (n = 14), melanoma (n = 11), breast cancer (n = 31), colorectal cancer (n = 20) and mesothelioma (n = 61) were studied. Also, serum samples collected retrospectively from patients with metastatic melanoma (n = 12) and NSCLC (n = 34) were ELISA assayed to quantify circulating NETs and IL-8. NETs were detected in six different human cancer types with wide individual variation in terms of tissue density and distribution. At least in NSCLC, bladder cancer and metastatic melanoma, NET density positively correlated with IL-8 protein expression and inversely correlated with CD8 + T-cell densities. In a series of serum samples from melanoma and NSCLC patients, a positive correlation between circulating NETs and IL-8 was found. In conclusion, NETs are detectable in formalin-fixed human biopsy samples from solid tumours and in the circulation of cancer patients with a considerable degree of individual variation. NETs show a positive association with IL-8 and a trend towards a negative association with CD8 + tumour-infiltrating lymphocytes. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland., (© 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.)

Specific mechanisms by which tumor-infiltrating lymphocytes (TIL) become dysfunctional remain poorly understood. Here, we employed a two-pronged approach using single-cell mass cytometry and tissue imaging technologies to dissect TILs from 25 patients with resectable and 35 patients with advanced non-small cell lung cancer (NSCLC). We identified a burned-out CD8 + TIL subset (Ebo) that specifically accumulated within the tumor microenvironment (TME) but not in adjacent nontumoral tissues. Ebo showed the highest expression of proliferation and activation markers but produced the lowest amount of IFNγ and were the most apoptotic CD8 + TIL subset. Using a humanized patient-derived tumor xenograft model, we demonstrated that Ebo expansion occurred within the TME in a PD-1/B7-H1 pathway-dependent manner. Ebo abundance in baseline tumor tissues was associated with resistance to anti-PD therapy in patients with NSCLC. Our study identifies a dysfunctional TIL subset, with distinct features from previously described exhausted T cells, and implies strategies to overcome immunotherapy resistance. SIGNIFICANCE: We identified a highly proliferative, overactivated, and apoptotic dysfunctional CD8 + tumor-infiltrating subpopulation that is functionally distinct from previously described exhausted T cells. This population is expanded in lung cancer tissues in a PD-1/B7-H1-dependent manner, and its abundance is associated with resistance to cancer immunotherapy, thus becoming a potential tissue biomarker. This article is highlighted in the In This Issue feature, p. 1601 ., (©2021 American Association for Cancer Research.)

Purpose: To analyze the distribution, associated immune contexture, and clinical significance of human leukocyte antigen (HLA) class-I and HLA class-II subunits in non-small cell lung cancer (NSCLC)., Experimental Design: Using spatially resolved and quantitative multiplexed immunofluorescence we studied the tumor/stromal tissue distribution, cancer cell-specific defects, and clinicopathologic/survival associations of β2 microglobulin (β2M), HLA-A, and HLA-B,-C heavy chains, as well as HLA class-II β chain in >700 immunotherapy-naïve NSCLCs from four independent cohorts. Genomic analysis of HLA genes in NSCLC was performed using two publicly available cohorts., Results: Cancer cell-specific downregulation of HLA markers was identified in 30.4% of cases. β2M was downregulated in 9.8% (70/714), HLA-A in 9% (65/722), HLA-B,-C in 12.1% (87/719), and HLA class-II in 17.7% (127/717) of evaluable samples. Concurrent downregulation of β2M, HLA-B,-C, and HLA class-II was commonly identified. Deleterious mutations in HLA genes were detected in <5% of lung malignancies. Tumors with cancer cell-specific β2M downregulation displayed reduced T cells and increased natural killer (NK)-cell infiltration. Samples with cancer cell HLA-A downregulation displayed modest increase in CD8 + T cells and NK-cell infiltration. Samples with cancer cell-selective HLA-B,-C or HLA class-II downregulation displayed reduced T cells and NK-cell infiltration. There was limited association of the markers with clinicopathologic variables and KRAS/EGFR mutations. Cancer cell-selective downregulation of the HLA subunits was associated with shorter overall survival., Conclusions: Our results reveal frequent and differential defects in HLA class-I and HLA class-II protein subunit expression in immunotherapy-naïve NSCLCs associated with distinct tumor microenvironment composition and patient survival., (©2021 American Association for Cancer Research.)

One of the most important mechanisms by which cancer fosters its own development is the generation of an immune microenvironment that inhibits or impairs antitumor immune responses. A cancer permissive immune microenvironment is present in a large proportion of the patients with cancer who do not respond to immunotherapy approaches intended to trigger preexisting antitumor immune responses, for instance, immune checkpoint blockade. High circulating levels of IL8 in patients with cancer quite accurately predict those who will not benefit from checkpoint-based immunotherapy. IL8 has been reported to favor cancer progression and metastases via different mechanisms, including proangiogenesis and the maintenance of cancer stem cells, but its ability to attract and functionally modulate neutrophils and macrophages is arguably one of the most important factors. IL8 does not only recruit neutrophils to tumor lesions, but also triggers the extrusion of neutrophil extracellular traps (NET). The relevance and mechanisms underlying the contribution of both neutrophils and NETs to cancer development and progression are starting to be uncovered and include both direct effects on cancer cells and changes in the tumor microenvironment, such as facilitating metastasis, awakening micrometastases from dormancy, and facilitating escape from cytotoxic immune cells. Blockade of IL8 or its receptors (CXCR1 and CXCR2) is being pursued in drug development, and clinical trials alone or in combination with anti-PD-L1 checkpoint inhibitors are already ongoing., (©2020 American Association for Cancer Research.)

Purpose: Imaging mass cytometry (IMC) is among the first tools with the capacity for multiplex analysis of more than 40 targets, which provides a novel approach to biomarker discovery. Here, we used IMC to characterize the tumor microenvironment of patients with metastatic melanoma who received immunotherapy in efforts to find indicative factors of treatment response. In spite of the new power of IMC, the image analysis aspects are still limited by the challenges of cell segmentation., Experimental Design: Here, rather than segment, we performed image analysis using a newly designed version of the AQUA software to measure marker intensity in molecularly defined compartments: tumor cells, stroma, T cells, B cells, and macrophages. IMC data were compared with quantitative immunofluorescence (QIF) and digital spatial profiling., Results: Validation of IMC results for immune markers was confirmed by regression with additional multiplexing methods and outcome assessment. Multivariable analyses by each compartment revealed significant associations of 12 markers for progression-free survival and seven markers for overall survival (OS). The most compelling indicative biomarker, beta2-microglobulin (B2M), was confirmed by correlation with OS by QIF in the discovery cohort and validated in an independent published cohort profiled by mRNA expression., Conclusions: Using digital image analysis based on pixel colocalization to assess IMC data allowed us to quantitively measure 25 markers simultaneously on formalin-fixed, paraffin-embedded tissue microarray samples. In addition to showing high concordance with other multiplexing technologies, we identified a series of potentially indicative biomarkers for immunotherapy in metastatic melanoma, including B2M., (©2021 American Association for Cancer Research.)

Poly-ADP-ribose polymerase (PARP) inhibitors are active against cells and tumors with defects in homology-directed repair as a result of synthetic lethality. PARP inhibitors (PARPi) have been suggested to act by either catalytic inhibition or by PARP localization in chromatin. In this study, we treat BRCA1 mutant cells derived from a patient with triple negative breast cancer and control cells for three weeks with veliparib, a PARPi, to determine if treatment with this drug induces increased levels of mutations and/or an inflammatory response. We show that long-term treatment with PARPi induces an inflammatory response in HCC1937 BRCA1 mutant cells. The levels of chromatin-bound PARP1 in the BRCA1 mutant cells correlate with significant upregulation of inflammatory genes and activation of the cyclic GMP-AMP synthase (cGAS)/signaling effector stimulator of interferon genes (STING pathway). In contrast, an increased mutational load is induced in BRCA1-complemented cells treated with a PARPi. Our results suggest that long-term PARP inhibitor treatment may prime both BRCA1 mutant and wild-type tumors for positive responses to immune checkpoint blockade, but by different underlying mechanisms., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Anti-PD-(L)1 therapy represents a turning point in lung cancer immunotherapy, moving from previously ineffective enhancer strategies to immune checkpoints as standard first- and second-line therapies. This unprecedented success highlights the importance of mechanisms to escape immune attack, such PD-1/PD-L1 axis, and emphasize the importance to better understand the tumor immune microenvironment. Analyzing the specifics of immune response against lung tumor cells and how malignant cells progressively adapt to this pressure may help to understand which are the key aspects to guide the development of new therapeutic strategies. Here we review the past and present of clinical lung cancer immunotherapy and give a perspective for the future development based on emerging biological insights.

Purpose: Only a minority of patients with advanced non-small cell lung cancer (NSCLC) truly benefits from single-agent PD-1 checkpoint blockade, and more robust predictive biomarkers are needed., Experimental Design: We assessed tumor samples from 67 immunotherapy-treated NSCLC cases represented in a tissue microarray, 53 of whom had pretreatment samples and received monotherapy. Using GeoMx Digital Spatial Profiling System (NanoString Technologies), we quantified 39 immune parameters simultaneously in four tissue compartments defined by fluorescence colocalization [tumor (panCK + ), leucocytes (CD45 + ), macrophages (CD68 + ), and nonimmune stroma]., Results: A total of 156 protein variables were generated per case. In the univariate unadjusted analysis, we found 18 markers associated with outcome in spatial context, five of which remained significant after multiplicity adjustment. In the multivariate analysis, high levels of CD56 and CD4 measured in the CD45 compartment were the only markers that were predictive for all clinical outcomes, including progression-free survival (PFS, HR: 0.24, P = 0.006; and HR: 0.31, P = 0.011, respectively), and overall survival (OS, HR: 0.26, P = 0.014; and HR: 0.23, P = 0.007, respectively). Then, using an orthogonal method based on multiplex immunofluorescence and cell counting (inForm), we validated that high CD56 + immune cell counts in the stroma were associated with PFS and OS in the same cohort., Conclusions: This pilot scale discovery study shows the potential of the digital spatial profiling technology in the identification of spatially informed biomarkers of response to PD-1 checkpoint blockade in NSCLC. We identified a number of relevant candidate immune predictors in spatial context that deserve validation in larger independent cohorts., (©2020 American Association for Cancer Research.)

Immune checkpoint inhibitors (ICI) have revolutionized the management of advanced non-small cell lung cancer (NSCLC). However, most pivotal phase III trials systematically excluded patients with active brain metastases, precluding the generalization of the results. Although theoretically restricted from crossing the blood-brain barrier, the novel pharmacokinetic/pharmacodynamic profiles of anti-PD-1/PD-L1 drugs have prompted studies to evaluate their activity in patients with NSCLC with active central nervous system (CNS) involvement. Encouraging results have suggested that ICI could be active in the CNS in selected patients with driver-negative advanced NSCLC with high PD-L1 expression and low CNS disease burden. Single-agent CNS response rates around 30% have been reported. Beyond this particular setting, anti-PD-1/PD-L1 antibodies have been evaluated in patients receiving local therapy for brain metastases (BM), addressing concerns about potential neurologic toxicity risks associated with radiotherapy, more specifically, radionecrosis (RN). Accordingly, a variety of clinical and imaging strategies are being appropriately developed to evaluate tumor response and to rule out pseudoprogression or radionecrosis. Our purpose is to critically summarize the advances regarding the role of systemic anti-PD-1/PD-L1 antibodies for the treatment of NSCLC BM. Data were collected from the PubMed database, reference lists, and abstracts from the latest scientific meetings. Recent reports suggest anti-PD-1/PD-L1 agents are active in a subset of patients with NSCLC with BM showing acceptable toxicity. These advances are expected to change soon the management of these patients but additional research is required to address concerns regarding radionecrosis and the appropriate sequencing of local and systemic therapy combinations., (©2020 American Association for Cancer Research.)

In the past 10 years, a deeper understanding of the immune landscape of cancers, including immune evasion processes, has allowed the development of a new class of agents. The reactivation of host antitumor immune response offers the potential for long-term survival benefit in a portion of patients with thoracic malignancies. The advent of programmed cell death protein 1/programmed death ligand-1 immune checkpoint inhibitors (ICIs), both as single agents and in combination with chemotherapy, and more recently, the combination of ICI, anti-programmed cell death protein 1, and anticytotoxic T-lymphocyte antigen 4 antibody, have led to breakthrough therapeutic advances for patients with advanced NSCLC, and to a lesser extent, patients with SCLC. Encouraging activity has recently emerged in pretreated patients with thymic carcinoma (TC). Conversely, in malignant pleural mesothelioma, pivotal positive signs of activity have not been fully confirmed in randomized trials. The additive effects of chemoradiation and immunotherapy suggested intriguing potential for therapeutic synergy with combination strategies. This has led to the introduction of ICI consolidation therapy in stage III NSCLC, creating a platform for future therapeutic developments in earlier-stage disease. Despite the definitive clinical benefit observed with ICI, primary and acquired resistance represent well-known biological phenomena, which may affect the therapeutic efficacy of these agents. The development of innovative strategies to overcome ICI resistance, standardization of new patterns of ICI progression, identification of predictive biomarkers of response, optimal treatment duration, and characterization of ICI efficacy in special populations, represent crucial issues to be adequately addressed, with the aim of improving the therapeutic benefit of ICI in patients with thoracic malignancies. In this article, an international panel of experts in the field of thoracic malignancies discussed these topics, evaluating currently available scientific evidence, with the final aim of providing clinical recommendations, which may guide oncologists in their current practice and elucidate future treatment strategies and research priorities., (Copyright © 2020 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Serum interleukin-8 (IL-8) levels and tumor neutrophil infiltration are associated with worse prognosis in advanced cancers. Here, using a large-scale retrospective analysis, we show that elevated baseline serum IL-8 levels are associated with poor outcome in patients (n = 1,344) with advanced cancers treated with nivolumab and/or ipilimumab, everolimus or docetaxel in phase 3 clinical trials, revealing the importance of assessing serum IL-8 levels in identifying unfavorable tumor immunobiology and as an independent biomarker in patients receiving immune-checkpoint inhibitors.

Background: We did a phase 2 trial of pembrolizumab in patients with non-small-cell lung cancer (NSCLC) or melanoma with untreated brain metastases to determine the activity of PD-1 blockade in the CNS. Interim results were previously published, and we now report an updated analysis of the full NSCLC cohort., Methods: This was an open-label, phase 2 study of patients from the Yale Cancer Center (CT, USA). Eligible patients were at least 18 years of age with stage IV NSCLC with at least one brain metastasis 5-20 mm in size, not previously treated or progressing after previous radiotherapy, no neurological symptoms or corticosteroid requirement, and Eastern Cooperative Oncology Group performance status less than two. Modified Response Evaluation Criteria in Solid Tumors (mRECIST) criteria was used to evaluate CNS disease; systemic disease was not required for participation. Patients were treated with pembrolizumab 10 mg/kg intravenously every 2 weeks. Patients were in two cohorts: cohort 1 was for those with PD-L1 expression of at least 1% and cohort 2 was patients with PD-L1 less than 1% or unevaluable. The primary endpoint was the proportion of patients achieving a brain metastasis response (partial response or complete response, according to mRECIST). All treated patients were analysed for response and safety endpoints. This study is closed to accrual and is registered with ClinicalTrials.gov, NCT02085070., Findings: Between March 31, 2014, and May 21, 2018, 42 patients were treated. Median follow-up was 8·3 months (IQR 4·5-26·2). 11 (29·7% [95% CI 15·9-47·0]) of 37 patients in cohort 1 had a brain metastasis response. There were no responses in cohort 2. Grade 3-4 adverse events related to treatment included two patients with pneumonitis, and one each with constitutional symptoms, colitis, adrenal insufficiency, hyperglycaemia, and hypokalaemia. Treatment-related serious adverse events occurred in six (14%) of 42 patients and were pneumonitis (n=2), acute kidney injury, colitis, hypokalaemia, and adrenal insufficiency (n=1 each). There were no treatment-related deaths., Interpretation: Pembrolizumab has activity in brain metastases from NSCLC with PD-L1 expression at least 1% and is safe in selected patients with untreated brain metastases. Further investigation of immunotherapy in patients with CNS disease from NSCLC is warranted., Funding: Merck and the Yale Cancer Center., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objectives: The interaction between the immune system and tumor cells is an important feature for the prognosis and treatment of cancer. Multiplex immunohistochemistry (mIHC) and multiplex immunofluorescence (mIF) analyses are emerging technologies that can be used to help quantify immune cell subsets, their functional state, and their spatial arrangement within the tumor microenvironment., Methods: The Society for Immunotherapy of Cancer (SITC) convened a task force of pathologists and laboratory leaders from academic centers as well as experts from pharmaceutical and diagnostic companies to develop best practice guidelines for the optimization and validation of mIHC/mIF assays across platforms., Results: Representative outputs and the advantages and disadvantages of mIHC/mIF approaches, such as multiplexed chromogenic IHC, multiplexed immunohistochemical consecutive staining on single slide, mIF (including multispectral approaches), tissue-based mass spectrometry, and digital spatial profiling are discussed., Conclusions: mIHC/mIF technologies are becoming standard tools for biomarker studies and are likely to enter routine clinical practice in the near future. Careful assay optimization and validation will help ensure outputs are robust and comparable across laboratories as well as potentially across mIHC/mIF platforms. Quantitative image analysis of mIHC/mIF output and data management considerations will be addressed in a complementary manuscript from this task force., Competing Interests: Competing interests: JMT has served on an advisory board, consulting and received research funding from Bristol-Myers Squibb; is a consultant and an advisory board member for Merck and Co., Inc., Kenilworth, New Jersey, USA, Amgen and AstraZeneca. MA is a founder, consultant and board member of IONpath. SG (Gnjatic) is a co-inventor on an issued patent that is filed through Icahn School of Medicine at Mount Sinai (ISMMS) and non-exclusively licensed to Caprion; reports consultancy/advisory fees from Merck and Co., Inc., Kenilworth, New Jersey, USA, Neon Therapeutics and OncoMed Pharmaceutical; and has received research funding from Agenus, Bristol-Myers Squibb, Genentech, Immune Design, Janssen R&D, Pfizer, Regeneron and Takeda Pharmaceutical. CVH is a stockholder of Bristol-Myers Squibb. TJH has received research support from Bristol-Myers Squibb and General Electric. JJ is an employee of Merck Sharp & Dohme, a subsidiary of Merck & Co., Inc., Kenilworth, New Jersey, USA and stockowner of Merck & Co., Inc., Kenilworth, New Jersey, USA; and a stockholder of Regeneron Pharmaceuticals, Illumina and Google. MCR is an employee and stockholder of AstraZeneca. DLR is a consultant, advisor, or serves on a scientific advisory board for Amgen, AstraZeneca, Biocept, Bristol-Myers Squibb, Cell Signaling Technology, Cepheid, Daiichi Sankyo, GlaxoSmithKline, Invicro Konica Minolta, Merck and Co., Inc., Kenilworth, New Jersey, USA, NanoString, PerkinElmer, Ultivue and Ventana; is a founder and equity holder of PixelGear; has received research support or instrument support from AstraZeneca, Cepheid, Eli Lilly, Navigate BioPharma/Novartis, NextCure, Ultivue, Akoya Biosciences/PerkinElmer, NanoString and Ventana; and has received royalty from Rarecyte. JRC is an employee and unvested stock options holder of AstraZeneca. KAS has received consultant honoraria from Clinica Alemana de Santiago, Celgene, Moderna Therapeutics, Pierre-Fabre Research Institute, Shattuck Labs, AbbVie, AstraZeneca, EMD Serono, Ono Pharmaceuticals, Dynamo Therapeutics and Takeda Pharmaceutical; speaker honoraria from Merck & Co., Inc., Kenilworth, New Jersey, USA, Takeda Pharmaceutical, The PeerView Institute and Fluidigm; and research funding from Genoptix/Navigate BioPharma (Novartis), Tioma Therapeutics (formerly Vasculox), Tesaro, Moderna, Takeda Pharmaceutical, Surface Oncology, Pierre-Fabre Research Institute, Merck & Co., Inc., Kenilworth, New Jersey, USA, Bristol-Myers Squibb, AstraZeneca and Eli Lilly. ECS is an employee of Jounce Therapeutics. CSF is an employee of Roche Diagnostics GmbH. KK is an employee and shareholder of Roche. SJR receives research grant support from Bristol-Myers Squibb, Merck and Co., Inc., Kenilworth, New Jersey, USA, Kite/Gilead and Affimed Inc. ES is an employee of AstraZeneca. KES is an employee and stockholder of AstraZeneca; and a spouse of an employee of Arcellx. MJS is an employee and stockholder of AstraZeneca. MTT has served on an advisory board for Myriad Genetics, Seattle Genetics and Novartis; and has presented as a speaker for NanoString. IIW has served on an advisory board for as well as received research support from Genetech/Roche, Bayer AG, Bristol-Myers Squibb, AstraZeneca, Pfizer, HTG Molecular Diagnostics and Merck and Co., Inc., Kenilworth, New Jersey, USA; has served on an advisory board for Asuragen, GlaxoSmithKline, Guardant Health and Merck Sharp & Dohme, a subsidiary of Merck & Co., Inc., Kenilworth, New Jersey, USA; has received research support from DEPArray/Menarini Silicon Biosystems, Adaptive Biotechnologies, Adaptimmune, EMD Serono, Takeda Pharmaceutical, Amgen, Karus Therapeutics, Johnson & Johnson, Iovance Biotherapeutics, 4D pharma, Novartis, Akoya Biosciences; and has presented as a speaker for Genetech/Roche, Pfizer and Merck Sharp & Dohme, a subsidiary of Merck & Co., Inc., Kenilworth, New Jersey, USA. CBB has served on the scientific advisory boards at Bristol-Myers Squibb, Roche, PrimeVax and Halio DX; is a stockholder of PrimeVax and holds the patent US20180322632A1., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)