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Background: The response rate to immune checkpoint inhibitors targeting programmed cell death 1 (PD-1) receptor is 13%-18% for patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). Detailed understanding of the tumor immune microenvironment (TIME) is crucial in order to explain and improve this response rate. HNSCCs arise at various anatomical locations including the oral cavity, hypopharynx, larynx and oropharynx. Studies directly comparing immune infiltration between anatomical sites are scarce. Since the distinct locations could drive deviating microenvironments, we questioned whether the immune composition varies across these HNSCC sites., Methods: Here, we characterized the TIME of 76 fresh tumor specimens using flow cytometry and performed single-cell RNA-sequencing on nine head and neck tumor samples., Results: We found major differences in the composition of the TIME between patients. When comparing anatomical sites: tumors originating from the oral cavity had higher T cell infiltrates than tumors from other anatomical sites. The percentage of tumor-infiltrating T-lymphocytes positive for the immune checkpoint PD-1 varied considerably between patients, with the highest fraction of PD-1+ T cells found in larynx squamous cell carcinomas (SCCs). While we had hypothesized that the anatomical sites of tumor origin would drive sample clustering, our data showed that the type of TIME was more dominant and was particularly driven by the fraction of T cells positive for PD-1. Moreover, a high proportion of PD-1+ CD8+ T cells associated with an improved overall survival. Using single-cell RNA-sequencing, we observed that PD-1 expression was highest in the CD8-ENTPD1 tissue resident memory T cell/exhausted T cell and CD4-CXCL13 type 1 T helper cell clusters., Conclusions: We found that oral cavity SCCs had the highest frequencies of T cells. We also observed considerable interpatient heterogeneity for PD-1 on T cells, with noticeably higher frequencies of PD-1+ CD4+ T helper cells in larynx SCCs. Within the entire cohort, a higher fraction of CD8+ T cells positive for PD-1 was linked to improved overall survival. Whether the fraction of PD-1+ T cells within the TIME enables immune checkpoint inhibitor response prediction for patients with head and neck cancer remains to be determined., Competing Interests: Competing interests: None declared., (© 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: Chemoimmunotherapy is a standard treatment for triple-negative breast cancer (TNBC), however, the impacts of different chemotherapies on T-cell populations, which could correlate with clinical activity, are not known. Quantifying T-cell populations with flow cytometry and T-cell receptor (TCR) immunosequencing may improve our understanding of how chemoimmunotherapy affects T-cell subsets, and to what extent clonal shifts occur during treatment. TCR immunosequencing of intratumoral T cells may facilitate the identification and monitoring of putatively tumor-reactive T-cell clones within the blood., Methods: Blood and tumor biopsies were collected from patients with metastatic TNBC enrolled in a phase Ib clinical trial of first or second-line pembrolizumab with paclitaxel or capecitabine. Using identical biospecimen processing protocols, blood samples from a cohort of patients treated for early-stage breast cancer were obtained for comparison. Treatment-related immunological changes in peripheral blood and intratumoral T cells were characterized using flow cytometry and TCR immunosequencing. Clonal proliferation rates of T cells were compared based on intratumoral enrichment., Results: When combined with pembrolizumab, paclitaxel and capecitabine resulted in similar time-dependent lymphodepletions across measured peripheral T-cell subsets. Their effects were more modest than that observed following curative-intent dose-dense anthracycline and cyclophosphamide (ddAC) (average fold-change in CD3 + cells, capecitabine: -0.42, paclitaxel: -0.56, ddAC: -1.21). No differences in T-cell clonality or richness were observed following capecitabine or paclitaxel-based treatments. Regression modeling identified differences in the emergence of novel T-cell clones that were not detected at baseline (odds compared with ddAC, capecitabine: 0.292, paclitaxel: 0.652). Pembrolizumab with paclitaxel or capecitabine expanded T-cell clones within tumors; however, these clones did not always expand within the blood. Proliferation rates within the blood were similar between clones that were enriched and those that were not enriched within tumors., Conclusion: Chemoimmunotherapy for metastatic TNBC with pembrolizumab and capecitabine or paclitaxel resulted in similar peripheral T-cell subset lymphodepletion without altering T-cell clonal diversity. Regression modeling methods are applicable in immune monitoring studies, such as this to identify the odds of novel T-cell clones emerging during treatment, and proliferation rates of tumor-enriched T-cell clones., Competing Interests: Competing interests: YK: Research support from Bristol Myers Squibb (BMS), GlaxoSmithKline, Shimadzu. WLR: Research support from Galectin Therapeutics, BMS, GlaxoSmithKline, Mi. NA Therapeutics, Inhibrx, Veana Therapeutics, Shimadzu, OncoSec Medical, Turn Biotechnologies, CanWell Pharma, Aeglea Biotherapeutics, and Calibr. Patents/Royalties: Galectin Therapeutics. Advisory Boards: Nektar Therapeutics, Vesselon. NP: Financial interest: Adaptive Biotechnologies. PAF: Financial interest: Adaptive Biotechnologies. WJU: Safety advisory board: Astra Zeneca. HLM: Research support: Merck, Lilly, BMS. Advisory/consultancy Merck, Lilly, Spectrum Pharmaceuticals, Amgen, Immunomedics, Pfizer, Genentech, BMS, Genomic Health, ZIOPHARM Oncology; Travel/expenses: Merck, Spectrum Pharmaceuticals, Lilly, Amgen, Immunomedics, Pfizer, Genentech Puma Biotechnology; Speaker Bureau: Lilly. Institutional funding: Merck, Lilly, BMS, ZIOPHARM Oncology DBP: Research support: BMS, Merck, Brooklyn ImmunoTherapeutics. Advisory Boards: BMS, Merck, Syndax, Nektar, Puma, Nanostring, Genetech, Brooklyn Immunotherapeutics, Sanofi, Biotheranostics, NGMBio, Lilly. Speaker Bureau: Genentech, Novartis., (© 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: Immune checkpoint blockade (ICB) using anti-CTLA-4 and anti-PD-1/PD-L1 has revolutionized the treatment of advanced cancer. However, ICB is effective for only a small fraction of patients, and biomarkers such as expression of PD-L1 in tumor or serum levels of CXCL11 have suboptimal sensitivity and specificity. Exposure-response (E-R) relationships have been observed with other therapeutic monoclonal antibodies. There are many factors influencing E-R relationships, yet several studies have shown that trough levels of anti-PD-1/PD-L1 correlated with clinical outcomes. However, the potential utility of anti-CTLA-4 levels as a biomarker remains unknown., Methods: Serum was obtained at trough levels at weeks 7 and 12 (after doses 2 and 4) from patients with advanced melanoma who received ipilimumab alone (3 mg/kg every 3 weeks for four treatments) via an expanded access program (NCT00495066). We have successfully established a proteomics assay to measure the concentration of ipilimumab in serum using an liquid chromatography with tandem mass spectrometry-based nanosurface and molecular-orientation limited proteolysis (nSMOL) approach. Serum samples from 38 patients were assessed for trough levels of ipilimumab by the nSMOL assay., Results: We found that trough levels of ipilimumab were higher in patients who developed immune-related adverse events but did not differ based on the presence or absence of disease progression. We found that patients with higher trough levels of ipilimumab had better overall survival when grouped based on ipilimumab trough levels. Trough levels of ipilimumab were inversely associated with pretreatment serum levels of CXCL11, a predictive biomarker we previously identified, and soluble CD25 (sCD25), a prognostic biomarker for advanced melanoma, as well as C reactive protein (CRP) and interleukin (IL)-6 levels at week 7., Conclusions: Our results suggest that trough levels of ipilimumab may be a useful biomarker for the long-term survival of patients with advanced melanoma treated with ipilimumab. The association of ipilimumab trough levels with pretreatment serum levels of CXCL11 and sCD25 is suggestive of a baseline-driven E-R relationship, and the association of ipilimumab trough levels with on-treatment levels of CRP and IL-6 is suggestive of response-driven E-R relationship. Our findings highlight the potential utility of trough levels of ipilimumab as a biomarker., Trial Registration Number: NCT00495066., Competing Interests: Competing interests: YK: research support from Bristol Myers Squibb (BMS), GlaxoSmithKline, and Shimadzu. NI and TS: employees of Shimadzu Scientific Instruments. BDC: research support from AstraZeneca, Clinigen, and Galectin Therapeutics; patents/royalties: none; advisory boards: Clinigen, Nektar, and Merck. WJU: research support from BMS; royalties: data safety monitoring board for AstraZeneca. BDP: research support from Heat Biologics and Shimadzu; advisory boards: Bayer and UnitedHealthcare. WLR: research support from Galectin Therapeutics, BMS, GlaxoSmithKline, MiNA Therapeutics, Inhibrx, Veana Therapeutics, Aeglea Biotherapeutics, Shimadzu, OncoSec, and Calibr; patents/licensing fees: Galectin Therapeutics; advisory boards: Nektar Therapeutics and Vesselon., (© 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.)

Background: PD-1/PD-L1 engagement and overexpression of galectin-3 (Gal-3) are critical mechanisms of tumor-induced immune suppression that contribute to immunotherapy resistance. We hypothesized that Gal-3 blockade with belapectin (GR-MD-02) plus anti-PD-1 (pembrolizumab) would enhance tumor response in patients with metastatic melanoma (MM) and head and neck squamous cell carcinoma (HNSCC)., Methods: We performed a phase I dose escalation study of belapectin+pembrolizumab in patients with advanced MM or HNSCC (NCT02575404). Belapectin was administered at 2, 4, or 8 mg/kg IV 60 min before pembrolizumab (200 mg IV every 3 weeks for five cycles). Responding patients continued pembrolizumab monotherapy for up to 17 cycles. Main eligibility requirements were a functional Eastern Cooperative Oncology Group status of 0-2, measurable or assessable disease, and no active autoimmune disease. Prior T-cell checkpoint antibody therapy was permitted., Results: Objective response was observed in 50% of MM (7/14) and and 33% of HNSCC (2/6) patients. Belapectin+pembrolizumab was associated with fewer immune-mediated adverse events than anticipated with pembrolizumab monotherapy. There were no dose-limiting toxicities for belapectin within the dose range investigated. Significantly increased effector memory T-cell activation and reduced monocytic myeloid-derived suppressor cells (M-MDSCs) were observed in responders compared with non-responders. Increased baseline expression of Gal-3+ tumor cells and PD-1+CD8+ T cells in the periphery correlated with response as did higher serum trough levels of pembrolizumab., Conclusions: Belapectin+pembrolizumab therapy has activity in MM and HNSCC. Increased Gal-3 expression, expansion of effector memory T cells, and decreased M-MDSCs correlated with clinical response. Further investigation is planned., Competing Interests: Competing interests: WLR: research support from Galectin Therapeutics, Bristol Myers Squibb (BMS), Merck, Tesaro/GlaxoSmithKline, MiNA Therapeutics, Inhibrx, Veana Therapeutics, Aeglea Biotherapeutics, Shimadzu, OncoSec, and Calibr. Patents/licensing fees: Galectin Therapeutics. Advisory boards: Nektar Therapeutics, Vesselon. BC: research support from AstraZeneca, Clinigen, and Galectin Therapeutics. Advisory Boards: Replimmune, Clinigen, BMS, and Merck. Leidner: research support from BMS. Advisory boards: Merck, Oncolys, Sanofi, and Regeneron. YW: advisory boards: Roche, Amgen, Alexion, and AstraZeneca. NI and TS: employees of Shimadzu Scientific Instruments. Traber: stockholder of Galectin Therapeutics; employee and stockholder for Selecta Biosciences. EZ, JRH, and HS: employees and stockholders of Galectin Therapeutics. All other authors had no relevant disclosures., (© 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.)

Since the publication of the Society for Immunotherapy of Cancer's (SITC) original cancer immunotherapy biomarkers resource document, there have been remarkable breakthroughs in cancer immunotherapy, in particular the development and approval of immune checkpoint inhibitors, engineered cellular therapies, and tumor vaccines to unleash antitumor immune activity. The most notable feature of these breakthroughs is the achievement of durable clinical responses in some patients, enabling long-term survival. These durable responses have been noted in tumor types that were not previously considered immunotherapy-sensitive, suggesting that all patients with cancer may have the potential to benefit from immunotherapy. However, a persistent challenge in the field is the fact that only a minority of patients respond to immunotherapy, especially those therapies that rely on endogenous immune activation such as checkpoint inhibitors and vaccination due to the complex and heterogeneous immune escape mechanisms which can develop in each patient. Therefore, the development of robust biomarkers for each immunotherapy strategy, enabling rational patient selection and the design of precise combination therapies, is key for the continued success and improvement of immunotherapy. In this document, we summarize and update established biomarkers, guidelines, and regulatory considerations for clinical immune biomarker development, discuss well-known and novel technologies for biomarker discovery and validation, and provide tools and resources that can be used by the biomarker research community to facilitate the continued development of immuno-oncology and aid in the goal of durable responses in all patients., Competing Interests: Competing interests: SHL – Consulting: Amgen, Merck, Genmab, Xencor, Bristol-Myers Squibb, Regeneron; Funding support: Bristol-Myers Squibb, Merck, Vaccinex; Contracted research: Astellas, Merck, Xencor, Boehringer Ingelheim, Kite Pharma, Vedanta. SB – Employee: Roche Tissue Diagnostics. SG – Funding: Bristol-Myers Squibb, Rexahn, Incyte, Novartis, LSK BioPharma, Five Prime, Mirati, QED Bioscience, Debiopharm, Merck, Pfizer, AstraZeneca, MedImmune, Clovis, and Immunocore; Spouse stock ownership: Salarius Pharmaceuticals. BAH – Research funding: Merck, GlaxoSmithKline, Tempest Therapeutics, Leap Therapeutics, A*STAR Singapore, Sanofi; Consulting fees: Novartis, Merck, G1 Therapeutics. SJ – President and owner: ZellNet Consulting, Inc. TOK – Owner: Biolab Services; Employee: Immodulon Therapeutics. YK – Travel funds: AgonOx, Beckman Coulter; Consulting: Curiox; Research and travel funding: Shimadzu Corporation; Research funding from Bristol-Myers Squibb. YDM – President and owner: FlowKnowHow LLC; Business relationship ZellNet Consulting, Inc. RDN – Shareholder: Eli Lilly, Bristol-Myers Squibb. TS – Employee: Regeneron; IP rights: Regeneron; Sockholder: Regeneron. HTM – Scientific advisory board: Cytek, Inc., Caris Life Sciences; Stockholder: BD Biosciences; Spouse employed by: AbbVie Inc. KD, JCJBG, AWL, CM, SRS, YZ – Nothing to disclose. SITC Staff: AK, BL, LL, RL, SMW – Nothing to disclose., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Background: Checkpoint inhibitors can induce profound anticancer responses, but programmed cell death protein-1 (PD-1) inhibition monotherapy has shown minimal activity in prostate cancer. A published report showed that men with prostate cancer who were resistant to the second-generation androgen receptor inhibitor enzalutamide had increased programmed death-ligand 1 (PD-L1) expression on circulating antigen-presenting cells. We hypothesized that the addition of PD-1 inhibition in these patients could induce a meaningful cancer response., Methods: We evaluated enzalutamide plus the PD-1 inhibitor pembrolizumab in a single-arm phase II study of 28 men with metastatic castration-resistant prostate cancer (mprogressing on enzalutamide alone. Pembrolizumab 200 mg intravenous was given every 3 weeks for four doses with enzalutamide. The primary endpoint was prostate-specific antigen (PSA) decline of ≥50%. Secondary endpoints were objective response, PSA progression-free survival (PFS), time to subsequent treatment, and time to death. Baseline tumor biopsies were obtained when feasible, and samples were sequenced and evaluated for the expression of PD-L1, microsatellite instability (MSI), mutational and neoepitope burdens., Results: Five (18%) of 28 patients had a PSA decline of ≥50%. Three (25%) of 12 patients with measurable disease at baseline achieved an objective response. Of the five responders, two continue with PSA and radiographic response after 39.3 and 37.8 months. For the entire cohort, median follow-up was 37 months, and median PSA PFS time was 3.8 months (95% CI: 2.8 to 9.9 months). Time to subsequent treatment was 7.21 months (95% CI: 5.1 to 11.1 months). Median overall survival for all patients was 21.9 months (95% CI: 14.7 to 28 .4 months), versus 41.7 months (95% CI: 22.16 to not reached (NR)) in the responders. Of the three responders with baseline biopsies, one had MSI high disease with mutations consistent with DNA-repair defects. None had detectable PD-L1 expression., Conclusions: Pembrolizumab has activity in mCRPC when added to enzalutamide. Responses were deep and durable and did not require tumor PD-L1 expression or DNA-repair defects., Trial Registration Number: clinicaltrials.gov (NCT02312557)., Competing Interests: Competing interests: JG has received research funding from Astellas/Medivation, Merck, Sanofi, Janssen Biotech, and travel support from Sanofi. CD has received research funding from Bristol Myers Squibb (BMS). He has received consulting fees from BMS, Merck, AstraZeneca (AZ) and Medimmune. He has patents licensed to AZ, BMS and Medimmune. WLR has received research grants, consulting fees, and/or royalties from Bristol-Myers Squibb, Merck, Galectin Therapeutics, and Nektar Therapeutics. TMB has research funding from Astellas/Medivation, Alliance Foundation Trials, Boehringer Ingelheim, Corcept Therapeutics, Endocyte Inc, Janssen Research & Development, OncoGenex, Sotio, and Theraclone Sciences/OncoResponse. TMB receives consulting fees from AbbVie, AstraZeneca, Astellas Pharma, Bayer, Boehringer Ingelheim, Clovis Oncology, GlaxoSmithKline, Janssen Biotech, Janssen Japan, Merck, and Pfizer and has stock ownership in Salarius Pharmaceuticals, and Arvinas Inc. JJA, GVT, and RS have no conflicts. RCB is a co-owner of Third Coast Therapeutics Inc, which has an option to license patents of which he is an owner. AEM has a sponsored research agreement with AstraZeneca and has received reagents from Genetech. JJA has received consulting fees from Merck., (© 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.)