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Pancreatic ductal adenocarcinoma (PDA) is characterized by low cytotoxic lymphocytes, abundant immune-suppressive cells, and resistance to immune checkpoint inhibitors (ICI). Preclinical PDA models showed the HDAC inhibitor entinostat reduced myeloid cell immunosuppression, sensitizing tumors to ICI therapy. This phase II study combined entinostat with nivolumab (PD1 inhibitor) in patients with advanced PDA (NCT03250273). Patients received entinostat 5 mg orally once weekly for 14-day lead-in, followed by entinostat and nivolumab. The primary endpoint was the objective response rate (ORR) by RECIST v1.1. Secondary endpoints included safety, duration of response, progression free-survival and overall survival. Between November 2017 and November 2020, 27 evaluable patients were enrolled. Three showed partial responses (11% ORR, 95% CI, 2.4%-29.2%) with a median response duration of 10.2 months. Median progression-free survival (PFS) and overall survival (OS) were, respectively, 1.89 (95% CI, 1.381-2.301) and 2.729 (95% CI, 1.841-5.622) months. Grade ≥3 treatment-related adverse events occurred in 19 patients (63%), including decreased lymphocyte count, anemia, hypoalbuminemia, and hyponatremia. As exploratory analysis, peripheral and tumor immune profiles changes were assessed using CyTOF, mIHC, and RNA-seq. Entinostat increased dendritic cell activation and maturation. Gene expression analysis revealed an enrichment in inflammatory response pathways with combination treatment. Although the primary endpoint was not met, entinostat and nivolumab showed durable responses in a small subset of PDA patients. Myeloid cell immunomodulation supported the preclinical hypothesis, providing a basis for future combinatorial therapies to enhance clinical benefits in PDA., Competing Interests: Competing interests M.B. served on advisory boards for AstraZeneca, Incyte. C.B.B is an employee of, and holds equity in, Akoya Biosciences, Inc. W.J.H. reports patent royalties from Rodeo/Amgen; grants from Sanofi, NeoTX, and Riboscience; speaking/travel honoraria from Exelixis and Standard BioTools. MY receives grant/research support (to Johns Hopkins) from Bristol-Myers Squibb, Exelixis, Incyte, and Genentech; receives honoraria from Genentech, Exelixis, AstraZeneca, Replimune, Hepion, Lantheus; MY is the co-inventor of patents pertaining to cancer vaccines and is a cofounder with equity of Adventris Pharmaceuticals, all outside of the submitted work. E.J.F is on the scientific advisory board for Resistance Bio, has consulted for Merck and Mestag Therapeutics, and receives research grants from Roche/Genetech and Abbvie Inc. L.M.C. has received reagent support from Cell Signaling Technologies, Syndax Pharmaceuticals, Inc., ZielBio, Inc., and Hibercell, Inc.; holds sponsored research agreements with Syndax Pharmaceuticals, Hibercell, Inc., Prospect Creek Foundation, Lustgarten Foundation for Pancreatic Cancer Research, Susan G. Komen Foundation, and the National Foundation for Cancer Research; is on the Advisory Board for Dispatch Biotherapeutics, Carisma Therapeutics, Inc., CytomX Therapeutics, Inc., Shasqi, Kineta, Inc., Hibercell, Inc., Cell Signaling Technologies, Inc., Alkermes, Inc., Raska Pharma, Inc., NextCure, Guardian Bio, AstraZeneca Partner of Choice Network (OHSU Site Leader), Genenta Sciences, Pio Therapeutics Pty Ltd., and Lustgarten Foundation for Pancreatic Cancer Research Therapeutics Working Group, Inc. E.J. reports other support from Abmeta, other support from Adventris, personal fees from Achilles, personal fees from DragonFly, non-financial support from Parker Institute, personal fees from Surge, grants from Lustgarten, grants from Genentech, personal fees from Mestag, personal fees from Medical Home Group, non-financial support from BMS, grants from Break Through Cancer, personal fees from CPRIT, personal fees from Neuvogen, non-financial support from HDT Bio, and personal fees from NeoTx outside the submitted work. N.S.A. receives grant/research support (to Johns Hopkins) from Bristol-Myers Squibb. L.D., J.N.D., L.C., D.N.S., J.A.T., S.C., N.G., A.H., C.T., R.W., J.L., S.M., B.C., A.S., D.A.G., S.Y. declare no competing interest related to this study., (© 2024. The Author(s).)

Tumor molecular data sets are becoming increasingly complex, making it nearly impossible for humans alone to effectively analyze them. Here, we demonstrate the power of using machine learning (ML) to analyze a single-cell, spatial, and highly multiplexed proteomic data set from human pancreatic cancer and reveal underlying biological mechanisms that may contribute to clinical outcomes. We designed a multiplex immunohistochemistry antibody panel to compare T-cell functionality and spatial localization in resected tumors from treatment-naïve patients with localized pancreatic ductal adenocarcinoma (PDAC) with resected tumors from a second cohort of patients treated with neoadjuvant agonistic CD40 (anti-CD40) monoclonal antibody therapy. In total, nearly 2.5 million cells from 306 tissue regions collected from 29 patients across both cohorts were assayed, and over 1,000 tumor microenvironment (TME) features were quantified. We then trained ML models to accurately predict anti-CD40 treatment status and disease-free survival (DFS) following anti-CD40 therapy based on TME features. Through downstream interpretation of the ML models' predictions, we found anti-CD40 therapy reduced canonical aspects of T-cell exhaustion within the TME, as compared with treatment-naïve TMEs. Using automated clustering approaches, we found improved DFS following anti-CD40 therapy correlated with an increased presence of CD44+CD4+ Th1 cells located specifically within cellular neighborhoods characterized by increased T-cell proliferation, antigen experience, and cytotoxicity in immune aggregates. Overall, our results demonstrate the utility of ML in molecular cancer immunology applications, highlight the impact of anti-CD40 therapy on T cells within the TME, and identify potential candidate biomarkers of DFS for anti-CD40-treated patients with PDAC., (©2024 American Association for Cancer Research.)

Tumor molecular datasets are becoming increasingly complex, making it nearly impossible for humans alone to effectively analyze them. Here, we demonstrate the power of using machine learning to analyze a single-cell, spatial, and highly multiplexed proteomic dataset from human pancreatic cancer and reveal underlying biological mechanisms that may contribute to clinical outcome. A novel multiplex immunohistochemistry antibody panel was used to audit T cell functionality and spatial localization in resected tumors from treatment-naive patients with localized pancreatic ductal adenocarcinoma (PDAC) compared to a second cohort of patients treated with neoadjuvant agonistic CD40 (αCD40) monoclonal antibody therapy. In total, nearly 2.5 million cells from 306 tissue regions collected from 29 patients across both treatment cohorts were assayed, and more than 1,000 tumor microenvironment (TME) features were quantified. We then trained machine learning models to accurately predict αCD40 treatment status and disease-free survival (DFS) following αCD40 therapy based upon TME features. Through downstream interpretation of the machine learning models' predictions, we found αCD40 therapy to reduce canonical aspects of T cell exhaustion within the TME, as compared to treatment-naive TMEs. Using automated clustering approaches, we found improved DFS following αCD40 therapy to correlate with the increased presence of CD44 + CD4 + Th1 cells located specifically within cellular spatial neighborhoods characterized by increased T cell proliferation, antigen-experience, and cytotoxicity in immune aggregates. Overall, our results demonstrate the utility of machine learning in molecular cancer immunology applications, highlight the impact of αCD40 therapy on T cells within the TME, and identify potential candidate biomarkers of DFS for αCD40-treated patients with PDAC., Competing Interests: R.H.V. is an inventor on licensed patents relating to cancer cellular immunotherapy and cancer vaccines, and mutant Kras specific T cell receptors; has received consulting fees from BMS; and receives royalties from Children’s Hospital Boston for a licensed research-only monoclonal antibody and from the University of Pennsylvania for licensed research cell lines. J.A.N. receives consulting fees from Leica Biosystems and research support from Natera. B.M.W. receives research funding from AstraZeneca, Celgene/BMS, Eli Lilly, Novartis, and Revolution Medicines, and consulting for Celgene, GRAIL, Ipsen, Mirati, Third Rock Ventures unrelated to the current work. C.B.B. is an employee of, and holds equity in, Akoya Biosciences, Inc. K.T.B. receives royalties from the University of Pennsylvania for licensed research cell lines and has received consulting fees from Guidepoint. L.M.C. has received reagent support from Cell Signaling Technologies, Syndax Pharmaceuticals, Inc., ZielBio, Inc., and Hibercell, Inc.; holds sponsored research agreements with Syndax Pharmaceuticals, Hibercell, Inc., Prospect Creek Foundation, Lustgarten Foundation for Pancreatic Cancer Research, Susan G. Komen Foundation, and the National Foundation for Cancer Research; is on the Advisory Board for Carisma Therapeutics, Inc., CytomX Therapeutics, Inc., Kineta, Inc., Hibercell, Inc., Cell Signaling Technologies, Inc., Alkermes, Inc., Raska Pharma, Inc., NextCure, Guardian Bio, AstraZeneca Partner of Choice Network (OHSU Site Leader), Genenta Sciences, Pio Therapeutics Pty Ltd., and Lustgarten Foundation for Pancreatic Cancer Research Therapeutics Working Group, Inc.

Cyclic immunohistochemistry (cycIHC) uses sequential rounds of colorimetric immunostaining and imaging for quantitative mapping of location and number of cells of interest. Additionally, cycIHC benefits from the speed and simplicity of brightfield microscopy, making the collection of entire tissue sections and slides possible at a trivial cost compared to other high dimensional imaging modalities. However, large cycIHC datasets currently require an expert data scientist to concatenate separate open-source tools for each step of image pre-processing, registration, and segmentation, or the use of proprietary software. Here, we present a unified and user-friendly pipeline for processing, aligning, and analyzing cycIHC data - Cyclic Analysis of Single-Cell Subsets and Tissue Territories (CASSATT). CASSATT registers scanned slide images across all rounds of staining, segments individual nuclei, and measures marker expression on each detected cell. Beyond straightforward single cell data analysis outputs, CASSATT explores the spatial relationships between cell populations. By calculating the log odds of interaction frequencies between cell populations within tissues and tissue regions, this pipeline helps users identify populations of cells that interact-or do not interact-at frequencies that are greater than those occurring by chance. It also identifies specific neighborhoods of cells based on the assortment of neighboring cell types that surround each cell in the sample. The presence and location of these neighborhoods can be compared across slides or within distinct regions within a tissue. CASSATT is a fully open source workflow tool developed to process cycIHC data and will allow greater utilization of this powerful staining technique., (© 2023 International Clinical Cytometry Society.)

Purpose: Chronic graft-versus-host disease (cGVHD) remains the major cause of late morbidity after allogeneic hematopoietic cell transplantation. Colony-stimulating factor 1 receptor (CSF-1R)-dependent macrophages promote cGVHD fibrosis, and their elimination in preclinical studies ameliorated cGVHD. Axatilimab is a humanized monoclonal antibody that inhibits CSF-1R signaling and restrains macrophage development., Patients and Methods: This phase I (phI)/phase II (phII) open-label study (ClinicalTrials.gov identifier: NCT03604692) evaluated safety, tolerability, and efficacy of axatilimab in patients age ≥ 6 years with active cGVHD after ≥ 2 prior systemic therapy lines. Primary objectives in phI were to identify the optimal biologic and recommended phII dose and in phII to evaluate the overall (complete and partial) response rate (ORR) at the start of treatment cycle 7., Results: Forty enrolled patients (17 phI; 23 phII) received at least one axatilimab dose. In phI, a dose of 3 mg/kg given once every 4 weeks met the optimal biologic dose definition. Two dose-limiting toxicities occurred at the 3 mg/kg dose given once every 2 weeks. At least one treatment-related adverse event (TRAE) was observed in 30 patients with grade ≥ 3 TRAEs in eight patients, the majority known on-target effects of CSF-1R inhibition. No cytomegalovirus reactivations occurred. With the 50% ORR at cycle 7 day 1, the phII cohort met the primary efficacy end point. Furthermore, the ORR in the first six cycles, an end point supporting regulatory approvals, was 82%. Responses were seen in all affected organs regardless of prior therapy. Fifty-eight percent of patients reported significant improvement in cGVHD-related symptoms using the Lee Symptom Scale. On-target activity of axatilimab was suggested by the decrease in skin CSF-1R-expressing macrophages., Conclusion: Targeting profibrotic macrophages with axatilimab is a therapeutically promising novel strategy with a favorable safety profile for refractory cGVHD., Competing Interests: Carrie L. KitkoConsulting or Advisory Role: Horizon TherapeuticsTravel, Accommodations, Expenses: Mallinckrodt Mukta AroraEmployment: AmgenStock and Other Ownership Interests: AmgenResearch Funding: Syndax (Inst), Kadmon (Inst), Pharmacyclics (Inst) Zachariah DeFilippConsulting or Advisory Role: Kadmon, Omeros, Incyte, MorphoSysResearch Funding: Incyte, REGiMMUNE, Taiho Oncology Mohammad Abu ZaidStock and Other Ownership Interests: Pieris PharmaceuticalsConsulting or Advisory Role: Syndax, Ossium HealthResearch Funding: Syndax (Inst), Pharmacyclics (Inst), Janssen (Inst), Incyte (Inst), AlloVir (Inst)Open Payments Link: https://openpaymentsdata.cms.gov/physician/1350343 Vedran RadojcicEmployment: Syndax Pharmaceuticals, IncStock and Other Ownership Interests: Syndax Pharmaceuticals, IncConsulting or Advisory Role: Regeneron, AllakosOpen Payments Link: https://openpaymentsdata.cms.gov/physician/114735 Courtney B. BettsOther Relationship: Akoya Biosciences Lisa M. CoussensEmployment: Oregon Health & Science University (OHSU)Honoraria: Lustgarten Foundation for Pancreatic Cancer Research, Syndax, Carisma Therapeutics, Verseau Therapeutics, Inc, Scientific Advisory Board, Zymeworks, CytomX Therapeutics, Inc, Kineta Inc, Starr Cancer Consortium, Therapeutics Working Group:, Susan G Komen Foundation, Komen Scholar, AACR: Cancer Immunology ResearchConsulting or Advisory Role: Cell Signaling Technologies, Pharmacyclics, CytomX Therapeutics, Carisma Therapeutics, Verseau Therapeutics, Zymeworks, Kineta, Inc, AbbVie, Shasqi Inc, HiberCell, Alkermes, AstraZeneca Partner of Choice Network, OHSU site leader:, Genenta Science, Susan G Komen Foundation, Komen Scholar, Syndax, PDX Pharmaceuticals, Inc, Pio Therapeutics Pty LtdResearch Funding: Syndax, Pharmacyclics, Cell Signaling Technologies, Innate Pharma, Acerta Pharma (Inst), Susan G. Komen for the Cure (Inst), ZellBio, Inc, HiberCellOther Relationship: Prospect Creek Foundation, Lustgarten Foundation for Pancreatic Cancer Research, (P30) Koch Institute for Integrated Cancer Research, Massachusetts Institute of Technology (2012-present; honorarium), (P30) Salk Institute Cancer Center (2016-2020; honorarium), Bloomberg-Kimmel Institute for Cancer Immunotherapy, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins (2016-present; honorarium), Dana Farber Cancer Center Breast SPORE (2017-present; honorarium), (P30) Dana Farber/Harvard Cancer Center (2019-present; honorarium), (P30) University of California, San Diego Moores Cancer Center (2019-present; honorarium), Cancer Research Institute (CRI; 2013-present; unpaid), The V Foundation for Cancer Research (2013-present; unpaid), Starr Cancer Consortium (2011-present, honorarium), Therapeutics Working Group (2019-present; paid), NIH/NCI-Frederick National Laboratory Advisory Committee (FNLAC; 2016-present; daily honorarium), Susan G Komen Foundation, Komen Scholar (2020-2023; honorarium), (P50) Dana Farber Cancer Center Breast SPORE, (P30) The Jackson Laboratory Cancer Center, (P01) Columbia University Medical Center, Prostate P01, (P50) MDACC GI SPORE, American Association for Cancer Research, AACR: Cancer Discovery, Cancer Cell, National Foundation for Cancer Research Michael L. MeyersEmployment: SyndaxLeadership: SyndaxStock and Other Ownership Interests: Syndax, Nuvalent, IncConsulting or Advisory Role: Syndax, Nuvalent, IncPatents, Royalties, Other Intellectual Property: Syndax, Nuvalent, Inc Peter OrdentlichEmployment: SyndaxLeadership: SyndaxStock and Other Ownership Interests: SyndaxConsulting or Advisory Role: Patrys, Twentyeight-Seven TherapeuticsPatents, Royalties, Other Intellectual Property: Issued patents and patents pending Christine QuarantoEmployment: Syndax, Aerovate TherapeuticsStock and Other Ownership Interests: Syndax Pharmaceuticals, Aerovate Therapeutics Aaron SchmittEmployment: Syndax Pharmaceuticals IncStock and Other Ownership Interests: Syndax Yu GuEmployment: Syndax, AstraZenecaStock and Other Ownership Interests: Syndax, AstraZeneca Bruce R. BlazarStock and Other Ownership Interests: BlueRock Therapeutics, Tmunity Therapeutics, Inc, Magenta TherapeuticsConsulting or Advisory Role: BlueRock Therapeutics, Magenta Therapeutics, Obsidian Therapeutics, Editas MedicineResearch Funding: BlueRock Therapeutics, Rheos Medicines, Équilibre Biopharmaceuticals Corp, Carisma TherapeuticsPatents, Royalties, Other Intellectual Property: Inducible regulatory T-cell generation for hematopoietic cell transplants (UMN Z09026), US 9,228,172, TALEN-based gene correction, Patent No. 9,393,257, Generation of natural killer cells and lymphoid tissue inducer–like (LTI-like) NK-22 cells, 9,862,928, Method for correcting a genetic sequence, 10,648,002Travel, Accommodations, Expenses: Incyte, Magenta Therapeutics, Rheos Medicines Amandeep SalhotraConsulting or Advisory Role: Kadmon, Syros Pharmaceuticals, SobiResearch Funding: Bristol Myers Squibb Iskra PusicConsulting or Advisory Role: Kadmon, Incyte, Syndax Madan JagasiaEmployment: Iovance BiotherapeuticsStock and Other Ownership Interests: Iovance BiotherapeuticsConsulting or Advisory Role: Kadmon Stephanie J. LeeHonoraria: Wolters Kluwer, PERConsulting or Advisory Role: EMD Serono, Pfizer, 4SC, Mallinckrodt/Therakos, Almirall Hermal GmbH, Rain Therapeutics, Kadmon, EquilliumResearch Funding: Kadmon, Amgen, Bristol Myers Squibb, EMD Serono, Incyte, Syndax, Pfizer, AstraZenecaPatents, Royalties, Other Intellectual Property: Patent pending for high-affinity T-cell receptors that target the Merkel polyomavirusOther Relationship: National Marrow Donor Program, Society for Investigative Dermatology (SID)No other potential conflicts of interest were reported.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with poor 5-year survival rates, necessitating identification of novel therapeutic targets. Elucidating the biology of the tumor immune microenvironment (TiME) can provide vital insights into mechanisms of tumor progression. In this study, we developed a quantitative image processing platform to analyze sequential multiplexed IHC data from archival PDAC tissue resection specimens. A 27-plex marker panel was employed to simultaneously phenotype cell populations and their functional states, followed by a computational workflow to interrogate the immune contextures of the TiME in search of potential biomarkers. The PDAC TiME reflected a low-immunogenic ecosystem with both high intratumoral and intertumoral heterogeneity. Spatial analysis revealed that the relative distance between IL10+ myelomonocytes, PD-1+ CD4+ T cells, and granzyme B+ CD8+ T cells correlated significantly with survival, from which a spatial proximity signature termed imRS was derived that correlated with PDAC patient survival. Furthermore, spatial enrichment of CD8+ T cells in lymphoid aggregates was also linked to improved survival. Altogether, these findings indicate that the PDAC TiME, generally considered immuno-dormant or immunosuppressive, is a spatially nuanced ecosystem orchestrated by ordered immune hierarchies. This new understanding of spatial complexity may guide novel treatment strategies for PDAC., Significance: Quantitative image analysis of PDAC specimens reveals intertumoral and intratumoral heterogeneity of immune populations and identifies spatial immune architectures that are significantly associated with disease prognosis., (©2022 The Authors; Published by the American Association for Cancer Research.)

Despite significant recent advances in precision medicine, pancreatic ductal adenocarcinoma (PDAC) remains near uniformly lethal. Although immune-modulatory therapies hold promise to meaningfully improve outcomes for patients with PDAC, the development of such therapies requires an improved understanding of the immune evasion mechanisms that characterize the PDAC microenvironment. Here, we show that cancer cell-intrinsic glutamic-oxaloacetic transaminase 2 (GOT2) shapes the immune microenvironment to suppress antitumor immunity. Mechanistically, we find that GOT2 functions beyond its established role in the malate-aspartate shuttle and promotes the transcriptional activity of nuclear receptor peroxisome proliferator-activated receptor delta (PPARδ), facilitated by direct fatty acid binding. Although GOT2 is dispensable for cancer cell proliferation in vivo, the GOT2-PPARδ axis promotes spatial restriction of both CD4+ and CD8+ T cells from the tumor microenvironment. Our results demonstrate a noncanonical function for an established mitochondrial enzyme in transcriptional regulation of immune evasion, which may be exploitable to promote a productive antitumor immune response., Significance: Prior studies demonstrate the important moonlighting functions of metabolic enzymes in cancer. We find that the mitochondrial transaminase GOT2 binds directly to fatty acid ligands that regulate the nuclear receptor PPARδ, and this functional interaction critically regulates the immune microenvironment of pancreatic cancer to promote tumor progression. See related commentary by Nwosu and di Magliano, p. 2237.. This article is highlighted in the In This Issue feature, p. 2221., (©2022 The Authors; Published by the American Association for Cancer Research.)

Myocardial infarction (MI) triggers an inflammatory response that transitions from pro-inflammatory to reparative over time. Restoring sympathetic nerves in the heart after MI prevents arrhythmias. This study investigated if reinnervation altered the immune response after MI. This study used quantitative multiplex immunohistochemistry to identify the immune cells present in the heart 2 weeks after ischemia-reperfusion. Two therapeutics stimulated reinnervation, preventing arrhythmias and shifting the immune response from inflammatory to reparative, with fewer pro-inflammatory macrophages and more regulatory T cells and reparative macrophages. Treatments did not alter macrophage phenotype in vitro, which suggested reinnervation contributed to the altered immune response., Competing Interests: This work was supported by the OCTRI Biomedical Innovation Program, the OHSU Bioscience Innovation Program, the M.J. Murdock Charitable Trust and National Institutes of Health grant R01 HL093056. Dr Gardner was supported by the American Heart Association (19POST34460031) and the National Institutes of Health (NIH) (grant T32HL094294). Ms Brooks was supported by NIH (grants UL1GM118964). Dr Sepe was supported by NIH (grant T32HL094294). Dr Habecker was supported by NIH (R01 HL093056). Dr Coussens was supported by NIH (1U01 CA224012, U2C CA233280), the Susan G Komen Foundation, the Knight Cancer Institute, and the Brenden-Colson Center for Pancreatic Care at OHSU. Drs Habecker and Gardner are the co-inventors of technology (ISP) that was used in this research, and that OHSU has licensed to NervGen Pharma Corp. This potential conflict of interest has been reviewed and managed by OHSU. Dr Coussens has been a paid consultant for Cell Signaling Technologies; has received reagent and/or research support from Plexxikon Inc, Pharmacyclics, Inc, Acerta Pharma, LLC, Deciphera Pharmaceuticals, LLC, Genentech, Inc, Roche Glycart AG, Syndax Pharmaceuticals Inc, Innate Pharma, NanoString Technologies, and Cell Signaling Technologies; has been a member of the Scientific Advisory Boards of Syndax Pharmaceuticals, Carisma Therapeutics, Zymeworks, Inc, Verseau Therapeutics, Cytomix Therapeutics, Inc., Kineta Inc, Hibercell, Inc, Cell Signaling Technologies, Alkermes Inc, PDX Pharmaceuticals, Genenta Sciences, and Pio Therapeutics Py Ltd; has been a member of the Lustgarten Therapeutics Advisory working group; and has been a site lead for the AstraZeneca Partner of Choice Network. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2022 The Authors.)

Purpose: Programmed cell death-1 (PD-1) receptor inhibitors have shown efficacy in head and neck squamous cell carcinoma (HNSCC), but treatment failure or secondary resistance occurs in most patients. In preclinical murine carcinoma models, inhibition of Bruton's tyrosine kinase (BTK) induces myeloid cell reprogramming that subsequently bolsters CD8+ T cell responses, resulting in enhanced antitumor activity. This phase 2, multicenter, open-label, randomized study evaluated pembrolizumab (anti-PD-1 monoclonal antibody) plus acalabrutinib (BTK inhibitor) in recurrent or metastatic HNSCC., Patients and Methods: Patients received pembrolizumab 200 mg intravenously every 3 weeks, alone or in combination with acalabrutinib 100 mg orally twice daily. Safety and overall response rate (ORR) were co-primary objectives. The secondary objectives were progression-free survival (PFS) and overall survival., Results: Seventy-six patients were evaluated (pembrolizumab, n = 39; pembrolizumab + acalabrutinib, n = 37). Higher frequencies of grade 3-4 treatment-emergent adverse events (AE; 65% vs. 39%) and serious AEs (68% vs. 31%) were observed with combination therapy versus monotherapy. ORR was 18% with monotherapy versus 14% with combination therapy. Median PFS was 2.7 [95% confidence interval (CI), 1.4-6.8] months in the combination arm and 1.7 (95% CI, 1.4-4.0) months in the monotherapy arm. The study was terminated due to lack of clinical benefit with combination treatment. To assess how tumor immune contexture was affected by therapy in patients with pre- and post-treatment biopsies, spatial proteomic analyses were conducted that revealed a trend toward increased CD45+ leukocyte infiltration of tumors from baseline at day 43 with pembrolizumab (monotherapy, n = 5; combination, n = 2), which appeared to be higher in combination-treated patients; however, definitive conclusions could not be drawn due to limited sample size., Conclusions: Despite lack of clinical efficacy, immune subset analyses suggest that there are additive effects of this combination; however, the associated toxicity limits the feasibility of combination treatment with pembrolizumab and acalabrutinib in patients with recurrent or metastatic HNSCC., (©2021 The Authors; Published by the American Association for Cancer Research.)

Mechanisms of therapeutic resistance and vulnerability evolve in metastatic cancers as tumor cells and extrinsic microenvironmental influences change during treatment. To support the development of methods for identifying these mechanisms in individual people, here we present an omic and multidimensional spatial (OMS) atlas generated from four serial biopsies of an individual with metastatic breast cancer during 3.5 years of therapy. This resource links detailed, longitudinal clinical metadata that includes treatment times and doses, anatomic imaging, and blood-based response measurements to clinical and exploratory analyses, which includes comprehensive DNA, RNA, and protein profiles; images of multiplexed immunostaining; and 2- and 3-dimensional scanning electron micrographs. These data report aspects of heterogeneity and evolution of the cancer genome, signaling pathways, immune microenvironment, cellular composition and organization, and ultrastructure. We present illustrative examples of how integrative analyses of these data reveal potential mechanisms of response and resistance and suggest novel therapeutic vulnerabilities., Competing Interests: D.S. is employed by Quantitative Imaging Systems. L.M.C. is a paid consultant for Cell Signaling Technologies, Shasqi, and AbbVie; received reagent and/or research support from Plexxikon, Pharmacyclics, Acerta Pharma, Deciphera Pharmaceuticals, Genentech, Roche Glycart AG, Syndax Pharmaceuticals, Innate Pharma, and NanoString Technologies; and is a member of the scientific advisory boards of Syndax Pharmaceuticals, Carisma Therapeutics, Zymeworks, Verseau Therapeutics, Cytomix Therapeutics, and Kineta. G.B.M. has licensed technologies to Myriad Genetics and NanoString; is on the SAB or is a consultant to Amphista, AstraZeneca, Chrysallis Biotechnology, GSK, ImmunoMET, Ionis, Lilly, PDX Pharmaceuticals, Signalchem Lifesciences, Symphogen, Tarveda, Turbine, and Zentalis Pharmaceuticals; and has stock/options/financial interests in Catena Pharmaceuticals, ImmunoMet, SignalChem, and Tarveda. J.W.G. has licensed technologies to Abbott Diagnostics, Zorro Bio, and PDX Pharmaceuticals; has ownership positions in Convergent Genomics, Health Technology Innovations, Zorro Bio, and PDX Pharmaceuticals; serves as a paid consultant to New Leaf Ventures; has received research support from Thermo Fisher Scientific (formerly FEI), Zeiss, Miltenyi Biotech, Cepheid (Danaher), Quantitative Imaging, Health Technology Innovations, and Micron Technologies; and owns stock in Abbott Diagnostics, AbbVie, Alphabet, Amazon, Amgen, Apple, General Electric, Gilead, Intel, Microsoft, Nvidia, and Zimmer Biomet., (© 2022 The Authors.)

Purpose: CD40 activation is a novel clinical opportunity for cancer immunotherapy. Despite numerous active clinical trials with agonistic CD40 monoclonal antibodies (mAb), biological effects and treatment-related modulation of the tumor microenvironment (TME) remain poorly understood., Patients and Methods: Here, we performed a neoadjuvant clinical trial of agonistic CD40 mAb (selicrelumab) administered intravenously with or without chemotherapy to 16 patients with resectable pancreatic ductal adenocarcinoma (PDAC) before surgery followed by adjuvant chemotherapy and CD40 mAb., Results: The toxicity profile was acceptable, and overall survival was 23.4 months (95% confidence interval, 18.0-28.8 months). Based on a novel multiplexed immunohistochemistry platform, we report evidence that neoadjuvant selicrelumab leads to major differences in the TME compared with resection specimens from treatment-naïve PDAC patients or patients given neoadjuvant chemotherapy/chemoradiotherapy only. For selicrelumab-treated tumors, 82% were T-cell enriched, compared with 37% of untreated tumors ( P = 0.004) and 23% of chemotherapy/chemoradiation-treated tumors ( P = 0.012). T cells in both the TME and circulation were more active and proliferative after selicrelumab. Tumor fibrosis was reduced, M2-like tumor-associated macrophages were fewer, and intratumoral dendritic cells were more mature. Inflammatory cytokines/sec CXCL10 and CCL22 increased systemically after selicrelumab., Conclusions: This unparalleled examination of CD40 mAb therapeutic mechanisms in patients provides insights for design of subsequent clinical trials targeting CD40 in cancer., (©2021 The Authors; Published by the American Association for Cancer Research.)

Immunotherapies targeting aspects of T cell functionality are efficacious in many solid tumors, but pancreatic ductal adenocarcinoma (PDAC) remains refractory to these treatments. Deeper understanding of the PDAC immune ecosystem is needed to identify additional therapeutic targets and predictive biomarkers for therapeutic response and resistance monitoring. To address these needs, we quantitatively evaluated leukocyte contexture in 135 human PDACs at single-cell resolution by profiling density and spatial distribution of myeloid and lymphoid cells within histopathologically defined regions of surgical resections from treatment-naive and presurgically (neoadjuvant)-treated patients and biopsy specimens from metastatic PDAC. Resultant data establish an immune atlas of PDAC heterogeneity, identify leukocyte features correlating with clinical outcomes, and, through an in silico study, provide guidance for use of PDAC tissue microarrays to optimally measure intratumoral immune heterogeneity. Atlas data have direct applicability as a reference for evaluating immune responses to investigational neoadjuvant PDAC therapeutics where pretherapy baseline specimens are not available. SIGNIFICANCE: We provide a phenotypic and spatial immune atlas of human PDAC identifying leukocyte composition at steady state and following standard neoadjuvant therapies. These data have broad utility as a resource that can inform on leukocyte responses to emerging therapies where baseline tissues were not acquired. This article is highlighted in the In This Issue feature, p. 1861 ., (©2021 American Association for Cancer Research.)

Introduction: The programmed death-ligand 1 (PD-L1) immune checkpoint inhibitors, atezolizumab and durvalumab, have received regulatory approval for the first-line treatment of patients with extensive-stage SCLC. Nevertheless, when used in combination with platinum-based chemotherapy, these PD-L1 inhibitors only improve overall survival by 2 to 3 months. This may be due to the observation that less than 20% of SCLC tumors express PD-L1 at greater than 1%. Evaluating the composition and abundance of checkpoint molecules in SCLC may identify molecules beyond PD-L1 that are amenable to therapeutic targeting., Methods: We analyzed RNA-sequencing data from SCLC cell lines (n = 108) and primary tumor specimens (n = 81) for expression of 39 functionally validated inhibitory checkpoint ligands. Furthermore, we generated tissue microarrays containing SCLC cell lines and patient with SCLC specimens to confirm expression of these molecules by immunohistochemistry. We annotated patient outcomes data, including treatment response and overall survival., Results: The checkpoint protein B7-H6 (NCR3LG1) exhibited increased protein expression relative to PD-L1 in cell lines and tumors (p < 0.05). Higher B7-H6 protein expression correlated with longer progression-free survival (p = 0.0368) and increased total immune infiltrates (CD45+) in patients. Furthermore, increased B7-H6 gene expression in SCLC tumors correlated with a decreased activated natural killer cell gene signature, suggesting a complex interplay between B7-H6 expression and immune signature in SCLC., Conclusions: We investigated 39 inhibitory checkpoint molecules in SCLC and found that B7-H6 is highly expressed and associated with progression-free survival. In addition, 26 of 39 immune checkpoint proteins in SCLC tumors were more abundantly expressed than PD-L1, indicating an urgent need to investigate additional checkpoint targets for therapy in addition to PD-L1., (Published by Elsevier Inc.)

Competing Interests: The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO’s conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/cci/author-center. Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments). Carl R. PelzConsulting or Advisory Role, Immediate Family Member: NovartisCharles D. LopezConsulting or Advisory Role: Boston Scientific, Celgene, Boston Biomedical, Pfizer, Exelixis, Astellas Pharma Research Funding: Taiho Pharmaceutical Travel, Accommodations, Expenses: RenovoRxLisa M. CoussensEmployment: Oregon Health & Science University (OHSU) Honoraria: Prospect Creek Foundation, Lustgarten Foundation for Pancreatic Cancer Research, Syndax Pharmaceuticals, Inc: External Advisory Board, Carisma Therapeutics Inc: Scientific Advisory Board, Verseau Therapeutics, Inc, Scientific Advisory Board, Zymeworks, Inc, Scientific Advisory Board, CytomX Therapeutics, Inc, Kineta Inc, (P30) Koch Institute for Integrated Cancer Research, Massachusetts Inst. of Tech, (P30) Salk Institute Cancer Center, Bloomberg-Kimmel Institute for Cancer Immunotherapy, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Dana Farber Cancer Center Breast SPORE, (P30) Dana Farber/Harvard Cancer Center, (P30) University of California, San Diego Moores Cancer Center, Starr Cancer Consortium, Lustgarten Foundation for Pancreatic Cancer Research, Therapeutics Working Group, NIH/NCI-Frederick National Laboratory Advisory Committee (FNLAC), Susan G Komen Foundation, Komen Scholar Consulting or Advisory Role: Cell Signaling Technologies, Pharmacyclics, CytomX Therapeutics, Syndax, Carisma Therapeutics, Verseau Therapeutics, Zymeworks, Kineta, Inc, Abbvie, Shasqi Inc Research Funding: Syndax Pharmaceuticals Inc, Pharmacyclics, Cell Signaling Technologies, Innate Pharma, Deciphera Travel, Accommodations, Expenses: Cell Signaling Technologies, AstraZeneca, Pharmacyclics, Verseau, Carisma Therapeutics, CytomX Therapeutics, Zymeworks Other Relationship: Prospect Creek Foundation, Lustgarten Foundation for Pancreatic Cancer Research, (P30) Koch Institute for Integrated Cancer Research, Massachusetts Inst. of Tech. (2012-present; honorarium), (P30) Salk Institute Cancer Center: (2016-2020; honorarium), Bloomberg-Kimmel Institute for Cancer Immunotherapy, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins: (2016-present; honorarium), Dana Farber Cancer Center Breast SPORE: (2017-present; honorarium), (P30) Dana Farber/Harvard Cancer Center: (2019-present; honorarium), (P30) University of California, San Diego Moores Cancer Center (2019-present; honorarium), Cancer Research Institute (CRI): (2013-present; unpaid), The V Foundation for Cancer Research): (2013-present; unpaid), Starr Cancer Consortium: (2011-present, honorarium), Lustgarten Foundation for Pancreatic Cancer Research, Therapeutics Working Group: (2019-present; paid), NIH/NCI-Frederick National Laboratory Advisory Committee (FNLAC): (2016-present; daily honorarium), Susan G Komen Foundation, Komen Scholar (2020 – 2023; honorarium)Rosalie C. SearsConsulting or Advisory Role: Novartis No other potential conflicts of interest were reported

In June 2015, the U.S. Supreme Court made a landmark decision to legalize marriage for same-sex couples amid nationwide debate and media coverage of this controversial issue. Using a content analysis of newspaper articles and television transcripts (N = 286) from top news outlets, this study examines the frames used in news coverage of same-sex marriage before and after the decision and tone of coverage by frame and medium. Findings suggest that frames and tone differed by medium, with television generally presenting more negative coverage and print more positive coverage. Results also suggest that some coverage frames were more negative than others and that the dominant frames of coverage differed from pre- to post-decision. This study helps improve our understanding of how the public was informed before and after a historic decision and illuminates the differences between frame and tone of coverage by medium, and by medium over time.

Lactation insufficiency is variously defined and includes the inability to produce milk, not producing enough milk to exclusively meet infant growth requirements, and pathological interruption of lactation (e.g., mastitis). Of women with intent-to-breastfeed, lactation insufficiency has been estimated to affect 38%-44% of newly postpartum women, likely contributing to the nearly 60% of infants that are not breastfed according to the World Health Organization's guidelines. To date, research and clinical practice aimed at improving feeding outcomes have focused on hospital lactation support and education, with laudable results. However, researchers' reports of recent rodent studies concerning fundamental lactation biology have suggested that the underlying pathologies of lactation insufficiency may be more nuanced than is currently appreciated. In this article, we identify mucosal biology of the breast and lactation-specific liver biology as two under-researched aspects of lactation physiology. Specifically, we argue that further scientific inquiry into reproductive state-dependent regulation of immunity in the human breast will reveal insights into novel immune based requirements for healthy lactation. Additionally, our synthesis of the literature supports the hypothesis that the liver is an essential player in lactation-highlighting the potential that pathologies of the liver may also be associated with lactation insufficiency. More research into these biologic underpinnings of lactation is anticipated to provide new avenues to understand and treat lactation insufficiency.

Biomarker assessments of tumor specimens is widely used in cancer research to audit tumor cell intrinsic as well as tumor cell extrinsic features including the diversity of immune, stromal, and mesenchymal cells. To comprehensively and quantitatively audit the tumor-immune microenvironment (TiME), we developed a novel multiplex immunohistochemistry (mIHC) platform and computational image processing workflow using a single formalin-fixed paraffin-embedded (FFPE) tissue section. Herein, we validated this platform using nine matched primary newly diagnosed and recurrent head and neck squamous cell carcinoma (HNSCC) sections sequentially subjected to immunodetection with a panel of 29 antibodies identifying malignant tumor cells, and 17 distinct leukocyte lineages and their functional states. Image cytometric analysis was applied to interpret chromogenic signals from digitally scanned and coregistered light microscopy-based images enabling identification and quantification of individual tumor cells, structural features, immune cell phenotypes and their functional state. In agreement with our previous study via a 12-plex imaging mIHC platform, myeloid-inflamed status in newly diagnosed primary tumors associated with significantly short progression free survival, independent of lymphoid-inflamed status. Spatial distribution of tumor and immune cell lineages in TiME was also examined and revealed statistically significant CD8 + T cell exclusion from tumor nests, whereas regulatory T cells and myeloid cells, when present in close proximity to tumor cells, highly associated with rapid cancer recurrence. These findings indicate presence of differential immune-spatial profiles in newly diagnosed and recurrent HNSCC, and establish the robustness of the 29-plex mIHC platform and associated analytics for quantitative analysis of single tissue sections revealing longitudinal TiME changes., (© 2020 Elsevier Inc. All rights reserved.)

Background: Women diagnosed with breast cancer within 5 years postpartum (PPBC) have poorer prognosis than age matched nulliparous women, even after controlling for clinical variables known to impact disease outcomes. Through rodent modeling, the poor prognosis of PPBC has been attributed to physiologic mammary gland involution, which shapes a tumor promotional microenvironment through induction of wound-healing-like programs including myeloid cell recruitment. Previous studies utilizing immune compromised mice have shown that blocking prostaglandin synthesis reduces PPBC tumor progression in a tumor cell extrinsic manner. Given the reported roles of prostaglandins in myeloid and T cell biology, and the established importance of these immune cell populations in dictating tumor growth, we investigate the impact of involution on shaping the tumor immune milieu and its mitigation by ibuprofen in immune competent hosts., Methods: In a syngeneic (D2A1) orthotopic Balb/c mouse model of PPBC, we characterized the impact of mammary gland involution and ibuprofen treatment on the immune milieu in tumors and draining lymph nodes utilizing flow cytometry, multiplex IHC, lipid mass spectroscopy and cytokine arrays. To further investigate the impact of ibuprofen on programming myeloid cell populations, we performed RNA-Seq on in vivo derived mammary myeloid cells from ibuprofen treated and untreated involution group mice. Further, we examined direct effects of ibuprofen through in vitro bone marrow derived myeloid cell cultures., Results: Tumors implanted into the mammary involution microenvironment grow more rapidly and display a distinct immune milieu compared to tumors implanted into glands of nulliparous mice. This milieu is characterized by increased presence of immature monocytes and reduced numbers of T cells and is reversed upon ibuprofen treatment. Further, ibuprofen treatment enhances Th1 associated cytokines as well as promotes tumor border accumulation of T cells. Safety studies demonstrate ibuprofen does not impede gland involution, impact subsequent reproductive success, nor promote auto-reactivity as detected through auto-antibody and naïve T cell priming assays., Conclusions: Ibuprofen administration during the tumor promotional microenvironment of the involuting mammary gland reduces overall tumor growth and enhances anti-tumor immune characteristics while avoiding adverse autoimmune reactions. In sum, these studies implicate beneficial prophylactic use of ibuprofen during the pro-tumorigenic window of mammary gland involution.

The mammary gland is not classically considered a mucosal organ, although it exhibits some features common to mucosal tissues. Notably, the mammary epithelium is contiguous with the external environment, is exposed to bacteria during lactation, and displays antimicrobial features. Nonetheless, immunological hallmarks predictive of mucosal function have not been demonstrated in the mammary gland, including immune tolerance to foreign Ags under homeostasis. This inquiry is important, as mucosal immunity in the mammary gland may assure infant and women's health during lactation. Further, such mucosal immune programs may protect mammary function at the expense of breast cancer promotion via decreased immune surveillance. In this study, using murine models, we evaluated mammary specific mucosal attributes focusing on two reproductive states at increased risk for foreign and self-antigen exposure: lactation and weaning-induced involution. We find a baseline mucosal program of RORγT + CD4 + T cells that is elevated within lactating and involuting mammary glands and is extended during involution to include tolerogenic dendritic cell phenotypes, barrier-supportive antimicrobials, and immunosuppressive Foxp3 + CD4 + T cells. Further, we demonstrate suppression of Ag-dependent CD4 + T cell activation, data consistent with immune tolerance. We also find Ag-independent accumulation of memory RORγT + Foxp3 + CD4 + T cells specifically within the involution mammary gland consistent with an active immune process. Overall, these data elucidate strong mucosal immune programs within lactating and involuting mammary glands. Our findings support the classification of the mammary gland as a temporal mucosal organ and open new avenues for exploration into breast pathologic conditions, including compromised lactation and breast cancer., (Copyright © 2018 by The American Association of Immunologists, Inc.)

How mechanical cues from the extracellular environment are translated biochemically to modulate the effects of TGF-β on myofibroblast differentiation remains a crucial area of investigation. We report here that the focal adhesion protein, Hic-5 (also known as TGFB1I1), is required for the mechanically dependent generation of stress fibers in response to TGF-β. Successful generation of stress fibers promotes the nuclear localization of the transcriptional co-factor MRTF-A (also known as MKL1), and this correlates with the mechanically dependent induction of α smooth muscle actin (α-SMA) and Hic-5 in response to TGF-β. As a consequence of regulating stress fiber assembly, Hic-5 is required for the nuclear accumulation of MRTF-A and the induction of α-SMA as well as cellular contractility, suggesting a crucial role for Hic-5 in myofibroblast differentiation. Indeed, the expression of Hic-5 was transient in acute wounds and persistent in pathogenic scars, and Hic-5 colocalized with α-SMA expression in vivo. Taken together, these data suggest that a mechanically dependent feed-forward loop, elaborated by the reciprocal regulation of MRTF-A localization by Hic-5 and Hic-5 expression by MRTF-A, plays a crucial role in myofibroblast differentiation in response to TGF-β., (© 2016. Published by The Company of Biologists Ltd.)

Breast involution following pregnancy has been implicated in the high rates of metastasis observed in postpartum breast cancers; however, it is not clear how this remodeling process promotes metastasis. Here, we demonstrate that human postpartum breast cancers have increased peritumor lymphatic vessel density that correlates with increased frequency of lymph node metastases. Moreover, lymphatic vessel density was increased in normal postpartum breast tissue compared with tissue from nulliparous women. In rodents, mammary lymphangiogenesis was upregulated during weaning-induced mammary gland involution. Furthermore, breast cancer cells exposed to the involuting mammary microenvironment acquired prolymphangiogenic properties that contributed to peritumor lymphatic expansion, tumor size, invasion, and distant metastases. Finally, in rodent models of postpartum breast cancer, cyclooxygenase-2 (COX-2) inhibition during the involution window decreased normal mammary gland lymphangiogenesis, mammary tumor-associated lymphangiogenesis, tumor cell invasion into lymphatics, and metastasis. Our data indicate that physiologic COX-2-dependent lymphangiogenesis occurs in the postpartum mammary gland and suggest that tumors within this mammary microenvironment acquire enhanced prolymphangiogenic activity. Further, our results suggest that the prolymphangiogenic microenvironment of the postpartum mammary gland has potential as a target to inhibit metastasis and suggest that further study of the therapeutic efficacy of COX-2 inhibitors in postpartum breast cancer is warranted.

Postpartum mammary gland involution has been identified as tumor-promotional and is proposed to contribute to the increased rates of metastasis and poor survival observed in postpartum breast cancer patients. In rodent models, the involuting mammary gland microenvironment is sufficient to induce enhanced tumor cell growth, local invasion, and metastasis. Postpartum involution shares many attributes with wound healing, including upregulation of genes involved in immune responsiveness and infiltration of tissue by immune cells. In rodent models, treatment with non-steroidal anti-inflammatory drugs (NSAIDs) ameliorates the tumor-promotional effects of involution, consistent with the immune milieu of the involuting gland contributing to tumor promotion. Currently, immunotherapy is being investigated as a means of breast cancer treatment with the purpose of identifying ways to enhance anti-tumor immune responses. Here we review evidence for postpartum mammary gland involution being a uniquely defined 'hot-spot' of pro-tumorigenic immune cell infiltration, and propose that immunotherapy should be explored for prevention and treatment of breast cancers that arise in this environment.