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Three-dimensional (3D) cultures are a valuable platform to study acquired multicellular apoptotic resistance of cancer. We used spheroids of cell lines and actual tumor to study resistance to the proteasome inhibitor bortezomib in mesothelioma, a highly chemoresistant tumor. Spheroids from mesothelioma cell lines acquired resistance to bortezomib by failing to upregulate Noxa, a pro-apoptotic sensitizer BH3-only protein that acts by displacing Bim, a pro-apoptotic Bax/Bak-activator protein. Surprisingly, despite their resistance, spheroids also upregulated Bim and thereby acquired sensitivity to ABT-737, an inhibitor of anti-apoptotic Bcl-2 molecules. Analysis using BH3 profiling confirmed that spheroids acquired a dependence on anti-apoptotic Bcl-2 proteins and were 'primed for death'. We then studied spheroids grown from actual mesothelioma. ABT-737 was active in spheroids grown from those tumors (5/7, ∼70%) with elevated levels of Bim. Using immunocytochemistry of tissue microarrays of 48 mesotheliomas, we found that most (33, 69%) expressed elevated Bim. In conclusion, mesothelioma cells in 3D alter the expression of Bcl-2 molecules, thereby acquiring both apoptotic resistance and sensitivity to Bcl-2 blockade. Mesothelioma tumors ex vivo also show sensitivity to Bcl-2 blockade that may depend on Bim, which is frequently elevated in mesothelioma. Therefore, mesothelioma, a highly resistant tumor, may have an intrinsic sensitivity to Bcl-2 blockade that can be exploited therapeutically.

Background/objectives: Intrauterine metabolic reprogramming occurs in mothers with obesity during gestation, putting the offspring at high risk of developing obesity and associated metabolic disorders even before birth. We have generated a mouse model of maternal high-fat diet-induced obesity that recapitulates the metabolic changes seen in humans born to women with obesity., Methods: Here, we profiled and compared the metabolic characteristics of bone marrow cells of newly weaned 3-week-old offspring of dams fed either a high-fat (Off-HFD) or a regular diet (Off-RD). We utilized a state-of-the-art flow cytometry, and targeted metabolomics approach coupled with a Seahorse metabolic analyzer., Results: We revealed significant metabolic perturbation in the offspring of HFD-fed vs. RD-fed dams, including utilization of glucose primarily via oxidative phosphorylation. We also show a reduction in levels of amino acids, a phenomenon previously linked to bone marrow aging. Using flow cytometry, we found changes in the immune complexity of bone marrow cells and identified a unique B cell population expressing CD19 and CD11b in the bone marrow of three-week-old offspring of high-fat diet-fed mothers. Our data also revealed increased expression of Cyclooxygenase-2 (COX-2) on myeloid CD11b, and on CD11b hi B cells., Conclusions: Altogether, we demonstrate that the offspring of mothers with obesity show metabolic and immune changes in the bone marrow at a very young age and prior to any symptomatic metabolic disease., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Tumor-associated macrophages (TAMs) and other myelomonocytic cells are implicated in regulating responsiveness to immunotherapies, including immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis. We have developed an ex vivo high-throughput approach to discover modulators of macrophage-mediated T cell suppression, which can improve clinical outcomes of ICIs. We screened 1,430 Food and Drug Administration (FDA)-approved small-molecule drugs using a co-culture assay employing bone-marrow-derived macrophages (BMDMs) and splenic-derived T cells. This identified 57 compounds that disrupted macrophage-mediated T cell suppression. Seven compounds exerted prominent synergistic T cell expansion activity when combined with αPD-L1. These include four COX1/2 inhibitors and two myeloid cell signaling inhibitors. We demonstrate that the use of cyclooxygenase (COX)1/2 inhibitors in combination with αPD-L1 decreases tumor growth kinetics and enhances overall survival in triple-negative breast cancer (TNBC) tumor models in a CD8 + T cell-dependent manner. Altogether, we present a rationalized approach for identifying compounds that synergize with ICI to potentially enhance therapeutic outcomes for patients with solid tumors., Competing Interests: Declaration of interests S.V.M., L.M.C., D.T., S. Kumar, and A.D. are inventors on the following US Provisional patent application: Combination Therapy for Treatment of Solid Tumors, provisional filed on 04/10/2023, provisional patent application no. 63/495,246. S. Kumar received reagent support and funding from HiberCell, Inc. S. Kummar – consultant/advisory board: Boehringer Ingelheim, SpringWorks Therapeutics, Seagen, Bayer, Genome & Company, Harbour BioMed, BPGbio Therapeutics, Oxford BioTherapeutics, Mundibiopharma, Gilead, EcoR1, and Mirati; PathomIQ (co-founder), Cadila Pharmaceuticals (scientific advisor-spouse), and Arxeon (co-founder-spouse). 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 and HiberCell, Inc.; receives research support from the Prospect Creek Foundation, Lustgarten Foundation for Pancreatic Cancer Research, Susan G. Komen Foundation, and the National Foundation for Cancer Research; and is on the advisory board for Carisma Therapeutics, Inc., CytomX Therapeutics, Inc., Kineta, Inc., HiberCell, Inc., Cell Signaling Technologies, Inc., Alkermes, Inc., NextCure, Guardian Bio, Dispatch Biotherapeutics, 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. S.V.M. is on the Scientific Advisory Board of Cadila Pharmaceuticals Pvt. Ltd. and is a co-founder of Arxeon, Inc., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Advancements in imaging technologies have revolutionized our ability to deeply profile pathological tissue architectures, generating large volumes of imaging data with unparalleled spatial resolution. This type of data collection, namely, spatial proteomics, offers invaluable insights into various human diseases. Simultaneously, computational algorithms have evolved to manage the increasing dimensionality of spatial proteomics inherent in this progress. Numerous imaging-based computational frameworks, such as computational pathology, have been proposed for research and clinical applications. However, the development of these fields demands diverse domain expertise, creating barriers to their integration and further application. This review seeks to bridge this divide by presenting a comprehensive guideline. We consolidate prevailing computational methods and outline a roadmap from image processing to data-driven, statistics-informed biomarker discovery. Additionally, we explore future perspectives as the field moves toward interfacing with other quantitative domains, holding significant promise for precision care in immuno-oncology., (© The Author(s) 2024. Published by Oxford University Press.)

Virulent infectious agents such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and methicillin-resistant Staphylococcus aureus (MRSA) induce tissue damage that recruits neutrophils, monocyte, and macrophages, leading to T cell exhaustion, fibrosis, vascular leak, epithelial cell depletion, and fatal organ damage. Neutrophils, monocytes, and macrophages recruited to pathogen-infected lungs, including SARS-CoV-2-infected lungs, express phosphatidylinositol 3-kinase gamma (PI3Kγ), a signaling protein that coordinates both granulocyte and monocyte trafficking to diseased tissues and immune-suppressive, profibrotic transcription in myeloid cells. PI3Kγ deletion and inhibition with the clinical PI3Kγ inhibitor eganelisib promoted survival in models of infectious diseases, including SARS-CoV-2 and MRSA, by suppressing inflammation, vascular leak, organ damage, and cytokine storm. These results demonstrate essential roles for PI3Kγ in inflammatory lung disease and support the potential use of PI3Kγ inhibitors to suppress inflammation in severe infectious diseases.

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.)

Imaging mass cytometry (IMC) is a powerful technique capable of detecting over 30 markers on a single slide. It has been increasingly used for single-cell-based spatial phenotyping in a wide range of samples. However, it only acquires a rectangle field of view (FOV) with a relatively small size and low image resolution, which hinders downstream analysis. Here, we reported a highly practical dual-modality imaging method that combines high-resolution immunofluorescence (IF) and high-dimensional IMC on the same tissue slide. Our computational pipeline uses the whole-slide image (WSI) of IF as a spatial reference and integrates small-FOV IMC into a WSI of IMC. The high-resolution IF images enable accurate single-cell segmentation to extract robust high-dimensional IMC features for downstream analysis. We applied this method in esophageal adenocarcinoma of different stages, identified the single-cell pathology landscape via reconstruction of WSI IMC images, and demonstrated the advantage of the dual-modality imaging strategy., Competing Interests: Declaration of interests L.M.C. acknowledges consulting services for Cell Signaling Technologies, AbbVie, the Susan G. Komen Foundation, and Shasqi; has received reagent and/or research support from Cell Signaling Technologies, Syndax Pharmaceuticals, ZielBio, Inc., and Hibercell, Inc.; and participates in advisory boards for Carisma, CytomX, Kineta, Hibercell, Cell Signaling Technologies, Alkermes, Genenta Sciences, Pio Therapeutics Pty., Ltd., Raska Pharma, Inc., NextCure, Guardian Bio, the AstraZeneca Partner of Choice Network, the Lustgarten Foundation, and the NIH/NCI-Frederick National Laboratory Advisory Committee. D.B. and G.J.H. are cofounders of Suil Interactive, a company focused on the development of virtual reality tools for the visualization of spatial profiling data, and of Elyx, a company developing and commercializing spatial profiling technologies. They are listed as inventors on patent applications relating to the spatial profiling field, including WO2021105723A1 and WO2021116715A. R.C.F. holds patents related to Cytosponge-TFF3 and related assays that have been licensed by the Medical Research Council to Covidien (now Medtronic). R.C.F. is a cofounder and shareholder in an early detection and digital pathology company Cyted, Ltd., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Triple-negative breast cancer (TNBC) patients have a poor prognosis and few treatment options. Mouse models of TNBC are important for development of new therapies, however, few mouse models represent the complexity of TNBC. Here, we develop a female TNBC murine model by mimicking two common TNBC mutations with high co-occurrence: amplification of the oncogene MYC and deletion of the tumor suppressor PTEN. This Myc;Ptenfl model develops heterogeneous triple-negative mammary tumors that display histological and molecular features commonly found in human TNBC. Our research involves deep molecular and spatial analyses on Myc;Ptenfl tumors including bulk and single-cell RNA-sequencing, and multiplex tissue-imaging. Through comparison with human TNBC, we demonstrate that this genetic mouse model develops mammary tumors with differential survival and therapeutic responses that closely resemble the inter- and intra-tumoral and microenvironmental heterogeneity of human TNBC, providing a pre-clinical tool for assessing the spectrum of patient TNBC biology and drug response., (© 2023. Springer Nature Limited.)

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.)

T cell receptor repertoires can be profiled using next generation sequencing (NGS) to measure and monitor adaptive dynamical changes in response to disease and other perturbations. Genomic DNA-based bulk sequencing is cost-effective but necessitates multiplex target amplification using multiple primer pairs with highly variable amplification efficiencies. Here, we utilize an equimolar primer mixture and propose a single statistical normalization step that efficiently corrects for amplification bias post sequencing. Using samples analyzed by both our open protocol and a commercial solution, we show high concordance between bulk clonality metrics. This approach is an inexpensive and open-source alternative to commercial solutions., (© 2023. The Author(s).)

Age is among the main risk factors for cancer, and any cancer study in adults is faced with an aging tissue and organism. Yet, pre-clinical studies are carried out using young mice and are not able to address the impact of aging and associated comorbidities on disease biology and treatment outcomes. Here, we discuss the limitations of current mouse cancer models and suggest strategies for developing novel models to address these major gaps in knowledge and experimental approaches., Competing Interests: Declaration of interests K. Polyak serves on the scientific advisory boards of Novartis, Vividion Therapeutics, Ideya Biosciences, and Scorpion Therapeutics; holds equity options in Scorpion Therapeutics; has received honorarium from Astra-Zeneca, New Equilibrium Biosciences, and Roche in the past 12 months; and receives sponsored research funding from Novartis. K. Politi is coinventor on a patent for EGFRT790M mutation testing issued, licensed, and with royalties paid from Molecular Diagnostics/Memorial Sloan Kettering Cancer Center. She reports research funding to her institution from AstraZeneca, Roche/Genentech, Boehringer Ingelheim, and D2G Oncology, and consulting for AstraZeneca and Jannssen. K. Palucka is a cofounder of Guardian Bio and holds equity and receives research support from Guardian Bio. She is a member of the scientific advisory board and holds equity from Cue Biopharma. She received research support from Merck in the past. O.A. has received research support from Sanofi and Pacbio in the past. J.J.T. receives patent royalties from Fate Therapeutics. She has also received research support from H3 Biomedicine, Inc. L.M.C. reports consulting services for Cell Signaling Technologies, AbbVie, the Susan G. Komen Foundation, and Shasqi; has received reagent and/or research support from Cell Signaling Technologies, Syndax Pharmaceuticals, ZelBio, Inc., Hibercell, Inc., Acerta Pharma, Prospect Creek Foundation, the Susan G. Komen Foundation, and National Foundation for Cancer Research; and has participated in advisory boards for Syndax Pharmaceuticals, Carisma Therapeutics, Inc., CytomX Therapeutics, Inc., Kineta, Inc., Hibercell, Inc., Cell Signaling Technologies, Alkermes, Inc., Genenta Sciences, Pio Therapeutics, Pty., Ltd., PDX Pharmaceuticals, Inc., NextCure, Guardian Bio, the AstraZeneca Partner of Choice Network, the Lustgarten Foundation, and the NIH/NCI-Frederick National Laboratory Advisory Committee., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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.

Antigen-specific CD8 + T cell accumulation in tumors is a prerequisite for effective immunotherapy, and yet the mechanisms of lymphocyte transit are not well defined. Here we show that tumor-associated lymphatic vessels control T cell exit from tumors via the chemokine CXCL12, and intratumoral antigen encounter tunes CXCR4 expression by effector CD8 + T cells. Only high-affinity antigen downregulates CXCR4 and upregulates the CXCL12 decoy receptor, ACKR3, thereby reducing CXCL12 sensitivity and promoting T cell retention. A diverse repertoire of functional tumor-specific CD8 + T cells, therefore, exit the tumor, which limits the pool of CD8 + T cells available to exert tumor control. CXCR4 inhibition or loss of lymphatic-specific CXCL12 boosts T cell retention and enhances tumor control. These data indicate that strategies to limit T cell egress might be an approach to boost the quantity and quality of intratumoral T cells and thereby response to immunotherapy., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: In preclinical studies of pancreatic ductal adenocarcinoma (PDAC), ibrutinib improved the antitumor efficacy of the standard of care chemotherapy. This led to a phase 1b clinical trial to determine the safety, tolerability, and immunologic effects of ibrutinib treatment in patients with advanced PDAC., Methods: Previously untreated patients with PDAC were enrolled in a phase 1b clinical trial (ClinicalTrials.gov) to determine the safety, toxicity, and maximal tolerated dose of ibrutinib when administered with the standard regimen of gemcitabine and nab-paclitaxel. To study the immune response to ibrutinib alone, the trial included an immune response arm where patients were administered with ibrutinib daily for a week followed by ibrutinib combined with gemcitabine and nab-paclitaxel. Endoscopic ultrasonography-guided primary PDAC tumor biopsies and blood were collected before and after ibrutinib monotherapy. Changes in abundance and functional state of immune cells in the blood was evaluated by mass cytometry by time of flight and statistical scaffold analysis, while that in the local tumor microenvironment (TME) were assessed by multiplex immunohistochemistry. Changes in B-cell receptor and T-cell receptor repertoire were assessed by sequencing and analysis of clonality., Results: In the blood, ibrutinib monotherapy significantly increased the frequencies of activated inducible T cell costimulator + (ICOS + ) CD4 + T cells and monocytes. Within the TME, ibrutinib monotherapy led to a trend in decreased B-cell abundance but increased interleukin-10 + B-cell frequency. Monotherapy also led to a trend in increased mature CD208 + dendritic cell density, increased late effector (programmed cell death protein 1 (PD-1 - ) eomesodermin (EOMES + )) CD8 + T-cell frequency, with a concomitantly decreased dysfunctional (PD-1 + EOMES + ) CD8 + T-cell frequency. When ibrutinib was combined with chemotherapy, most of these immune changes were not observed. Patients with partial clinical responses had more diverse T and B cell receptor repertoires prior to therapy initiation., Conclusion: Ibrutinib monotherapy skewed the immune landscape both in the circulation and TME towards activated T cells, monocytes and DCs. These effects were not observed when combining ibrutinib with standard of care chemotherapy. Future studies may focus on other therapeutic combinations that augment the immunomodulatory effects of ibrutinib in solid tumors., Trial Registration Number: NCT02562898., Competing Interests: Competing interests: CDL reports research support from Taiho Pharmaceuticals, Servier Pharmaceuticals, Cardiff Pharmaceutical, Lilly/Loxo, AstraZeneca, and Roche/Genentech. MT reports consultancy/advisory role with Advance Medical, AstraZeneca, Bristol-Myers Squibb, EcoR1 Capital, Elicio Therapeutics, Fibrogen, GlaxoSmithKline, Immunovia, ISPEN, Karyopharm Therapeutics, Merck & Co, and Swedish Orphan Biovitrum; research funding from Celgene and Halozyme; other relationship(s) with Astellas Pharma Global Development. LMC reports consulting services for Cell Signaling Technologies, AbbVie, the Susan G Komen Foundation, and Shasqi, received reagent and/or research support from Cell Signaling Technologies, Syndax Pharmaceuticals, ZelBio, Hibercell, and Acerta Pharma, and has participated in advisory boards for Pharmacyclics, Syndax, Carisma, Verseau, CytomX, Kineta, Hibercell, Cell Signaling Technologies, Alkermes, Zymeworks, Genenta Sciences, Pio Therapeutics, PDX Pharmaceuticals, the AstraZeneca Partner of Choice Network, the Lustgarten Foundation, and the NIH/NCI-Frederick National Laboratory Advisory Committee. LF reports research support from AbbVie, Amgen, Bavarian Nordic, Bristol-Myers Squibb, Dendreon, Janssen, Merck, Roche/Genentech; ownership interests in Actym, Alector, Atreca, Bioatla, Bolt, Immunogenesis, Nutcracker, RAPT, Scribe, and Senti, unrelated to the work here. The other authors declare no conflicts of interest., (© 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.)

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.)

Systematically identifying synergistic combinations of targeted agents and immunotherapies for cancer treatments remains difficult. In this study, we integrated high-throughput and high-content techniques-an implantable microdevice to administer multiple drugs into different sites in tumors at nanodoses and multiplexed imaging of tumor microenvironmental states-to investigate the tumor cell and immunological response signatures to different treatment regimens. Using a mouse model of breast cancer, we identified effective combinations from among numerous agents within days. In vivo studies in three immunocompetent mammary carcinoma models demonstrated that the predicted combinations synergistically increased therapeutic efficacy. We identified at least five promising treatment strategies, of which the panobinostat, venetoclax and anti-CD40 triple therapy was the most effective in inducing complete tumor remission across models. Successful drug combinations increased spatial association of cancer stem cells with dendritic cells during immunogenic cell death, suggesting this as an important mechanism of action in long-term breast cancer control., (© 2022. The Author(s).)

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.)

As a transcription factor that promotes cell growth, proliferation, and apoptosis, c-MYC (MYC) expression in the cell is tightly controlled. Disruption of oncogenic signaling pathways in human cancers can increase MYC protein stability, due to altered phosphorylation ratios at two highly conserved sites, Threonine 58 (T58) and Serine 62 (S62). The T58 to Alanine mutant (T58A) of MYC mimics the stabilized, S62 phosphorylated, and highly oncogenic form of MYC. The S62A mutant is also stabilized, lacks phosphorylation at both Serine 62 and Threonine 58, and has been shown to be nontransforming in vitro. However, several regulatory proteins are reported to associate with MYC lacking phosphorylation at S62 and T58, and the role this form of MYC plays in MYC transcriptional output and in vivo oncogenic function is understudied. We generated conditional c-Myc knock-in mice in which the expression of wild-type MYC (MYCWT), the T58A mutant (MYCT58A), or the S62A mutant (MYCS62A) with or without expression of endogenous Myc is controlled by the T-cell-specific Lck-Cre recombinase. MYCT58A expressing mice developed clonal T-cell lymphomas with 100% penetrance and conditional knock-out of endogenous Myc accelerated this lymphomagenesis. In contrast, MYCS62A mice developed clonal T-cell lymphomas at a much lower penetrance, and the loss of endogenous MYC reduced the penetrance while increasing the appearance of a non-transgene driven B-cell lymphoma with splenomegaly. Together, our study highlights the importance of regulated phosphorylation of MYC at T58 and S62 for T-cell transformation., Implications: Dysregulation of phosphorylation at conserved T58 and S62 residues of MYC differentially affects T-cell development and lymphomagenesis., (©2022 The Authors; Published by the American Association for Cancer Research.)

Immune checkpoint inhibitors (ICIs) have revolutionized the clinical management of multiple tumours. However, only a few patients respond to ICIs, which has generated considerable interest in the identification of resistance mechanisms. One such mechanism reflects the ability of various oncogenic pathways, as well as stress response pathways required for the survival of transformed cells (a situation commonly referred to as 'non-oncogene addiction'), to support tumour progression not only by providing malignant cells with survival and/or proliferation advantages, but also by establishing immunologically 'cold' tumour microenvironments (TMEs). Thus, both oncogene and non-oncogene addiction stand out as promising targets to robustly inflame the TME and potentially enable superior responses to ICIs., (© 2022. Springer Nature Limited.)

Esophageal adenocarcinoma (EAC) develops from a chronic inflammatory environment across four stages: intestinal metaplasia, known as Barrett's esophagus, low- and high-grade dysplasia, and adenocarcinoma. Although the genomic characteristics of this progression have been well defined via large-scale DNA sequencing, the dynamics of various immune cell subsets and their spatial interactions in their tumor microenvironment remain unclear. Here, we applied a sequential multiplex immunohistochemistry (mIHC) platform with computational image analysis pipelines that allow for the detection of 10 biomarkers in one formalin-fixed paraffin-embedded (FFPE) tissue section. Using this platform and quantitative image analytics, we studied changes in the immune landscape during disease progression based on 40 normal and diseased areas from endoscopic mucosal resection specimens of chemotherapy treatment- naïve patients, including normal esophagus, metaplasia, low- and high-grade dysplasia, and adenocarcinoma. The results revealed a steady increase of FOXP3 + T regulatory cells and a CD163 + myelomonocytic cell subset. In parallel to the manual gating strategy applied for cell phenotyping, we also adopted a sparse subspace clustering (SSC) algorithm allowing the automated cell phenotyping of mIHC-based single-cell data. The algorithm successfully identified comparable cell types, along with significantly enriched FOXP3 T regulatory cells and CD163 + myelomonocytic cells as found in manual gating. In addition, SCC identified a new CSF1R + CD1C + myeloid lineage, which not only was previously unknown in this disease but also increases with advancing disease stages. This study revealed immune dynamics in EAC progression and highlighted the potential application of a new multiplex imaging platform, combined with computational image analysis on routine clinical FFPE sections, to investigate complex immune populations in tumor ecosystems., Competing Interests: LC reports consulting services for Cell Signaling Technologies, AbbVie, the Susan G Komen Foundation, and Shasqi, received reagent and/or research support from Cell Signaling Technologies, Syndax Pharmaceuticals, and Acerta Pharma, and has participated in advisory boards for Pharmacyclics, Syndax, Carisma, Verseau, CytomX, Kineta, Hibercell, Cell Signaling Technologies, Alkermes, Zymeworks, the AstraZeneca Partner of Choice Network, the Lustgarten Foundation, and the NIH/NCI-Frederick National Laboratory Advisory Committee. RF holds patents related to Cytosponge-TFF3 and related assays that have been licensed by the Medical Research Council to Covidien (now Medtronic). RF is a co-founder and shareholder in an early detection and digital pathology company Cyted Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sundaram, Kim, Jones, Sivagnanam, Tripathi, Miremadi, Di Pietro, Coussens, Fitzgerald, Chang and Zhuang.)

Survival improves when cancer is detected early. However, ~50% of cancers are at an advanced stage when diagnosed. Early detection of cancer or precancerous change allows early intervention to try to slow or prevent cancer development and lethality. To achieve early detection of all cancers, numerous challenges must be overcome. It is vital to better understand who is at greatest risk of developing cancer. We also need to elucidate the biology and trajectory of precancer and early cancer to identify consequential disease that requires intervention. Insights must be translated into sensitive and specific early detection technologies and be appropriately evaluated to support practical clinical implementation. Interdisciplinary collaboration is key; advances in technology and biological understanding highlight that it is time to accelerate early detection research and transform cancer survival.

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.)

Highly multiplexed tissue imaging makes detailed molecular analysis of single cells possible in a preserved spatial context. However, reproducible analysis of large multichannel images poses a substantial computational challenge. Here, we describe a modular and open-source computational pipeline, MCMICRO, for performing the sequential steps needed to transform whole-slide images into single-cell data. We demonstrate the use of MCMICRO on tissue and tumor images acquired using multiple imaging platforms, thereby providing a solid foundation for the continued development of tissue imaging software., (© 2021. The Author(s).)

There is increasing evidence that the spatial organization of cells within the tumor-immune microenvironment (TiME) of solid tumors influences survival and response to therapy in numerous cancer types. Here, we report results and demonstrate the applicability of quantitative single-cell spatial proteomics analyses in the TiME of primary and recurrent human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) tumors. Single-cell compositions of a nine patient, primary and recurrent (n = 18), HNSCC cohort is presented, followed by deeper investigation into the spatial architecture of the TiME and its relationship with clinical variables and progression free survival (PFS). Multiple spatial algorithms were used to quantify the spatial landscapes of immune cells within TiMEs and demonstrate that neoplastic tumor-immune cell spatial compartmentalization, rather than mixing, is associated with longer PFS. Mesenchymal (αSMA + ) cellular neighborhoods describe distinct immune landscapes associated with neoplastic tumor-immune compartmentalization and improved patient outcomes. Results from this investigation are concordant with studies in other tumor types, suggesting that trends in TiME cellular heterogeneity and spatial organization may be shared across cancers and may provide prognostic value in multiple cancer types., (© 2022. The Author(s).)

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.)

Background: Functional interactions between immune cells and neoplastic cells in the tumor immune microenvironment have been actively pursued for both biomarker discovery for patient stratification, as well as therapeutic anti-cancer targets to improve clinical outcomes. Although accumulating evidence indicates that intratumoral infiltration of immune cells has prognostic significance, limited information is available on the spatial infiltration patterns of immune cells within intratumoral regions. This study aimed to understand the intratumoral heterogeneity and spatial distribution of immune cell infiltrates associated with cell phenotypes and prognosis in head and neck squamous cell carcinoma (HNSCC)., Methods: A total of 88 specimens of oropharyngeal squamous cell carcinoma, categorized into discovery (n = 38) and validation cohorts (n = 51), were analyzed for immune contexture by multiplexed immunohistochemistry (IHC) and image cytometry-based quantification. Tissue segmentation was performed according to a mathematical morphological approach using neoplastic cell IHC images to dissect intratumoral regions into tumor cell nests versus intratumoral stroma., Results: Tissue segmentation revealed heterogeneity in intratumoral T cells, varying from tumor cell nest-polarized to intratumoral stroma-polarized distributions. Leukocyte composition analysis revealed higher ratios of T H 1/T H 2 in tumor cell nests with higher percentages of helper T cells, B cells, and CD66b + granulocytes within intratumoral stroma. A discovery and validation approach revealed a high density of programmed death receptor-1 (PD-1) + helper T cells in tumor cell nests as a negative prognostic factor for short overall survival. CD163 + tumor-associated macrophages (TAM) provided the strongest correlation with PD-1 + helper T cells, and cases with a high density of PD-1 + helper T cells and CD163 + TAM had a significantly shorter overall survival than other cases., Conclusion: This study reveals the significance of analyzing intratumoral cell nests and reports that an immune microenvironment with a high density of PD-1 + helper T cells in tumoral cell nests is a poor prognostic factor for HNSCC., Competing Interests: TT is a paid consultant for Ono Pharmaceutical and receives speaker fees from Merck Sharp & Dohme Corp, Ono Pharmaceutical, and Bristol-Myers Squibb. HO is an employee of SCREEN Holdings Co., Ltd. SSh is an employee of SCREEN Holdings Co., Ltd. EK is a paid consultant for Roche Diagnostics and receives speaker fees from Chugai Pharmaceutical. KI received research funding from the SCREEN Holdings Co., Ltd. LC is a paid consultant for Cell Signaling Technologies, AbbVie Inc., and Shasqi Inc., 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, is a member of the Scientific Advisory Boards of Syndax Pharmaceuticals, Carisma Therapeutics, Zymeworks, Inc, Verseau Therapeutics, Cytomix Therapeutics, Inc., Hibercell, Inc., Alkermes, Inc., Genenta Sciences, and Kineta Inc, and is a member of the Lustgarten Therapeutics Advisory working group, and the AstraZeneca Partner of Choice Network. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yoshimura, Tsujikawa, Mitsuda, Ogi, Saburi, Ohmura, Arai, Shibata, Thibault, Chang, Clayburgh, Yasukawa, Miyagawa-Hayashino, Konishi, Itoh, Coussens and Hirano.)

In a pilot study, we evaluated the feasibility of real-time deep analysis of serial tumor samples from triple negative breast cancer patients to identify mechanisms of resistance and treatment opportunities as they emerge under therapeutic stress engendered by poly-ADP-ribose polymerase (PARP) inhibitors (PARPi). In a BRCA-mutant basal breast cancer exceptional long-term survivor, a striking tumor destruction was accompanied by a marked infiltration of immune cells containing CD8 effector cells, consistent with pre-clinical evidence for association between STING mediated immune activation and benefit from PARPi and immunotherapy. Tumor cells in the exceptional responder underwent extensive protein network rewiring in response to PARP inhibition. In contrast, there were minimal changes in the ecosystem of a luminal androgen receptor rapid progressor, likely due to indifference to the effects of PARP inhibition. Together, identification of PARPi-induced emergent changes could be used to select patient specific combination therapies, based on tumor and immune state changes., (© 2021. The Author(s).)

Functionally significant proteins expressed by tumor macrophages have emerged as promising anti-cancer targets. In this issue of Cancer Cell, Sun et al. identify two FDA-approved agents that together safely reprogram tumor macrophages into potent anti-tumor effectors, demonstrating the power of engaging both immune system arms to fight cancer., Competing Interests: Declaration of interests Dr. Coussens receives sponsored research support from Syndax Pharmaceuticals Inc., Acerta Pharma, LLC, Prospect Creek Foundation, Lustgarten Foundation for Pancreatic Cancer Research, and Susan G. Komen Foundation. Dr. Coussens serves on advisory boards for Pharmacyclics, Inc., Syndax Pharmaceuticals, Inc., Carisma Therapeutics, Inc., Verseau Therapeutics, Inc., CytomX Therapeutics, Inc., Kineta, Inc., Hibercell, Inc., Cell Signaling Technologies, Alkermes, Inc., Zymeworks, Inc., AstraZeneca Partner of Choice Network, and Genenta Sciences, and is a paid consultant for Cell Signaling Technologies, AbbVie, Inc., and Shasqi, Inc., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.)

Background: First-line treatment of metastatic pancreatic ductal adenocarcinoma (PDAC) includes nab-paclitaxel/gemcitabine. Ibrutinib, a Bruton's tyrosine kinase inhibitor, exhibits antitumor activity through tumor microenvironment modulation. The safety and efficacy of first-line ibrutinib plus nab-paclitaxel/gemcitabine treatment in patients with PDAC were evaluated., Patients and Methods: RESOLVE (NCT02436668) was a phase III, randomized, double-blind, placebo-controlled study. Patients (histologically-confirmed PDAC; stage IV diagnosis ≥6 weeks of randomization; Karnofsky performance score ≥70) were randomized to once-daily oral ibrutinib (560 mg) or placebo plus nab-paclitaxel (125 mg/m 2 ) and gemcitabine (1000 mg/m 2 ). Primary endpoints were overall survival (OS) and investigator-assessed progression-free survival (PFS); overall response rate and safety were assessed., Results: In total, 424 patients were randomized (ibrutinib arm, n = 211; placebo arm, n = 213). Baseline characteristics were balanced across arms. After a median follow-up of 25 months, there was no significant difference in OS between ibrutinib plus nab-paclitaxel/gemcitabine versus placebo plus nab-paclitaxel/gemcitabine (median of 9.7 versus 10.8 months; P = 0.3225). PFS was shorter for ibrutinib plus nab-paclitaxel/gemcitabine compared with placebo plus nab-paclitaxel/gemcitabine (median 5.3 versus 6.0 months; P < 0.0001). Overall response rates were 29% and 42%, respectively (P = 0.0058). Patients in the ibrutinib arm had less time on treatment and received lower cumulative doses for all agents compared with the placebo arm. The most common grade ≥3 adverse events for ibrutinib versus placebo arms included neutropenia (24% versus 35%), peripheral sensory neuropathy (17% versus 8%), and anemia (16% versus 17%). Primary reasons for any treatment discontinuation were disease progression and adverse events., Conclusions: Ibrutinib plus nab-paclitaxel/gemcitabine did not improve OS or PFS for patients with PDAC. Safety was consistent with known profiles for these agents., Competing Interests: Disclosure MT: consultancy/advisory role with Advance Medical, AstraZeneca, Bristol-Myers Squibb (BMS), EcoR1 Capital, Elicio Therapeutics, FibroGen, Inc., GlaxoSmithKline, Immunovia, ISPEN, Karyopharm Therapeutics, Merck & Co., Inc., and Swedish Orphan Biovitrum; research funding from Celgene and Halozyme; other relationship(s) with Astellas Pharma Global Development, Inc. (DSMC). D-YO: consultancy/advisory role with ASLAN, AstraZeneca, Bayer, Genentech/Roche, Halozyme, Merck Serono, Novartis, Taiho, and Zymeworks; research funding from Array, AstraZeneca, and Eli Lilly. JT: consultancy/advisory role with Array Biopharma, AstraZeneca, Bayer, BeiGene, Biocartis, Boehringer Ingelheim, Chugai, F. Hoffmann-La Roche Ltd, Foundation Medicine, Genentech, Inc., Genmab A/S, HalioDX SAS, Halozyme, Imugene Limited, Inflection Biosciences Limited, Ipsen, Kura Oncology, Lilly, Merck Sharp & Dohme (MSD), Menarini, Merck Serono, Merrimack, Merus, Molecular Partners, Novartis, Peptomyc, Pfizer, Pharmacyclics LLC, an AbbVie Company, ProteoDesign SL, Rafael Pharmaceuticals, Roche Diagnostics, Sanofi, Seagen, Seattle Genetics, Servier, Symphogen, Taiho, and VCN Biosciences. MR: honoraria from Baxalta, Celgene, and Shire; consultancy/advisory role with Baxalta, Celgene, Eli Lilly, Novartis, Novocure, Pfizer, and Shire; research funding to Institution from Celgene; travel expenses from Celgene, AstraZeneca; other relationship(s) with AstraZeneca, Boston Pharmaceuticals, and Celgene. EVC: consultancy/advisory role with AstraZeneca, Bayer, BMS, Celgene, Lilly, MSD, Merck DGaA, Novartis, Roche, and Servier; research funding from Amgen, Bayer, Boehringer Ingelheim, BMS, Celgene, Ipsen, Lilly, Merck, Merck KGaA, Novartis, Roche, and Servier. AH: consultancy/advisory role with AbbVie, Ipsen, Merck, and Novartis; research funding from Ipsen. D-TW: speakers' bureau for AstraZeneca, BMS, Celgene, Eisai, Falk, Ipsen, Novartis, Roche, Servier, Shire Baxalta, and Sirtex; travel expenses from Bayer Health Pharma, Celgene, Ipsen, Novartis, and Sirtex. NS: honoraria from Eli Lilly, Merck Serono, MSD Oncology, and Pierre Fabre; consultancy/advisory role with AstraZeneca, Pfizer, and Servier; research funding from AstraZeneca, BMS, and Pfizer; travel expenses from AstraZeneca, BMS, Eli Lilly, Merck, and Roche. J-BB: honoraria from Amgen, AstraZeneca, Bayer, Celgene, Merck Serono, Mundipharma, Pierre Fabre, Roche, Sanofi, and Servier; consultancy/advisory role with Amgen, AstraZeneca, Bayer, Merck Serono, Pierre Fabre, and Servier; travel expenses from Amgen, Bayer, Celgene, Merck Serono, Roche, Sanofi, and Servier. H-MC: research funding from Astellas, Halozyme, Pharmacyclics LLC, an AbbVie Company, Senhwa Biosciences, Taiho Oncology. JM: consultancy/advisory role with Advance Medical, AstraZeneca, Bayer, Pierre-Fabre, Roche, Sanofi, Servier, Shire, and Sirtex; research funding from Amgen, Biocartis, Incyte, Merck, Nanostring, and Servier; patents, royalties, or other intellectual property with GAIS-42-patent P5020EP00. RG: honoraria from AAA, Amgen, Bayer, BMS, Ipsen, Lilly, Merck, MSD, Novartis, PharmaMar, Pfizer, Roche, and Sanofi; research funding from ARMO Biosciences, AstraZeneca, Pfizer, Novartis, Ipsen, Roche, Pharmacyclics LLC, an AbbVie Company, Boston Biomedicals, Merck, MSD, Amgen, Sanofi, Bayer, BMS, Boehringer, Sysmex, Gilead Sciences, Servier, Adacap, VCN, Lilly, PharmaMar; travel expenses from Ipsen, Merck, Novartis, and Servier. SL: consultancy/advisory role with Amgen, Lilly, Merck Serono, and Servier; research funding from Amgen and Merck Serono; speakers' bureau for BMS, Lilly, Merck Serono, Roche, and Servier. LMC: employment with Oregon Health & Science University; honoraria from Aduro Biotech, AstraZeneca, Carisma Therapeutics, Inc., Cell Signaling Technologies, CytomX Therapeutics, Inc., Jackson Laboratories, Seattle Genetics, Syndax Pharmaceuticals, Inc., Verseau Therapeutics, Inc., and Zymeworks, Inc.; consultancy/advisory role with Carisma Therapeutics Inc., Cell Signaling Technologies, CytomX Therapeutics, Inc., Syndax Pharmaceuticals, Inc., Verseau Therapeutics, Inc., and Zymeworks, Inc.; research funding (for profit) Acerta Pharma, Deciphera Pharmaceuticals, Innate Pharma, Roche Glycart and Parker Institute for Immunotherapy, and Syndax Pharmaceuticals, Inc.; patents, royalties, or other intellectual property with Oregon Health & Science University; other relationship(s) with Reagent from Reagent support from: Acerta Pharma, LLC, Cell Signaling Technologies, Deciphera Pharmaceuticals, Genentech/Roche Glycart AG, NanoString Technologies, Inc., Plexxikon, Inc, and Syndax Pharmaceuticals. LF: research funding from AbbVie, Bavarian Nordic, BMS, Dendreon, Janssen, Merck, Roche/Genentech. GC: employment with Pharmacyclics LLC, an AbbVie Company; stock ownership in AbbVie. DJ: employment with Pharmacyclics LLC, an AbbVie Company; leadership role with Pharmacyclics LLC, an AbbVie Company; stock ownership in AbbVie; patents, royalties, or other intellectual property with Pharmacyclics LLC, an AbbVie Company. TM: consultancy/advisory role with Advance Medical HCMS, Baxter, BioLineRX Ltd, Celgene SLU, Eisai, Genzyme, Incyte, IPSEN Pharma Lab. Menarini, Lab. Servier, Lilly, QED Therapeutics, MSD, Prime Oncology EU, QED Therapeutics Inc., Sanofi-Aventis; research funding from Agios, ASLAN, AstraZeneca, Bayer, Celgene, Genentech, Hallozyme, Immunomedics, Lilly, Merimarck, Millenium, Novartis, Pfizer, Pharmacyclics LLC, an AbbVie Company, and Roche; travel expenses from Servier and Incyte. LCT has declared no conflicts of interest., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Stromal stiffening accompanies malignancy, compromises treatment and promotes tumour aggression. Clarifying the molecular nature and the factors that regulate stromal stiffening in tumours should identify biomarkers to stratify patients for therapy and interventions to improve outcome. We profiled lysyl hydroxylase-mediated and lysyl oxidase-mediated collagen crosslinks and quantified the greatest abundance of total and complex collagen crosslinks in aggressive human breast cancer subtypes with the stiffest stroma. These tissues harbour the highest number of tumour-associated macrophages, whose therapeutic ablation in experimental models reduced metastasis, and decreased collagen crosslinks and stromal stiffening. Epithelial-targeted expression of the crosslinking enzyme, lysyl oxidase, had no impact on collagen crosslinking in PyMT mammary tumours, whereas stromal cell targeting did. Stromal cells in microdissected human tumours expressed the highest level of collagen crosslinking enzymes. Immunohistochemical analysis of biopsies from a cohort of patients with breast cancer revealed that stromal expression of lysyl hydroxylase 2, an enzyme that induces hydroxylysine aldehyde-derived collagen crosslinks and stromal stiffening, correlated significantly with disease specific mortality. The findings link tissue inflammation, stromal cell-mediated collagen crosslinking and stiffening to tumour aggression and identify lysyl hydroxylase 2 as a stromal biomarker.

Background: Characterization of breast cancer (BC) through the determination of conventional markers such as ER, PR, HER2, and Ki67 has been useful as a predictive and therapeutic tool. Also, assessment of tumor-infiltrating lymphocytes has been proposed as an important prognostic aspect to be considered in certain BC subtypes. However, there is still a need to identify additional biomarkers that could add precision in distinguishing therapeutic response of individual patients. To this end, we focused in the expression of interferon regulatory factor 8 (IRF8) in BC cells. IRF8 is a transcription factor which plays a well-determined role in myeloid cells and that seems to have multiple antitumoral roles: it has tumor suppressor functions; it acts downstream IFN/STAT1, required for the success of some therapeutic regimes, and its expression in neoplastic cells seems to depend on a cross talk between the immune contexture and the tumor cells. The goal of the present study was to examine the relationship between IRF8 with the therapeutic response and the immune contexture in BC, since its clinical significance in this type of cancer has not been thoroughly addressed., Methods: We identified the relationship between IRF8 expression and the clinical outcome of BC patients and validated IRF8 as predictive biomarker by using public databases and then performed in silico analysis. To correlate the expression of IRF8 with the immune infiltrate in BC samples, we performed quantitative multiplex immunohistochemistry., Results: IRF8 expression can precisely predict the complete pathological response to monoclonal antibody therapy or to select combinations of chemotherapy such as FAC (fluorouracil, adriamycin, and cytoxan) in ER-negative BC subtypes. Analysis of immune cell infiltration indicates there is a strong correlation between activated and effector CD8 + T cell infiltration and tumoral IRF8 expression., Conclusions: We propose IRF8 expression as a potent biomarker not only for prognosis, but also for predicting therapy response in ER-negative BC phenotypes. Its expression in neoplastic cells also correlates with CD8 + T cell activation and infiltration. Therefore, our results justify new efforts towards understanding mechanisms regulating IRF8 expression and how they can be therapeutically manipulated.

Purpose: Although abundant myeloid cell populations in the pancreatic ductal adenocarcinoma (PDAC) microenvironment have been postulated to suppress antitumor immunity, the composition of these populations, their spatial locations, and how they relate to patient outcomes are poorly understood., Experimental Design: To generate spatially resolved tumor and immune cell data at single-cell resolution, we developed two quantitative multiplex immunofluorescence assays to interrogate myeloid cells (CD15, CD14, ARG1, CD33, HLA-DR) and macrophages [CD68, CD163, CD86, IFN regulatory factor 5, MRC1 (CD206)] in the PDAC tumor microenvironment. Spatial point pattern analyses were conducted to assess the degree of colocalization between tumor cells and immune cells. Multivariable-adjusted Cox proportional hazards regression was used to assess associations with patient outcomes., Results: In a multi-institutional cohort of 305 primary PDAC resection specimens, myeloid cells were abundant, enriched within stromal regions, highly heterogeneous across tumors, and differed by somatic genotype. High densities of CD15 + ARG1 + immunosuppressive granulocytic cells and M2-polarized macrophages were associated with worse patient survival. Moreover, beyond cell density, closer proximity of M2-polarized macrophages to tumor cells was strongly associated with disease-free survival, revealing the clinical significance and biologic importance of immune cell localization within tumor areas., Conclusions: A diverse set of myeloid cells are present within the PDAC tumor microenvironment and are distributed heterogeneously across patient tumors. Not only the densities but also the spatial locations of myeloid immune cells are associated with patient outcomes, highlighting the potential role of spatially resolved myeloid cell subtypes as quantitative biomarkers for PDAC prognosis and therapy., (©2020 American Association for Cancer Research.)

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