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Purpose: Mismatch repair-deficient (dMMR) tumors have demonstrated favorable responses to immune checkpoint inhibition targeting PD-1. However, more in-depth identification of predictors of response could further refine patient selection for immunotherapy treatment., Patients and Methods: We undertook integrated evaluation performed on samples collected from 28 of 42 patients enrolled on the NCI-Molecular Analysis for Therapy Choice arm Z1D trial that evaluated PD-1 inhibition treatment with nivolumab in patients with noncolorectal dMMR tumors. Genomic analyses were performed using next-generation sequencing (NGS), whole-exome sequencing, and RNA sequencing and supplemented by multiplex immunofluorescence performed on tissue samples., Results: In this dMMR population, more extensive alterations of microsatellites as assessed by measures of NGS were associated with clinical benefit and tumor mutational burden. RNA sequencing further revealed associations between clinical benefit and immune infiltration index. Gene sets enriched in patients with clinical benefit included IFN signaling, antigen processing, and PI3K-AKT-mTOR signaling, whereas hedgehog signaling was found to be enriched in subjects lacking clinical benefit., Conclusions: These genomic data highlight the importance of immune infiltration and antigen presentation in dMMR tumors that respond to immune checkpoint blockade. In addition, they suggest that, even within a dMMR population, NGS-based measures of microsatellite instability could serve as biomarkers of immunotherapy response., (©2024 The Authors; Published by the American Association for Cancer Research.)

Hodgkin Reed-Sternberg (HRS) cells of classic Hodgkin lymphoma (cHL), like many solid tumors, elicit ineffective immune responses. However, patients with cHL are highly responsive to PD-1 blockade, which largely depends on HRS cell-specific retention of MHC class II and implicates CD4 + T cells and additional MHC class I-independent immune effectors. Here, we utilize single-cell RNA sequencing and spatial analysis to define shared circulating and microenvironmental features of the immune response to PD-1 blockade in cHL. Compared with non-responders, responding patients have more circulating CD4 + naïve and central memory T cells and B cells, as well as more diverse CD4 + T cell and B cell receptor repertoires. Importantly, a population of circulating and tumor-infiltrating IL1β + monocytes/macrophages is detectable in patients with cHL but not healthy donors, and a proinflammatory, tumor-promoting signature of these circulating IL1β + monocytes is associated with resistance to PD-1 blockade in cHL. Altogether, our findings reveal extensive immune rewiring and complementary roles of CD4 + T cells, B cells and IL1β + monocytes in the response to PD-1 blockade and suggest that these features can be captured with a peripheral blood test., Competing Interests: Competing interests: M.T. is a full-time employee of Astra Zeneca. K.L. is a full-time employee of PathAI. F.Z.C. is a full-time employee of AstraZeneca. J.O. is a full-time employee of Bristol Myers Squibb. P.A. consults for Merck, Bristol Myers Squibb, A.D.C. Therapeutics, GenMab, Enterome, Genentech/Roche, ATB Therapeutics, Foresight Diagnostics, AstraZeneca, MSD, Tessa Therapeutics, Regeneron, Xencor, and receives institutional research funding from Merck, Bristol Myers Squibb, Genentech/Roche, KITE/Gilead, Adaptive Biotechnologies, IGM, AstraZeneca, MSD, Aamed Therapeutics and honoraria from Merck. S.J.R. receives institutional research funding from Bristol Myers Squibb, KITE/Gilead and is an advisory board member of Immunitas Therapeutics. X.S.L. is the founder and CEO of GV20 Therapeutics, LLC. M.A.S. has received research funding from Bristol Myers Squibb, AstraZeneca, Bayer Abbvie, Genentech and Novartis and has served on advisory boards for Bristol Myers Squibb and AstraZeneca. The remaining authors declare no competing financial interests. No authors have non-financial competing interests., (© 2024. The Author(s).)

Purpose: Although immune checkpoint inhibitors (ICI) have extended survival in patients with non-small-cell lung cancer (NSCLC), acquired resistance (AR) to ICI frequently develops after an initial benefit. However, the mechanisms of AR to ICI in NSCLC are largely unknown., Methods: Comprehensive tumor genomic profiling, machine learning-based assessment of tumor-infiltrating lymphocytes, multiplexed immunofluorescence, and/or HLA-I immunohistochemistry (IHC) were performed on matched pre- and post-ICI tumor biopsies from patients with NSCLC treated with ICI at the Dana-Farber Cancer Institute who developed AR to ICI. Two additional cohorts of patients with intervening chemotherapy or targeted therapies between biopsies were included as controls., Results: We performed comprehensive genomic profiling and immunophenotypic characterization on samples from 82 patients with NSCLC and matched pre- and post-ICI biopsies and compared findings with a control cohort of patients with non-ICI intervening therapies between biopsies (chemotherapy, N = 32; targeted therapies, N = 89; both, N = 17). Putative resistance mutations were identified in 27.8% of immunotherapy-treated cases and included acquired loss-of-function mutations in STK11 , B2M , APC , MTOR , KEAP1 , and JAK1 / 2 ; these acquired alterations were not observed in the control groups. Immunophenotyping of matched pre- and post-ICI samples demonstrated significant decreases in intratumoral lymphocytes, CD3e + and CD8a + T cells, and PD-L1-PD1 engagement, as well as increased distance between tumor cells and CD8 + PD-1 + T cells. There was a significant decrease in HLA class I expression in the immunotherapy cohort at the time of AR compared with the chemotherapy ( P = .005) and the targeted therapy ( P = .01) cohorts., Conclusion: These findings highlight the genomic and immunophenotypic heterogeneity of ICI resistance in NSCLC, which will need to be considered when developing novel therapeutic strategies aimed at overcoming resistance.

Purpose: Eribulin modulates the tumor-immune microenvironment via cGAS-STING signaling in preclinical models. This non-randomized phase II trial evaluated the combination of eribulin and pembrolizumab in patients with soft-tissue sarcomas (STS)., Patients and Methods: Patients enrolled in one of three cohorts: leiomyosarcoma (LMS), liposarcomas (LPS), or other STS that may benefit from PD-1 inhibitors, including undifferentiated pleomorphic sarcoma (UPS). Eribulin was administered at 1.4 mg/m2 i.v. (days 1 and 8) with fixed-dose pembrolizumab 200 mg i.v. (day 1) of each 21-day cycle, until progression, unacceptable toxicity, or completion of 2 years of treatment. The primary endpoint was the 12-week progression-free survival rate (PFS-12) in each cohort. Secondary endpoints included the objective response rate, median PFS, safety profile, and overall survival (OS). Pretreatment and on-treatment blood specimens were evaluated in patients who achieved durable disease control (DDC) or progression within 12 weeks [early progression (EP)]. Multiplexed immunofluorescence was performed on archival LPS samples from patients with DDC or EP., Results: Fifty-seven patients enrolled (LMS, n = 19; LPS, n = 20; UPS/Other, n = 18). The PFS-12 was 36.8% (90% confidence interval: 22.5-60.4) for LMS, 69.6% (54.5-89.0) for LPS, and 52.6% (36.8-75.3) for UPS/Other cohorts. All 3 patients in the UPS/Other cohort with angiosarcoma achieved RECIST responses. Toxicity was manageable. Higher IFNα and IL4 serum levels were associated with clinical benefit. Immune aggregates expressing PD-1 and PD-L1 were observed in a patient that completed 2 years of treatment., Conclusions: The combination of eribulin and pembrolizumab demonstrated promising activity in LPS and angiosarcoma., (©2024 The Authors; Published by the American Association for Cancer Research.)

TP53 is the most frequently mutated gene across many cancers and is associated with shorter survival in lung adenocarcinoma (LUAD). To define how TP53 mutations affect the LUAD tumor microenvironment (TME), we constructed a multi-omic cellular and spatial tumor atlas of 23 treatment-naïve human lung tumors. We found that TP53 -mutant ( TP53 mut ) malignant cells lose alveolar identity and upregulate highly proliferative and entropic gene expression programs consistently across resectable LUAD patient tumors, genetically engineered mouse models, and cell lines harboring a wide spectrum of TP53 mutations. We further identified a multicellular tumor niche composed of SPP1 + macrophages and collagen-expressing fibroblasts that coincides with hypoxic, pro-metastatic expression programs in TP53 mut tumors. Spatially correlated angiostatic and immune checkpoint interactions, including CD274 - PDCD1 and PVR - TIGIT , are also enriched in TP53 mut LUAD tumors, which may influence response to checkpoint blockade therapy. Our methodology can be further applied to investigate mutation-specific TME changes in other cancers.

Highly multiplexed protein imaging is emerging as a potent technique for analyzing protein distribution within cells and tissues in their native context. However, existing cell annotation methods utilizing high-plex spatial proteomics data are resource intensive and necessitate iterative expert input, thereby constraining their scalability and practicality for extensive datasets. We introduce MAPS (Machine learning for Analysis of Proteomics in Spatial biology), a machine learning approach facilitating rapid and precise cell type identification with human-level accuracy from spatial proteomics data. Validated on multiple in-house and publicly available MIBI and CODEX datasets, MAPS outperforms current annotation techniques in terms of speed and accuracy, achieving pathologist-level precision even for typically challenging cell types, including tumor cells of immune origin. By democratizing rapidly deployable and scalable machine learning annotation, MAPS holds significant potential to expedite advances in tissue biology and disease comprehension., (© 2024. The Author(s).)

Immune checkpoint blockade (ICB) has become the standard of care for several solid tumors. Multiple combinatorial approaches have been studied to improve therapeutic efficacy. The combination of antiangiogenic agents and ICB has demonstrated efficacy in several cancers. To improve the mechanistic understanding of synergies with these treatment modalities, we performed screens of sera from long-term responding patients treated with ipilimumab and bevacizumab. We discovered a high-titer antibody response against EGF-like repeats and discoidin I-like domains protein 3 (EDIL3) that correlated with favorable clinical outcomes. EDIL3 is an extracellular protein, previously identified as a marker of poor prognosis in various malignancies. Our Tumor Immune Dysfunction and Exclusion analysis predicted that EDIL3 was associated with immune exclusion signatures for cytotoxic immune cell infiltration and nonresponse to ICB. Cancer-associated fibroblasts (CAF) were predicted as the source of EDIL3 in immune exclusion-related cells. Furthermore, The Cancer Genome Atlas Skin Cutaneous Melanoma (TCGA-SKCM) and CheckMate 064 data analyses correlated high levels of EDIL3 with increased pan-fibroblast TGFβ response, enrichment of angiogenic signatures, and induction of epithelial-to-mesenchymal transition. Our in vitro studies validated EDIL3 overexpression and TGFβ regulation in patient-derived CAFs. In pretreatment serum samples from patients, circulating levels of EDIL3 were associated with circulating levels of VEGF, and like VEGF, EDIL3 increased the angiogenic abilities of patient-derived tumor endothelial cells (TEC). Mechanistically, three-dimensional microfluidic cultures and two-dimensional transmigration assays with TEC endorsed EDIL3-mediated disruption of the lymphocyte function-associated antigen-1 (LFA-1)-ICAM-1 interaction as a possible means of T-cell exclusion. We propose EDIL3 as a potential target for improving the transendothelial migration of immune cells and efficacy of ICB therapy., (©2023 The Authors; Published by the American Association for Cancer Research.)

Diffuse large B-cell lymphoma (DLBCL) not otherwise specified is the most common aggressive non-Hodgkin lymphoma and a biologically heterogeneous disease. Despite the development of effective immunotherapies, the organization of the DLBCL tumor-immune microenvironment (TIME) remains poorly understood.We interrogated the intact TIME of 51 de novo DLBCLs with triplicate sampling to characterize 337 995 tumor and immune cells using a 27-plex antibody panel that captured cell lineage, architectural, and functional markers. We spatially assigned individual cells, identified local cell neighborhoods, and established their topographical organization in situ. We found that the organization of local tumor and immune cells can be modeled by 6 composite cell neighborhood types (CNTs). Differential CNT representation divided cases into 3 aggregate TIME categories: immune-deficient, dendritic cell-enriched (DC-enriched), and macrophage-enriched (Mac-enriched). Cases with immune-deficient TIMEs have tumor cell-rich CNTs, in which the few infiltrating immune cells are enriched near CD31+ vessels, in keeping with limited immune activity. Cases with DC-enriched TIMEs selectively include tumor cell-poor/immune cell-rich CNTs with high numbers of CD11c+ DCs and antigen-experienced T cells also enriched near CD31+ vessels, in keeping with increased immune activity. Cases with Mac-enriched TIMEs selectively include tumor cell-poor/immune cell-rich CNTs with high numbers of CD163+ macrophages and CD8 T cells throughout the microenvironment, accompanied by increased IDO-1 and LAG-3 and decreased HLA-DR expression and genetic signatures in keeping with immune evasion. Our findings reveal that the heterogenous cellular components of DLBCL are not randomly distributed but organized into CNTs that define aggregate TIMEs with distinct cellular, spatial, and functional features., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Atezolizumab with chemotherapy has shown improved progression-free and overall survival in patients with metastatic PD-L1 positive triple negative breast cancer (TNBC). Atezolizumab with anthracycline- and taxane-based neoadjuvant chemotherapy has also shown increased pathological complete response (pCR) rates in early TNBC. This trial evaluated neoadjuvant carboplatin and paclitaxel with or without atezolizumab in patients with clinical stages II-III TNBC. The co-primary objectives were to evaluate if chemotherapy and atezolizumab increase pCR rate and tumor infiltrating lymphocyte (TIL) percentage compared to chemotherapy alone in the mITT population. Sixty-seven patients (ages 25-78 years; median, 52 years) were randomly assigned - 22 patients to Arm A, and 45 to Arm B. Median follow up was 6.6 months. In the modified intent to treat population (all patients evaluable for the primary endpoints who received at least one dose of combination therapy), the pCR rate was 18.8% (95% CI 4.0-45.6%) in Arm A, and 55.6% (95% CI 40.0-70.4%) in Arm B (estimated treatment difference: 36.8%, 95% CI 8.5-56.6%; p = 0.018). Grade 3 or higher treatment-related adverse events occurred in 62.5% of patients in Arm A, and 57.8% of patients in Arm B. One patient in Arm B died from recurrent disease during the follow-up period. TIL percentage increased slightly from baseline to cycle 1 in both Arm A (mean ± SD: 0.6% ± 21.0%) and Arm B (5.7% ± 15.8%) (p = 0.36). Patients with pCR had higher median TIL percentages (24.8%) than those with non-pCR (14.2%) (p = 0.02). Although subgroup analyses were limited by the small sample size, PD-L1-positive patients treated with chemotherapy and atezolizumab had a pCR rate of 75% (12/16). The addition of atezolizumab to neoadjuvant carboplatin and paclitaxel resulted in a statistically significant and clinically relevant increased pCR rate in patients with clinical stages II and III TNBC. (Funded by National Cancer Institute)., (© 2022. The Author(s).)

MHC-II is known to be mainly expressed on the surface of antigen-presenting cells. Evidence suggests MHC-II is also expressed by cancer cells and may be associated with better immunotherapy responses. However, the role and regulation of MHC-II in cancer cells remain unclear. In this study, we leveraged data mining and experimental validation to elucidate the regulation of MHC-II in cancer cells and its role in modulating the response to immunotherapy. We collated an extensive collection of omics data to examine cancer cell-intrinsic MHC-II expression and its association with immunotherapy outcomes. We then tested the functional relevance of cancer cell-intrinsic MHC-II expression using a syngeneic transplantation model. Finally, we performed data mining to identify pathways potentially involved in the regulation of MHC-II expression, and experimentally validated candidate regulators. Analyses of preimmunotherapy clinical samples in the CheckMate 064 trial revealed that cancer cell-intrinsic MHC-II protein was positively correlated with more favorable immunotherapy outcomes. Comprehensive meta-analyses of multiomics data from an exhaustive collection of data revealed that MHC-II is heterogeneously expressed in various solid tumors, and its expression is particularly high in melanoma. Using a syngeneic transplantation model, we further established that melanoma cells with high MHC-II responded better to anti-PD-1 treatment. Data mining followed by experimental validation revealed the Hippo signaling pathway as a potential regulator of melanoma MHC-II expression. In summary, we identified the Hippo signaling pathway as a novel regulator of cancer cell-intrinsic MHC-II expression. These findings suggest modulation of MHC-II in melanoma could potentially improve immunotherapy response., (©2022 American Association for Cancer Research.)

Understanding the mechanisms of HIV tissue persistence necessitates the ability to visualize tissue microenvironments where infected cells reside; however, technological barriers limit our ability to dissect the cellular components of these HIV reservoirs. Here, we developed protein and nucleic acid in situ imaging (PANINI) to simultaneously quantify DNA, RNA, and protein levels within these tissue compartments. By coupling PANINI with multiplexed ion beam imaging (MIBI), we measured over 30 parameters simultaneously across archival lymphoid tissues from healthy or simian immunodeficiency virus (SIV)-infected nonhuman primates. PANINI enabled the spatial dissection of cellular phenotypes, functional markers, and viral events resulting from infection. SIV infection induced IL-10 expression in lymphoid B cells, which correlated with local macrophage M2 polarization. This highlights a potential viral mechanism for conditioning an immunosuppressive tissue environment for virion production. The spatial multimodal framework here can be extended to decipher tissue responses in other infectious diseases and tumor biology., Competing Interests: Declaration of interests G.P.N. and M.A. are cofounders and stockholders of Ionpath Inc., which manufactures the instrument used in this manuscript. G.P.N. and Y.G. are cofounders and stockholders of Akoya Biosciences, Inc. and inventors on patent US9909167. S.J.R. is a Scientific Advisory Board member for KITE/Gilead and Immunitas Therapeutics. S.J., X.R.-C., Y.B., and G.P.N. are inventors on pending patent US17433542 related in part to this work. The other authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Patients with non-small-cell lung cancer (NSCLC) that is resistant to PD-1 and PD-L1 (PD[L]-1)-targeted therapy have poor outcomes. Studies suggest that radiotherapy could enhance antitumour immunity. Therefore, we investigated the potential benefit of PD-L1 (durvalumab) and CTLA-4 (tremelimumab) inhibition alone or combined with radiotherapy., Methods: This open-label, multicentre, randomised, phase 2 trial was done by the National Cancer Institute Experimental Therapeutics Clinical Trials Network at 18 US sites. Patients aged 18 years or older with metastatic NSCLC, an Eastern Cooperative Oncology Group performance status of 0 or 1, and progression during previous PD(L)-1 therapy were eligible. They were randomly assigned (1:1:1) in a web-based system by the study statistician using a permuted block scheme (block sizes of three or six) without stratification to receive either durvalumab (1500 mg intravenously every 4 weeks for a maximum of 13 cycles) plus tremelimumab (75 mg intravenously every 4 weeks for a maximum of four cycles) alone or with low-dose (0·5 Gy delivered twice per day, repeated for 2 days during each of the first four cycles of therapy) or hypofractionated radiotherapy (24 Gy total delivered over three 8-Gy fractions during the first cycle only), 1 week after initial durvalumab-tremelimumab administration. Study treatment was continued until 1 year or until progression. The primary endpoint was overall response rate (best locally assessed confirmed response of a partial or complete response) and, along with safety, was analysed in patients who received at least one dose of study therapy. The trial is registered with ClinicalTrials.gov, NCT02888743, and is now complete., Findings: Between Aug 24, 2017, and March 29, 2019, 90 patients were enrolled and randomly assigned, of whom 78 (26 per group) were treated. This trial was stopped due to futility assessed in an interim analysis. At a median follow-up of 12·4 months (IQR 7·8-15·1), there were no differences in overall response rates between the durvalumab-tremelimumab alone group (three [11·5%, 90% CI 1·2-21·8] of 26 patients) and the low-dose radiotherapy group (two [7·7%, 0·0-16·3] of 26 patients; p=0·64) or the hypofractionated radiotherapy group (three [11·5%, 1·2-21·8] of 26 patients; p=0·99). The most common grade 3-4 adverse events were dyspnoea (two [8%] in the durvalumab-tremelimumab alone group; three [12%] in the low-dose radiotherapy group; and three [12%] in the hypofractionated radiotherapy group) and hyponatraemia (one [4%] in the durvalumab-tremelimumab alone group vs two [8%] in the low-dose radiotherapy group vs three [12%] in the hypofractionated radiotherapy group). Treatment-related serious adverse events occurred in one (4%) patient in the durvalumab-tremelimumab alone group (maculopapular rash), five (19%) patients in the low-dose radiotherapy group (abdominal pain, diarrhoea, dyspnoea, hypokalemia, and respiratory failure), and four (15%) patients in the hypofractionated group (adrenal insufficiency, colitis, diarrhoea, and hyponatremia). In the low-dose radiotherapy group, there was one death from respiratory failure potentially related to study therapy., Interpretation: Radiotherapy did not increase responses to combined PD-L1 plus CTLA-4 inhibition in patients with NSCLC resistant to PD(L)-1 therapy. However, PD-L1 plus CTLA-4 therapy could be a treatment option for some patients. Future studies should refine predictive biomarkers in this setting., Funding: The US National Institutes of Health and the Dana-Farber Cancer Institute., Competing Interests: Declaration of interests JDS declares research support paid to their institution from Merck, BMS, Regeneron, Debiopharm, and the NCI; consulting or participation on a scientific advisory board, and travel fees and payment for lectures from Genentech, Immunitas, Debiopharm, BMS, Nanobiotix, Tilos, AstraZeneca, LEK, Catenion, ACI Clinical, Astellas, Stimit, and Merck; expert witness fees from Pearson Doyle Mohre and Pastis, Kline and Specter, and Heidell, Pittoni, Murphy and Bach; stock options from Immunitas; and equity in Doximity. SMA declares research support paid to her institution from Merck, Exilixis, Abbvie, Kura Oncology, Amgen, and Nektar. RDG declares support for the present manuscript from a grant from the NCI of the National Institutes of Health (NIH); grants or contracts (to his institution) from Pfizer, Mirati, Daiichi Sankyo, Jounce Therapeutics, Helsinn, BMS, Merck, Janssen, and Takeda; honoraria from Rockpointe CME, Targeted Oncology, OncLive, and the Society for Immunotherapy of Cancer; reimbursement for travel for meetings from Pfizer and AstraZeneca; participation on advisory boards for Mirati, Daiichi Sankyo, AstraZeneca, Sanofi, Oncocyte, Jazz Pharmaceuticals, BluePrint Medicines, and Pfizer; and that he is co-chair of the Hoosier Cancer Research Network Thoracic Clinical Trial Working Group. CL declares honorarium for chairing the data and safety monitoring board for Delcath. JH declares participation on advisory boards for Bayer and Merck, and research funding from Merck, Boston Biomedical, Treos Bio, Senhwa Biosciences, Bayer, Incyte, TriOncology, Seattle Genetics, Hutchison MediPharma, Pionyr Immunotherapeutics, Trovogene, Taiho Pharmaceutical, Effector Therapeutics, and G1 Therapeutics. JLA declares research support from Pfizer; scientific advisory board membership for the Lustgarten Foundation, Stand Up to Cancer, Moleculin Biotech, Bessor Pharma, and Fujifilm; stock options from Bessor Pharma and Moleculin Biotech; and honoraria for data and safety monitoring board membership from Panbela Therapeutics and the Pancreatic Cancer Action Network. SKJ declares grant funding to her institution from the NCI and the Cancer Therapy Evaluation Program, and is a consultant and adjudication committee member for Merck, IMX Medical, and Syntactx. NVU has consulted for QED, Ipsen, Taiho, Incyte, AstraZeneca, and Astellas, and declares research funding from Taiho, Eli Lilly, Ipsen, and EMD Serono, and long position holdings in Natera and Exact Sciences. KLS is a member of the data and safety monitoring committee for the Case Comprehensive Cancer Center. JMJ declares consulting for Foundation Medicine. HP declares research grants or funding to their institution from Adlai Nortye USA, Alpine Immune Sciences, Ambrx, Amgen, Aprea Therapeutics, ArrayBioPharma, Bayer, BeiGene, BJ Bioscience, BMS, Daiichi Pharmaceutical, Eli Lilly, Elicio Therapeutics, EMD Serono, Exelixis, Genentech, Gilead Sciences, GlaxoSmithKline, Gossamer Bio, Hoffman-LaRoche, Hutchison MediPharma, ImmuneOncia Therapeutics, Incyte, Jounce Therapeutics, Mabspace Biosciences, MacroGenics, Medimmune, Medivation, Merck, Millennium, Mirati Therapeutics, Novartis Pharmaceuticals, Oncologie, Pfizer, PsiOxus Therapeutics, Puma Biotechnology, Regeneron Pharmaceuticals, RePare Therapeutics, Seattle Genetics, Synermore Biologics, Taiho Pharmaceutical, TopAlliance Biosciences, TurningPoint Therapeutics, Vedanta Biosciences, and Xencor, and reimbursement for meetings or travel from Daiichi Sankyo and Vedanta. LCV declares consulting fees for Janssen, BMS, Takeda, Jazz, and Daiichi Sankyo. JLW declares research support from the NCI of the NIH. RHM declares research support from ViewRay and AstraZeneca; scientific advisory board participation for ViewRay and AstraZeneca; and expert witness fees from the US Attorney's Office Northern District of New York. MarMA has consulted for Genentech, BMS, Merck, AstraZeneca, Maverick, Blueprint Medicine, Syndax, Ariad/Takeda, Nektar, Gritstone, ArcherDX, Mirati, NextCure, Novartis, EMD Serono, and Panvaxal/NovaRx; declares research funding (to their institute) from AstraZeneca, Lilly, Genentech, BMS, and Merck; and declares participation on a data safety monitoring board and advisory board for BMS and Apollomics. SJR declares research support from BMS, Merck, Affimed, and KITE/Gilead, is on the Scientific Advisory Board of Immunitas Therapeutics, and has equity in Immunitas Therapeutics. AMM declares research support from Merck, BMS, Transgene, Incyte, Trisalus, Genentech, and the NIH; consulting fees from Zosano; advisory board participation for BMS, AstraZeneca, Incyte, and Dynavax; and stock options in MultiplexThera. CJW declares equity in BioNTech, U24 research support from the NCI of the NIH, and research funding from Pharmacyclics. FSH declares research support from the NCI of the NIH, BMS, Novartis, and Genentech; royalties or licenses from BMS and Novartis; consulting fees from BMS, EMD Serono, Surface, Sanofi, Genentech, Gossamer, Trillium, Immunocore, Merck, Novartis, Compass Therapeutics, Pieris, Bioentre, Iovance, Catalym, and Amgen; patents for the methods for treating MHC class I polypeptide-related sequence A disorders (number 20100111973; with royalties paid), tumour antigens and uses thereof (number 7250291; issued), angiopoiten-2 biomarkers predictive of anti-immune checkpoint response (number 20170248603; pending), compositions and methods for the identification, assessment, prevention, and treatment of melanoma using PD-L1 isoforms (number 20160340407; pending), therapeutic peptides (number 20160046716; pending), therapeutic peptides (number 20140004112; pending), therapeutic peptides (number 20170022275; pending), therapeutic peptides (number 20170008962; pending), therapeutic peptides (number 9402905; issued), methods of using pembrolizumab and trebananib (pending), vaccine compositions and methods for restoring NKG2D pathway function against cancers (number 10279021; issued), antibodies that bind to MHC class I polypeptide-related sequence A (number 10106611; issued), and anti-galectin antibody biomarkers predictive of anti-immune checkpoint and anti-angiogenesis responses (number 20170343552; pending); data safety monitoring board and advisory board participation for Aduro and Checkpoint Therapeutics; scientific advisory board leadership for Bicara and Apricity; and stock options in Checkpoint Therapeutics, Pionyr, Apricity, and Bicara. AL is currently an employee of Bristol Myers Squibb. All other authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Purpose: Whole-exome (WES) and RNA sequencing (RNA-seq) are key components of cancer immunogenomic analyses. To evaluate the consistency of tumor WES and RNA-seq profiling platforms across different centers, the Cancer Immune Monitoring and Analysis Centers (CIMAC) and the Cancer Immunologic Data Commons (CIDC) conducted a systematic harmonization study., Experimental Design: DNA and RNA were centrally extracted from fresh frozen and formalin-fixed paraffin-embedded non-small cell lung carcinoma tumors and distributed to three centers for WES and RNA-seq profiling. In addition, two 10-plex HapMap cell line pools with known mutations were used to evaluate the accuracy of the WES platforms., Results: The WES platforms achieved high precision (> 0.98) and recall (> 0.87) on the HapMap pools when evaluated on loci using > 50× common coverage. Nonsynonymous mutations clustered by tumor sample, achieving an index of specific agreement above 0.67 among replicates, centers, and sample processing. A DV200 > 24% for RNA, as a putative presequencing RNA quality control (QC) metric, was found to be a reliable threshold for generating consistent expression readouts in RNA-seq and NanoString data. MedTIN > 30 was likewise assessed as a reliable RNA-seq QC metric, above which samples from the same tumor across replicates, centers, and sample processing runs could be robustly clustered and HLA typing, immune infiltration, and immune repertoire inference could be performed., Conclusions: The CIMAC collaborating laboratory platforms effectively generated consistent WES and RNA-seq data and enable robust cross-trial comparisons and meta-analyses of highly complex immuno-oncology biomarker data across the NCI CIMAC-CIDC Network., (©2020 American Association for Cancer Research.)

Immune checkpoint blockade (ICB) therapy revolutionized cancer treatment, but many patients with impaired MHC-I expression remain refractory. Here, we combined FACS-based genome-wide CRISPR screens with a data-mining approach to identify drugs that can upregulate MHC-I without inducing PD-L1. CRISPR screening identified TRAF3, a suppressor of the NFκB pathway, as a negative regulator of MHC-I but not PD-L1. The Traf3 -knockout gene expression signature is associated with better survival in ICB-naïve patients with cancer and better ICB response. We then screened for drugs with similar transcriptional effects as this signature and identified Second Mitochondria-derived Activator of Caspase (SMAC) mimetics. We experimentally validated that the SMAC mimetic birinapant upregulates MHC-I, sensitizes cancer cells to T cell-dependent killing, and adds to ICB efficacy. Our findings provide preclinical rationale for treating tumors expressing low MHC-I expression with SMAC mimetics to enhance sensitivity to immunotherapy. The approach used in this study can be generalized to identify other drugs that enhance immunotherapy efficacy. SIGNIFICANCE: MHC-I loss or downregulation in cancer cells is a major mechanism of resistance to T cell-based immunotherapies. Our study reveals that birinapant may be used for patients with low baseline MHC-I to enhance ICB response. This represents promising immunotherapy opportunities given the biosafety profile of birinapant from multiple clinical trials. This article is highlighted in the In This Issue feature, p. 1307 ., (©2021 American Association for Cancer Research.)

Purpose: Prospective human data are lacking regarding safety, efficacy, and immunologic impacts of different radiation doses administered with combined PD-L1/CTLA-4 blockade., Patients and Methods: We performed a multicenter phase II study randomly assigning patients with metastatic microsatellite stable colorectal cancer to repeated low-dose fractionated radiation (LDFRT) or hypofractionated radiation (HFRT) with PD-L1/CTLA-4 inhibition. The primary endpoint was response outside the radiation field. Correlative samples were analyzed using multiplex immunofluorescence (IF), IHC, RNA/T-cell receptor (TCR) sequencing, cytometry by time-of-flight (CyTOF), and Olink., Results: Eighteen patients were evaluable for response. Median lines of prior therapy were four (range, 1-7). Sixteen patients demonstrated toxicity potentially related to treatment (84%), and 8 patients had grade 3-4 toxicity (42%). Best response was stable disease in 1 patient with out-of-field tumor shrinkage. Median overall survival was 3.8 months (90% confidence interval, 2.3-5.7 months). Correlative IF and RNA sequencing (RNA-seq) revealed increased infiltration of CD8 + and CD8 + /PD-1 + /Ki-67 + T cells in the radiation field after HFRT. LDFRT increased foci of micronuclei/primary nuclear rupture in two subjects. CyTOF and RNA-seq demonstrated significant declines in multiple circulating immune populations, particularly in patients receiving HFRT. TCR sequencing revealed treatment-associated changes in T-cell repertoire in the tumor and peripheral blood., Conclusions: We demonstrate the feasibility and safety of adding LDFRT and HFRT to PD-L1/CTLA-4 blockade. Although the best response of stable disease does not support the use of concurrent PD-L1/CTLA-4 inhibition with HFRT or LDFRT in this population, biomarkers provide support that both LDFRT and HFRT impact the local immune microenvironment and systemic immunogenicity that can help guide future studies., (©2021 American Association for Cancer Research.)

Mucosal melanoma is a rare form of melanoma which arises from melanocytes in the mucosal membranes and can be effectively treated with immune checkpoint blockade (ICB). However, response rates in mucosal melanoma are lower than those observed for cutaneous melanomas. Targeted sequencing of up to 447 genes (OncoPanel) was performed on tumors from all mucosal melanoma patients seen at the Dana-Farber Cancer Institute from 2011 until March 2019. We identified a total of 46 patients who received ICB with both tumor-genotype and ICB response data available. Within this cohort of patients, 16 (35%) had durable clinical benefit (DCB) to their first line of ICB. The average mutational burden/megabase was 6.23 and did not correlate with tumor response to ICB. Patients with KIT aberrations had a higher DCB rate compared with patients with wildtype KIT (71 vs. 28%), but this was not found to be statistically significant. For comparison, we analyzed tumor genotypes from an additional 50 mucosal melanoma tumors and 189 cutaneous melanoma tumors. The most frequent mutations in mucosal melanoma were in SF3B1 (27%), KIT (18%), and NF1 (17%), a pattern that is distinct from cutaneous melanomas. In addition, there were genetic differences observed based upon the site of origin of the mucosal melanoma. Our findings explore clinical features of response in patients with mucosal melanoma treated with ICB and demonstrate a low mutational burden that does not correlate with response. In addition, the lack of significant association between the genetic aberrations tested and response to ICB indicates the need for further exploration in this patient population., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

T-cell/histiocyte-rich large B-cell lymphoma (TCRLBCL) is an aggressive variant of diffuse large B-cell lymphoma (DLBCL) characterized by rare malignant B cells within a robust but ineffective immune cell infiltrate. The mechanistic basis of immune escape in TCRLBCL is poorly defined and not targeted therapeutically. We performed a genetic and quantitative spatial analysis of the PD-1/PD-L1 pathway in a multi-institutional cohort of TCRLBCLs and found that malignant B cells harbored PD-L1/PD-L2 copy gain or amplification in 64% of cases, which was associated with increased PD-L1 expression (P = .0111). By directed and unsupervised spatial analyses of multiparametric cell phenotypic data within the tumor microenvironment, we found that TCRLBCL is characterized by tumor-immune "neighborhoods" in which malignant B cells are surrounded by exceptionally high numbers of PD-L1-expressing TAMs and PD-1+ T cells. Furthermore, unbiased clustering of spatially resolved immune signatures distinguished TCRLBCL from related subtypes of B-cell lymphoma, including classic Hodgkin lymphoma (cHL) and DLBCL-NOS. Finally, we observed clinical responses to PD-1 blockade in 3 of 5 patients with relapsed/refractory TCRLBCL who were enrolled in clinical trials for refractory hematologic malignancies (NCT03316573; NCT01953692), including 2 complete responses and 1 partial response. Taken together, these data implicate PD-1 signaling as an immune escape pathway in TCRLBCL and also support the potential utility of spatially resolved immune signatures to aid the diagnostic classification and immunotherapeutic prioritization of diverse tumor types., (© 2021 by The American Society of Hematology.)

We performed harmonized molecular and clinical analysis on 1,048 melanomas and discovered markedly different global genomic properties among subtypes (BRAF, (N)RAS, NF1, triple wild-type (TWT)), subtype-specific preferences for secondary driver genes and active mutational processes previously unreported in melanoma. Secondary driver genes significantly enriched in specific subtypes reflected preferential dysregulation of additional pathways, such as induction of transforming growth factor-β signaling in BRAF melanomas and inactivation of the SWI/SNF complex in (N)RAS melanomas, and select co-mutation patterns coordinated selective response to immune checkpoint blockade. We also defined the mutational landscape of TWT melanomas and revealed enrichment of DNA-repair-defect signatures in this subtype, which were associated with transcriptional downregulation of key DNA-repair genes, and may revive previously discarded or currently unconsidered therapeutic modalities for genomically stratified melanoma patient subsets. Broadly, harmonized meta-analysis of melanoma whole exomes revealed distinct molecular drivers that may point to multiple opportunities for biological and therapeutic investigation.

Importance: Novel approaches are needed to improve outcomes in patients with squamous cell carcinoma of the oral cavity. Neoadjuvant immunotherapy given prior to surgery and combining programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) immune checkpoint inhibitors are 2 strategies to enhance antitumor immune responses that could be of benefit., Design, Setting, and Participants: In this randomized phase 2 clinical trial conducted at 1 academic center, 29 patients with untreated squamous cell carcinoma of the oral cavity (≥T2, or clinically node positive) were enrolled between 2016 to 2019., Interventions: Treatment was administered with nivolumab, 3 mg/kg, weeks 1 and 3, or nivolumab and ipilimumab (ipilimumab, 1 mg/kg, given week 1 only). Patients had surgery 3 to 7 days following cycle 2., Main Outcomes and Measures: Safety and volumetric response determined using bidirectional measurements. Secondary end points included pathologic and objective response, progression-free survival (PFS), and overall survival. Multiplex immunofluorescence was used to evaluate primary tumor immune markers., Results: Fourteen patients were randomized to nivolumab (N) and 15 patients to nivolumab/ipilimumab (N+I) (mean [SD] age, 62 [12] years; 18 men [62%] and 11 women [38%]). The most common subsite was oral tongue (n = 16). Baseline clinical staging included patients with T2 (n = 20) or greater (n = 9) T stage and 17 patients (59%) with node-positive disease. Median time from cycle 1 to surgery was 19 days (range, 7-21 days); there were no surgical delays. There were toxic effects at least possibly related to study treatment in 21 patients, including grade 3 to 4 events in 2 (N), and 5 (N+I) patients. One patient died of conditions thought unrelated to study treatment (postoperative flap failure, stroke). There was evidence of response in both the N and N+I arms (volumetric response 50%, 53%; pathologic downstaging 53%, 69%; RECIST response 13%, 38%; and pathologic response 54%, 73%, respectively). Four patients had major/complete pathologic response greater than 90% (N, n = 1; N+I, n = 3). With 14.2 months median follow-up, 1-year progression-free survival was 85% and overall survival was 89%., Conclusions and Relevance: Treatment with N and N+I was feasible prior to surgical resection. We observed promising rates of response in both arms, supporting further neoadjuvant studies with these agents., Trial Registration: ClinicalTrials.gov Identifier: NCT02919683.

PD-1 blockade is highly effective in classical Hodgkin lymphomas (cHLs), which exhibit frequent copy-number gains of CD274 (PD-L1) and PDC1LG2 (PD-L2) on chromosome 9p24.1. However, in this largely MHC-class-I-negative tumor, the mechanism of action of anti-PD-1 therapy remains undefined. We utilized the complementary approaches of T cell receptor (TCR) sequencing and cytometry by time-of-flight analysis to obtain a peripheral immune signature of responsiveness to PD-1 blockade in 56 patients treated in the CheckMate 205 phase II clinical trial (NCT02181738). Anti-PD-1 therapy was most effective in patients with a diverse baseline TCR repertoire and an associated expansion of singleton clones during treatment. CD4 + , but not CD8 + , TCR diversity significantly increased during therapy, most strikingly in patients who had achieved complete responses. Additionally, patients who responded to therapy had an increased abundance of activated natural killer cells and a newly identified CD3 - CD68 + CD4 + GrB + subset. These studies highlight the roles of recently expanded, clonally diverse CD4 + T cells and innate effectors in the efficacy of PD-1 blockade in cHL.

Ancillary testing during the initial workup of acute myeloid leukemia (AML) is largely performed using aspirated materials. We utilized multiplex immunofluorescence (MIF) imaging with digital image analysis to perform an in situ analysis of the microenvironment in NPM1-mutated AML using diagnostic bone marrow biopsy tissues (N = 17) and correlated these findings with diagnostic next-generation sequencing (NGS, N = 17), flow cytometry (FC, N = 14), and first remission (CR1) NPM1-specific molecular MRD (n = 16) data. The total CD3-positive T-cell percentages correlated positively between FC and MIF (r = 0.53, p = 0.05), but were significantly lower by MIF (1.62% vs. 3.4%, p = 0.009). The percentage of mutant NPM1-positive (NPM1c+) cells ranged from 9.7 to 90.8% (median 45.4%) and did not correlate with the NPM1 mutant allele fraction by NGS (p > 0.05). The percentage of CD34+/NPM1c+ cells ranged from 0 to 1.8% (median 0.07%). The percentage of NPM1c+ cells correlated inversely (34% vs. 62%, p = 0.03), while the percentages of CD3-/NPM1c- cells (64% vs. 35%, p = 0.03), and specifically CD3-/CD4-/NPM1c- cells (26% vs. 13%, p = 0.04), correlated positively with subsequent MRD. Discordances between MIF and FC/NGS data suggest that aspirate materials are likely an imperfect reflection of the core biopsy tissue. Furthermore, increased numbers of NPM1 wild-type cells within the microenvironment at diagnosis correlate with the subsequent presence of MRD.

Mechanisms of chimeric antigen receptor (CAR) T cell-mediated antitumor immunity and toxicity remain poorly characterized because few studies examine the intact tumor microenvironment (TME) following CAR T cell infusion. Axicabtagene ciloleucel is an autologous anti-CD19 CAR T cell therapy approved for patients with large B cell lymphoma. We devised multiplex immunostaining and ISH assays to interrogate CAR T cells and other immune cell infiltrates in biopsies of diffuse large B cell lymphoma following axicabtagene ciloleucel infusion. We found that a majority of intratumoral CAR T cells expressed markers of T cell activation but, unexpectedly, constituted ≤5% of all T cells within the TME 5 days or more after therapy. Large numbers of T cells without CAR were also activated within the TME after axicabtagene ciloleucel infusion; these cells were positive for Ki-67, IFN-γ, granzyme B (GzmB), and/or PD-1 and were found at the highest levels in biopsies with CAR T cells. Additionally, non-CAR immune cells were the exclusive source of IL-6, a cytokine associated with cytokine release syndrome, and were found at their highest numbers in biopsies with CAR T cells. These data suggest that intratumoral CAR T cells are associated with non-CAR immune cell activation within the TME with both beneficial and pathological effects.

Classic Hodgkin lymphoma (cHL) is a tumor composed of rare, atypical, germinal center-derived B cells (Hodgkin Reed-Sternberg [HRS] cells) embedded within a robust but ineffective inflammatory milieu. The cHL tumor microenvironment (TME) is compartmentalized into "niches" rich in programmed cell death-1 ligand (PD-L1)-positive HRS cells and tumor-associated macrophages (TAMs), which associate with PD-1-positive T cells to suppress antitumor immunity via PD-L1/PD-1 signaling. Despite the exquisite sensitivity of cHL to PD-1 checkpoint blockade, most patients eventually relapse and need therapeutic alternatives. Using multiplex immunofluorescence microscopy with digital image analysis, we found that cHL is highly enriched for non-T-regulatory, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)-positive T cells (compared with reactive lymphoid tissues) that outnumber PD-1-positive and lymphocyte-activating gene-3 (LAG-3)-positive T cells. In addition, T cells touching HRS cells are more frequently positive for CTLA-4 than for PD-1 or LAG-3. We further found that HRS cells, and a subset of TAMs, are positive for the CTLA-4 ligand CD86 and that the fractions of T cells and TAMs that are CTLA-4-positive and CD86-positive, respectively, are greater within a 75 μm HRS cell niche relative to areas outside this region (CTLA-4, 38% vs 18% [P = .0001]; CD86, 38% vs 24% [P = .0007]). Importantly, CTLA-4-positive cells are present, and focally contact HRS cells, in recurrent cHL tumors following a variety of therapies, including PD-1 blockade. These results implicate CTLA-4:CD86 interactions as a component of the immunologically privileged niche surrounding HRS cells and raise the possibility that patients with cHL refractory to PD-1 blockade may benefit from CTLA-4 blockade., (© 2019 by The American Society of Hematology.)

Combination anti-cytotoxic T lymphocyte antigen 4 (CTLA-4) and anti-programmed cell death protein 1 (PD-1) therapy promotes antitumor immunity and provides superior benefit to patients with advanced-stage melanoma compared with either therapy alone. T cell immunity requires recognition of antigens in the context of major histocompatibility complex (MHC) class I and class II proteins by CD8 + and CD4 + T cells, respectively. We examined MHC class I and class II protein expression on tumor cells from previously untreated melanoma patients and correlated the results with transcriptional and genomic analyses and with clinical response to anti-CTLA-4, anti-PD-1, or combination therapy. Most (>50% of cells) or complete loss of melanoma MHC class I membrane expression was observed in 78 of 181 cases (43%), was associated with transcriptional repression of HLA-A , HLA-B , HLA-C , and B2M , and predicted primary resistance to anti-CTLA-4, but not anti-PD-1, therapy. Melanoma MHC class II membrane expression on >1% cells was observed in 55 of 181 cases (30%), was associated with interferon-γ (IFN-γ) and IFN-γ-mediated gene signatures, and predicted response to anti-PD-1, but not anti-CTLA-4, therapy. We conclude that primary response to anti-CTLA-4 requires robust melanoma MHC class I expression. In contrast, primary response to anti-PD-1 is associated with preexisting IFN-γ-mediated immune activation that includes tumor-specific MHC class II expression and components of innate immunity when MHC class I is compromised. The benefits of combined checkpoint blockade may be attributable, in part, to distinct requirements for melanoma-specific antigen presentation to initiate antitumor immunity., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Chromosome instability and aneuploidies occur very frequently in human embryos, impairing proper embryogenesis and leading to cell cycle arrest, loss of cell viability, and developmental failures in 50-80% of cleavage-stage embryos. This high frequency of cellular extinction events represents a significant experimental obstacle challenging analyses of individual cells isolated from human preimplantation embryos. We carried out single cell expression profiling of 241 individual cells recovered from 32 human embryos during the early and late stages of viable human blastocyst (VHB) differentiation. Classification of embryonic cells was performed solely based on expression patterns of human pluripotency-associated transcripts ( HPAT ), which represent a family of primate-specific transposable element-derived lincRNAs highly expressed in human embryonic stem cells and regulating nuclear reprogramming and pluripotency induction. We then validated our findings by analyzing transcriptomes of 1,708 individual cells recovered from more than 100 human embryos and 259 mouse cells from more than 40 mouse embryos at different stages of preimplantation embryogenesis. HPAT 's expression-guided spatiotemporal reconstruction of human embryonic development inferred from single-cell expression analysis of VHB differentiation enabled identification of telomerase-positive embryonic cells co-expressing key pluripotency regulatory genes and genetic markers of three major lineages. Follow-up validation analyses confirmed the emergence in human embryos prior to lineage segregation of telomerase-positive cells co-expressing genetic markers of multiple lineages. Observations reported in this contribution support the hypothesis of a developmental pathway of creation embryonic lineages and extraembryonic tissues from telomerase-positive pre-lineage cells manifesting multi-lineage precursor phenotype.

Background: Given the demonstrated utility of Third Generation Sequencing [Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT)] long reads in many studies, a comprehensive analysis and comparison of their data quality and applications is in high demand. Methods: Based on the transcriptome sequencing data from human embryonic stem cells, we analyzed multiple data features of PacBio and ONT, including error pattern, length, mappability and technical improvements over previous platforms. We also evaluated their application to transcriptome analyses, such as isoform identification and quantification and characterization of transcriptome complexity, by comparing the performance of size-selected PacBio, non-size-selected ONT and their corresponding Hybrid-Seq strategies (PacBio+Illumina and ONT+Illumina). Results: PacBio shows overall better data quality, while ONT provides a higher yield. As with data quality, PacBio performs marginally better than ONT in most aspects for both long reads only and Hybrid-Seq strategies in transcriptome analysis. In addition, Hybrid-Seq shows superior performance over long reads only in most transcriptome analyses. Conclusions: Both PacBio and ONT sequencing are suitable for full-length single-molecule transcriptome analysis. As this first use of ONT reads in a Hybrid-Seq analysis has shown, both PacBio and ONT can benefit from a combined Illumina strategy. The tools and analytical methods developed here provide a resource for future applications and evaluations of these rapidly-changing technologies., Competing Interests: Competing interests: No competing interests were disclosed.

Genetic risk factors contribute to asymptomatic versus symptomatic visceral leishmaniasis (VL) outcomes following infection with Leishmania infantum. We therefore carried out a family-based (n = 918 post-quality control fully genotyped and phenotyped individuals) candidate gene study for symptomatic VL or asymptomatic delayed-type hypersensitivity (DTH) skin test phenotypes in highly endemic neighborhoods of northeast Brazil. A total of 248 SNPs were genotyped in 42 genes selected as candidates on the basis of prior genetic, immunological, and transcriptional profiling studies. The most significant association with the VL phenotype was with SNP rs6785358 (P = 5.7e-04; p corrected = 0.026) 3.8 kb upstream of TGFBR2, the gene encoding the type 2 receptor for transforming growth factor beta (TGFβ). A second inhibitory member of the TGBβ superfamily signaling pathway, SMAD7, was associated with the DTH phenotype (SNP rs7238442: P = 0.001; p corrected = 0.051). The most significant association for the DTH phenotype was with SNP rs10800309 (P = -8.4e-06; p corrected = 3.9e-04) situated 3.1 kb upstream of FCGR2A, the gene encoding the low-affinity IIa receptor for the Fc fragment of IgG. Overall, our results imply a role for IgG-mediated inflammation in determining DTH associated with asymptomatic infection and contribute to growing evidence that the TGFβ pathway is important in the immunopathogenesis of VL., (© 2017 John Wiley & Sons Ltd/University College London.)

Infection with the protozoan Leishmania infantum can lead to asymptomatic infection and protective immunity, or to the progressive and potentially fatal disease visceral leishmaniasis (VL). Published studies show host genetic background determines in part whether infected individuals will develop a symptomatic or asymptomatic outcome. The purpose of the current study was to fine map chromosome regions previously linked with risk for symptomatic (chromosome 9) or asymptomatic (chromosomes 15 and 19) manifestations of L. infantum infection. We conducted a family-based genetic study of VL and asymptomatic infection (detected by a DTH skin test) with a final post quality control sample of 961 individuals with full genotype and phenotype information from highly endemic neighborhoods of northeast Brazil. A total of 5485 SNPs under the linkage peaks on chromosomes 9, 15 and 19 were genotyped. No strong SNP associations were observed for the DTH phenotype. The most significant associations with the VL phenotype were with SNP rs1470217 (p=5.9e-05; pcorrected=0.057) on chromosome 9, and with SNP rs8107014 (p=1.4e-05; pcorrected=0.013) on chromosome 19. SNP rs1470217 is situated in a 180kb intergenic region between TMEM215 (Transmembrane protein 215) and APTX (Aprataxin). SNP rs8107014 lies in the intron between exons 26 and 27 of a 34 exon transcript (ENST00000204005) of LTBP4, (Latent transforming growth factor-beta-binding protein 4a). The latter supports growing evidence that the transforming growth factor-beta pathway is important in the immunopathogenesis of VL., Competing Interests: The authors declare they have no competing interests., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Nivolumab plus ipilimumab (aCTLA-4/aPD-1) combination therapy has significantly improved clinical outcomes in patients with metastatic melanoma, with 50%-60% of patients responding to treatment, but predictors of response are poorly characterized. We hypothesized that circulating cytokines and peripheral white blood cells may predict response to therapy and evaluated 15 cytokines and complete blood counts (CBC with differentials) from 89 patients with advanced melanoma treated with combination therapy from three points in time: pre-treatment, one month and approximately three months after starting therapy. Clinical endpoints evaluated included durable clinical benefit (DCB), progression-free survival (PFS), and overall survival (OS). A parsimonious predictive model was developed to identify cytokines predictors of response to combination therapy. In this study, we found that pre-treatment, patients with DCB had higher IL-23, lower CXCL6, and lower IL-10 levels. Lower NLR one month after starting therapy predicted better PFS and OS, primarily driven by an increase in absolute lymphocytes. A multivariate model demonstrated that baseline CXCL6, IL-10, IL-23 were independent predictors of therapy response, and the combined model has reached an area under the curve (AUC) of 0.79 in prediction of response to combination therapy. Our study identified baseline CXCL6, IL-23, and IL-10 as predictors of response to aCTLA4/aPD1 combination therapy among patients with metastatic melanoma. This study also provides a framework for identifying patients who are likely to respond to combination ICB, as well as a subset of patients with high risk of developing resistance and are thus in need of alternative therapeutic options, such as clinical trials.

We developed an innovative hybrid sequencing approach, IDP-fusion, to detect fusion genes, determine fusion sites and identify and quantify fusion isoforms. IDP-fusion is the first method to study gene fusion events by integrating Third Generation Sequencing long reads and Second Generation Sequencing short reads. We applied IDP-fusion to PacBio data and Illumina data from the MCF-7 breast cancer cells. Compared with the existing tools, IDP-fusion detects fusion genes at higher precision and a very low false positive rate. The results show that IDP-fusion will be useful for unraveling the complexity of multiple fusion splices and fusion isoforms within tumorigenesis-relevant fusion genes., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

IFN regulatory factor 6 (IRF6) is a transcription factor that, in mammals, is required for the differentiation of skin, breast epithelium, and oral epithelium. However, the transcriptional targets that mediate these effects are currently unknown. In zebrafish and frog embryos, Irf6 is necessary for differentiation of the embryonic superficial epithelium, or periderm. Here we use microarrays to identify genes that are expressed in the zebrafish periderm and whose expression is inhibited by a dominant-negative variant of Irf6 (dnIrf6). These methods identify Grainyhead-like 3 (Grhl3), an ancient regulator of the epidermal permeability barrier, as acting downstream of Irf6. In human keratinocytes, IRF6 binds conserved elements near the GRHL3 [corrected] promoter. We show that one of these elements has enhancer activity in human keratinocytes and zebrafish periderm, suggesting that Irf6 directly stimulates Grhl3 expression in these tissues. Simultaneous inhibition of grhl1 and grhl3 disrupts periderm differentiation in zebrafish, and, intriguingly, forced grhl3 expression restores periderm markers in both zebrafish injected with dnIrf6 and frog embryos depleted of Irf6. Finally, in Irf6-deficient mouse embryos, Grhl3 expression in the periderm and oral epithelium is virtually absent. These results indicate that Grhl3 is a key effector of Irf6 in periderm differentiation.

The protozoan parasite Trypanosoma brucei switches its variant surface glycoprotein (VSG) to subvert its mammalian hosts' immune responses. The T. brucei genome contains as many as 1600 VSG genes (VSGs), but most are silent noncoding pseudogenes. Only one functional VSG, located in a telomere-linked expression site, is transcribed at a time. Silent VSGs are copied into a VSG expression site through gene conversion. Truncated gene conversion events can generate new mosaic VSGs with segments of sequence identity to other VSGs. To examine the VSG family sub-structure within which these events occur, we combined the available VSG sequences and annotations with scripted BLAST searches to map the relationships among VSGs in the T. brucei genome. Clusters of related VSGs were visualized in 2- and 3-dimensions for different N- and C-terminal regions. Five types of N-termini (N1-N5) were observed, within which gene recombinational events are likely to occur, often with fully-coding 'functional' or 'atypical'VSGs centrally located between more dissimilar VSGs. Members of types N1, N3 and N4 are most closely related in the middle of the N-terminal region, whereas type N2 members are more similar near the N-terminus. Some preference occurs in pairing between specific N- and C-terminal types. Statistical analyses indicated no overall tendency for more related VSGs to be located closer in the genome than less related VSGs, although exceptions were noted. Many potential mosaic gene formation events within each N-terminal type were identified, contrasted by only one possible mosaic gene formation between N-terminal types (N1 and N2). These data suggest that mosaic gene formation is a major contributor to the overall VSG diversity, even though gene recombinational events between members of different N-terminal types occur only rarely., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Visceral leishmaniasis (VL) is endemic in large cities in Brazil, including Natal. We determined the prevalence of asymptomatic human infection with Leishmania infantum chagasi and associated environmental risks around Natal. Infection was detected by Leishmania skin test (LST) and anti-leishmanial antibodies in humans and anti-leishmanial antibodies in dogs. Amongst 345 humans, 24.6% were seropositive, and 38.6% were LST-positive. Prevalence of positive serology was similar in both sexes and across all ages. However, positive LST responses increased with age, suggesting that LST is long-lasting and cumulative. Multinomial logistic analysis showed that LST response varied with location (P = 0.007) and that males were more frequently LST-positive (P = 0.027). Indicators of lower socioeconomic status associated significantly with human infection. Furthermore, there was geographic coincidence of seropositive humans and dogs (r = 0.7926, P = 0.011). These data suggest that dog and human L. i. chagasi infection are intimately interrelated in environmental conditions associated with low income.

The Leishmania species cause a variety of human disease syndromes. Methods for diagnosis and species differentiation are insensitive and many require invasive sampling. Although quantitative PCR (qPCR) methods are reported for leishmania detection, no systematic method to quantify parasites and determine the species in clinical specimens is established. We developed a serial qPCR strategy to identify and rapidly differentiate Leishmania species and quantify parasites in clinical or environmental specimens. SYBR green qPCR is mainly employed, with corresponding TaqMan assays for validation. The screening primers recognize kinetoplast minicircle DNA of all Leishmania species. Species identification employs further qPCR set(s) individualized for geographic regions, combining species-discriminating probes with melt curve analysis. The assay was sufficient to detect Leishmania parasites, make species determinations, and quantify Leishmania spp. in sera, cutaneous biopsy specimens, or cultured isolates from subjects from Bangladesh or Brazil with different forms of leishmaniasis. The multicopy kinetoplast DNA (kDNA) probes were the most sensitive and useful for quantification based on promastigote standard curves. To test their validity for quantification, kDNA copy numbers were compared between Leishmania species, isolates, and life stages using qPCR. Maxicircle and minicircle copy numbers differed up to 6-fold between Leishmania species, but the differences were smaller between strains of the same species. Amastigote and promastigote leishmania life stages retained similar numbers of kDNA maxi- or minicircles. Thus, serial qPCR is useful for leishmania detection and species determination and for absolute quantification when compared to a standard curve from the same Leishmania species.

Objectives: This study describes parasite kinetics in the blood of visceral leishmaniasis patients treated with liposomal amphotericin B (L-AmB) or a preformed fat emulsion of amphotericin B (ApL) using real-time quantitative PCR (qPCR)., Methods: Forty-six patients were treated with a single dose (15 mg/kg of body weight) of either L-AmB (n = 13) or ApL (n = 33). qPCR was used to estimate parasite kinetics by detection of Leishmania donovani DNA using kinetoplast DNA-specific primers in peripheral blood samples using an absolute quantification method., Results: The mean parasite load decreased from baseline (day 0) values of 894.07 and 980.48 to 71.72 and 211.52 parasite genomes/mL at day 7 in L-AmB and ApL groups, respectively, and at day 30 these further declined to 8.30 and 133.98 parasite genomes/mL, respectively. At day 30 post-treatment evaluation, the decline in parasite load was significantly greater (P = 0.024) with L-AmB compared with ApL. Four of 33 patients in the ApL group failed treatment (1 primary failure and 3 relapses) with the presence of parasites, whereas all patients in the L-AmB group were cured at 6 month follow-up., Conclusions: qPCR can be a tool to measure parasite dynamics accurately and provide a marker to measure the efficacy of various drugs. It can be used as a test of cure, allowing us to do away with invasive and risky methods such as splenic or bone marrow aspiration.

Genetic variation in the transcription factor interferon regulatory factor 6 (IRF6) causes and contributes risk for oral clefting disorders. We hypothesized that genes regulated by IRF6 are also involved in oral clefting disorders. We used five criteria to identify potential IRF6 target genes; differential gene expression in skin taken from wild-type and Irf6-deficient murine embryos, localization to the Van der Woude syndrome 2 (VWS2) locus at 1p36-1p32, overlapping expression with Irf6, presence of a conserved predicted-binding site in the promoter region, and a mutant murine phenotype that was similar to the Irf6 mutant mouse. Previously, we observed altered expression for 573 genes; 13 were located in the murine region syntenic to the VWS2 locus. Two of these genes, Wdr65 and Stratifin, met 4 of 5 criteria. Wdr65 was a novel gene that encoded a predicted protein of 1,250 amino acids with two WD domains. As potential targets for Irf6 regulation, we hypothesized that disease-causing mutations will be found in WDR65 and Stratifin in individuals with VWS or VWS-like syndromes. We identified a potentially etiologic missense mutation in WDR65 in a person with VWS who does not have an exonic mutation in IRF6. The expression and mutation data were consistent with the hypothesis that WDR65 was a novel gene involved in oral clefting.