Radiomic analysis of patient and inter-organ heterogeneity in response to immunotherapies and BRAF targeted therapy in metastatic melanoma.

Background: Variability in treatment response may be attributable to organ-level heterogeneity in tumor lesions. Radiomic analysis of medical images can elucidate non-invasive biomarkers of clinical outcome. Organ-specific radiomic comparison across immunotherapies and targeted therapies has not been previously reported. Methods: We queried UPMC Hillman Cancer Center registry for patients with metastatic melanoma (MEL) treated with immune checkpoint inhibitors (ICI) (anti-PD1/CTLA4 [ipilimumab+nivolumab; I+N] or anti-PD1 monotherapy) or BRAF targeted therapy. Best overall response was measured using RECIST v1.1. Lesions were segmented into discrete volume-of-interest with 400 radiomics features extracted. Overall and organ-specific machine-learning models were constructed to predict disease control (DC) versus progressive disease (PD) using XGBoost. Results: 291 MEL patients were identified, including 242 ICI (91 I+N, 151 PD1) and 49 BRAF. 667 metastases were analyzed, including 541 ICI (236 I+N, 305 PD1) and 126 BRAF. Across cohorts, baseline demographics included 39-47% female, 24-29% M1C, 24-46% M1D, and 61-80% with elevated LDH. Among patients experiencing DC, the organs with the greatest reduction were liver (-88%±12%, I+N; mean±S.E.M.) and lung (-72%±8%, I+N). For patients with multiple same-organ target lesions, the highest inter-lesion heterogeneity was observed in brain among patients who received ICI while no intra-organ heterogeneity was observed in BRAF. 267 patients were kept for radiomic modeling, including 221 ICI (86 I+N, 135 PD1) and 46 BRAF. Models consisting of optimized radiomic signatures classified DC/PD across I+N (AUC=0.85) and PD1 (0.71) and within individual organ sites (AUC=0.72∼0.94). Integration of clinical variables improved the models' performance. Comparison of models between treatments and across organ sites suggested mostly non-overlapping DC or PD features. Skewness, kurtosis, and informational measure of correlation (IMC) were among the radiomic features shared between overall response models. Kurtosis and IMC were also utilized by multiple organ-site models. Conclusions: Differential organ-specific response was observed across BRAF and ICI with within organ heterogeneity observed for ICI but not for BRAF. Radiomic features of organ-specific response demonstrated little overlap. Integrating clinical factors with radiomics improves the prediction of disease course outcome and prediction of tumor heterogeneity.

Autologous HER2-specific CAR T cells after lymphodepletion for advanced sarcoma: a phase 1 trial.

In this prospective, interventional phase 1 study for individuals with advanced sarcoma, we infused autologous HER2-specific chimeric antigen receptor T cells (HER2 CAR T cells) after lymphodepletion with fludarabine (Flu) ± cyclophosphamide (Cy): 1 × 108 T cells per m2 after Flu (cohort A) or Flu/Cy (cohort B) and 1 × 108 CAR+ T cells per m2 after Flu/Cy (cohort C). The primary outcome was assessment of safety of one dose of HER2 CAR T cells after lymphodepletion. Determination of antitumor responses was the secondary outcome. Thirteen individuals were treated in 14 enrollments, and seven received multiple infusions. HER2 CAR T cells expanded after 19 of 21 infusions. Nine of 12 individuals in cohorts A and B developed grade 1-2 cytokine release syndrome. Two individuals in cohort C experienced dose-limiting toxicity with grade 3-4 cytokine release syndrome. Antitumor activity was observed with clinical benefit in 50% of individuals treated. The tumor samples analyzed showed spatial heterogeneity of immune cells and clustering by sarcoma type and by treatment response. Our results affirm HER2 as a CAR T cell target and demonstrate the safety of this therapeutic approach in sarcoma. ClinicalTrials.gov registration: NCT00902044 .

Th2 Cells Are Associated with Tumor Recurrence Following Radiation.

The curative treatment of multiple solid tumors, including head and neck squamous cell carcinoma (HNSCC), utilizes radiation. The outcomes for HPV/p16-negative HNSCC are significantly worse than HPV/p16-positive tumors, with increased radiation resistance leading to worse locoregional recurrence (LRR) and ultimately death. This study analyzed the relationship between immune function and outcomes following radiation in HPV/p16-negative tumors to identify mechanisms of radiation resistance and prognostic immune biomarkers. A discovery cohort of 94 patients with HNSCC treated uniformly with surgery and adjuvant radiation and a validation cohort of 97 similarly treated patients were utilized. Tumor immune infiltrates were derived from RNAseq gene expression. The immune cell types significantly associated with outcomes in the discovery cohort were examined in the independent validation cohort. A positive association between high Th2 infiltration and LRR was identified in the discovery cohort and validated in the validation cohort. Tumor mutations in CREBBP/EP300 and CASP8 were significantly associated with Th2 infiltration. A pathway analysis of genes correlated with Th2 cells revealed the potential repression of the antitumor immune response and the activation of BRCA1-associated DNA damage repair in multiple cohorts. The Th2 infiltrates were enriched in the HPV/p16-negative HNSCC tumors and associated with LRR and mutations in CASP8, CREBBP/EP300, and pathways previously shown to impact the response to radiation.

Facts and Hopes in Using Omics to Advance Combined Immunotherapy Strategies.

The field of oncology has been transformed by immune checkpoint inhibitors (ICI) and other immune-based agents; however, many patients do not receive a durable benefit. While biomarker assessments from pivotal ICI trials have uncovered certain mechanisms of resistance, results thus far have only scraped the surface. Mechanisms of resistance are as complex as the tumor microenvironment (TME) itself, and the development of effective therapeutic strategies will only be possible by building accurate models of the tumor-immune interface. With advancement of multi-omic technologies, high-resolution characterization of the TME is now possible. In addition to sequencing of bulk tumor, single-cell transcriptomic, proteomic, and epigenomic data as well as T-cell receptor profiling can now be simultaneously measured and compared between responders and nonresponders to ICI. Spatial sequencing and imaging platforms have further expanded the dimensionality of existing technologies. Rapid advancements in computation and data sharing strategies enable development of biologically interpretable machine learning models to integrate data from high-resolution, multi-omic platforms. These models catalyze the identification of resistance mechanisms and predictors of benefit in ICI-treated patients, providing scientific foundation for novel clinical trials. Moving forward, we propose a framework by which in silico screening, functional validation, and clinical trial biomarker assessment can be used for the advancement of combined immunotherapy strategies.

Identification of tumor-intrinsic drivers of immune exclusion in acral melanoma.

Acral melanoma (AM) has distinct characteristics as compared with cutaneous melanoma and exhibits poor response to immune checkpoint inhibitors (ICIs). Tumor-intrinsic mechanisms of immune exclusion have been identified in many cancers but less studied in AM. We characterized clinically annotated tumors from patients diagnosed with AM at our institution in correlation with ICI response using whole transcriptome RNAseq, whole exome sequencing, CD8 immunohistochemistry, and multispectral immunofluorescence imaging. A defined interferon-γ-associated T cell-inflamed gene signature was used to categorize tumors into non-T cell-inflamed and T cell-inflamed phenotypes. In combination with AM tumors from two published studies, we systematically assessed the immune landscape of AM and detected differential gene expression and pathway activation in a non-T cell-inflamed tumor microenvironment (TME). Two single-cell(sc) RNAseq AM cohorts and 11 bulk RNAseq cohorts of various tumor types were used for independent validation on pathways associated with lack of ICI response. In total, 892 specimens were included in this study. 72.5% of AM tumors showed low expression of the T cell-inflamed gene signature, with 23.9% of total tumors categorized as the non-T cell-inflamed phenotype. Patients of low CD3+CD8+PD1+ intratumoral T cell density showed poor prognosis. We identified 11 oncogenic pathways significantly upregulated in non-T cell-inflamed relative to T cell-inflamed TME shared across all three acral cohorts (MYC, HGF, MITF, VEGF, EGFR, SP1, ERBB2, TFEB, SREBF1, SOX2, and CCND1). scRNAseq analysis revealed that tumor cell-expressing pathway scores were significantly higher in low versus high T cell-infiltrated AM tumors. We further demonstrated that the 11 pathways were enriched in ICI non-responders compared with responders across cancers, including AM, cutaneous melanoma, triple-negative breast cancer, and non-small cell lung cancer. Pathway activation was associated with low expression of interferon stimulated genes, suggesting suppression of antigen presentation. Across the 11 pathways, fatty acid synthase and CXCL8 were unifying downstream target molecules suggesting potential nodes for therapeutic intervention. A unique set of pathways is associated with immune exclusion and ICI resistance in AM. These data may inform immunotherapy combinations for immediate clinical translation.

Identification of tumor-intrinsic drivers of immune exclusion in acral melanoma.

Background: Acral melanoma (AM) has distinct characteristics as compared to cutaneous melanoma and exhibits poor response to immune checkpoint inhibitors (ICI). Tumor-intrinsic mechanisms of immune exclusion have been identified in many cancers but less studied in AM. Methods: We characterized clinically annotated tumors from patients diagnosed with AM at our institution in correlation with ICI response using whole transcriptome RNAseq, whole exome sequencing, CD8 immunohistochemistry, and multispectral immunofluorescence imaging. A defined interferon-γ-associated T cell-inflamed gene signature was used to categorize tumors into non-T cell-inflamed and T cell-inflamed phenotypes. In combination with AM tumors from two published studies, we systematically assessed the immune landscape of AM and detected differential gene expression and pathway activation in a non-T cell-inflamed tumor microenvironment (TME). Two single-cell(sc) RNAseq AM cohorts and 11 bulk RNAseq cohorts of various tumor types were used for independent validation on pathways associated with lack of ICI response. In total, 892 specimens were included in this study. Results: 72.5% of AM tumors showed low expression of the T cell-inflamed gene signature, with 23.9% of total tumors categorized as the non-T cell-inflamed phenotype. Patients of low CD3 + CD8 + PD1 + intratumoral T cell density showed poor prognosis. We identified 11 oncogenic pathways significantly upregulated in non-T cell-inflamed relative to T cell-inflamed TME shared across all three acral cohorts (MYC, HGF, MITF, VEGF, EGFR, SP1, ERBB2, TFEB, SREBF1, SOX2, and CCND1). scRNAseq analysis revealed that tumor cell-expressing pathway scores were significantly higher in low vs high T cell-infiltrated AM tumors. We further demonstrated that the 11 pathways were enriched in ICI non-responders compared to responders across cancers, including acral melanoma, cutaneous melanoma, triple-negative breast cancer, and non-small cell lung cancer. Pathway activation was associated with low expression of interferon stimulated genes, suggesting suppression of antigen presentation. Across the 11 pathways, fatty acid synthase and CXCL8 were unifying downstream target molecules suggesting potential nodes for therapeutic intervention. Conclusions: A unique set of pathways is associated with immune exclusion and ICI resistance in AM. These data may inform immunotherapy combinations for immediate clinical translation.

Tumor cell p38 inhibition to overcome immunotherapy resistance.

Patients with tumors that do not respond to immune-checkpoint inhibition often harbor a non-T cell-inflamed tumor microenvironment, characterized by the absence of IFN-γ-associated CD8+ T cell and dendritic cell activation. Understanding the molecular mechanisms underlying immune exclusion in non-responding patients may enable the development of novel combination therapies. p38 MAPK is a known regulator of dendritic and myeloid cells however a tumor-intrinsic immunomodulatory role has not been previously described. Here we identify tumor cell p38 signaling as a therapeutic target to potentiate anti-tumor immunity and overcome resistance to immune-checkpoint inhibitors (ICI). Molecular analysis of tumor tissues from patients with human papillomavirus-negative head and neck squamous carcinoma reveals a p38-centered network enriched in non-T cell-inflamed tumors. Pan-cancer single-cell RNA analysis suggests that p38 activation may be an immune-exclusion mechanism across multiple tumor types. P38 knockdown in cancer cell lines increases T cell migration, and p38 inhibition plus ICI in preclinical models shows greater efficacy compared to monotherapies. In a clinical trial of patients refractory to PD1/L1 therapy, pexmetinib, a p38 inhibitor, plus nivolumab demonstrated deep and durable clinical responses. Targeting of p38 with anti-PD1 has the potential to induce the T cell-inflamed phenotype and overcome immunotherapy resistance.

A Novel Humanized Immune Stroma PDX Cancer Model for Therapeutic Studies.

Standard preclinical human tumor models lack a human tumor stroma. However, as stroma contributes to therapeutic resistance, the lack of human stroma may make current models less stringent for testing new therapies. To address this, using patient-derived tumor cells, patient derived cancer-associated mesenchymal stem/progenitor cells, and human endothelial cells, we created a Human Stroma-Patient Derived Xenograft (HS-PDX) tumor model. HS-PDX, compared to the standard PDX model, demonstrate greater resistance to targeted therapy and chemotherapy, and better reflect patient response to therapy. Furthermore, HS-PDX can be grown in mice with humanized bone marrow to create humanized immune stroma patient-derived xenograft (HIS-PDX) models. The HIS-PDX model contains human connective tissues, vascular and immune cell infiltrates. RNA sequencing analysis demonstrated a 94-96% correlation with primary human tumor. Using this model, we demonstrate the impact of human tumor stroma on response to CAR-T cell therapy and immune checkpoint inhibitor therapy. We show an immunosuppressive role for human tumor stroma and that this model can be used to identify immunotherapeutic combinations to overcome stromally mediated immunosuppression. Combined, our data confirm a critical role for human stoma in therapeutic response and indicate that HIS-PDX can be an important tool for preclinical drug testing. Statement of Significance: We developed a tumor model with human stromal, vascular, and immune cells. This model mirrors patient response to chemotherapy, targeted therapy, and immunotherapy, and can be used to study therapy resistance.

Partial tumor irradiation plus pembrolizumab in treating large advanced solid tumor metastases.

BACKGROUNDWe previously demonstrated the safety of stereotactic body radiotherapy followed by pembrolizumab (SBRT+P) in patients with advanced solid tumors. This phase I clinical trial was expanded to study the safety of partial tumor irradiation (partial-Rx). We assessed irradiated local failure (LF) and clinical outcomes with correlations to biomarkers including CD8+ T cell radiomics score (RS) and circulating cytokines.METHODSPatients received SBRT to 2-4 metastases and pembrolizumab for up to 7 days after SBRT. Tumors measuring up to 65 cc received the full radiation dose (complete-Rx), whereas tumors measuring more than 65 cc received partial-Rx. Landmark analysis was used to assess the relationship between tumor response and overall survival (OS). Multivariable analysis was performed for RS and circulating cytokines.RESULTSIn the combined (expansion plus original) cohort, 97 patients (219 metastases) were analyzed and received SBRT+P. Forty-six (47%) patients received at least 1 partial-Rx treatment. There were 7 (7.2%)dose-limiting toxicities (DLTs). 1-year LF was 7.6% overall, and 13.3% and 5.4% for partial-Rx and complete-Rx tumors, respectively (HR 2.32, 95% CI 0.90-5.97, P = 0.08). The overall, unirradiated, and irradiated objective response rates were 22%, 12%, and 34%, respectively. Irradiated tumor response to SBRT+P was associated with prolonged OS; 1-year OS was 71% (responders), 42% (mixed-responders), and 0% (nonresponders) (P < 0.01). High-RS was significantly associated with improved LF, progression-free survival (PFS), and OS. Elevated circulating IL-8 was independently associated with inferior PFS and OS.CONCLUSIONSBRT+P is safe in patients with large, advanced solid tumors. Additional studies are warranted to assess noninferiority of complete versus partial irradiation of tumors in the setting of immunotherapy.TRIAL REGISTRATIONClinicaltrials.gov NCT02608385FUNDINGMerck Investigator Studies Program; Hillman Fellows for Innovative Cancer Research Program; NIH grants UM1CA186690-06, P50CA254865-01A1, P30CA047904-32, and R01DE031729-01A1.

BET Inhibition Sensitizes Immunologically Cold Rb-Deficient Prostate Cancer to Immune Checkpoint Blockade.

Non-T-cell-inflamed immunologically "cold" tumor microenvironments (TME) are associated with poor responsiveness to immune checkpoint blockade (ICB) and can be sculpted by tumor cell genomics. Here, we evaluated how retinoblastoma (Rb) tumor-suppressor loss-of-function (LOF), one of the most frequent alterations in human cancer and associated with lineage plasticity, poor prognosis, and therapeutic outcomes, alters the TME, and whether therapeutic strategies targeting the molecular consequences of Rb loss enhance ICB efficacy. We performed bioinformatics analysis to elucidate the impact of endogenous Rb LOF on the immune TME in human primary and metastatic tumors. Next, we used isogenic murine models of Rb-deficient prostate cancer for in vitro and in vivo mechanistic studies to examine how Rb loss and bromodomain and extraterminal (BET) domain inhibition (BETi) reprograms the immune landscape, and evaluated in vivo therapeutic efficacy of BETi, singly and in combination with ICB and androgen deprivation therapy. Rb loss was enriched in non-T-cell-inflamed tumors, and Rb-deficient murine tumors demonstrated decreased immune infiltration in vivo. The BETi JQ1 increased immune infiltration into the TME through enhanced tumor cell STING/NF-κB activation and type I IFN signaling within tumor cells, resulting in differential macrophage and T-cell-mediated tumor growth inhibition and sensitization of Rb-deficient prostate cancer to ICB. BETi can reprogram the immunologically cold Rb-deficient TME via STING/NF-κB/IFN signaling to sensitize Rb-deficient prostate cancer to ICB. These data provide the mechanistic rationale to test combinations of BETi and ICB in clinical trials of Rb-deficient prostate cancer.

Randomized, Double-Blind, Placebo-Controlled Trial of MUC1 Peptide Vaccine for Prevention of Recurrent Colorectal Adenoma.

PURPOSE: To assess whether MUC1 peptide vaccine produces an immune response and prevents subsequent colon adenoma formation. PATIENTS AND METHODS: Multicenter, double-blind, placebo-controlled randomized trial in individuals age 40 to 70 with diagnosis of an advanced adenoma ≤1 year from randomization. Vaccine was administered at 0, 2, and 10 weeks with a booster injection at week 53. Adenoma recurrence was assessed ≥1 year from randomization. The primary endpoint was vaccine immunogenicity at 12 weeks defined by anti-MUC1 ratio ≥2.0. RESULTS: Fifty-three participants received the MUC1 vaccine and 50 placebo. Thirteen of 52 (25%) MUC1 vaccine recipients had a ≥2-fold increase in MUC1 IgG (range, 2.9-17.3) at week 12 versus 0/50 placebo recipients (one-sided Fisher exact P < 0.0001). Of 13 responders at week 12, 11 (84.6%) responded to a booster injection at week 52 with a ≥2-fold increase in MUC1 IgG measured at week 55. Recurrent adenoma was observed in 31 of 47 (66.0%) in the placebo group versus 27 of 48 (56.3%) in the MUC1 group [adjusted relative risk (aRR), 0.83; 95% confidence interval (CI), 0.60-1.14; P = 0.25]. Adenoma recurrence occurred in 3/11 (27.3%) immune responders at week 12 and week 55 (aRR, 0.41; 95% CI, 0.15-1.11; P = 0.08 compared with placebo). There was no difference in serious adverse events. CONCLUSIONS: An immune response was observed only in vaccine recipients. Adenoma recurrence was not different than placebo, but a 38% absolute reduction in adenoma recurrence compared with placebo was observed in participants who had an immune response at week 12 and with the booster injection.

Targeting Cbl-b in cancer immunotherapy.

Cancer immunotherapy with immune-checkpoint blockade has improved the outcomes of patients with various malignancies, yet a majority do not benefit or develop resistance. To address this unmet need, efforts across the field are targeting additional coinhibitory receptors, costimulatory proteins, and intracellular mediators that could prevent or bypass anti-PD1 resistance mechanisms. The CD28 costimulatory pathway is necessary for antigen-specific T cell activation, though prior CD28 agonists did not translate successfully to clinic due to toxicity. Casitas B lymphoma-b (Cbl-b) is a downstream, master regulator of both CD28 and CTLA-4 signaling. This E3 ubiquitin ligase regulates both innate and adaptive immune cells, ultimately promoting an immunosuppressive tumor microenvironment (TME) in the absence of CD28 costimulation. Recent advances in pharmaceutical screening and computational biology have enabled the development of novel platforms to target this once 'undruggable' protein. These platforms include DNA encoded library screening, allosteric drug targeting, small-interfering RNA inhibition, CRISPR genome editing, and adoptive cell therapy. Both genetic knock-out models and Cbl-b inhibitors have been shown to reverse immunosuppression in the TME, stimulate cytotoxic T cell activity, and promote tumor regression, findings augmented with PD1 blockade in experimental models. In translating Cbl-b inhibitors to clinic, we propose specific gene expression profiles that may identify patient populations most likely to benefit. Overall, novel Cbl-b inhibitors provide antigen-specific immune stimulation and are a promising therapeutic tool in the field of immuno-oncology.

Old Dog, New Trick: A Tumor-Intrinsic Role for PD-1 in Chemoresistant Tumor Subclones.

SUMMARY: Programmed cell death protein 1 (PD-1) is a well-known driver of immunosuppression and lymphocyte-associated disease progression. Increasing evidence suggests a tumor-intrinsic role for PD-1 in promoting chemoresistance via stem-like features. Moving forward, a recent study implies a novel antitumor mechanism for PD-1 inhibition. See related article by Rotolo et al., p. 621.

Inherited cancer predisposing mutations in patients with therapy-related myeloid neoplasms.

Some patients with therapy-related myeloid neoplasms (t-MN) may have unsuspected inherited cancer predisposition syndrome (CPS). We propose a set of clinical criteria to identify t-MN patients with high risk of CPS (HR-CPS). Among 225 t-MN patients with an antecedent non-myeloid malignancy, our clinical criteria identified 52 (23%) HR-CPS patients. Germline whole-exome sequencing identified pathogenic or likely pathogenic variants in 10 of 27 HR-CPS patients compared to 0 of 9 low-risk CPS patients (37% vs. 0%, p = 0.04). These simple clinical criteria identify t-MN patients most likely to benefit from genetic testing for inherited CPS.

Treatment of peanut allergy and colitis in mice via the intestinal release of butyrate from polymeric micelles.

The microbiome modulates host immunity and aids the maintenance of tolerance in the gut, where microbial and food-derived antigens are abundant. Yet modern dietary factors and the excessive use of antibiotics have contributed to the rising incidence of food allergies, inflammatory bowel disease and other non-communicable chronic diseases associated with the depletion of beneficial taxa, including butyrate-producing Clostridia. Here we show that intragastrically delivered neutral and negatively charged polymeric micelles releasing butyrate in different regions of the intestinal tract restore barrier-protective responses in mouse models of colitis and of peanut allergy. Treatment with the butyrate-releasing micelles increased the abundance of butyrate-producing taxa in Clostridium cluster XIVa, protected mice from an anaphylactic reaction to a peanut challenge and reduced disease severity in a T-cell-transfer model of colitis. By restoring microbial and mucosal homoeostasis, butyrate-releasing micelles may function as an antigen-agnostic approach for the treatment of allergic and inflammatory diseases.

Wilms tumor reveals DNA repair gene hyperexpression is linked to lack of tumor immune infiltration.

BACKGROUND: A T cell-rich tumor microenvironment has been associated with improved clinical outcome and response to immune checkpoint blockade therapies in several adult cancers. Understanding the mechanisms for lack of immune cell infiltration in tumors is critical for expanding immunotherapy efficacy. To gain new insights into the mechanisms of poor tumor immunogenicity, we turned to pediatric cancers, which are generally unresponsive to checkpoint blockade. METHODS: RNA sequencing and clinical data were obtained for Wilms tumor, rhabdoid tumor, osteosarcoma, and neuroblastoma from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database, and adult cancers from The Cancer Genome Atlas (TCGA). Using an 18-gene tumor inflammation signature (TIS) representing activated CD8+ T cells, we identified genes inversely correlated with the signature. Based on these results, adult tumors were also analyzed, and immunofluorescence was performed on metastatic melanoma samples to assess the MSH2 relationship to anti-programmed cell death protein-1 (PD-1) efficacy. RESULTS: Among the four pediatric cancers, we observed the lowest TIS scores in Wilms tumor. TIS scores were lower in Wilms tumors compared with matched normal kidney tissues, arguing for loss of endogenous T cell infiltration. Pathway analysis of genes upregulated in Wilms tumor and anti-correlated with TIS revealed activated pathways involved DNA repair. The majority of adult tumors in TCGA also showed high DNA repair scores associated with low TIS. Melanoma samples from an independent cohort revealed an inverse correlation between MSH2+ tumor cells and CD8+ T cells. Additionally, melanomas with high MSH2+ tumor cell numbers were largely non-responders to anti-PD-1 therapy. CONCLUSIONS: Increased tumor expression of DNA repair genes is associated with a less robust immune response in Wilms tumor and the majority of TCGA tumor types. Surprisingly, the negative relationship between DNA repair score and TIS remained strong across TCGA when correcting for mutation count, indicating a potential role for DNA repair genes outside of preventing the accumulation of mutations. While loss of DNA repair machinery has been associated with carcinogenesis and mutational antigen generation, our results suggest that hyperexpression of DNA repair genes might be prohibitive for antitumor immunity, arguing for pharmacologic targeting of DNA repair as a potential therapeutic strategy.

Next steps for clinical translation of adenosine pathway inhibition in cancer immunotherapy.

Increasing evidence supports targeting the adenosine pathway in immuno-oncology with several clinical programs directed at adenosine A2 receptor (A2AR, A2BR), CD73 and CD39 in development. Through a cyclic-AMP-mediated intracellular cascade, adenosine shifts the cytokine and cellular profile of the tumor microenvironment away from cytotoxic T cell inflammation toward one of immune tolerance. A perpetuating cycle of tumor cell proliferation, tissue injury, dysregulated angiogenesis, and hypoxia promote adenosine accumulation via ATP catabolism. Adenosine receptor (eg, A2AR, A2BR) stimulation of both the innate and adaptive cellular precursors lead to immunosuppressive phenotypic differentiation. Preclinical work in various tumor models with adenosine receptor inhibition has demonstrated restoration of immune cell function and tumor regression. Given the broad activity but known limitations of anti-programmed cell death protein (PD1) therapy and other checkpoint inhibitors, ongoing studies have sought to augment the successful outcomes of anti-PD1 therapy with combinatorial approaches, particularly adenosine signaling blockade. Preliminary data have demonstrated an optimal safety profile and enhanced overall response rates in several early phase clinical trials with A2AR and more recently CD73 inhibitors. However, beneficial outcomes for both monotherapy and combinations have been mostly lower than expected based on preclinical studies, indicating a need for more nuanced patient selection or biomarker integration that might predict and optimize patient outcomes. In the context of known immuno-oncology biomarkers such as tumor mutational burden and interferon-associated gene expression, a comparison of adenosine-related gene signatures associated with clinical response indicates an underlying biology related to immunosuppression, angiogenesis, and T cell inflammation. Importantly, though, adenosine associated gene expression may point to a unique intratumoral phenotype independent from IFN-γ related pathways. Here, we discuss the cellular and molecular mechanisms of adenosine-mediated immunosuppression, preclinical investigation of adenosine signaling blockade, recent response data from clinical trials with A2AR, CD73, CD39 and PD1/L1 inhibitors, and ongoing development of predictive gene signatures to enhance combinatorial immune-based therapies.

Host-Microbiota Interactions in the Esophagus During Homeostasis and Allergic Inflammation.

BACKGROUND & AIMS: Microbiota composition and mechanisms of host-microbiota interactions in the esophagus are unclear. We aimed to uncover fundamental information about the esophageal microbiome and its potential significance to eosinophilic esophagitis (EoE). METHODS: Microbiota composition, transplantation potential, and antibiotic responsiveness in the esophagus were established via 16S ribosomal RNA sequencing. Functional outcomes of microbiota colonization were assessed by RNA sequencing analysis of mouse esophageal epithelium and compared with the human EoE transcriptome. The impact of dysbiosis was assessed using a preclinical model of EoE. RESULTS: We found that the murine esophagus is colonized with diverse microbial communities within the first month of life. The esophageal microbiota is distinct, dominated by Lactobacillales, and demonstrates spatial heterogeneity as the proximal and distal esophagus are enriched in Bifidobacteriales and Lactobacillales, respectively. Fecal matter transplantation restores the esophageal microbiota, demonstrating that the local environment drives diversity. Microbiota colonization modifies esophageal tissue morphology and gene expression that is enriched in pathways associated with epithelial barrier function and overlapping with genes involved in EoE, including POSTN, KLK5, and HIF1A. Finally, neonatal antibiotic treatment reduces the abundance of Lactobacillales and exaggerates type 2 inflammation in the esophagus. Clinical data substantiated loss of esophageal Lactobacillales in EoE compared with controls. CONCLUSIONS: The esophagus has a unique microbiome with notable differences between its proximal and distal regions. Fecal matter transplantation restores the esophageal microbiome. Antibiotic-induced dysbiosis exacerbates disease in a murine model of EoE. Collectively, these data establish the composition, transplantation potential, antibiotic responsiveness, and host-microbiota interaction in the esophagus and have implications for gastrointestinal health and disease.

Investigating immune and non-immune cell interactions in head and neck tumors by single-cell RNA sequencing.

Head and neck squamous cell carcinoma (HNSCC) is characterized by complex relations between stromal, epithelial, and immune cells within the tumor microenvironment (TME). To enable the development of more efficacious therapies, we aim to study the heterogeneity, signatures of unique cell populations, and cell-cell interactions of non-immune and immune cell populations in 6 human papillomavirus (HPV)+ and 12 HPV- HNSCC patient tumor and matched peripheral blood specimens using single-cell RNA sequencing. Using this dataset of 134,606 cells, we show cell type-specific signatures associated with inflammation and HPV status, describe the negative prognostic value of fibroblasts with elastic differentiation specifically in the HPV+ TME, predict therapeutically targetable checkpoint receptor-ligand interactions, and show that tumor-associated macrophages are dominant contributors of PD-L1 and other immune checkpoint ligands in the TME. We present a comprehensive single-cell view of cell-intrinsic mechanisms and cell-cell communication shaping the HNSCC microenvironment.