Randomized Placebo-Controlled, Biomarker-Stratified Phase Ib Microbiome Modulation in Melanoma: Impact of Antibiotic Preconditioning on Microbiome and Immunity.

Gut-microbiota modulation shows promise in improving immune-checkpoint blockade (ICB) response; however, precision biomarker-driven, placebo-controlled trials are lacking. We performed a multicenter, randomized placebo-controlled, biomarker-stratified phase I trial in patients with ICB-naïve metastatic melanoma using SER-401, an orally delivered Firmicutes-enriched spore formulation. Fecal microbiota signatures were characterized at baseline; patients were stratified by high versus low Ruminococcaceae abundance prior to randomization to the SER-401 arm (oral vancomycin-preconditioning/SER-401 alone/nivolumab + SER-401), versus the placebo arm [placebo antibiotic/placebo microbiome modulation (PMM)/nivolumab + PMM (NCT03817125)]. Analysis of 14 accrued patients demonstrated that treatment with SER-401 + nivolumab was safe, with an objective response rate of 25% in the SER-401 arm and 67% in the placebo arm (though the study was under-powered related to poor accrual during the COVID-19 pandemic). Translational analyses demonstrated that vancomycin preconditioning was associated with the disruption of the gut microbiota and impaired immunity, with incomplete recovery at ICB administration (particularly in patients with high baseline Ruminococcaceae). These results have important implications for future microbiome modulation trials. SIGNIFICANCE: This first-of-its-kind, placebo-controlled, randomized biomarker-driven microbiome modulation trial demonstrated that vancomycin + SER-401 and anti-PD-1 are safe in melanoma patients. Although limited by poor accrual during the pandemic, important insights were gained via translational analyses, suggesting that antibiotic preconditioning and interventional drug dosing regimens should be carefully considered when designing such trials.

Histologic and Genomic Analysis of Conjunctival SCC in African and American Cohorts Reveal UV Light and HPV Signatures and High Tumor Mutation Burden.

Purpose: Conjunctival squamous cell carcinoma (conjSCC) is more prevalent and aggressive in sub-Saharan African countries compared with the rest of the world. This study aims to compare the genomic, immunophenotypic, and histologic features between patients from the United States and Ethiopia, to identify etiopathogenic mechanisms and unveil potential treatment strategies. Methods: We compared histologic features and mutational profiles using whole exome sequencing, high-risk human papillomavirus (HPV) status, PD-L1 expression, and tumor-infiltrating lymphocytes in conjSCC tumors of patients from Ethiopia (ETH; n = 25) and the United States (from MD Anderson [the MDA cohort]; n = 29). Genomic alterations were compared with SCCs from other anatomic sites using data from The Cancer Genome Atlas. Results: Solar elastosis was seen in 78% of ETH and 10% of MDA samples. Thicker tumors had higher density of CD8+ and CD3+ cells. HPV status was similar between the cohorts (ETH = 21% and MDA = 28%). The mean tumor mutation burden (TMB) was significantly higher in conjSCC (3.01/Mb, log10) and cutaneous SCC compared other SCC subtypes. ETH samples had higher TMB compared to the MDA cohort (3.34 vs. 2.73). Mutations in genes associated with ultraviolet light (UV) signature were most frequently encountered (SBS7b = 74% and SBS7a = 72%), with higher prevalence in the ETH cohort, whereas SBS2 and SBS13 signatures were more common among MDA HPV+ conjSCCs. Conclusions: Our findings suggest that UV exposure may play a major role in conjSCC, with a higher prevalence in the ETH cohort compared with the MDA cohort, where HPV also contributes.

Phenotypic switch in mycosis fungoides: A tertiary cancer center experience.

BACKGROUND: Changes in immunophenotype in mycosis fungoides (MF) are rarely reported, making this phenomenon a diagnostic challenge with unclear significance for the disease's biological behavior. This study examines a large series of MF patients who exhibited a phenotype switch (PS) and analyzes their clinical and histopathologic characteristics. DESIGN: Institutional files were searched for MF cases exhibiting PS between 2010 and 2020. Clinical, follow-up, and histopathological data were collected. RESULTS: Forty-two biopsies from 32 patients (13 women and 19 men, median age 67.5) showed PS. Eight patients (25 %) experienced multiple PS during their disease course. The median time for PS was 22 months from the initial diagnosis. In 5 cases tested, identical TCR clone peaks were detected in the immunophenotypically distinct lesions. Median follow-up was 14.5 months. Among deceased patients, median time from MF diagnosis to PS was 20.6 months, while among the patients who were still alive, median time was 44.1 months. CONCLUSION: MF biopsies can show PS during the course of the disease and may indicate a change in clinical behavior. 28.1 % of patients displayed more than one PS, further indicating high plasticity of MF cells. No obvious association was found between PS and therapy initiation or response. Features that appeared to portend a worse clinical course were earlier PS in the course of the disease and PS from CD4-/CD8-to CD8+, and CD8+ to CD4-/CD8-. Awareness of this phenomenon is crucial to avoid misdiagnosing phenotypically distinct lymphomas as second primaries and to alert clinicians about potential changes in the disease's clinical course.

Gene expression profiling and multiplex immunofluorescence analysis of bullous pemphigoid immune-related adverse event reveal upregulation of toll-like receptor 4/complement-induced innate immune response and increased density of TH 1 T-cells.

BACKGROUND: Immune checkpoint inhibitor (ICI)-based cancer therapies cause a variety of cutaneous immune-related adverse events (irAEs) including immunobullous skin eruptions like bullous pemphigoid (BP). However, little is known about the underlying immunopathogenic drivers of these reactions, and understanding the unique gene expression profile and immune composition of BP-irAE remains a critical knowledge gap in the field of oncodermatology/oncodermatopathology. METHODS: BP-irAE (n = 8) and de novo BP control (n = 8) biopsy samples were subjected to gene expression profiling using the NanoString® Technologies nCounter PanCancer Immune Profiling Panel. Multiplex immunofluorescence (mIF) studies using markers for T-cells (CD3 and CD8), T helper 1 (TH 1) cells (Tbet), TH 2 cells (Gata3), TH 17 cells (RORγT), and regulatory T-cells (Tregs; FoxP3) were further evaluated using InForm® image analysis. RESULTS: Compared with de novo BP controls, BP-irAE samples exhibited upregulation of 30 mRNA transcripts (p < 0.025), including toll-like receptor 4 (TLR4) and genes associated with complement activation, and downregulation of 89 mRNA transcripts (p < 0.025), including genes associated with TH 2, TH 17, and B-cell immune response. BP-irAE demonstrated a greater density of Tbet+ (TH 1) cells in the dermis (p = 0.004) and fewer Tregs in the blister floor (p = 0.028) when compared with that of de novo control BP samples. CONCLUSIONS: BP-irAE exhibited activation of the TLR4/complement-driven classical innate immune response pathway, with dermal TH 1 immune cell polarization and decreased Tregs in the blister floor. TLR/complement signaling may underlie the immunopathogenesis of BP-irAE.

Multiplatform Analysis of Intratumoral PTEN Heterogeneity in Melanoma.

Loss of protein expression of the tumor suppressor PTEN is associated with increased cancer aggressiveness, decreased tumor immune infiltration, and resistance to immune and targeted therapies in melanoma. We assessed a unique cohort of eight melanoma samples with focal loss of PTEN protein expression to understand the features and mechanisms of PTEN loss in this disease. We compared the PTEN-negative (PTEN[-]) areas to their adjacent PTEN-positive (PTEN[+]) areas using DNA sequencing, DNA methylation, RNA expression, digital spatial profiling, and immunohistochemical platforms. Variations or homozygous deletions of PTEN were identified in PTEN(-) areas that were not detected in the adjacent PTEN(+) areas in three cases (37.5%), but no clear genomic or DNA methylation basis for loss was identified in the remaining PTEN(-) samples. RNA expression data from two independent platforms identified a consistent increase in chromosome segregation gene expression in PTEN(-) versus adjacent PTEN(+) areas. Proteomic analysis showed a relative paucity of tumor-infiltrating lymphocytes in PTEN(-) versus adjacent PTEN(+) areas. The findings add to our understanding of potential molecular intratumoral heterogeneity in melanoma and the features associated with the loss of PTEN protein in this disease.

Intestinal toxicity to CTLA-4 blockade driven by IL-6 and myeloid infiltration.

Immune checkpoint blockade (ICB) has revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by immune-related adverse events (irAEs). Limited understanding of irAE mechanisms hampers development of approaches to mitigate their damage. To address this, we examined whether mice gained sensitivity to anti-CTLA-4 (αCTLA-4)-mediated toxicity upon disruption of gut homeostatic immunity. We found αCTLA-4 drove increased inflammation and colonic tissue damage in mice with genetic predisposition to intestinal inflammation, acute gastrointestinal infection, transplantation with a dysbiotic fecal microbiome, or dextran sodium sulfate administration. We identified an immune signature of αCTLA-4-mediated irAEs, including colonic neutrophil accumulation and systemic interleukin-6 (IL-6) release. IL-6 blockade combined with antibiotic treatment reduced intestinal damage and improved αCTLA-4 therapeutic efficacy in inflammation-prone mice. Intestinal immune signatures were validated in biopsies from patients with ICB colitis. Our work provides new preclinical models of αCTLA-4 intestinal irAEs, mechanistic insights into irAE development, and potential approaches to enhance ICB efficacy while mitigating irAEs.

Obesity Is Associated with Altered Tumor Metabolism in Metastatic Melanoma.

PURPOSE: Overweight/obese (OW/OB) patients with metastatic melanoma unexpectedly have improved outcomes with immune checkpoint inhibitors (ICI) and BRAF-targeted therapies. The mechanism(s) underlying this association remain unclear, thus we assessed the integrated molecular, metabolic, and immune profile of tumors, as well as gut microbiome features, for associations with patient body mass index (BMI). EXPERIMENTAL DESIGN: Associations between BMI [normal (NL < 25) or OW/OB (BMI ≥ 25)] and tumor or microbiome characteristics were examined in specimens from 782 patients with metastatic melanoma across 7 cohorts. DNA associations were evaluated in The Cancer Genome Atlas cohort. RNA sequencing from 4 cohorts (n = 357) was batch corrected and gene set enrichment analysis (GSEA) by BMI category was performed. Metabolic profiling was conducted in a subset of patients (x = 36) by LC/MS, and in flow-sorted melanoma tumor cells (x = 37) and patient-derived melanoma cell lines (x = 17) using the Seahorse XF assay. Gut microbiome features were examined in an independent cohort (n = 371). RESULTS: DNA mutations and copy number variations were not associated with BMI. GSEA demonstrated that tumors from OW/OB patients were metabolically quiescent, with downregulation of oxidative phosphorylation and multiple other metabolic pathways. Direct metabolite analysis and functional metabolic profiling confirmed decreased central carbon metabolism in OW/OB metastatic melanoma tumors and patient-derived cell lines. The overall structure, diversity, and taxonomy of the fecal microbiome did not differ by BMI. CONCLUSIONS: These findings suggest that the host metabolic phenotype influences melanoma metabolism and provide insight into the improved outcomes observed in OW/OB patients with metastatic melanoma treated with ICIs and targeted therapies. See related commentary by Smalley, p. 5.

Neoadjuvant relatlimab and nivolumab in resectable melanoma.

Relatlimab and nivolumab combination immunotherapy improves progression-free survival over nivolumab monotherapy in patients with unresectable advanced melanoma1. We investigated this regimen in patients with resectable clinical stage III or oligometastatic stage IV melanoma (NCT02519322). Patients received two neoadjuvant doses (nivolumab 480 mg and relatlimab 160 mg intravenously every 4 weeks) followed by surgery, and then ten doses of adjuvant combination therapy. The primary end point was pathologic complete response (pCR) rate2. The combination resulted in 57% pCR rate and 70% overall pathologic response rate among 30 patients treated. The radiographic response rate using Response Evaluation Criteria in Solid Tumors 1.1 was 57%. No grade 3-4 immune-related adverse events were observed in the neoadjuvant setting. The 1- and 2-year recurrence-free survival rate was 100% and 92% for patients with any pathologic response, compared to 88% and 55% for patients who did not have a pathologic response (P = 0.005). Increased immune cell infiltration at baseline, and decrease in M2 macrophages during treatment, were associated with pathologic response. Our results indicate that neoadjuvant relatlimab and nivolumab induces a high pCR rate. Safety during neoadjuvant therapy is favourable compared to other combination immunotherapy regimens. These data, in combination with the results of the RELATIVITY-047 trial1, provide further confirmation of the efficacy and safety of this new immunotherapy regimen.

Society for Immunotherapy of Cancer (SITC) clinical practice guideline on immunotherapy for the treatment of nonmelanoma skin cancer.

Nonmelanoma skin cancers (NMSCs) are some of the most commonly diagnosed malignancies. In general, early-stage NMSCs have favorable outcomes; however, a small subset of patients develop resistant, advanced, or metastatic disease, or aggressive subtypes that are more challenging to treat successfully. Recently, immune checkpoint inhibitors (ICIs) have been approved by the US Food and Drug Administration (FDA) for the treatment of Merkel cell carcinoma (MCC), cutaneous squamous cell carcinoma (CSCC), and basal cell carcinoma (BCC). Although ICIs have demonstrated activity against NMSCs, the routine clinical use of these agents may be more challenging due to a number of factors including the lack of predictive biomarkers, the need to consider special patient populations, the management of toxicity, and the assessment of atypical responses. With the goal of improving patient care by providing expert guidance to the oncology community, the Society for Immunotherapy of Cancer (SITC) convened a multidisciplinary panel of experts to develop a clinical practice guideline (CPG). The expert panel drew on the published literature as well as their own clinical experience to develop recommendations for healthcare professionals on important aspects of immunotherapeutic treatment for NMSCs, including staging, biomarker testing, patient selection, therapy selection, post-treatment response evaluation and surveillance, and patient quality of life (QOL) considerations, among others. The evidence- and consensus-based recommendations in this CPG are intended to provide guidance to cancer care professionals treating patients with NMSCs.

Androgen receptor blockade promotes response to BRAF/MEK-targeted therapy.

Treatment with therapy targeting BRAF and MEK (BRAF/MEK) has revolutionized care in melanoma and other cancers; however, therapeutic resistance is common and innovative treatment strategies are needed1,2. Here we studied a group of patients with melanoma who were treated with neoadjuvant BRAF/MEK-targeted therapy ( NCT02231775 , n = 51) and observed significantly higher rates of major pathological response (MPR; ≤10% viable tumour at resection) and improved recurrence-free survival (RFS) in female versus male patients (MPR, 66% versus 14%, P = 0.001; RFS, 64% versus 32% at 2 years, P = 0.021). The findings were validated in several additional cohorts2-4 of patients with unresectable metastatic melanoma who were treated with BRAF- and/or MEK-targeted therapy (n = 664 patients in total), demonstrating improved progression-free survival and overall survival in female versus male patients in several of these studies. Studies in preclinical models demonstrated significantly impaired anti-tumour activity in male versus female mice after BRAF/MEK-targeted therapy (P = 0.006), with significantly higher expression of the androgen receptor in tumours of male and female BRAF/MEK-treated mice versus the control (P = 0.0006 and P = 0.0025). Pharmacological inhibition of androgen receptor signalling improved responses to BRAF/MEK-targeted therapy in male and female mice (P = 0.018 and P = 0.003), whereas induction of androgen receptor signalling (through testosterone administration) was associated with a significantly impaired response to BRAF/MEK-targeted therapy in male and female patients (P = 0.021 and P < 0.0001). Together, these results have important implications for therapy.

Short-term treatment with multi-drug regimens combining BRAF/MEK-targeted therapy and immunotherapy results in durable responses in Braf-mutated melanoma.

Targeted and immunotherapy regimens have revolutionized the treatment of advanced melanoma patients. Despite this, only a subset of patients respond durably. Recently, combination strategies of BRAF/MEK inhibitors with immune checkpoint inhibitor monotherapy (α-CTLA-4 or α-PD-1) have increased the rate of durable responses. Based on evidence from our group and others, these therapies appear synergistic, but at the cost of significant toxicity. We know from other treatment paradigms (e.g. hematologic malignancies) that combination strategies with multi-drug regimens (>4 drugs) are associated with more durable disease control. To better understand the mechanism of these improved outcomes, and to identify and prioritize new strategies for testing, we studied several multi-drug regimens combining BRAF/MEK targeted therapy and immunotherapy combinations in a Braf-mutant murine melanoma model (BrafV600E/Pten-/- ). Short-term treatment with α-PD-1 and α-CTLA-4 monotherapies were relatively ineffective, while treatment with α-OX40 demonstrated some efficacy [17% of mice with no evidence of disease, (NED), at 60-days]. Outcomes were improved in the combined α-OX40/α-PD-1 group (42% NED). Short-term treatment with quadruplet therapy of immunotherapy doublets in combination with targeted therapy [dabrafenib and trametinib (DT)] was associated with excellent tumor control, with 100% of mice having NED after combined DT/α-CTLA-4/α-PD-1 or DT/α-OX40/α-PD-1. Notably, tumors from mice in these groups demonstrated a high proportion of effector memory T cells, and immunologic memory was maintained with tumor re-challenge. Together, these data provide important evidence regarding the potential utility of multi-drug therapy in treating advanced melanoma and suggest these models can be used to guide and prioritize combinatorial treatment strategies.

Impact of Next-generation Sequencing on Interobserver Agreement and Diagnosis of Spitzoid Neoplasms.

Atypical Spitzoid melanocytic tumors are diagnostically challenging. Many studies have suggested various genomic markers to improve classification and prognostication. We aimed to assess whether next-generation sequencing studies using the Tempus xO assay assessing mutations in 1711 cancer-related genes and performing whole transcriptome mRNA sequencing for structural alterations could improve diagnostic agreement and accuracy in assessing neoplasms with Spitzoid histologic features. Twenty expert pathologists were asked to review 70 consultation level cases with Spitzoid features, once with limited clinical information and again with additional genomic information. There was an improvement in overall agreement with additional genomic information. Most significantly, there was increase in agreement of the diagnosis of conventional melanoma from moderate (κ=0.470, SE=0.0105) to substantial (κ=0.645, SE=0.0143) as measured by an average Cohen κ. Clinical follow-up was available in all 70 cases which substantiated that the improved agreement was clinically significant. Among 3 patients with distant metastatic disease, there was a highly significant increase in diagnostic recognition of the cases as conventional melanoma with genomics (P<0.005). In one case, none of 20 pathologists recognized a tumor with BRAF and TERT promoter mutations associated with fatal outcome as a conventional melanoma when only limited clinical information was provided, whereas 60% of pathologists correctly diagnosed this case when genomic information was also available. There was also a significant improvement in agreement of which lesions should be classified in the Spitz category/WHO Pathway from an average Cohen κ of 0.360 (SE=0.00921) to 0.607 (SE=0.0232) with genomics.

Gut microbiota signatures are associated with toxicity to combined CTLA-4 and PD-1 blockade.

Treatment with combined immune checkpoint blockade (CICB) targeting CTLA-4 and PD-1 is associated with clinical benefit across tumor types, but also a high rate of immune-related adverse events. Insights into biomarkers and mechanisms of response and toxicity to CICB are needed. To address this, we profiled the blood, tumor and gut microbiome of 77 patients with advanced melanoma treated with CICB, with a high rate of any ≥grade 3 immune-related adverse events (49%) with parallel studies in pre-clinical models. Tumor-associated immune and genomic biomarkers of response to CICB were similar to those identified for ICB monotherapy, and toxicity from CICB was associated with a more diverse peripheral T-cell repertoire. Profiling of gut microbiota demonstrated a significantly higher abundance of Bacteroides intestinalis in patients with toxicity, with upregulation of mucosal IL-1ß in patient samples of colitis and in pre-clinical models. Together, these data offer potential new therapeutic angles for targeting toxicity to CICB.

Multiplex Tissue Imaging Harmonization: A Multicenter Experience from CIMAC-CIDC Immuno-Oncology Biomarkers Network.

PURPOSE: The Cancer Immune Monitoring and Analysis Centers - Cancer Immunologic Data Commons (CIMAC-CIDC) network supported by the NCI Cancer Moonshot initiative was established to provide correlative analyses for clinical trials in cancer immunotherapy, using state-of-the-art technology. Fundamental to this initiative is implementation of multiplex IHC assays to define the composition and distribution of immune infiltrates within tumors in the context of their potential role as biomarkers. A critical unanswered question involves the relative fidelity of such assays to reliably quantify tumor-associated immune cells across different platforms. EXPERIMENTAL DESIGN: Three CIMAC sites compared across their laboratories: (i) image analysis algorithms, (ii) image acquisition platforms, (iii) multiplex staining protocols. Two distinct high-dimensional approaches were employed: multiplexed IHC consecutive staining on single slide (MICSSS) and multiplexed immunofluorescence (mIF). To eliminate variables potentially impacting assay performance, we completed a multistep harmonization process, first comparing assay performance using independent protocols followed by the integration of laboratory-specific protocols and finally, validating this harmonized approach in an independent set of tissues. RESULTS: Data generated at the final validation step showed an intersite Spearman correlation coefficient (r) of ≥0.85 for each marker within and across tissue types, with an overall low average coefficient of variation ≤0.1. CONCLUSIONS: Our results support interchangeability of protocols and platforms to deliver robust, and comparable data using similar tissue specimens and confirm that CIMAC-CIDC analyses may therefore be used with confidence for statistical associations with clinical outcomes largely independent of site, antibody selection, protocol, and platform across different sites.

Resolution of tissue signatures of therapy response in patients with recurrent GBM treated with neoadjuvant anti-PD1.

The response of patients with recurrent glioblastoma multiforme to neoadjuvant immune checkpoint blockade has been challenging to interpret due to the inter-patient and intra-tumor heterogeneity. We report on a comparative analysis of tumor tissues collected from patients with recurrent glioblastoma and high-risk melanoma, both treated with neoadjuvant checkpoint blockade. We develop a framework that uses multiplex spatial protein profiling, machine learning-based image analysis, and data-driven computational models to investigate the pathophysiological and molecular factors within the tumor microenvironment that influence treatment response. Using melanoma to guide the interpretation of glioblastoma analyses, we interrogate the protein expression in microscopic compartments of tumors, and determine the correlates of cytotoxic CD8+ T cells, tumor growth, treatment response, and immune cell-cell interaction. This work reveals similarities shared between glioblastoma and melanoma, immunosuppressive factors that are unique to the glioblastoma microenvironment, and potential co-targets for enhancing the efficacy of neoadjuvant immune checkpoint blockade.