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Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer with a ∼50% response rate to immune checkpoint blockade (ICB) therapy. To identify predictive biomarkers, we integrated bulk and single-cell RNA sequencing (RNA-seq) with spatial transcriptomics from a cohort of 186 samples from 116 patients, including bulk RNA-seq from 14 matched pairs pre- and post-ICB. In nonresponders, tumors show evidence of increased tumor proliferation, neuronal stem cell markers, and IL1. Responders have increased type I/II interferons and preexisting tissue resident (Trm) CD8 or Vδ1 γδ T cells that functionally converge with overlapping antigen-specific transcriptional programs and clonal expansion of public T-cell receptors. Spatial transcriptomics demonstrated colocalization of T cells with B and dendritic cells, which supply chemokines and costimulation. Lastly, ICB significantly increased clonal expansion or recruitment of Trm and Vδ1 cells in tumors specifically in responders, underscoring their therapeutic importance. These data identify potential clinically actionable biomarkers and therapeutic targets for MCC. Significance: MCC serves as a model of ICB response. We utilized the largest-to-date, multimodal MCC dataset (n = 116 patients) to uncover unique tumor-intrinsic properties and immune circuits that predict response. We identified CD8 Trm and Vδ1 T cells as clinically actionable mediators of ICB response in major histocompatibility complex-high and -low MCCs, respectively., (©2024 American Association for Cancer Research.)

Background: Cigarette smoke exposure has been linked to systemic immune dysfunction, including for B-cell and immunoglobulin (Ig) production, and poor outcomes in patients with ovarian cancer. No study has evaluated the impact of smoke exposure across the life-course on B-cell infiltration and Ig abundance in ovarian tumors., Methods: We measured markers of B and plasma cells and Ig isotypes using multiplex immunofluorescence on 395 ovarian cancer tumors in the Nurses' Health Study (NHS)/NHSII. We conducted beta-binomial analyses evaluating odds ratios (OR) and 95% confidence intervals (CI) for positivity of immune markers by cigarette exposure among cases and Cox proportional hazards models to evaluate hazard ratios (HR) and 95% CI for developing tumors with low (

Background: Depression is associated with a higher ovarian cancer risk. Prior work suggests that depression can lead to systemic immune suppression, which could potentially alter the anti-tumor immune response., Methods: We evaluated the association of pre-diagnosis depression with features of the anti-tumor immune response, including T and B cells and immunoglobulins, among women with ovarian tumor tissue collected in three studies, the Nurses' Health Study (NHS; n = 237), NHSII (n = 137) and New England Case-Control Study (NECC; n = 215). Women reporting depressive symptoms above a clinically relevant cut-point, antidepressant use, or physician diagnosis of depression at any time prior to diagnosis of ovarian cancer were considered to have pre-diagnosis depression. Multiplex immunofluorescence was performed on tumor tissue microarrays to measure immune cell infiltration. In pooled analyses, we estimated odds ratios (OR) and 95% confidence intervals (CI) for the positivity of tumor immune cells using a beta-binomial model comparing those with and without depression. We used Bonferroni corrections to adjust for multiple comparisons., Results: We observed no statistically significant association between depression status and any immune markers at the Bonferroni corrected p-value of 0.0045; however, several immune markers were significant at a nominal p-value of 0.05. Specifically, there were increased odds of having recently activated cytotoxic (CD3 + CD8 + CD69 + ) and exhausted-like T cells (CD3 + Lag3 + ) in tumors of women with vs. without depression (OR = 1.36, 95 %CI = 1.09-1.69 and OR = 1.24, 95 %CI = 1.01-1.53, respectively). Associations were comparable when considering high grade serous tumors only (comparable ORs = 1.33, 95 %CI = 1.05-1.69 and OR = 1.25, 95 %CI = 0.99-1.58, respectively). There were decreased odds of having tumor infiltrating plasma cells (CD138 + ) in women with vs. without depression (OR = 0.54, 95 %CI = 0.33-0.90), which was similar among high grade serous carcinomas, although not statistically significant. Depression was also related to decreased odds of having naïve and memory B cells (CD20 + : OR = 0.54, 95 %CI = 0.30-0.98) and increased odds of IgG (OR = 1.22, 95 %CI = 0.97-1.53) in high grade serous carcinomas., Conclusion: Our results provide suggestive evidence that depression may influence ovarian cancer outcomes through changes in the tumor immune microenvironment, including increasing T cell activation and exhaustion and reducing antibody-producing B cells. Further studies with clinical measures of depression and larger samples are needed to confirm these results., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer. Although cSCC contributes to substantial morbidity and mortality in high-risk individuals, deployment of otherwise effective chemoprevention of cSCC is limited by toxicities. Our systematic computational drug repurposing screen predicted that selumetinib, a MAPK (mitogen-activated protein kinase) kinase inhibitor (MEKi), would reverse transcriptional signatures associated with cSCC development, consistent with our genomic analysis implicating MEK as a chemoprevention target. Although systemic MEKi suppresses the formation of cSCC in mice, systemic MEKi can cause severe adverse effects. Here, we report the development of a metabolically labile MEKi, NFX-179, designed to potently and selectively suppress the MAPK pathway in the skin before rapid metabolism in the systemic circulation. NFX-179 was identified on the basis of its biochemical and cellular potency, selectivity, and rapid metabolism upon systemic absorption. In our ultraviolet-induced cSCC mouse model, topical application of NFX-179 gel reduced the formation of new cSCCs by an average of 60% at doses of 0.1% and greater at 28 days. We further confirmed the localized nature of these effects in an additional split-mouse randomized controlled study where suppression of cSCC was observed only in drug-treated areas. No toxicities were observed. NFX-179 inhibits the growth of human SCC cell lines in a dose-dependent manner, and topical NFX-179 application penetrates human skin and inhibits MAPK signaling in human cSCC explants. Together, our data provide a compelling rationale for using topical MEK inhibition through the application of NFX-179 gel as an effective strategy for cSCC chemoprevention.

Background: Despite the immunogenic nature of many ovarian tumors, treatment with immune checkpoint therapies has not led to substantial improvements in ovarian cancer survival. To advance population-level research on the ovarian tumor immune microenvironment, it is critical to understand methodologic issues related to measurement of immune cells on tissue microarrays (TMA) using multiplex immunofluorescence (mIF) assays., Methods: In two prospective cohorts, we collected formalin-fixed, paraffin-embedded ovarian tumors from 486 cases and created seven TMAs. We measured T cells, including several sub-populations, and immune checkpoint markers on the TMAs using two mIF panels. We used Spearman correlations, Fisher exact tests, and multivariable-adjusted beta-binomial models to evaluate factors related to immune cell measurements in TMA tumor cores., Results: Between-core correlations of intratumoral immune markers ranged from 0.52 to 0.72, with more common markers (e.g., CD3+, CD3+CD8+) having higher correlations. Correlations of immune cell markers between the whole core, tumor area, and stromal area were high (range 0.69-0.97). In multivariable-adjusted models, odds of T-cell positivity were lower in clear cell and mucinous versus type II tumors (ORs, 0.13-0.48) and, for several sub-populations, were lower in older tissue (sample age > 30 versus ≤ 10 years; OR, 0.11-0.32)., Conclusions: Overall, high correlations between cores for immune markers measured via mIF support the use of TMAs in studying ovarian tumor immune infiltration, although very old samples may have reduced antigenicity., Impact: Future epidemiologic studies should evaluate differences in the tumor immune response by histotype and identify modifiable factors that may alter the tumor immune microenvironment., (©2023 American Association for Cancer Research.)

Introduction: In clear cell renal cell carcinoma (ccRCC), tumor-associated macrophage (TAM) induction of CD8+T cells into a terminally exhausted state has been implicated as a major mechanism of immunotherapy resistance, but a deeper biological understanding is necessary., Methods: Primary ccRCC tumor samples were obtained from 97 patients between 2004 and 2018. Multiplex immunofluorescence using lymphoid and myeloid markers was performed in seven regions of interest per patient across three predefined zones, and geospatial analysis was performed using Ripley's K analysis, a methodology adapted from ecology., Results: Clustering of CD163+M2 like TAMs into the stromal compartment at the tumor-stroma interface was associated with worse clinical stage (tumor/CD163+nK(75): stage I/II: 4.4 (IQR -0.5 to 5.1); stage III: 1.4 (IQR -0.3 to 3.5); stage IV: 0.6 (IQR -2.1 to 2.1); p=0.04 between stage I/II and stage IV), and worse overall survival (OS) and cancer-specific survival (CSS) (tumor/CD163+nK(75): median OS-hi=149 months, lo=86 months, false-discovery rate (FDR)-adj. Cox p<0.001; median CSS-hi=174 months, lo=85 months; FDR-adj. Cox p<0.001). An RNA-seq differential gene expression score was developed using this geospatial metric, and was externally validated in multiple independent cohorts of patients with ccRCC including: TCGA KIRC, and the IMmotion151, IMmotion150, and JAVELIN Renal 101 clinical trials. In addition, this CD163+ geospatial pattern was found to be associated with a higher TIM-3+ proportion of CD8+T cells, indicative of terminal exhaustion (tumor-core: 0.07 (IQR 0.04-0.14) vs 0.40 (IQR 0.15-0.66), p=0.05)., Conclusions: Geospatial clustering of CD163+M2 like TAMs into the stromal compartment at the tumor-stromal interface was associated with poor clinical outcomes and CD8+T cell terminal exhaustion., Competing Interests: Competing interests: The corresponding author certifies that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (ie. employment/affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: NC, YK, AB, AC, GJK, AH, JN, CM-S, DS-V, ENK, NL-B, PR, MF, DJ, LW, JD, JKT, JD, PMA, and AAH have no disclosures; BJM is an NCCN Kidney Cancer Panel Member and an advisor for Merck; PES is an NCCN Bladder and Penile Cancer Panel Member and Vice-Chair; JJM is Associate Center Director at Moffitt Cancer Center, has ownership interest in Aleta Biotherapeutics, CG Oncology, Turnstone Biologics, Ankyra Therapeutics, and AffyImmune Therapeutics, and is a paid consultant/paid advisory board member for ONCoPEP, CG Oncology, Mersana Therapeutics, Turnstone Biologics, Vault Pharma, Ankyra Therapeutics, AffyImmune Therapeutics, UbiVac, Vycellix, and Aleta Biotherapeutics., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Exposure to cigarette smoke, particularly in early life, is modestly associated with ovarian cancer risk and may impact systemic immunity and the tumor immune response. However, no studies have evaluated whether cigarette smoke exposure impacts the ovarian tumor immune microenvironment., Methods: Participants in the Nurses' Health Study (NHS) and NHSII reported on early life exposure to cigarette smoke and personal smoking history on questionnaires (n = 165,760). Multiplex immunofluorescence assays were used to measure markers of T cells and immune checkpoints in tumor tissue from 385 incident ovarian cancer cases. We used Cox proportional hazards models to evaluate HRs and 95% confidence intervals (CI) for developing ovarian tumors with a low (

Background: Tumor-infiltrating lymphocytes (TIL) confer a survival benefit among patients with ovarian cancer; however, little work has been conducted in racially diverse cohorts., Methods: The current study investigated racial differences in the tumor immune landscape and survival of age- and stage-matched non-Hispanic Black and non-Hispanic White women with high-grade serous ovarian carcinoma (HGSOC) enrolled in two population-based studies (n = 121 in each racial group). We measured TILs (CD3+), cytotoxic T cells (CD3+CD8+), regulatory T cells (CD3+FoxP3+), myeloid cells (CD11b+), and neutrophils (CD11b+CD15+) via multiplex immunofluorescence. Multivariable Cox proportional hazard regression was used to estimate the association between immune cell abundance and survival overall and by race., Results: Overall, higher levels of TILs, cytotoxic T cells, myeloid cells, and neutrophils were associated with better survival in the intratumoral and peritumoral region, irrespective of tissue compartment (tumor, stroma). Improved survival was noted for T-regulatory cells in the peritumoral region and in the stroma of the intratumoral region, but no association for intratumoral T-regulatory cells. Despite similar abundance of immune cells across racial groups, associations with survival among non-Hispanic White women were consistent with the overall findings, but among non-Hispanic Black women, most associations were attenuated and not statistically significant., Conclusions: Our results add to the existing evidence that a robust immune infiltrate confers a survival advantage among women with HGSOC; however, non-Hispanic Black women may not experience the same survival benefit as non-Hispanic White women with HGSOC., Impact: This study contributes to our understanding of the immunoepidemiology of HGSOC in diverse populations., (©2022 American Association for Cancer Research.)

Background: Clear cell renal cell carcinoma (ccRCC) tumors have low frequencies of genetic alterations compared with other malignancies, but very high levels of immune cell infiltration and favorable response rates to immunotherapy. Currently, the interplay between specific ccRCC somatic mutations and immune infiltration pattern is unclear., Objective: To analyze the associations between common ccRCC somatic mutations and immune cell infiltration patterns within the tumor immune microenvironment (TIME)., Design, Setting, and Participants: The study included tumor samples (24 primary and 24 metastatic) from 48 patients with stage IV ccRCC. Targeted sequencing was performed for well-characterized recurrent somatic mutations in ccRCC, with the analysis focusing on the six most common ones: VHL, BAP1, PBRM1, SETD2, TP53, and KDM5C. For each sample, multiplex immunofluorescence (IF) was performed in lymphoid and myeloid panels, for seven regions of interest in three zones (tumor core, stroma, and tumor-stroma interface). IF-derived cellular densities were compared across patients, stratified by their somatic mutation status, using a linear mixed-model analysis. External validation was pursued using RNA-seq enrichment scoring from three large external data sources., Results and Limitations: Tumors with SETD2 mutations demonstrated significantly decreased levels of FOXP3+ T cells in the tumor core, stroma, and tumor-stroma interface. PBRM1 mutations were associated with decreased FOXP3+ T cells in the tumor core. Primary KDM5C mutations were associated with significantly increased CD206+ macrophage tumor infiltration in the tumor core. A computational method estimating immune cell types in the TIME using bulk RNA-seq data, xCell scoring, failed to validate associations from the IF analysis in large external data sets. A major limitation of the study is the relatively small patient population studied., Conclusions: This study provides evidence that common somatic mutations in ccRCC, such as SETD2, PBRM1, and KDM5C, are associated with distinct immune infiltration patterns within the TIME., Patient Summary: In this study, we analyzed tumor samples from patients with metastatic kidney cancer to determine whether common genetic mutations that arise from the cancer cells are associated with the density of immune cells found within those tumors. We found several distinct immune cell patterns that were associated with specific genetic mutations. These findings provide insight into the interaction between cancer genetics and the immune system in kidney cancer., (Copyright © 2021 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Immune modulation is considered a hallmark of cancer initiation and progression. The recent development of immunotherapies has ushered in a new era of cancer treatment. These therapeutics have led to revolutionary breakthroughs; however, the efficacy of immunotherapy has been modest and is often restricted to a subset of patients. Hence, identification of which cancer patients will benefit from immunotherapy is essential. Multiplex immunofluorescence (mIF) microscopy allows for the assessment and visualization of the tumor immune microenvironment (TIME). The data output following image and machine learning analyses for cell segmenting and phenotyping consists of the following information for each tumor sample: the number of positive cells for each marker and phenotype(s) of interest, number of total cells, percent of positive cells for each marker, and spatial locations for all measured cells. There are many challenges in the analysis of mIF data, including many tissue samples with zero positive cells or "zero-inflated" data, repeated measurements from multiple TMA cores or tissue slides per subject, and spatial analyses to determine the level of clustering and co-localization between the cell types in the TIME. In this review paper, we will discuss the challenges in the statistical analysis of mIF data and opportunities for further research.

Immune infiltration is typically quantified using cellular density, not accounting for cellular clustering. Tumor-associated macrophages (TAM) activate oncogenic signaling through paracrine interactions with tumor cells, which may be better reflected by local cellular clustering than global density metrics. Using multiplex immunohistochemistry and digital pathologic analysis we quantified cellular density and cellular clustering for myeloid cell markers in 129 regions of interest from 55 samples from 35 patients with metastatic ccRCC. CD68+ cells were found to be clustered with tumor cells and dispersed from stromal cells, while CD163+ and CD206+ cells were found to be clustered with stromal cells and dispersed from tumor cells. CD68+ density was not associated with OS, while high tumor/CD68+ cell clustering was associated with significantly worse OS. These novel findings would not have been identified if immune infiltrate was assessed using cellular density alone, highlighting the importance of including spatial analysis in studies of immune cell infiltration of tumors. Significance: Increased clustering of CD68+ TAMs and tumor cells was associated with worse overall survival for patients with metastatic ccRCC. This effect would not have been identified if immune infiltrate was assessed using cell density alone, highlighting the importance of including spatial analysis in studies of immune cell infiltration of tumors., Competing Interests: The corresponding author certifies that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (ie. employment/affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: NHC, GJK, AH, AMA, LZ, JN, JC, SF, MF, JD, SM, CM, EK, PMA, and YK have no disclosures; BJM is an NCCN Kidney Cancer Panel Member; PES is an NCCN Bladder and Penile Cancer Panel Member and Vice-Chair; JM is an Associate Center Director at Moffitt Cancer Center, has ownership interest in Fulgent Genetics, Inc., Aleta Biotherapeutics, Inc., Cold Genesys, Inc., Myst Pharma, Inc., and Tailored Therapeutics, Inc., and is a consultant/advisory board member for ONCoPEP, Inc., Cold Genesys, Inc., Morphogenesis, Inc., Mersana Therapeutics, Inc., GammaDelta Therapeutics, Ltd., Myst Pharma, Inc., Tailored Therapeutics, Inc., Verseau Therapeutics, Inc., Iovance Biotherapeutics, Inc., Vault Pharma, Inc., Noble Life Sciences Partners, Fulgent Genetics, Inc., UbiVac, LLC, Vycellix, Inc., and Aleta Biotherapeutics, Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials.