Apoptosis Pathway-Associated Proteins Are Frequently Expressed in Melanoma: A Study of 32 Cases With Focus on Acral Lentiginous Melanoma.

ABSTRACT: Acral lentiginous melanoma (ALM) is an aggressive type of cutaneous melanoma (CM) that arises on palms, soles, and nail units. ALM is rare in White population, but it is relatively more frequent in dark-skinned populations. There is an unmet need to develop new personalized and more effective treatments strategies for ALM. Increased expression of antiapoptotic proteins (ie, BCL2, MCL1) has been shown to contribute to tumorigenesis and therapeutic resistance in multiple tumor types and has been observed in a subset of ALM and mucosal melanoma cell lines in vivo and in vitro. However, little is known about their expression and clinical significance in patients with ALM. Thus, we assessed protein expression of BCL2, MCL1, BIM, and BRAF V600E by immunohistochemistry in 32 melanoma samples from White and Hispanic populations, including ALM and non-ALM (NALM). BCL2, MCL1, and BIM were expressed in both ALM and NALM tumors, and no significant differences in expression of any of these proteins were detected between the groups, in our relatively small cohort. There were no significant associations between protein expression and BRAF V600E status, overall survival, or ethnicity. In summary, ALM and NALM demonstrate frequent expressions of apoptosis-related proteins BCL2, MCL1, and BIM. Our findings suggest that patients with melanoma, including ALM, may be potential candidates for apoptosis-directed therapies.

Differential Upregulation of Th1/Th17-Associated Proteins and PD-L1 in Granulomatous Mycosis Fungoides.

Granulomatous Mycosis Fungoides (GMF) is a rare form of mycosis fungoides (MF) characterized by a granulomatous infiltrate associated with the neoplastic lymphoid population and is considered to have a worse prognosis compared with regular MF. The upregulation of the T helper (Th) axis, especially Th17, plays an important role in the pathogenesis of several inflammatory/infectious granulomatous cutaneous diseases, but its role in GMF is still not elucidated to date. In this study, we evaluated the immunohistochemical expression of Th1 (Tbet), Th2 (GATA-3), Th17 (RORγT), T regulatory (Foxp3), and immune checkpoint (IC) (PD-1 and PD-L1) markers in a cohort of patients with GMF and MF with large cell transformation (MFLCT). Skin biopsies from 49 patients (28 GMF and 21 MFLCT) were studied. Patients with GMF were associated with early clinical stage (p = 0.036) and lower levels of lactate dehydrogenase (p = 0.042). An increased percentage of cells positive for Tbet (p = 0.017), RORγT (p = 0.001), and PD-L1 (p = 0.011) was also observed among the GMF specimens, while a stronger PD-1 intensity was detected in cases of MFLCT. In this cohort, LCT, RORγT < 10%, Foxp3 < 10%, age, and advanced stage were associated with worse overall survival (OS) in univariate analysis. GMF demonstrated Th1 (cellular response) and Th17 (autoimmunity) phenotype, seen in early MF and granulomatous processes, respectively, which may be related to the histopathological appearance and biological behavior of GMF. Further studies involving larger series of cases and more sensitive techniques are warranted.

Nectin-4 expression in a subset of cutaneous adnexal carcinomas: A potential target for therapy with enfortumab vedotin.

BACKGROUND: Enfortumab vedotin (EV) is an antibody-drug conjugate directed against Nectin-4 that is used to treat urothelial carcinoma. Nectin-4 is inherently expressed in the skin and adnexal structures. Since therapeutic options for cutaneous adnexal carcinomas are limited, we sought to evaluate Nectin-4 expression in adnexal carcinomas and benign adnexal neoplasms to identify tumors that are potentially targetable with EV. METHODS: Eight sebaceous carcinomas (seven periocular and one lymph node metastasis), eight digital papillary adenocarcinomas, seven squamoid eccrine ductal carcinomas, eight poromas, eight trichilemmomas, and seven sebaceous adenomas were subjected to immunohistochemical staining for anti-Nectin-4 antibody. H-scores for Nectin-4 expression were calculated. RESULTS: Benign adnexal neoplasms had a significantly lower mean (±SD) Nectin-4 H-score (142.6 ± 39.1) than did the adnexal carcinomas (198 ± 90.8; p = 0.006). Nectin-4 was expressed in 91% (21/23) of adnexal carcinomas. Sebaceous carcinomas frequently exhibited high expression of Nectin-4 (88% [7/8]), with a mean (±SD) H-score (258.1 ± 58.4) significantly higher than those for digital papillary adenocarcinomas (197.5 ± 52.5; p = 0.035) and squamoid eccrine ductal carcinomas (131.4 ± 114.1; p = 0.031). Sebaceous carcinomas also had significantly higher H-scores than did sebaceous adenomas (186.4 ± 25.0; p = 0.013). CONCLUSIONS: Increased Nectin-4 expression in a subset of cutaneous adnexal carcinomas, particularly sebaceous carcinomas, reveals that EV is a potential therapeutic option for these tumors.

PRAME and LEF1 in Combined Deep Penetrating Nevus and Combined Blue Nevus: Utility and Pitfalls.

ABSTRACT: Deep penetrating nevi (DPN), particularly those showing combined features, or combined deep penetrating nevi (CDPN), may show histopathological resemblance to blue nevus (BN) and melanoma. Preferentially Expressed Antigen in MElanoma (PRAME) is a marker that helps distinguish melanoma from benign melanocytic lesions. Lymphoid enhancer-binding factor 1 (LEF1) has been proposed to be used in conjunction with ß-catenin for diagnosis of DPN. The immunohistochemical expression of PRAME and LEF1 was evaluated in 10 DPN (including 6 CDPN and 2 DPN-like proliferations with atypical features), 16 BN (including combined and cellular BN), and 2 melanomas with features of DPN or BN. PRAME was negative in most DPN (n = 10/10, n = 9/10, one case with discrepancy between readers) and all BN (n = 16/16), while the 2 melanomas included were positive (n = 2/2). All DPN were positive for LEF1 (n = 9/9) while only a subset of BN were positive (n = 6/16, P = 0.0028; n = 5/16, P = 0.001, per both readers). LEF1 seemed to be easier to interpret than ß-catenin because of its nuclear pattern of expression. The expression of LEF1 in the regular nevus component of combined BN presents a potential pitfall in practice because it may lead to misinterpretation of LEF1 as positive in the BN component of the lesion. However, a subset (approximately one-third) of combined BN seemed to show true LEF1 expression. Taking into account pitfalls in interpretation, the combinatorial panel of PRAME and LEF1, in addition to conventional histopathological features, may be useful to distinguish CDPN from combined BN and other benign and malignant mimics.

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.

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.

Dietary fiber and probiotics influence the gut microbiome and melanoma immunotherapy response.

Gut bacteria modulate the response to immune checkpoint blockade (ICB) treatment in cancer, but the effect of diet and supplements on this interaction is not well studied. We assessed fecal microbiota profiles, dietary habits, and commercially available probiotic supplement use in melanoma patients and performed parallel preclinical studies. Higher dietary fiber was associated with significantly improved progression-free survival in 128 patients on ICB, with the most pronounced benefit observed in patients with sufficient dietary fiber intake and no probiotic use. Findings were recapitulated in preclinical models, which demonstrated impaired treatment response to anti­programmed cell death 1 (anti­PD-1)­based therapy in mice receiving a low-fiber diet or probiotics, with a lower frequency of interferon-γ­positive cytotoxic T cells in the tumor microenvironment. Together, these data have clinical implications for patients receiving ICB for cancer.

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.

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.

PARP and CDK4/6 Inhibitor Combination Therapy Induces Apoptosis and Suppresses Neuroendocrine Differentiation in Prostate Cancer.

We analyzed the efficacy and mechanistic interactions of PARP inhibition (PARPi; olaparib) and CDK4/6 inhibition (CDK4/6i; palbociclib or abemaciclib) combination therapy in castration-resistant prostate cancer (CRPC) and neuroendocrine prostate cancer (NEPC) models. We demonstrated that combined olaparib and palbociblib or abemaciclib treatment resulted in synergistic suppression of the p-Rb1-E2F1 signaling axis at the transcriptional and posttranslational levels, leading to disruption of cell-cycle progression and inhibition of E2F1 gene targets, including genes involved in DDR signaling/damage repair, antiapoptotic BCL-2 family members (BCL-2 and MCL-1), CDK1, and neuroendocrine differentiation (NED) markers in vitro and in vivo In addition, olaparib + palbociclib or olaparib + abemaciclib combination treatment resulted in significantly greater growth inhibition and apoptosis than either single agent alone. We further showed that PARPi and CDK4/6i combination treatment-induced CDK1 inhibition suppressed p-S70-BCL-2 and increased caspase cleavage, while CDK1 overexpression effectively prevented the downregulation of p-S70-BCL-2 and largely rescued the combination treatment-induced cytotoxicity. Our study defines a novel combination treatment strategy for CRPC and NEPC and demonstrates that combination PARPi and CDK4/6i synergistically promotes suppression of the p-Rb1-E2F1 axis and E2F1 target genes, including CDK1 and NED proteins, leading to growth inhibition and increased apoptosis in vitro and in vivo Taken together, our results provide a molecular rationale for PARPi and CDK4/6i combination therapy and reveal mechanism-based clinical trial opportunities for men with NEPC.

Tilsotolimod with Ipilimumab Drives Tumor Responses in Anti-PD-1 Refractory Melanoma.

Many patients with advanced melanoma are resistant to immune checkpoint inhibition. In the ILLUMINATE-204 phase I/II trial, we assessed intratumoral tilsotolimod, an investigational Toll-like receptor 9 agonist, with systemic ipilimumab in patients with anti-PD-1- resistant advanced melanoma. In all patients, 48.4% experienced grade 3/4 treatment-emergent adverse events. The overall response rate at the recommended phase II dose of 8 mg was 22.4%, and an additional 49% of patients had stable disease. Responses in noninjected lesions and in patients expected to be resistant to ipilimumab monotherapy were observed. Rapid induction of a local IFNα gene signature, dendritic cell maturation and enhanced markers of antigen presentation, and T-cell clonal expansion correlated with clinical response. A phase III clinical trial with this combination (NCT03445533) is ongoing. SIGNIFICANCE: Despite recent developments in advanced melanoma therapies, most patients do not experience durable responses. Intratumoral tilsotolimod injection elicits a rapid, local type 1 IFN response and, in combination with ipilimumab, activates T cells to promote clinical activity, including in distant lesions and patients not expected to respond to ipilimumab alone.This article is highlighted in the In This Issue feature, p. 1861.

Clinical, molecular, metabolic, and immune features associated with oxidative phosphorylation in melanoma brain metastases.

BACKGROUND: Recently, we showed that melanoma brain metastases (MBMs) are characterized by increased utilization of the oxidative phosphorylation (OXPHOS) metabolic pathway compared to melanoma extracranial metastases (ECMs). MBM growth was inhibited by a potent direct OXPHOS inhibitor, but observed toxicities support the need to identify alternative therapeutic strategies. Thus, we explored the features associated with OXPHOS to improve our understanding of the pathogenesis and potential therapeutic vulnerabilities of MBMs. METHODS: We applied an OXPHOS gene signature to our cohort of surgically resected MBMs that had undergone RNA-sequencing (RNA-seq) (n = 88). Clustering by curated gene sets identified MBMs with significant enrichment (High-OXPHOS; n = 21) and depletion (Low-OXPHOS; n = 25) of OXPHOS genes. Clinical data, RNA-seq analysis, and immunohistochemistry were utilized to identify significant clinical, molecular, metabolic, and immune associations with OXPHOS in MBMs. Preclinical models were used to further compare melanomas with High- and Low-OXPHOS and for functional validation. RESULTS: High-OXPHOS MBMs were associated with shorter survival from craniotomy compared to Low-OXPHOS MBMs. High-OXPHOS MBMs exhibited an increase in glutamine metabolism, and treatment with the glutaminase inhibitor CB839 improved survival in mice with MAPKi-resistant, High-OXPHOS intracranial xenografts. High-OXPHOS MBMs also exhibited a transcriptional signature of deficient immune activation, which was reversed in B16-F10 intracranial tumors with metformin treatment, an OXPHOS inhibitor. CONCLUSIONS: OXPHOS is associated with distinct clinical, molecular, metabolic, and immune phenotypes in MBMs. These associations suggest rational therapeutic strategies for further testing to improve outcomes in MBM patients.

The Glutaminase Inhibitor CB-839 (Telaglenastat) Enhances the Antimelanoma Activity of T-Cell-Mediated Immunotherapies.

Immune-checkpoint inhibitors and adoptive tumor-infiltrating lymphocyte (TIL) therapies have profoundly improved the survival of patients with melanoma. However, a majority of patients do not respond to these agents, and many responders experience disease relapse. Although numerous innovative treatments are being explored to offset the limitations of these agents, novel therapeutic combinations with immunotherapies have the potential to improve patient responses. In this study, we evaluated the antimelanoma activity of immunotherapy combinations with Telaglenastat (CB-839), a potent glutaminase inhibitor (GLSi) that has favorable systemic tolerance. In in vitro TIL:tumor coculture studies, CB-839 treatment improved the cytotoxic activity of autologous TILs on patient-derived melanoma cells. CB-839 treatment decreased the conversion of glutamine to alpha-ketoglutarate (αKGA) more potently in tumor cells versus TILs in these cocultures. These results suggest that CB-839 may improve immune function in a tumor microenvironment by differentially altering tumor and immune cell metabolism. In vivo CB-839 treatment activated melanoma antigen-specific T cells and improved their tumor killing activity in an immune-competent mouse model of adoptive T-cell therapy. Additionally, the combination of CB-839 with anti-PD1 or anti-CTLA4 antibodies increased tumor infiltration by effector T cells and improved the antitumor activity of these checkpoint inhibitors in a high mutation burden mouse melanoma model. Responsiveness to these treatments was also accompanied by an increase of interferon gamma (IFNγ)-associated gene expression in the tumors. Together, these results provide a strong rationale for combining CB-839 with immune therapies to improve efficacy of these treatments against melanoma.

A phase II study of the insulin-like growth factor type I receptor inhibitor IMC-A12 in patients with metastatic uveal melanoma.

Uveal melanoma is a rare and aggressive malignancy and up to half of all patients will develop metastatic disease despite the effective treatment of the primary tumor. Insulin-like growth factors I/II play a fundamental role in the cell migration, proliferation, and apoptosis. IMC-A12, a mAb specifically targets insulin-like growth factor type I receptor, has shown promise in preclinical studies. We performed a multicenter phase II study for patients with metastatic uveal melanoma administered IMC-A12 10 mg/kg IV every two weeks until disease progression or unacceptable toxicity. The primary endpoint was objective response (proportion of patients with complete or partial response), and secondary endpoints were disease control rate, progression-free survival, and overall survival. A total of 18 patients enrolled in this study (10 males and eight females) with a median age. Ten patients (55%) had stable disease, seven patients (38%) had progression as best overall response. No partial response or complete response was observed; however, the disease control rate, defined as complete response + partial response + stable disease ≥3 months, was 50%. Median progression-free survival was 3.1 months, and median overall survival was 13.8 months. Adverse events of any grade occurred in 13 patients (72.2%). Treatment-related grade 3 adverse events were rare, and there were no grade 4 or 5 related adverse events. IMC-A12 was very well tolerated, however, showed limited clinical activity in uveal melanoma as a single agent. Due to its low toxicity profile it could be studied in combination with other pathway-specific agents.

Spatially resolved analyses link genomic and immune diversity and reveal unfavorable neutrophil activation in melanoma.

Complex tumor microenvironmental (TME) features influence the outcome of cancer immunotherapy (IO). Here we perform immunogenomic analyses on 67 intratumor sub-regions of a PD-1 inhibitor-resistant melanoma tumor and 2 additional metastases arising over 8 years, to characterize TME interactions. We identify spatially distinct evolution of copy number alterations influencing local immune composition. Sub-regions with chromosome 7 gain display a relative lack of leukocyte infiltrate but evidence of neutrophil activation, recapitulated in The Cancer Genome Atlas (TCGA) samples, and associated with lack of response to IO across three clinical cohorts. Whether neutrophil activation represents cause or consequence of local tumor necrosis requires further study. Analyses of T-cell clonotypes reveal the presence of recurrent priming events manifesting in a dominant T-cell clonotype over many years. Our findings highlight the links between marked levels of genomic and immune heterogeneity within the physical space of a tumor, with implications for biomarker evaluation and immunotherapy response.

T-Cell Repertoire in Combination with T-Cell Density Predicts Clinical Outcomes in Patients with Merkel Cell Carcinoma.

The integrity of the immune system represents a pivotal risk factor and prognostic biomarker for Merkel cell carcinoma. A higher density of tumor-associated T cells correlates with improved Merkel cell carcinoma-specific survival, but the prognostic importance of the T-cell infiltrate reactivity is unknown. We evaluated the T-cell receptor repertoire associated with 72 primary Merkel cell carcinomas and correlated metrics of the T-cell receptor repertoire with clinicopathologic characteristics and patient outcomes. We showed that a high Simpson's Dominance index (SDom) was significantly associated with fewer metastases (P = 0.01), lower stage at presentation (P = 0.02), lower final stage at last follow-up (P = 0.05), and longer time to first lymph node metastasis (P = 0.04). These correlations were mostly preserved in the Merkel cell polyomavirus-negative subgroup. Combining SDom with CD3+ or CD8+ T-cell density revealed three distinct prognostic groups with respect to disease-specific survival. Patients with both high SDom and high CD3+ or CD8+ T-cell density had markedly improved disease-specific survival compared with patients with low SDom and low CD3+ or CD8+ T-cell density (P = 0.002 and P = 0.03, respectively). Patients with either high SDom or high CD3+ or CD8+ had intermediate disease-specific survival. Our findings demonstrate that the quality of the tumor-associated T-cell infiltrate informs patient prognosis in primary Merkel cell carcinoma beyond the T-cell density.

B cells and tertiary lymphoid structures promote immunotherapy response.

Treatment with immune checkpoint blockade (ICB) has revolutionized cancer therapy. Until now, predictive biomarkers1-10 and strategies to augment clinical response have largely focused on the T cell compartment. However, other immune subsets may also contribute to anti-tumour immunity11-15, although these have been less well-studied in ICB treatment16. A previously conducted neoadjuvant ICB trial in patients with melanoma showed via targeted expression profiling17 that B cell signatures were enriched in the tumours of patients who respond to treatment versus non-responding patients. To build on this, here we performed bulk RNA sequencing and found that B cell markers were the most differentially expressed genes in the tumours of responders versus non-responders. Our findings were corroborated using a computational method (MCP-counter18) to estimate the immune and stromal composition in this and two other ICB-treated cohorts (patients with melanoma and renal cell carcinoma). Histological evaluation highlighted the localization of B cells within tertiary lymphoid structures. We assessed the potential functional contributions of B cells via bulk and single-cell RNA sequencing, which demonstrate clonal expansion and unique functional states of B cells in responders. Mass cytometry showed that switched memory B cells were enriched in the tumours of responders. Together, these data provide insights into the potential role of B cells and tertiary lymphoid structures in the response to ICB treatment, with implications for the development of biomarkers and therapeutic targets.

Variable Expression of MSH6 in Endometrial Carcinomas With Intact Mismatch Repair and With MLH1 Loss Due to MLH1 Methylation.

Immunohistochemistry for mismatch repair proteins MLH1, MSH2, MSH6, and PMS2 is an effective screen to detect individuals at risk for Lynch syndrome. College of American Pathologists guidelines stipulate that protein expression should be reported as present versus absent, as most patients with germline mutations in a mismatch repair gene have complete loss of protein expression in tumor cells. A similar approach is employed to screen for cancer patients eligible for immune checkpoint blockade. This "all or none" interpretive approach ignores substantial evidence that mismatch repair may be more finely regulated by other mechanisms. We have observed clinically that MSH6 expression is variable, even in carcinomas that are overall considered positive for MSH6 expression. A proof-of-principle study was therefore designed to more rigorously quantify the protein expression of MSH6 and its binding partner, MSH2, using image analysis applied to age-matched endometrioid grade 2 subsets that were either mismatch repair intact or MLH1-deficient due to MLH1 gene methylation. In both endometrioid groups, MSH6 expression was significantly lower than MSH2 expression. MSH6 expression increased in higher grade, mismatch repair intact serous carcinomas, but it was still significantly lower than that for MSH2. MSH2 expression was consistently high across the 3 different tumor groups. These results suggest that MSH6 expression is subject to wide fluctuations in expression, even when overall its expression is considered intact. While such fluctuations are likely not relevant for Lynch syndrome screening, they may be more impactful when considering patients eligible for immune checkpoint blockade.