Spatially organized multicellular immune hubs in human colorectal cancer.

Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd individuals. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across tumors from affected individuals and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T cell-attracting chemokines. By identifying interacting cellular programs, we reveal the logic underlying spatially organized immune-malignant cell networks.

The Human and Mouse Enteric Nervous System at Single-Cell Resolution.

The enteric nervous system (ENS) coordinates diverse functions in the intestine but has eluded comprehensive molecular characterization because of the rarity and diversity of cells. Here we develop two methods to profile the ENS of adult mice and humans at single-cell resolution: RAISIN RNA-seq for profiling intact nuclei with ribosome-bound mRNA and MIRACL-seq for label-free enrichment of rare cell types by droplet-based profiling. The 1,187,535 nuclei in our mouse atlas include 5,068 neurons from the ileum and colon, revealing extraordinary neuron diversity. We highlight circadian expression changes in enteric neurons, show that disease-related genes are dysregulated with aging, and identify differences between the ileum and proximal/distal colon. In humans, we profile 436,202 nuclei, recovering 1,445 neurons, and identify conserved and species-specific transcriptional programs and putative neuro-epithelial, neuro-stromal, and neuro-immune interactions. The human ENS expresses risk genes for neuropathic, inflammatory, and extra-intestinal diseases, suggesting neuronal contributions to disease.

Molecular Pathways of Colon Inflammation Induced by Cancer Immunotherapy.

Checkpoint blockade with antibodies specific for the PD-1 and CTLA-4 inhibitory receptors can induce durable responses in a wide range of human cancers. However, the immunological mechanisms responsible for severe inflammatory side effects remain poorly understood. Here we report a comprehensive single-cell analysis of immune cell populations in colitis, a common and severe side effect of checkpoint blockade. We observed a striking accumulation of CD8 T cells with highly cytotoxic and proliferative states and no evidence of regulatory T cell depletion. T cell receptor (TCR) sequence analysis demonstrated that a substantial fraction of colitis-associated CD8 T cells originated from tissue-resident populations, explaining the frequently early onset of colitis symptoms following treatment initiation. Our analysis also identified cytokines, chemokines, and surface receptors that could serve as therapeutic targets for colitis and potentially other inflammatory side effects of checkpoint blockade.

Targeting TBK1 to overcome resistance to cancer immunotherapy.

Despite the success of PD-1 blockade in melanoma and other cancers, effective treatment strategies to overcome resistance to cancer immunotherapy are lacking1,2. Here we identify the innate immune kinase TANK-binding kinase 1 (TBK1)3 as a candidate immune-evasion gene in a pooled genetic screen4. Using a suite of genetic and pharmacological tools across multiple experimental model systems, we confirm a role for TBK1 as an immune-evasion gene. Targeting TBK1 enhances responses to PD-1 blockade by decreasing the cytotoxicity threshold to effector cytokines (TNF and IFNγ). TBK1 inhibition in combination with PD-1 blockade also demonstrated efficacy using patient-derived tumour models, with concordant findings in matched patient-derived organotypic tumour spheroids and matched patient-derived organoids. Tumour cells lacking TBK1 are primed to undergo RIPK- and caspase-dependent cell death in response to TNF and IFNγ in a JAK-STAT-dependent manner. Taken together, our results demonstrate that targeting TBK1 is an effective strategy to overcome resistance to cancer immunotherapy.

Factors Affecting Recurrence and Survival for Patients with High-Risk Stage II Melanoma.

BACKGROUND: In the current era of effective adjuvant therapies and de-escalation of surgery, distinguishing which patients with high-risk stage II melanoma are at increased risk of recurrence after excision of the primary lesion is essential to determining appropriate treatment and surveillance plans. METHODS: A single-center retrospective study analyzed patients with stage IIB or IIC melanoma. Demographic and tumor data were collected, and genomic analysis of formalin-fixed, paraffin-embedded tissue samples was performed via an internal next-generation sequencing (NGS) platform (SNaPshot). The end points examined were relapse-free survival (RFS), distant metastasis-free survival (DMFS), overall survival (OS), and melanoma-specific survival (MSS). Uni- and multivariable Cox regressions were performed to calculate the hazard ratios. RESULTS: The study included 92 patients with a median age of 69 years and a male/female ratio of 2:1. A Breslow depth greater than 4 mm, a higher mitotic rate, an advanced T stage, and a KIT mutation had a negative impact on RFS. A primary lesion in the head and neck, a mitotic rate exceeding 10 mitoses per mm2, a CDH1 mutation, or a KIT mutation was significantly associated with a shorter DMFS. Overall survival was significantly lower with older age at diagnosis and a higher mitotic rate. An older age at diagnosis also had a negative impact on MSS. CONCLUSION: Traditional histopathologic factors and specific tumor mutations displayed a significant correlation with disease recurrence and survival for patients with high-risk stage II melanoma. This study supported the use of genomic testing of high-risk stage II melanomas for prognostic prediction and risk stratification.

Benefit and toxicity of programmed death-1 blockade vary by ethnicity in patients with advanced melanoma: an international multicentre observational study.

BACKGROUND: Programmed cell death receptor-1 (PD-1) monotherapy is a standard treatment for advanced cutaneous melanoma, but its efficacy and toxicity are defined in white populations and remain poorly characterized in other ethnic groups, such as East Asian, Hispanic and African. OBJECTIVES: To determine the efficacy and toxicity of PD-1 monotherapy in different ethnic groups. METHODS: Clinical data for patients with unresectable or advanced melanoma treated with anti-PD-1 monotherapy between 2009 and 2019 were collected retrospectively from five independent institutions in the USA, Australia and China. Tumour response, survival and immune-related adverse events (irAEs) were compared by ethnicity (white vs. East Asian/Hispanic/African) across different melanoma subtypes: nonacral cutaneous (NAC)/unknown primary (UP) and acral/mucosal/uveal. RESULTS: In total, 1135 patients were included. White patients had significantly higher objective response rate (ORR) [54%, 95% confidence interval (CI) 50-57% vs. 20%, 95% CI 13-28%; adjusted P < 0·001] and longer progression-free survival (14·2 months, 95% CI 10·7-20·3 vs. 5·4 months, 95% CI 4·5-7·0; adjusted P < 0·001) than East Asian, Hispanic and African patients in the NAC and UP subtypes. White ethnicity remained independently associated with a higher ORR (odds ratio 4·10, 95% CI 2·48-6·81; adjusted P < 0·001) and longer PFS (hazard ratio 0·58, 95% CI 0·46-0·74; adjusted P < 0·001) in multivariate analyses after adjustment for age, sex, primary anatomical location, metastasis stage, baseline lactate dehydrogenase level, mutational status and prior systemic treatment. White and East Asian/Hispanic/African patients shared similar ORR and progression-free survival in acral/mucosal/uveal melanomas. Similar melanoma-subtype-specific ethnic discrepancies were observed in complete response rate and overall survival. White patients had higher rates of gastrointestinal irAEs but lower rates of endocrine, liver and other rare types of irAEs. These differences in irAEs by ethnicity were not attributable to varying melanoma subtypes. CONCLUSIONS: Ethnic discrepancy in clinical benefit is specific to melanoma subtype, and East Asian, Hispanic and African patients with NAC and UP melanomas have poorer clinical benefits than previously recognized. The ethnic discrepancy in toxicity observed across different melanoma subtypes warrants an ethnicity-based irAE surveillance strategy. More research is needed to elucidate the molecular and immunological determinants of these differences. What is already known about this topic? There is a great difference in response to immunotherapy between different subtypes of melanoma (cutaneous, mucosal, acral and uveal) in patients with advanced disease. What does this study add? Our data show for the first time that there are differences between different ethnic groups in terms of both response and toxicity to immunotherapy beyond the well-appreciated discrepancies due to melanoma subtype.

p63 and Brg1 control developmentally regulated higher-order chromatin remodelling at the epidermal differentiation complex locus in epidermal progenitor cells.

Chromatin structural states and their remodelling, including higher-order chromatin folding and three-dimensional (3D) genome organisation, play an important role in the control of gene expression. The role of 3D genome organisation in the control and execution of lineage-specific transcription programmes during the development and differentiation of multipotent stem cells into specialised cell types remains poorly understood. Here, we show that substantial remodelling of the higher-order chromatin structure of the epidermal differentiation complex (EDC), a keratinocyte lineage-specific gene locus on mouse chromosome 3, occurs during epidermal morphogenesis. During epidermal development, the locus relocates away from the nuclear periphery towards the nuclear interior into a compartment enriched in SC35-positive nuclear speckles. Relocation of the EDC locus occurs prior to the full activation of EDC genes involved in controlling terminal keratinocyte differentiation and is a lineage-specific, developmentally regulated event controlled by transcription factor p63, a master regulator of epidermal development. We also show that, in epidermal progenitor cells, p63 directly regulates the expression of the ATP-dependent chromatin remodeller Brg1, which binds to distinct domains within the EDC and is required for relocation of the EDC towards the nuclear interior. Furthermore, Brg1 also regulates gene expression within the EDC locus during epidermal morphogenesis. Thus, p63 and its direct target Brg1 play an essential role in remodelling the higher-order chromatin structure of the EDC and in the specific positioning of this locus within the landscape of the 3D nuclear space, as required for the efficient expression of EDC genes in epidermal progenitor cells during skin development.

Lhx2 differentially regulates Sox9, Tcf4 and Lgr5 in hair follicle stem cells to promote epidermal regeneration after injury.

The Lhx2 transcription factor plays essential roles in morphogenesis and patterning of ectodermal derivatives as well as in controlling stem cell activity. Here, we show that during murine skin morphogenesis, Lhx2 is expressed in the hair follicle (HF) buds, whereas in postnatal telogen HFs Lhx2(+) cells reside in the stem cell-enriched epithelial compartments (bulge, secondary hair germ) and co-express selected stem cell markers (Sox9, Tcf4 and Lgr5). Remarkably, Lhx2(+) cells represent the vast majority of cells in the bulge and secondary hair germ that proliferate in response to skin injury. This is functionally important, as wound re-epithelization is significantly retarded in heterozygous Lhx2 knockout (+/-) mice, whereas anagen onset in the HFs located closely to the wound is accelerated compared with wild-type mice. Cell proliferation in the bulge and the number of Sox9(+) and Tcf4(+) cells in the HFs closely adjacent to the wound in Lhx2(+/-) mice are decreased in comparison with wild-type controls, whereas expression of Lgr5 and cell proliferation in the secondary hair germ are increased. Furthermore, acceleration of wound-induced anagen development in Lhx2(+/-) mice is inhibited by administration of Lgr5 siRNA. Finally, Chip-on-chip/ChIP-qPCR and reporter assay analyses identified Sox9, Tcf4 and Lgr5 as direct Lhx2 targets in keratinocytes. These data strongly suggest that Lhx2 positively regulates Sox9 and Tcf4 in the bulge cells, and promotes wound re-epithelization, whereas it simultaneously negatively regulates Lgr5 in the secondary hair germ and inhibits HF cycling. Thus, Lhx2 operates as an important regulator of epithelial stem cell activity in the skin response to injury.

Cryoablation and post-progression immune checkpoint inhibition in metastatic melanoma: a phase II trial.

Image-guided percutaneous cryoablation is an established minimally invasive oncologic treatment. We hypothesized that cryoablation may modify the immune microenvironment through direct modulation of the tumor, thereby generating an anti-tumor response in tumors refractory to immune checkpoint inhibition (ICI). In this non-randomized phase II single-center study (NCT03290677), subjects with unresectable melanoma progressing on ICI underwent cryoablation of an enlarging metastasis, and ICI was continued for a minimum of two additional cycles. The primary endpoints were safety, feasibility and tumor response in non-ablated lesions. From May 2018 through July 2020, 17 patients were treated on study. The study met its primary endpoints with the combination strategy found to be safe and feasible with an objective response rate of 23.5% and disease control rate of 41% (4 partial response, 3 stable disease). Our data support further study of this synergistic therapeutic approach.

Single-cell transcriptomic analyses reveal distinct immune cell contributions to epithelial barrier dysfunction in checkpoint inhibitor colitis.

Immune checkpoint inhibitor (ICI) therapy has revolutionized oncology, but treatments are limited by immune-related adverse events, including checkpoint inhibitor colitis (irColitis). Little is understood about the pathogenic mechanisms driving irColitis, which does not readily occur in model organisms, such as mice. To define molecular drivers of irColitis, we used single-cell multi-omics to profile approximately 300,000 cells from the colon mucosa and blood of 13 patients with cancer who developed irColitis (nine on anti-PD-1 or anti-CTLA-4 monotherapy and four on dual ICI therapy; most patients had skin or lung cancer), eight controls on ICI therapy and eight healthy controls. Patients with irColitis showed expanded mucosal Tregs, ITGAEHi CD8 tissue-resident memory T cells expressing CXCL13 and Th17 gene programs and recirculating ITGB2Hi CD8 T cells. Cytotoxic GNLYHi CD4 T cells, recirculating ITGB2Hi CD8 T cells and endothelial cells expressing hypoxia gene programs were further expanded in colitis associated with anti-PD-1/CTLA-4 therapy compared to anti-PD-1 therapy. Luminal epithelial cells in patients with irColitis expressed PCSK9, PD-L1 and interferon-induced signatures associated with apoptosis, increased cell turnover and malabsorption. Together, these data suggest roles for circulating T cells and epithelial-immune crosstalk critical to PD-1/CTLA-4-dependent tolerance and barrier function and identify potential therapeutic targets for irColitis.

Disrupting CD38-driven T cell dysfunction restores sensitivity to cancer immunotherapy.

A central problem in cancer immunotherapy with immune checkpoint blockade (ICB) is the development of resistance, which affects 50% of patients with metastatic melanoma1,2. T cell exhaustion, resulting from chronic antigen exposure in the tumour microenvironment, is a major driver of ICB resistance3. Here, we show that CD38, an ecto-enzyme involved in nicotinamide adenine dinucleotide (NAD+) catabolism, is highly expressed in exhausted CD8+ T cells in melanoma and is associated with ICB resistance. Tumour-derived CD38hiCD8+ T cells are dysfunctional, characterised by impaired proliferative capacity, effector function, and dysregulated mitochondrial bioenergetics. Genetic and pharmacological blockade of CD38 in murine and patient-derived organotypic tumour models (MDOTS/PDOTS) enhanced tumour immunity and overcame ICB resistance. Mechanistically, disrupting CD38 activity in T cells restored cellular NAD+ pools, improved mitochondrial function, increased proliferation, augmented effector function, and restored ICB sensitivity. Taken together, these data demonstrate a role for the CD38-NAD+ axis in promoting T cell exhaustion and ICB resistance, and establish the efficacy of CD38 directed therapeutic strategies to overcome ICB resistance using clinically relevant, patient-derived 3D tumour models.

Fucosylation of HLA-DRB1 regulates CD4+ T cell-mediated anti-melanoma immunity and enhances immunotherapy efficacy.

Immunotherapy efficacy is limited in melanoma, and combinations of immunotherapies with other modalities have yielded limited improvements but also adverse events requiring cessation of treatment. In addition to ineffective patient stratification, efficacy is impaired by paucity of intratumoral immune cells (itICs); thus, effective strategies to safely increase itICs are needed. We report that dietary administration of L-fucose induces fucosylation and cell surface enrichment of the major histocompatibility complex (MHC)-II protein HLA-DRB1 in melanoma cells, triggering CD4+ T cell-mediated increases in itICs and anti-tumor immunity, enhancing immune checkpoint blockade responses. Melanoma fucosylation and fucosylated HLA-DRB1 associate with intratumoral T cell abundance and anti-programmed cell death protein 1 (PD1) responder status in patient melanoma specimens, suggesting the potential use of melanoma fucosylation as a strategy for stratifying patients for immunotherapies. Our findings demonstrate that fucosylation is a key mediator of anti-tumor immunity and, importantly, suggest that L-fucose is a powerful agent for safely increasing itICs and immunotherapy efficacy in melanoma.

Immune Responses in Checkpoint Myocarditis Across Heart, Blood, and Tumor.

Immune checkpoint inhibitors (ICIs) are widely used anti-cancer therapies that can cause morbid and potentially fatal immune-related adverse events (irAEs). ICI-related myocarditis (irMyocarditis) is uncommon but has the highest mortality of any irAE. The pathogenesis of irMyocarditis and its relationship to anti-tumor immunity remain poorly understood. We sought to define immune responses in heart, tumor, and blood during irMyocarditis and identify biomarkers of clinical severity by leveraging single-cell (sc)RNA-seq coupled with T cell receptor (TCR) sequencing, microscopy, and proteomics analysis of 28 irMyocarditis patients and 23 controls. Our analysis of 284,360 cells from heart and blood specimens identified cytotoxic T cells, inflammatory macrophages, conventional dendritic cells (cDCs), and fibroblasts enriched in irMyocarditis heart tissue. Additionally, potentially targetable, pro-inflammatory transcriptional programs were upregulated across multiple cell types. TCR clones enriched in heart and paired tumor tissue were largely non-overlapping, suggesting distinct T cell responses within these tissues. We also identify the presence of cardiac-expanded TCRs in a circulating, cycling CD8 T cell population as a novel peripheral biomarker of fatality. Collectively, these findings highlight critical biology driving irMyocarditis and putative biomarkers for therapeutic intervention.

Ultrasensitive Detection of Circulating LINE-1 ORF1p as a Specific Multicancer Biomarker.

Improved biomarkers are needed for early cancer detection, risk stratification, treatment selection, and monitoring treatment response. Although proteins can be useful blood-based biomarkers, many have limited sensitivity or specificity for these applications. Long INterspersed Element-1 (LINE-1) open reading frame 1 protein (ORF1p) is a transposable element protein overexpressed in carcinomas and high-risk precursors during carcinogenesis with negligible expression in normal tissues, suggesting ORF1p could be a highly specific cancer biomarker. To explore ORF1p as a blood-based biomarker, we engineered ultrasensitive digital immunoassays that detect mid-attomolar (10-17 mol/L) ORF1p concentrations in plasma across multiple cancers with high specificity. Plasma ORF1p shows promise for early detection of ovarian cancer, improves diagnostic performance in a multianalyte panel, provides early therapeutic response monitoring in gastroesophageal cancers, and is prognostic for overall survival in gastroesophageal and colorectal cancers. Together, these observations nominate ORF1p as a multicancer biomarker with potential utility for disease detection and monitoring. SIGNIFICANCE: The LINE-1 ORF1p transposon protein is pervasively expressed in many cancers and is a highly specific biomarker of multiple common, lethal carcinomas and their high-risk precursors in tissue and blood. Ultrasensitive ORF1p assays from as little as 25 µL plasma are novel, rapid, cost-effective tools in cancer detection and monitoring. See related commentary by Doucet and Cristofari, p. 2502. This article is featured in Selected Articles from This Issue, p. 2489.

Combined tumor and immune signals from genomes or transcriptomes predict outcomes of checkpoint inhibition in melanoma.

Immune checkpoint blockade (CPB) improves melanoma outcomes, but many patients still do not respond. Tumor mutational burden (TMB) and tumor-infiltrating T cells are associated with response, and integrative models improve survival prediction. However, integrating immune/tumor-intrinsic features using data from a single assay (DNA/RNA) remains underexplored. Here, we analyze whole-exome and bulk RNA sequencing of tumors from new and published cohorts of 189 and 178 patients with melanoma receiving CPB, respectively. Using DNA, we calculate T cell and B cell burdens (TCB/BCB) from rearranged TCR/Ig sequences and find that patients with TMBhigh and TCBhigh or BCBhigh have improved outcomes compared to other patients. By combining pairs of immune- and tumor-expressed genes, we identify three gene pairs associated with response and survival, which validate in independent cohorts. The top model includes lymphocyte-expressed MAP4K1 and tumor-expressed TBX3. Overall, RNA or DNA-based models combining immune and tumor measures improve predictions of melanoma CPB outcomes.

Type 2 immunity is maintained during cancer-associated adipose tissue wasting.

OBJECTIVES: Cachexia is a systemic metabolic disorder characterized by loss of fat and muscle mass, which disproportionately impacts patients with gastrointestinal malignancies such as pancreatic cancer. While the immunologic shifts contributing to the development of other adipose tissue (AT) pathologies such as obesity have been well described, the immune microenvironment has not been studied in the context of cachexia. METHODS: We performed bulk RNA-sequencing, cytokine arrays, and flow cytometry to characterize the immune landscape of visceral AT (VAT) in the setting of pancreatic and colorectal cancers. RESULTS: The cachexia inducing factor IL-6 is strongly elevated in the wasting VAT of cancer bearing mice, but the regulatory type 2 immune landscape which characterizes healthy VAT is maintained. Pathologic skewing toward Th1 and Th17 inflammation is absent. Similarly, the VAT of patients with colorectal cancer is characterized by a Th2 signature with abundant IL-33 and eotaxin-2, albeit also with high levels of IL-6. CONCLUSIONS: Wasting AT during the development of cachexia may not undergo drastic changes in immune composition like those seen in obese AT. Our approach provides a framework for future immunologic analyses of cancer associated cachexia.

Radiological dynamics and SITC-defined resistance types of advanced melanoma during anti-PD-1 monotherapy: an independent single-blind observational study on an international cohort.

BACKGROUND: Although the Society for Immunotherapy of Cancer (SITC) Immunotherapy Resistance Taskforce recently defined primary and secondary resistance to anti-programmed cell death protein 1 (anti-PD-1) therapy, there is lack of real-world data regarding differences in these resistance subtypes with respect to radiological dynamics and clinical manifestations. METHODS: We performed single-blind re-evaluations of radiological images by independent radiologists on a retrospectively assembled cohort of patients with advanced melanoma (n=254, median follow-up 31 months) receiving anti-PD-1 monotherapy at Massachusetts General Hospital and Peking University Cancer Hospital. Radiological characteristics and timing at multiple crucial time points were analyzed and correlated with each other and with survival. Primary and secondary resistance was defined as per the SITC Immunotherapy Resistance Taskforce definitions. RESULTS: The most significant target lesion measurement change took place within the first 3 months after anti-PD-1 initiation. Patients with stable disease with versus without tumor shrinkage at the initial evaluation exhibited distinct disease trajectory, as the rate of further upgrade to a partial or complete remission (CR/PR) was 44% and 0%, respectively. Eleven per cent of PR patients ultimately achieved a CR. In multivariate analyses, deeper response depth was independently associated with a more limited progression pattern, fewer involved organs, lower tumor burden, slower growth rate at disease progression (PD) (all p≤0.001), and longer post-progression survival (PPS) (bivariate analysis, p=0.005). Compared with primary resistance, secondary resistance was associated with less widespread PD pattern, lower tumor burden and slower tumor growth (all p≤0.001). Patients with secondary resistance were less likely to receive further systemic therapy (28% vs 57%, p<0.001) yet had significantly better PPS (HR 0.503, 95% CI 0.288 to 0.879, p=0.02). CONCLUSIONS: Radiological dynamics were variable, yet significantly correlated with survival outcomes. SITC-defined primary and secondary resistance are distinct clinical manifestations in patients with melanoma, suggesting the possibility of resistance-type-based therapeutic decision-making and clinical trial design, once further validated by future prospective studies.

Early Use of High-Dose Glucocorticoid for the Management of irAE Is Associated with Poorer Survival in Patients with Advanced Melanoma Treated with Anti-PD-1 Monotherapy.

PURPOSE: Programmed cell death receptor-1 (PD-1) inhibitors are frontline therapy in advanced melanoma. Severe immune-related adverse effects (irAEs) often require immunosuppressive treatment with glucocorticoids (GCCs), but GCC use and its correlation with patient survival outcomes during anti-PD-1 monotherapy remains unclear. EXPERIMENTAL DESIGN: In this multicenter retrospective analysis, patients treated with anti-PD-1 monotherapy between 2009 and 2019 and detailed GCC use, data were identified from five independent cohorts, with median follow-up time of 206 weeks. IrAEs were tracked from the initiation of anti-PD-1 until disease progression, initiation of a new therapy, or last follow-up. Correlations between irAEs, GCC use, and survival outcomes were analyzed. RESULTS: Of the entire cohort of 947 patients, 509 (54%) developed irAEs. In the MGH cohort [irAE(+) n = 90], early-onset irAE (within 8 weeks of anti-PD-1 initiation) with high-dose GCC use (≥60-mg prednisone equivalent once a day) was independently associated with poorer post-irAE PFS/OS (progression-free survival/overall survival) [post-irAE PFS: HR, 5.37; 95% confidence interval (CI), 2.10-13.70; P < 0.001; post-irAE OS: HR, 5.95; 95% CI, 2.20-16.09; P < 0.001] compared with irAEs without early high-dose GCC use. These findings were validated in the combined validation cohort [irAE(+) n = 419, post-irAE PFS: HR, 1.69; 95% CI, 1.04-2.76; P = 0.04; post-irAE OS: HR, 1.97; 95% CI, 1.15-3.39; P = 0.01]. Similar findings were also observed in the 26-week landmark analysis for post-irAE-PFS but not for post-irAE-OS. A sensitivity analysis using accumulated GCC exposure as the measurement achieved similar results. CONCLUSIONS: Early high-dose GCC use was associated with poorer PFS and OS after irAE onset. Judicious use of GCC early during anti-PD-1 monotherapy should be considered. Further prospective randomized control clinical trials designed to explore alternative irAE management options are warranted.

Evolution of delayed resistance to immunotherapy in a melanoma responder.

Despite initial responses1-3, most melanoma patients develop resistance4 to immune checkpoint blockade (ICB). To understand the evolution of resistance, we studied 37 tumor samples over 9 years from a patient with metastatic melanoma with complete clinical response to ICB followed by delayed recurrence and death. Phylogenetic analysis revealed co-evolution of seven lineages with multiple convergent, but independent resistance-associated alterations. All recurrent tumors emerged from a lineage characterized by loss of chromosome 15q, with post-treatment clones acquiring additional genomic driver events. Deconvolution of bulk RNA sequencing and highly multiplexed immunofluorescence (t-CyCIF) revealed differences in immune composition among different lineages. Imaging revealed a vasculogenic mimicry phenotype in NGFRhi tumor cells with high PD-L1 expression in close proximity to immune cells. Rapid autopsy demonstrated two distinct NGFR spatial patterns with high polarity and proximity to immune cells in subcutaneous tumors versus a diffuse spatial pattern in lung tumors, suggesting different roles of this neural-crest-like program in different tumor microenvironments. Broadly, this study establishes a high-resolution map of the evolutionary dynamics of resistance to ICB, characterizes a de-differentiated neural-crest tumor population in melanoma immunotherapy resistance and describes site-specific differences in tumor-immune interactions via longitudinal analysis of a patient with melanoma with an unusual clinical course.

Bone morphogenetic protein antagonist noggin promotes skin tumorigenesis via stimulation of the Wnt and Shh signaling pathways.

Bone morphogenetic proteins (BMPs) play pivotal roles in the regulation of skin development. To study the role of BMPs in skin tumorigenesis, BMP antagonist noggin was used to generate keratin 14-targeted transgenic mice. In contrast to wild-type mice, transgenic mice developed spontaneous hair follicle-derived tumors, which resemble human trichofolliculoma. Global gene expression profiles revealed that in contrast to anagen hair follicles of wild-type mice, tumors of transgenic mice showed stage-dependent increases in the expression of genes encoding the selected components of Wnt and Shh pathways. Specifically, expression of the Wnt ligands increased at the initiation stage of tumor formation, whereas expression of the Wnt antagonist and tumor suppressor Wnt inhibitory factor-1 decreased, as compared with fully developed tumors. In contrast, expression of the components of Shh pathway increased in fully developed tumors, as compared with the tumor placodes. Consistent with the expression data, pharmacological treatment of transgenic mice with Wnt and Shh antagonists resulted in the stage-dependent inhibition of tumor initiation, and progression, respectively. Furthermore, BMP signaling stimulated Wnt inhibitory factor-1 expression and promoter activity in cultured tumor cells and HaCaT keratinocytes, as well as inhibited Shh expression, as compared with the corresponding controls. Thus, tumor suppressor activity of the BMPs in skin epithelium depends on the local concentrations of noggin and is mediated at least in part via stage-dependent antagonizing of Wnt and Shh signaling pathways.