Publications by authors named "James S Wilmott"

100 Publications

The deacylase SIRT5 supports melanoma viability by influencing chromatin dynamics.

J Clin Invest 2021 May 4. Epub 2021 May 4.

Department of Biomedical Engineering, University of Michigan, Ann Arbor, United States of America.

Cutaneous melanoma remains the most lethal skin cancer, and ranks third among all malignancies in terms of years of life lost. Despite the advent of immune checkpoint and targeted therapies, only roughly half of patients with advanced melanoma achieves a durable remission. SIRT5 is a member of the sirtuin family of protein deacylases that regulate metabolism and other biological processes. Germline Sirt5 deficiency is associated with mild phenotypes in mice. Here we show that SIRT5 is required for proliferation and survival across all cutaneous melanoma genotypes tested, as well as uveal melanoma, a genetically distinct melanoma subtype that arises in the eye and is incurable once metastatic. Likewise, SIRT5 is required for efficient tumor formation by melanoma xenografts and in an autochthonous mouse Braf;Pten-driven melanoma model. Via metabolite and transcriptomic analyses, we find that SIRT5 is required to maintain histone acetylation and methylation levels in melanoma cells, thereby promoting proper gene expression. SIRT5-dependent genes notably include MITF, a key lineage-specific survival oncogene in melanoma, and the c-MYC proto-oncogene. SIRT5 may represent a novel, druggable genotype-independent addiction in melanoma.
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http://dx.doi.org/10.1172/JCI138926DOI Listing
May 2021

Targeting NK Cells to Enhance Melanoma Response to Immunotherapies.

Cancers (Basel) 2021 Mar 17;13(6). Epub 2021 Mar 17.

Melanoma Institute Australia, The University of Sydney, Sydney 2006, Australia.

Natural killer (NK) cells are a key component of an innate immune system. They are important not only in initiating, but also in augmenting adaptive immune responses. NK cell activation is mediated by a carefully orchestrated balance between the signals from inhibitory and activating NK cell receptors. NK cells are potent producers of proinflammatory cytokines and are also able to elicit strong antitumor responses through secretion of perforin and granzyme B. Tumors can develop many mechanisms to evade NK cell antitumor responses, such as upregulating ligands for inhibitory receptors, secreting anti-inflammatory cytokines and recruiting immunosuppressive cells. Enhancing NK cell responses will likely augment the effectiveness of immunotherapies, and strategies to accomplish this are currently being evaluated in clinical trials. A comprehensive understanding of NK cell biology will likely provide additional opportunities to further leverage the antitumor effects of NK cells. In this review, we therefore sought to highlight NK cell biology, tumor evasion of NK cells and clinical trials that target NK cells.
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http://dx.doi.org/10.3390/cancers13061363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8002669PMC
March 2021

γδ T Cells in Merkel Cell Carcinomas Have a Proinflammatory Profile Prognostic of Patient Survival.

Cancer Immunol Res 2021 Mar 5. Epub 2021 Mar 5.

Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria, Australia.

Merkel cell carcinomas (MCC) are immunogenic skin cancers associated with viral infection or UV mutagenesis. To study T-cell infiltrates in MCC, we analyzed 58 MCC lesions from 39 patients using multiplex-IHC/immunofluorescence (m-IHC/IF). CD4 or CD8 T cells comprised the majority of infiltrating T lymphocytes in most tumors. However, almost half of the tumors harbored prominent CD4/CD8 double-negative (DN) T-cell infiltrates (>20% DN T cells), and in 12% of cases, DN T cells represented the majority of T cells. Flow cytometric analysis of single-cell suspensions from fresh tumors identified DN T cells as predominantly Vδ2 γδ T cells. In the context of γδ T-cell inflammation, these cells expressed PD-1 and LAG3, which is consistent with a suppressed or exhausted phenotype, and CD103, which indicates tissue residency. Furthermore, single-cell RNA sequencing (scRNA-seq) identified a transcriptional profile of γδ T cells suggestive of proinflammatory potential. T-cell receptor (TCR) analysis confirmed clonal expansion of Vδ1 and Vδ3 clonotypes, and functional studies using cloned γδ TCRs demonstrated restriction of these for CD1c and MR1 antigen-presenting molecules. On the basis of a 13-gene γδ T-cell signature derived from scRNA-seq analysis, gene-set enrichment on bulk RNA-seq data showed a positive correlation between enrichment scores and DN T-cell infiltrates. An improved disease-specific survival was evident for patients with high enrichment scores, and complete responses to anti-PD-1/PD-L1 treatment were observed in three of four cases with high enrichment scores. Thus, γδ T-cell infiltration may serve as a prognostic biomarker and should be explored for therapeutic interventions.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0817DOI Listing
March 2021

Tumour gene expression signature in primary melanoma predicts long-term outcomes.

Nat Commun 2021 02 18;12(1):1137. Epub 2021 Feb 18.

Cambridge Cancer Centre, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK.

Adjuvant systemic therapies are now routinely used following resection of stage III melanoma, however accurate prognostic information is needed to better stratify patients. We use differential expression analyses of primary tumours from 204 RNA-sequenced melanomas within a large adjuvant trial, identifying a 121 metastasis-associated gene signature. This signature strongly associated with progression-free (HR = 1.63, p = 5.24 × 10) and overall survival (HR = 1.61, p = 1.67 × 10), was validated in 175 regional lymph nodes metastasis as well as two externally ascertained datasets. The machine learning classification models trained using the signature genes performed significantly better in predicting metastases than models trained with clinical covariates (p = 7.03 × 10), or published prognostic signatures (p < 0.05). The signature score negatively correlated with measures of immune cell infiltration (ρ = -0.75, p < 2.2 × 10), with a higher score representing reduced lymphocyte infiltration and a higher 5-year risk of death in stage II melanoma. Our expression signature identifies melanoma patients at higher risk of metastases and warrants further evaluation in adjuvant clinical trials.
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http://dx.doi.org/10.1038/s41467-021-21207-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7893180PMC
February 2021

G9a Inhibition Enhances Checkpoint Inhibitor Blockade Response in Melanoma.

Clin Cancer Res 2021 May 15;27(9):2624-2635. Epub 2021 Feb 15.

QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia.

Purpose: G9a histone methyltransferase exerts oncogenic effects in several tumor types and its inhibition promotes anticancer effects. However, the impact on checkpoint inhibitor blockade response and the utility of G9a or its target genes as a biomarker is poorly studied. We aimed to examine whether G9a inhibition can augment the efficacy of checkpoint inhibitor blockade and whether , a G9a target gene, can predict treatment response.

Experimental Design: Clinical potential of LC3B as a biomarker of checkpoint inhibitor blockade was assessed using patient samples including tumor biopsies and circulating tumor cells from liquid biopsies. Efficacy of G9a inhibition to enhance checkpoint inhibitor blockade was examined using a mouse model.

Results: Patients with melanoma who responded to checkpoint inhibitor blockade were associated with not only a higher level of tumor LC3B but also a higher proportion of cells expressing LC3B. A higher expression of or LC3B protein was associated with longer survival and lower incidence of acquired resistance to checkpoint inhibitor blockade, suggesting LC3B as a potential predictive biomarker. We demonstrate that G9a histone methyltransferase inhibition is able to not only robustly induce LC3B level to augment the efficacy of checkpoint inhibitor blockade, but also induces melanoma cell death.

Conclusions: Checkpoint inhibitor blockade response is limited to a subset of the patient population. These results have implications for the development of LC3B as a predictive biomarker of checkpoint inhibitor blockade to guide patient selection, as well as G9a inhibition as a strategy to extend the proportion of patients responding to immunotherapy.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-3463DOI Listing
May 2021

Genetic drivers of non-cutaneous melanomas: Challenges and opportunities in a heterogeneous landscape.

Exp Dermatol 2021 Jan 17. Epub 2021 Jan 17.

Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia.

Non-cutaneous melanomas most frequently involve the uveal tract and mucosal membranes, including the conjunctiva. In contrast to cutaneous melanoma, they often present at an advanced clinical stage, are associated with worse clinical outcomes and show poorer responses to immunotherapy. The mutational load within most non-cutaneous melanomas reflects their lower ultraviolet light (UV) exposure. The genetic drivers within non-cutaneous melanomas are heterogeneous. Within ocular melanomas, posterior uveal tract melanomas typically harbour one of two distinct, sets of driver mutations and alterations of clinical and biological significance. In contrast to posterior uveal tract melanomas, anterior uveal tract melanomas of the iris and conjunctival melanomas frequently carry both a higher mutational burden and specific mutations linked with UV exposure. The genetic drivers in iris melanomas more closely resemble those of the posterior uveal tract, whereas conjunctival melanomas harbour similar genetic driver mutations to cutaneous melanomas. Mucosal melanomas occur in sun-shielded sites including sinonasal and oral cavities, nasopharynx, oesophagus, genitalia, anus and rectum, and their mutational landscape is frequently associated with a dominant process of spontaneous deamination and infrequent presence of UV mutation signatures. Genetic drivers of mucosal melanomas are diverse and vary with anatomic location. Further understanding of the causes of already identified recurrent molecular events in non-cutaneous melanomas, identification of additional drivers in specific subtypes, integrative multi-omics analyses and analysis of the tumor immune microenvironment will expand knowledge in this field. Furthermore, such data will likely uncover new therapeutic strategies which will lead to improved clinical outcomes in non-cutaneous melanoma patients.
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http://dx.doi.org/10.1111/exd.14287DOI Listing
January 2021

Whole-genome sequencing of acral melanoma reveals genomic complexity and diversity.

Nat Commun 2020 10 16;11(1):5259. Epub 2020 Oct 16.

Center for Rare Melanomas, University of Colorado Cancer Center, Aurora, Colorado, USA.

To increase understanding of the genomic landscape of acral melanoma, a rare form of melanoma occurring on palms, soles or nail beds, whole genome sequencing of 87 tumors with matching transcriptome sequencing for 63 tumors was performed. Here we report that mutational signature analysis reveals a subset of tumors, mostly subungual, with an ultraviolet radiation signature. Significantly mutated genes are BRAF, NRAS, NF1, NOTCH2, PTEN and TYRP1. Mutations and amplification of KIT are also common. Structural rearrangement and copy number signatures show that whole genome duplication, aneuploidy and complex rearrangements are common. Complex rearrangements occur recurrently and are associated with amplification of TERT, CDK4, MDM2, CCND1, PAK1 and GAB2, indicating potential therapeutic options.
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http://dx.doi.org/10.1038/s41467-020-18988-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7567804PMC
October 2020

CD155 on Tumor Cells Drives Resistance to Immunotherapy by Inducing the Degradation of the Activating Receptor CD226 in CD8 T Cells.

Immunity 2020 10;53(4):805-823.e15

Institute of Pathology, University Hospital Bonn, University of Bonn, Bonn, Germany.

The activating receptor CD226 is expressed on lymphocytes, monocytes, and platelets and promotes anti-tumor immunity in pre-clinical models. Here, we examined the role of CD226 in the function of tumor-infiltrating lymphocytes (TILs) and resistance to immunotherapy. In murine tumors, a large proportion of CD8 TILs had decreased surface expression of CD226 and exhibited features of dysfunction, whereas CD226 TILs were highly functional. This correlation was seen also in TILs isolated from HNSCC patients. Mutation of CD226 at tyrosine 319 (Y319) led to increased CD226 surface expression, enhanced anti-tumor immunity and improved efficacy of immune checkpoint blockade (ICB). Mechanistically, tumor-derived CD155, the ligand for CD226, initiated phosphorylation of Y319 by Src kinases, thereby enabling ubiquitination of CD226 by CBL-B, internalization, and proteasomal degradation. In pre-treatment samples from melanoma patients, CD226CD8 T cells correlated with improved progression-free survival following ICB. Our findings argue for the development of therapies aimed at maintaining the expression of CD226.
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http://dx.doi.org/10.1016/j.immuni.2020.09.010DOI Listing
October 2020

The tumour immune landscape and its implications in cutaneous melanoma.

Pigment Cell Melanoma Res 2021 May 1;34(3):529-549. Epub 2020 Oct 1.

Melanoma Institute Australia, The University of Sydney, Sydney, Australia.

The field of tumour immunology has rapidly advanced in the last decade, leading to the advent of effective immunotherapies for patients with advanced cancers. This highlights the critical role of the immune system in determining tumour development and outcome. The tumour immune microenvironment (TIME) is highly heterogeneous, and the interactions between tumours and the immune system are vastly complex. Studying immune cell function in the TIME will provide an improved understanding of the mechanisms underpinning these interactions. This review examines the role of immune cell populations in the TIME based on their phenotype, function and localisation, as well as contextualising their position in the dynamic relationship between tumours and the immune system. We discuss the function of immune cell populations, examine their impact on patient outcome and highlight gaps in current understanding of their roles in the TIME, both in cancers in general and specifically in melanoma. Studying the TIME by evaluating both pro-tumour and anti-tumour effects may elucidate the conditions which lead to tumour growth and metastasis or immune-mediated tumour regression. Moreover, an in-depth understanding of these conditions could contribute to improved prognostication, more effective use of current immunotherapies and guide the development of novel treatment strategies and therapies.
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http://dx.doi.org/10.1111/pcmr.12926DOI Listing
May 2021

Tumor Mutation Burden and Structural Chromosomal Aberrations Are Not Associated with T-cell Density or Patient Survival in Acral, Mucosal, and Cutaneous Melanomas.

Cancer Immunol Res 2020 11 11;8(11):1346-1353. Epub 2020 Sep 11.

Melanoma Institute Australia, The University of Sydney, North Sydney, New South Wales, Australia.

Tumor mutation burden (TMB) has been proposed as a key determinant of immunogenicity in several cancers, including melanoma. The evidence presented thus far, however, is often contradictory and based mostly on RNA-sequencing data for the quantification of immune cell phenotypes. Few studies have investigated TMB across acral, mucosal, and cutaneous melanoma subtypes, which are known to have different TMB. It is also unknown whether chromosomal structural mutations [structural variant (SV) mutations] contribute to the immunogenicity in acral and mucosal melanomas where such aberrations are common. We stained 151 cutaneous and 35 acral and mucosal melanoma patient samples using quantitative IHC and correlated immune infiltrate phenotypes with TMB and other genomic profiles. TMB and SVs did not correlate with the densities of CD8 lymphocytes, CD103 tumor-resident T cells (Trm), CD45RO cells, and other innate and adaptive immune cell subsets in cutaneous and acral/mucosal melanoma tumors, respectively, including in analyses restricted to the site of disease and in a validation cohort. In 43 patients with stage III treatment-naïve cutaneous melanoma, we found that the density of immune cells, particularly Trm, was significantly associated with patient survival, but not with TMB. Overall, TMB and chromosomal structural aberrations are not associated with protective antitumor immunity in treatment-naïve melanoma.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0835DOI Listing
November 2020

Integration of Digital Pathologic and Transcriptomic Analyses Connects Tumor-Infiltrating Lymphocyte Spatial Density With Clinical Response to BRAF Inhibitors.

Front Oncol 2020 14;10:757. Epub 2020 May 14.

Department of Surgery, Massachusetts General Hospital, Boston, MA, United States.

Metastatic melanoma is one of the most immunogenic malignancies due to its high rate of mutations and neoantigen formation. Response to BRAF inhibitors (BRAFi) may be determined by intratumoral immune activation within melanoma metastases. To evaluate whether CD8+ T cell infiltration and distribution within melanoma metastases can predict clinical response to BRAFi, we developed a methodology to integrate immunohistochemistry with automated image analysis of CD8+ T cell position. CD8+ distribution patterns were correlated with gene expression data to identify and quantify "hot" areas within a tumor. Furthermore, the relative activation of CD8+cells, based on transcriptomic analysis, and their relationship to other CD8+ T cells and non-CD8+ cells within the tumor suggested a less crowded distribution of cells around activated CD8+ T cells. Furthermore, the relative activation of these CD8+ T cells was associated with improved clinical outcomes and decreased tumor cell proliferation. This study demonstrates the potential of digital pathomics to incorporate immune cell spatial distribution within metastases and RNAseq analysis to predict clinical response to BRAF inhibition in metastatic melanoma.
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http://dx.doi.org/10.3389/fonc.2020.00757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247820PMC
May 2020

Whole genome landscapes of uveal melanoma show an ultraviolet radiation signature in iris tumours.

Nat Commun 2020 05 15;11(1):2408. Epub 2020 May 15.

QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia.

Uveal melanoma (UM) is the most common intraocular tumour in adults and despite surgical or radiation treatment of primary tumours, ~50% of patients progress to metastatic disease. Therapeutic options for metastatic UM are limited, with clinical trials having little impact. Here we perform whole-genome sequencing (WGS) of 103 UM from all sites of the uveal tract (choroid, ciliary body, iris). While most UM have low tumour mutation burden (TMB), two subsets with high TMB are seen; one driven by germline MBD4 mutation, and another by ultraviolet radiation (UVR) exposure, which is restricted to iris UM. All but one tumour have a known UM driver gene mutation (GNAQ, GNA11, BAP1, PLCB4, CYSLTR2, SF3B1, EIF1AX). We identify three other significantly mutated genes (TP53, RPL5 and CENPE).
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http://dx.doi.org/10.1038/s41467-020-16276-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229209PMC
May 2020

Tumor CD155 Expression Is Associated with Resistance to Anti-PD1 Immunotherapy in Metastatic Melanoma.

Clin Cancer Res 2020 07 28;26(14):3671-3681. Epub 2020 Apr 28.

Immunology in Cancer and Infection Laboratory, QIMR Berghofer Medical Research Institute, Queensland, Australia.

Purpose: Resistance to anti-PD1-based immune checkpoint blockade (ICB) remains a problem for the treatment of metastatic melanoma. Tumor cells as well as host myeloid cells can express the immune checkpoint ligand CD155 to regulate immune cell function. However, the effect of tumor CD155 on the immune context of human melanoma has not been well described. This observational study characterizes tumor CD155 ligand expression by metastatic melanoma tumors and correlates results with differences in immune cell features and response to ICB.

Experimental Design: Pretreatment tumor specimens, from 155 patients with metastatic melanoma treated with ICB and from 50 patients treated with BRAF/MEK-directed targeted therapy, were assessed for CD155 expression by IHC. Intratumor T-cell features were analyzed using multiplex-immunohistofluorescence for CD8, PD1, and SOX10. Correlations were made between CD155 tumor level and bulk tumor RNA sequencing results, as well as clinical RECIST response and progression-free survival.

Results: High pretreatment CD155 tumor levels correlated with high parenchymal PD1CD8/CD8 T-cell ratios (PD1) and poor response to anti-PD1 therapy. In PDL1 negative tumors, high CD155 tumor expression was associated with patients who had poor response to combination anti-PD1/CTLA4 therapy.

Conclusions: Our findings are the first to suggest that tumor CD155 supports an increase in the fraction of PD1CD8 T cells in anti-PD1 refractory melanoma tumors and, further, that targeting the CD155 pathway might improve response to anti-PD1 therapy for patients with metastatic melanoma.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3925DOI Listing
July 2020

Transcriptional downregulation of MHC class I and melanoma de- differentiation in resistance to PD-1 inhibition.

Nat Commun 2020 04 20;11(1):1897. Epub 2020 Apr 20.

Macquarie University, Sydney, NSW, Australia.

Transcriptomic signatures designed to predict melanoma patient responses to PD-1 blockade have been reported but rarely validated. We now show that intra-patient heterogeneity of tumor responses to PD-1 inhibition limit the predictive performance of these signatures. We reasoned that resistance mechanisms will reflect the tumor microenvironment, and thus we examined PD-1 inhibitor resistance relative to T-cell activity in 94 melanoma tumors collected at baseline and at time of PD-1 inhibitor progression. Tumors were analyzed using RNA sequencing and flow cytometry, and validated functionally. These analyses confirm that major histocompatibility complex (MHC) class I downregulation is a hallmark of resistance to PD-1 inhibitors and is associated with the MITF/AXL de-differentiated phenotype and cancer-associated fibroblast signatures. We demonstrate that TGFß drives the treatment resistant phenotype (MITF/AXL) and contributes to MHC class I downregulation in melanoma. Combinations of anti-PD-1 with drugs that target the TGFß signaling pathway and/or which reverse melanoma de-differentiation may be effective future therapeutic strategies.
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http://dx.doi.org/10.1038/s41467-020-15726-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7171183PMC
April 2020

Replacement and desmoplastic histopathological growth patterns in cutaneous melanoma liver metastases: frequency, characteristics, and robust prognostic value.

J Pathol Clin Res 2020 07 18;6(3):195-206. Epub 2020 Apr 18.

Department of Translational Research, Institut Curie, Paris, France.

Among visceral metastatic sites, cutaneous melanoma (CM) metastasises initially to the liver in ~14-20% of cases. Liver metastases in CM patients are associated with both poor prognosis and poor response to immunotherapy. Histopathological growth patterns (HGPs) of liver metastases of the replacement and desmoplastic type, particularly from colorectal cancer and uveal melanoma (UM), may impart valuable biological and prognostic information. Here, we have studied HGP in 43 CM liver metastases resected from 42 CM patients along with other prognostic factors from three institutions. The HGPs (replacement, desmoplastic, pushing) were scored at the metastasis-liver interface with two algorithms: (1) 100% desmoplastic growth pattern (dHGP) and any (≥1%) replacement pattern (any-rHGP) and (2) >50% dHGP, >50% rHGP or mixed (<50% dHGP and/or rHGP, pushing HGP). For 1 patient with 2 metastases, an average was taken to obtain 1 final HGP yielding 42 observations from 42 patients. 22 cases (52%) had 100% dHGP whereas 20 (48%) had any replacement. Cases with rHGP demonstrated vascular co-option/angiotropism. With the development of liver metastasis, only rHGP (both algorithms), male gender and positive resection margins predicted diminished overall survival (p = 0.00099 and p = 0.0015; p = 0.034 and p = 0.024 respectively). On multivariate analysis, only HGP remained significant. 7 of 42 (17%) patients were alive with disease and 21 (50%) died with follow-up after liver metastases ranging from 1.8 to 42.2 months (mean: 20.4 months, median: 19.0 months). 14 (33%) patients with previously-treated metastatic disease had no evidence of disease at last follow up. In conclusion, we report for the first time replacement and desmoplastic HGPs in CM liver metastases and their prognostic value, as in UM and other solid cancers. Of particular importance, any rHGP significantly predicted diminished overall survival while 100% dHGP correlated with increased survival. These results contribute to a better understanding of the biology of CM liver metastases and potentially may be utilised in managing patients with these metastases.
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http://dx.doi.org/10.1002/cjp2.161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7339161PMC
July 2020

Temporal and spatial modulation of the tumor and systemic immune response in the murine Gl261 glioma model.

PLoS One 2020 2;15(4):e0226444. Epub 2020 Apr 2.

Bill Walsh Translational Cancer Research Laboratory, Kolling Institute, The University of Sydney Northern Clinical School and Northern Sydney Local Health District, St Leonards, NSW, Australia.

Glioblastoma, the most aggressive form of glioma, has a 5-year survival rate of <5%. While radiation and immunotherapies are routinely studied in the murine Gl261 glioma model, little is known about its inherent immune response. This study quantifies the temporal and spatial localization of immune cell populations and mediators during glioma development. Eight-week old male C57Bl/6 mice were orthotopically inoculated with 1x106 Gl261 cells and tumor morphology, local and systemic immune cell populations, and plasma cytokines/chemokines assessed at day 0, 1, 3, 7, 14, and 21 post-inoculation by magnetic resonance imaging, chromogenic immunohistochemistry, multiplex immunofluorescent immunohistochemistry, flow cytometry and multiplex immunoassay respectively. From day 3 tumors were distinguishable with >30% Ki67 and increased tissue vascularization (p<0.05). Increasing tumor proliferation/malignancy and vascularization were associated with significant temporal changes in immune cell populations within the tumor (p<0.05) and systemic compartments (p = 0.02 to p<0.0001). Of note, at day 14 16/24 plasma cytokine/chemokines levels decreased coinciding with an increase in tumor cytotoxic T cells, natural killer and natural killer/T cells. Data derived provide baseline characterization of the local and systemic immune response during glioma development. They reveal that type II macrophages and myeloid-derived suppressor cells are more prevalent in tumors than regulatory T cells, highlighting these cell types for further therapeutic exploration.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0226444PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7117758PMC
July 2020

Close proximity of immune and tumor cells underlies response to anti-PD-1 based therapies in metastatic melanoma patients.

Oncoimmunology 2020 16;9(1):1659093. Epub 2019 Oct 16.

Melanoma Institute Australia, The University of Sydney, Sydney, Australia.

Immune checkpoint blockade has greatly improved the clinical outcomes of many patients with metastatic melanoma, however, almost half do not respond. Whether the interspatial distribution of immune and tumor cells predicts response to anti-PD-1-based therapies and patient outcomes in any cancer, including melanoma, is currently unknown. Here, we examined the spatial distribution of immune and tumor cells via multiplex immunofluorescence. Pre-treatment melanoma specimens from 27 patients ( = 18 responders; = 9 non-responders) treated with anti-PD-1 monotherapy and 34 patients ( = 22 responders; = 12 non-responders) treated with combined ipilimumab and anti-PD-1 immunotherapy were studied. Responders displayed significantly higher densities of CD8 tumor-infiltrating lymphocytes within a 20 µM distance from a melanoma cell compared to non-responders in both anti-PD-1 alone ( = .0024) and combination-treated patients ( = .0096), that were associated with improved progression-free survival for both therapies (anti-PD-1 = .0158; combination therapy = .0088). In multivariate analysis, the best model for 12-month progression-free survival for anti-PD-1 monotherapy included PD-L1 cells within proximity to tumor cells and intratumoral CD8 density (AUC = 0.80), and for combination therapy included CD8 cells in proximity to tumor cells, intratumoral PD-L1 density and LDH (AUC = 0.85). Assessment of the spatial distribution of immune cells in relation to tumor cells provides insight into their role in modulating immune response and highlights their potential role as predictors of response to anti-PD-1 based therapies.
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http://dx.doi.org/10.1080/2162402X.2019.1659093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959449PMC
October 2019

Molecular Profiling of Noncoding Mutations Distinguishes Nevoid Melanomas From Mitotically Active Nevi in Pregnancy.

Am J Surg Pathol 2020 03;44(3):357-367

Royal Prince Alfred Hospital.

The accurate recognition of subtle melanomas and their distinction from benign mimics is an oft-recurring diagnostic problem, critical for patient management. Melanomas that bear resemblance to benign nevi (so-called nevoid melanomas, NMs) and benign mitotically active nevi in pregnancy (MANP) are 2 lesions particularly prone to error. Molecular data, including analysis of noncoding regions, in MANP and NM are very limited. This study sought to identify differences in clinical, pathologic, and molecular characteristics between MANP and NMs to facilitate correct diagnosis and reduce the risk of overtreatment or undertreatment. Clinicopathologic characteristics of NM (n=18) and MANP (n=30) were evaluated, and mutation data were analyzed using next-generation sequencing for available cases in each group (NM, n=8; MANP, n=12). All MANP showed innocent histopathologic characteristics apart from increased mitotic activity, frequently in both superficial and deep parts of the lesion (median dermal mitotic rate: 2/mm, range: 1 to 7/mm). All cases of NM demonstrated a characteristic nevoid silhouette, subtle atypical architectural and cytologic features, and variable mitoses (median mitotic rate: 3/mm, range: 1 to 5/mm). Median NM tumor thickness was 1.4 mm. Four of 10 NM patients with follow-up had metastatic disease, including 3 patients who developed widespread metastases, with 1 disease-related death. No other recurrences have been identified (follow-up period: 24 to 60 mo). None of the 15 MANP patients with available follow-up had a recurrence. Most NMs harbored hotspot mutations in NRAS (6/8, 75%). Noncoding mutations were significantly more common in NMs than in MANP (median: 4 vs. 0, P=0.0014). Copy number alterations were infrequent but, when present, were seen in NMs (3/8 NMs vs. 0/12 MANP). All NMs but only 1 of 12 MANP had >1 abnormality in the noncoding regions. Similar to conventional common acquired nevi, MANP mostly harbored driver BRAF mutations, while activating NRAS mutations, noncoding mutations, and copy number alterations were rare. NM and MANP have subtle but recognizable distinguishing histopathologic characteristics that are underpinned by molecular differences. Mutation analysis of targeted noncoding mutations may assist in the diagnosis of difficult lesions.
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http://dx.doi.org/10.1097/PAS.0000000000001406DOI Listing
March 2020

The prognostic value of tumor mitotic rate in children and adolescents with cutaneous melanoma: A retrospective cohort study.

J Am Acad Dermatol 2020 Apr 2;82(4):910-919. Epub 2019 Nov 2.

Melanoma Institute Australia, Sydney, New South Wales, Australia; Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia; Department of Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Sydney, New South Wales, Australia. Electronic address:

Background: Mitotic rate is a strong predictor of outcome in adult patients with primary cutaneous melanoma, but for children and adolescent patients this is unknown.

Objective: We sought to assess the prognostic value of primary tumor mitotic rate in children and adolescents with primary melanoma.

Methods: This was a cohort study of 156 patients who were <20 years of age and who had clinically localized cutaneous melanoma. Patients <12 years of age were classified as children and those 12 to 19 years of age as adolescents. Clinicopathologic and outcome data were collected. Recurrence-free and melanoma-specific survival were calculated. Univariable and multivariable analyses were performed using Cox proportional hazard models.

Results: Thirteen of 156 patients (8%) were children. The mitotic rate was ≥1/mm in 104 patients (67%) and correlated with increasing Breslow thickness. A positive sentinel node was found in 23 of 61 patients (38%) in whom a sentinel lymph node biopsy specimen was obtained. The median follow-up was 61 months. Five-year melanoma-specific and recurrence-free survival rates were 91% and 84%, respectively. Mitotic rate was a stronger predictor of outcome than tumor thickness and was the only factor independently associated with recurrence-free survival.

Limitations: This research was conducted at a single institution and the sample size was small.

Conclusion: Mitotic rate is an independent predictor of recurrence-free survival in children and adolescents with clinically localized melanoma.
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http://dx.doi.org/10.1016/j.jaad.2019.10.065DOI Listing
April 2020

Macrophage-Derived CXCL9 and CXCL10 Are Required for Antitumor Immune Responses Following Immune Checkpoint Blockade.

Clin Cancer Res 2020 01 21;26(2):487-504. Epub 2019 Oct 21.

Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.

Purpose: Response rates to immune checkpoint blockade (ICB; anti-PD-1/anti-CTLA-4) correlate with the extent of tumor immune infiltrate, but the mechanisms underlying the recruitment of T cells following therapy are poorly characterized. A greater understanding of these processes may see the development of therapeutic interventions that enhance T-cell recruitment and, consequently, improved patient outcomes. We therefore investigated the chemokines essential for immune cell recruitment and subsequent therapeutic efficacy of these immunotherapies.

Experimental Design: The chemokines upregulated by dual PD-1/CTLA-4 blockade were assessed using NanoString-based analysis with results confirmed at the protein level by flow cytometry and cytometric bead array. Blocking/neutralizing antibodies confirmed the requirement for key chemokines/cytokines and immune effector cells. Results were confirmed in patients treated with immune checkpoint inhibitors using single-cell RNA-sequencing (RNA-seq) and paired survival analyses.

Results: The CXCR3 ligands, CXCL9 and CXCL10, were significantly upregulated following dual PD-1/CTLA-4 blockade and both CD8 T-cell infiltration and therapeutic efficacy were CXCR3 dependent. In both murine models and patients undergoing immunotherapy, macrophages were the predominant source of CXCL9 and their depletion abrogated CD8 T-cell infiltration and the therapeutic efficacy of dual ICB. Single-cell RNA-seq analysis of patient tumor-infiltrating lymphocytes (TIL) revealed that CXCL9/10/11 was predominantly expressed by macrophages following ICB and we identified a distinct macrophage signature that was associated with positive responses to ICB.

Conclusions: These data underline the fundamental importance of macrophage-derived CXCR3 ligands for the therapeutic efficacy of ICB and highlight the potential of manipulating this axis to enhance patient responses.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-1868DOI Listing
January 2020

Novel Immune Targets in Melanoma-Response.

Clin Cancer Res 2019 09;25(17):5424-5425

Melanoma Institute Australia, The University of Sydney, North Sydney, New South Wales, Australia.

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http://dx.doi.org/10.1158/1078-0432.CCR-19-1866DOI Listing
September 2019

Whole-genome landscape of mucosal melanoma reveals diverse drivers and therapeutic targets.

Nat Commun 2019 07 18;10(1):3163. Epub 2019 Jul 18.

Department of Pathology, University of California, San Francisco, CA, 94143, USA.

Knowledge of key drivers and therapeutic targets in mucosal melanoma is limited due to the paucity of comprehensive mutation data on this rare tumor type. To better understand the genomic landscape of mucosal melanoma, here we describe whole genome sequencing analysis of 67 tumors and validation of driver gene mutations by exome sequencing of 45 tumors. Tumors have a low point mutation burden and high numbers of structural variants, including recurrent structural rearrangements targeting TERT, CDK4 and MDM2. Significantly mutated genes are NRAS, BRAF, NF1, KIT, SF3B1, TP53, SPRED1, ATRX, HLA-A and CHD8. SF3B1 mutations occur more commonly in female genital and anorectal melanomas and CTNNB1 mutations implicate a role for WNT signaling defects in the genesis of some mucosal melanomas. TERT aberrations and ATRX mutations are associated with alterations in telomere length. Mutation profiles of the majority of mucosal melanomas suggest potential susceptibility to CDK4/6 and/or MEK inhibitors.
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http://dx.doi.org/10.1038/s41467-019-11107-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6639323PMC
July 2019

Neoadjuvant dabrafenib combined with trametinib for resectable, stage IIIB-C, BRAF mutation-positive melanoma (NeoCombi): a single-arm, open-label, single-centre, phase 2 trial.

Lancet Oncol 2019 07 3;20(7):961-971. Epub 2019 Jun 3.

Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia; Royal North Shore Hospital, St Leonards, Sydney, NSW, Australia.

Background: Adjuvant dabrafenib plus trametinib therapy improves relapse-free survival in patients with resected stage III melanoma. We aimed to ascertain the proportion of patients who would have a pathological response and a response according to Response Evaluation Criteria in Solid Tumors (RECIST) after neoadjuvant dabrafenib plus trametinib therapy for resectable clinical stage III melanoma.

Methods: NeoCombi was a single-arm, open-label, single-centre, phase 2 study done at Melanoma Institute Australia (Sydney, NSW, Australia). Eligible patients were adults (aged ≥18 years) with histologically confirmed, resectable, RECIST-measurable, clinical stage IIIB-C (American Joint Committee on Cancer [AJCC] 7th edition), BRAF-mutant melanoma, and had an Eastern Cooperative Oncology Group performance status of 1 or lower. Patients received 150 mg dabrafenib orally, twice daily, plus 2 mg trametinib orally, once daily, for 52 weeks (12 weeks of neoadjuvant therapy before complete resection of the pre-therapy tumour bed, and 40 weeks of adjuvant therapy thereafter). CT and PET scans were done at baseline and before resection. The primary outcomes were the proportion of patients achieving a complete pathological response and the proportion of patients achieving a response according to RECIST at week 12, analysed as per protocol. This trial is registered with ClinicalTrials.gov, NCT01972347, and follow-up of patients is ongoing.

Findings: Between Aug 20, 2014, and April 19, 2017, 40 patients were screened, of whom 35 eligible patients were enrolled, received neoadjuvant dabrafenib plus trametinib, and underwent resection. At the data cutoff (Sept 24, 2018), median follow-up was 27 months (IQR 21-36). At resection, 30 (86%) patients achieved a RECIST response; 16 (46%; 95% CI 29-63) had a complete response and 14 (40%; 24-58) had a partial response. Five patients (14%; 95% CI 5-30) had stable disease, and no patients progressed. After resection and pathological evaluation, all 35 patients achieved a pathological response, of whom 17 (49%; 95% CI 31-66) patients had a complete pathological response and 18 (51%; 95% CI 34-69) had a non-complete pathological response. Treatment-related serious adverse events occurred in six (17%) of 35 patients and grade 3-4 adverse events occurred in ten (29%) patients. No treatment-related deaths were reported.

Interpretation: Neoadjuvant dabrafenib plus trametinib therapy could be considered in the management of RECIST-measurable resectable stage III melanoma as it led to a high proportion of patients achieving a complete response according to RECIST and a high proportion of patients achieving a complete pathological response, with no progression during neoadjuvant therapy.

Funding: GlaxoSmithKline; Novartis; National Health and Medical Research Council, Australia; and Melanoma Institute Australia.
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http://dx.doi.org/10.1016/S1470-2045(19)30331-6DOI Listing
July 2019

LNK suppresses interferon signaling in melanoma.

Nat Commun 2019 05 20;10(1):2230. Epub 2019 May 20.

Cancer Science Institute of Singapore, National University of Singapore, Singapore, 117599, Singapore.

LNK (SH2B3) is a key negative regulator of JAK-STAT signaling which has been extensively studied in malignant hematopoietic diseases. We found that LNK is significantly elevated in cutaneous melanoma; this elevation is correlated with hyperactive signaling of the RAS-RAF-MEK pathway. Elevated LNK enhances cell growth and survival in adverse conditions. Forced expression of LNK inhibits signaling by interferon-STAT1 and suppresses interferon (IFN) induced cell cycle arrest and cell apoptosis. In contrast, silencing LNK expression by either shRNA or CRISPR-Cas9 potentiates the killing effect of IFN. The IFN-LNK signaling is tightly regulated by a negative feedback mechanism; melanoma cells exposed to IFN upregulate expression of LNK to prevent overactivation of this signaling pathway. Our study reveals an unappreciated function of LNK in melanoma and highlights the critical role of the IFN-STAT1-LNK signaling axis in this potentially devastating disease. LNK may be further explored as a potential therapeutic target for melanoma immunotherapy.
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http://dx.doi.org/10.1038/s41467-019-09711-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6527565PMC
May 2019

Recurrent hotspot SF3B1 mutations at codon 625 in vulvovaginal mucosal melanoma identified in a study of 27 Australian mucosal melanomas.

Oncotarget 2019 Jan 29;10(9):930-941. Epub 2019 Jan 29.

Melanoma Institute Australia, The University of Sydney, Sydney, Australia.

Introduction: Clinical outcomes for mucosal melanomas are often poor due to a lack of effective systemic drug therapies. Identifying driver genes in mucosal melanoma may enhance the understanding of disease pathogenesis and provide novel opportunities to develop effective therapies.

Results: Somatic variant analysis identified (6 of 27: 22%) as the most commonly mutated gene, followed by (3 of 27: 11%). Other less frequently mutated genes (4% otherwise stated) included (7%), (7%), , , , , , , and . Recurrent SF3B1 p.R625 hotspot mutations were exclusively detected in vulvovaginal (5 of 19: 26%) and anorectal melanomas (3 of 5:60%). The only other SF3B1 mutation was a p.C1123Y mutation that occurred in a conjunctival mucosal melanoma.-mutated patients were associated with shorter overall survival (OS; 34.9 months) and progression-free survival (PFS; 16.9 months) compared to non--mutated patients (OS: 79.7 months, log-rank = 0.1172; PFS: 35.7 months, log-rank = 0.0963).

Conclusion: Molecular subgroups of mucosal melanoma with mutations occurred predominantly in the vulvovaginal region. mutations may have a negative prognostic impact.

Methods: Formalin-fixed biopsies were collected from 27 pathologically-confirmed mucosal melanomas. Genomic DNA was isolated from the tumor tissue and sequenced using a novel dual-strand amplicon sequencing technique to determine the frequency and types of mutations across 45 target genes.
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http://dx.doi.org/10.18632/oncotarget.26584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398173PMC
January 2019

Prevalence and Cellular Distribution of Novel Immune Checkpoint Targets Across Longitudinal Specimens in Treatment-naïve Melanoma Patients: Implications for Clinical Trials.

Clin Cancer Res 2019 06 18;25(11):3247-3258. Epub 2019 Feb 18.

Melanoma Institute Australia, The University of Sydney, North Sydney, New South Wales, Australia.

Purpose: Immunotherapies targeting costimulating and coinhibitory checkpoint receptors beyond PD-1 and CTLA-4 have entered clinical trials. Little is known about the relative abundance, coexpression, and immune cells enriched for each specific drug target, limiting understanding of the biological basis of potential treatment outcomes and development of predictive biomarkers for personalized immunotherapy. We sought to assess the abundance of checkpoint receptors during melanoma disease progression and identify immune cells enriched for them. Multiplex immunofluorescence staining for immune checkpoint receptors (ICOS, GITR, OX40, PD-1, TIM-3, VISTA) was performed on 96 melanoma biopsies from 41 treatment-naïve patients, including patient-matched primary tumors, nodal metastases, and distant metastases. Mass cytometry was conducted on tumor dissociates from 18 treatment-naïve melanoma metastases to explore immune subsets enriched for checkpoint receptors.

Results: A small subset of tumor-infiltrating leukocytes expressed checkpoint receptors at any stage of melanoma disease. GITR and OX40 were the least abundant checkpoint receptors, with <1% of intratumoral T cells expressing either marker. ICOS, PD-1, TIM-3, and VISTA were most abundant, with TIM-3 and VISTA mostly expressed on non-T cells, and TIM-3 enriched on dendritic cells. Tumor-resident T cells (CD69/CD103/CD8) were enriched for TIGIT (>70%) and other coinhibitory but not costimulatory receptors. The proportion of GITR T cells decreased from primary melanoma (>5%) to lymph node (<1%, = 0.04) and distant metastases (<1%, = 0.0005).

Conclusions: This study provides the first comprehensive assessment of immune checkpoint receptor expression in any cancer and provides important data for rational selection of targets for trials and predictive biomarker development.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-4011DOI Listing
June 2019

Molecular Genomic Profiling of Melanocytic Nevi.

J Invest Dermatol 2019 08 14;139(8):1762-1768. Epub 2019 Feb 14.

Melanoma Institute Australia, The University of Sydney, New South Wales, Australia; Sydney Medical School, The University of Sydney, Sydney, New South Wales, Australia.

The benign melanocytic nevus is the most common tumor in humans and rarely transforms into cutaneous melanoma. Elucidation of the nevus genome is required to better understand the molecular steps of progression to melanoma. We performed whole genome sequencing on a series of 14 benign melanocytic nevi consisting of both congenital and acquired types. All nevi had driver mutations in the MAPK signaling pathway, either BRAF V600E or NRAS Q61R/L. No additional definite driver mutations were identified. Somatic mutations in nevi with higher mutation loads showed a predominance of mutational signatures 7a and 7b, consistent with UVR exposure, whereas nevi with lower mutation loads (including all three congenital nevi) had a predominance of the ubiquitous signatures 1 and 5. Two nevi had mutations in promoter regions predicted to bind E26 transformation-specific family transcription factors, as well as subclonal mutations in the TERT promoter. This paper presents whole genome data from melanocytic nevi. We confirm that UVR is involved in the etiology of a subset of nevi. This study also establishes that TERT promoter mutations are present in morphologically benign skin nevi in subclonal populations, which has implications regarding the interpretation of this emerging biomarker in sensitive assays.
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http://dx.doi.org/10.1016/j.jid.2018.12.033DOI Listing
August 2019

Distinct Immune Cell Populations Define Response to Anti-PD-1 Monotherapy and Anti-PD-1/Anti-CTLA-4 Combined Therapy.

Cancer Cell 2019 02;35(2):238-255.e6

Melanoma Institute Australia, The University of Sydney, Sydney, NSW 2065, Australia; Sydney Medical School, The University of Sydney, Sydney, NSW 2006, Australia. Electronic address:

Cancer immunotherapies provide survival benefits in responding patients, but many patients fail to respond. Identifying the biology of treatment response and resistance are a priority to optimize drug selection and improve patient outcomes. We performed transcriptomic and immune profiling on 158 tumor biopsies from melanoma patients treated with anti-PD-1 monotherapy (n = 63) or combined anti-PD-1 and anti-CTLA-4 (n = 57). These data identified activated T cell signatures and T cell populations in responders to both treatments. Further mass cytometry analysis identified an EOMESCD69CD45RO effector memory T cell phenotype that was significantly more abundant in responders to combined immunotherapy compared with non-responders (n = 18). The gene expression profile of this population was associated with longer progression-free survival in patients treated with single agent and greater tumor shrinkage in both treatments.
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http://dx.doi.org/10.1016/j.ccell.2019.01.003DOI Listing
February 2019

Author Correction: Tissue-resident memory CD8 T cells promote melanoma-immune equilibrium in skin.

Nature 2019 02;566(7745):E10

Department of Microbiology and Immunology, , The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.

Panel j was inadvertently labelled as panel k in the caption to Fig. 4. Similarly, 'Fig. 4k' should have been 'Fig. 4j' in the sentence beginning 'TNF-α-deficient gBT-I cells were…'. In addition, the surname of author Umaimainthan Palendira was misspelled 'Palendria'. These errors have been corrected online.
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http://dx.doi.org/10.1038/s41586-019-0958-0DOI Listing
February 2019

Integrated molecular and immunophenotypic analysis of NK cells in anti-PD-1 treated metastatic melanoma patients.

Oncoimmunology 2019;8(2):e1537581. Epub 2018 Oct 31.

Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia.

: Anti-PD-1 therapy has revolutionized the treatment and improved the survival of stage IV melanoma patients. However, almost half of the patients fail to respond due to immune evasive mechanism. A known mechanism is the downregulation of major histocompatibility complex (MHC) class I expression, which prevents T cell recognition of the tumor. This study determined the relationship between natural killer (NK) cell numbers and clinical response to anti-PD-1 therapy in metastatic melanoma. : Twenty-five anti-PD-1 treated metastatic melanoma patients were categorized into responders (complete response (CR)/partial response (PR)/stable disease (SD) ≥ 6 mo, n = 13) and non-responders (SD < 6 days/progressive disease (PD), n = 12) based on RECIST response. Whole transcriptome sequencing and multiplex immunofluorescent staining were performed on pre-treatment and on a subset of early during treatment tumor samples. Spatial distribution analysis was performed on multiplex immunofluorescent images to determine the proximity of NK cells to tumor cells. Flow cytometry was used to confirm NK phenotypes in lymph node metastases of treatment naïve melanoma patients (n = 5). Cytotoxic assay was performed using NK cells treated with anti-PD-1 or with isotype control and co-cultured with 3 different melanoma cell lines and with K562 cells (leukemia cell line). : Differential expression analysis identified nine upregulated NK cell specific genes (adjusted p < 0.05) in responding (n = 11) versus non-responding patients (n = 10). Immunofluorescent staining of biopsies confirmed a significantly higher density of intra- and peri-tumoral CD16+ and granzyme B + NK cells in responding patients (< 0.05). Interestingly, NK cells were in closer proximity to tumor cells in responding PD-1 treated patients compared to non-responding patients. Patients who responded to anti-PD-1 therapy, despite MHC class I loss had higher NK cell densities than patients with low MHC class I expression. Lastly, functional assays demonstrated PD-1 blockade induces an increase in NK cells' cytotoxicity. : A higher density of tumoral NK cells is associated with response to anti-PD-1 therapy. NK cells may play an important role in mediating response to anti-PD-1 therapy, including in a subset of tumors downregulating MHC class I expression.
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http://dx.doi.org/10.1080/2162402X.2018.1537581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343795PMC
October 2018