We thank Nocentini and colleagues for their interest in our recent work in which we reported on the prevalence and distribution of the expression of novel checkpoint targets in melanoma, as published in Clinical Cancer Research (1). In their letter, the authors state that anti-GITR clone 621, an antibody used in our CyTOF analysis, but not immunohistochemical experiments, may not be as sensitive for detecting GITR as other clones. To address possible differences in the sensitivity of different anti-GITR antibodies, we performed a direct comparison of GITR staining by anti-GITR clone 621 and anti-GITR clone DT5D3 (the clone recommended by Nocentini and colleagues) using flow cytometry and compared the levels of GITR detected on immune cells in three human melanoma tumor dissociates.We found that the average levels of GITR detected on immune cell subsets was comparable between the two clones, albeit with 2% average increase in GITR being detected on CD4+ T cells by clone DT5D3, and elevated levels of GITR seen on myeloid cells with clone 621 (Fig. 1A). We note, however, that there were differences in the level of GITR detected by the two clones within the same tumor sample in some cases (Fig. 1B). Nonetheless, the results provided from anti-GITR clone DT5D3 suggest that <1% of CD8 T cells, approximately 2%–3% of CD4 T cells, and approximately 1%–2% of total T cells in the tumor microenvironment express GITR (Fig. 1A), reaffirming our previous conclusion that GITR is minimally expressed on T cells in the tumor microenvironment. While we previously reported an average 2% expression of GITR on B cells and monocytes with anti-GITR clone 621, results utilizing clone DT5D3 show that GITR expression on these cells was <1%.In one of the three melanoma tumor dissociates, we found sufficient numbers of FoxP3+ CD25hi T-regulatory cells to compare levels of GITR detected by anti-GITR clone DT5D3 and anti-GITR clone 621. In this sample, the anti-GITR clone DT5D3 did detect a significantly higher proportion of GITR+ regulatory T cells than with the anti-GITR clone 621, and with better resolution of GITR+ cells (Fig. 1C). Indeed, anti-GITR clone DT5D3 resolved GITR-positive cells better than anti-GITR clone 621 on all immune cell subsets examined (Fig. 1D).In conclusion, while differences in GITR expression levels were observed between anti-GITR clone DT5D3 and anti-GITR clone 621, the overall results from both antibodies were comparable, with the notable exception of GITR staining on regulatory T cells. The results provided by anti-GITR clone DT5D3 reaffirm our conclusions on low level GITR expression in the tumor microenvironment in melanoma, with the exception that a higher proportion of regulatory T cells are likely to be positive for GITR, previously undetected by anti-GITR clone 621.See the original Letter to the Editor, p. 5422G. V. Long is a consultant/advisory board member for Aduro, Amgen, Array Biopharma, Bristol-Myers Squibb, Merck Sharpe & Dohme, Novartis, Oncosec, Pierre Fabre, and Roche. R.A. Scolyer is a consultant/advisory board member for Merck Sharp & Dohme, Novartis, Myriad, and NeraCare. No potential conflicts of interest were disclosed by the other authors.This work was supported by Melanoma Institute Australia, the New South Wales Department of Health, NSW Health Pathology, the National Health and Medical Research Council of Australia (NHMRC) and Cancer Institute NSW. J.S. Wilmott, R.A. Scolyer, and G. Long are supported by NHMRC Fellowships. U. Palendira and A. Ferguson are supported by the Cancer Council NSW (RG18-08). G. Long is supported by the Melanoma Foundation of the University of Sydney through the University of Sydney Medical Foundation. J. Edwards is supported by the Research Training Program and Sydney Catalyst scholarships at The University of Sydney.
Abstract Motivation: Gene annotation and pathway databases such as Gene Ontology and Kyoto Encyclopedia of Genes and Genomes are important tools in Gene Set Test (GST) that describe gene biological functions and associated pathways. GST aims to establish an association relationship between a gene set of interest and an annotation. Importantly, GST tests for over-representation of genes in an annotation term. One implicit assumption of GST is that the gene expression platform captures the complete or a very large proportion of the genome. However, this assumption is neither satisfied for the increasingly popular boutique array nor the custom designed gene expression profiling platform. Specifically, conventional GST is no longer appropriate due to the gene set selection bias induced during the construction of these platforms. Results: We propose bcGST, a bias-corrected Gene Set Test by introducing bias correction terms in the contingency table needed for calculating the Fisher’s Exact Test (FET). The adjustment method works by estimating the proportion of genes captured on the array with respect to the genome in order to assist filtration of annotation terms that would otherwise be falsely included or excluded. We illustrate the practicality of bcGST and its stability through multiple differential gene expression analyses in melanoma and TCGA cancer studies. Availability: The bcGST method is made available as a Shiny web application at http://shiny.maths.usyd.edu.au/bcGST/ Contact: kevin.wang@sydney.edu.au
9518 Background: Immune checkpoint blockade improves the survival of patients with metastatic melanoma, but many patients fail to respond to immunotherapy and the lack of accurate predictors of response or progression remains a major clinical problem. We investigated potential mechanisms of response and resistance to anti-PD1 +/- ipilimumab. Methods: 141 melanoma biopsies from advanced melanoma pts treated with anti-PD-1 monotherapy (n= 54), or anti-PD1 + ipilimumab (n= 51) were classified as responders (CR/PR/SD > 6 mo) or non-responders (SD≤6 mo/PD) based on RECIST. The transcriptomic and immunophenotypic profiles of 105 baseline (PRE) and 36 early-during treatment (EDT) tumor biopsies from pts treated with monotherapy (n= 33 responders, n= 21 non-responders) or anti-PD-1 + ipilimumab (n= 38 responders, n= 13 non-responders) were characterized via RNA sequencing and multiplex immunofluorescence. Results: Responders to monotherapy displayed increased expression of genes associated with a Type 1 interferon response, tissue-resident T-cells and drug targets (TIGIT, ADAR, ADORA2A, CD137, IDO1 and LAG3) (diff. p < 0.05). Genes unique to anti-PD-1 + ipilimumab responders included T-cell and NK-cell genes EOMES, CD48, CD96, and FASLG. Non-responders displayed significantly higher expression of genes associated with WNT signaling along with novel hypoxic and metabolic pathways, including CA9 and NABP1 (p < 0.05). Non-responders with high CD8/PD-L1 densities expressed novel immune drug targets (IDO1 expressed by 37% of monotherapy non-responders, ICOS (37%), TNFRSF9 (26%), LAG3 (16%), TIGIT (16%) and ADORA2A (16%)). In contrast, TIL-low tumors displayed a lack of expression of the aforementioned targets (42% of monotherapy and 86% of anti-PD-1 + ipilimumab non-responders). Conclusions: These findings demonstrate that combinations of novel drug targets may provide clinical benefits in non-responding and non-CR responding patients with high TILs. TIL-low non-responders may require modulation of WNT, hypoxic and metabolic pathways to overcome resistance, facilitating the development of novel synergistic drug targets.
Read moreA multidisciplinary working party established by Cancer Council Australia has recently produced up-to-date, evidence-based clinical practice guidelines for the management of melanoma and LM. Following selection of the most relevant clinical questions, a comprehensive literature search for relevant studies was conducted, followed by systematic review of these studies. Data were summarised and the evidence was assessed, leading to the development of recommendations. After public consultation and approval by the full guidelines working party, these recommendations were published on the Cancer Council Australia wiki platform (https://wiki.cancer.org.au/australia/Clinical_question:Effective_interventions_to_improve_outcomes_in_lentigo_maligna%3F). Main Recommendations: Surgical removal of LM remains the standard treatment, with 5- to 10-mm clinical margins when possible. While yet to be fully validated, the use of peri-operative reflectance confocal microscopy to assess margins should be considered where available. There is a lack of high-quality evidence to infer the most effective non-surgical treatment. When surgical removal of LM is not possible or refused, radiotherapy is recommended. When both surgery and radiotherapy are not appropriate or refused, topical imiquimod is the recommended treatment. Cryotherapy and laser therapy are not recommended for the treatment of LM.
Read moreAbstract 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.
Read more9558 Background: Desmoplastic melanoma (DM) is a rare subtype of melanoma characterized by a dense fibrous stroma, resistance to chemotherapy and no actionable driver mutation for targeted therapy. We investigated the efficacy of PD-1/L1 inhibitors and correlation with genetic landscape and tumor immune microenvironment in DM. Methods: Retrospective analysis of 1054 pts with melanoma treated with anti-PD-1/L1, resulting in 57 pts with unresectable or metastatic DM. Available baseline biopsies were analysed by digital quantitative immunohistochemistry (IHC) for CD8 and PD-L1 and by whole exome sequencing (WES), compared to available tissue from non-DM pts treated with anti-PD1/L1 at UCLA. Results: At a median follow up of 20 mo, 40 pts (70%, 95% CI 57-82) had an objective response by RECIST 1.1 criteria, including 18 (45%) CRs with no relapse observed to date. Responses were similar in DM subsets (23 pure, 29 mixed and 5 indeterminate). Kaplan-Meier estimated 1-year and 2-year overall survival were 85% (95% CI 78-98) and 74% (95% CI 64-89). WES revealed a median of 1282 (interquartile range 517-1692) non-synonymous somatic mutations per tumor in DM tumors (n = 17), significantly higher (p = 0.02) than the median of 462 (interquartile range 230-1150) in non-DM (n = 23). Mutations in NF-1 were the most common (13/17) followed by loss-of-function TP53 and ARID2, and > 82% of single nucleotide mutations were UV damage signatures. IHC analysis from 19 DM and 13 non-DM revealed a strikingly higher percentage of PD-L1 positive cells in the tumor parenchyma in DM (p = 0.04), highly associated with CD8 density and PD-L1 expression in the tumor invasive margins, indicating an active adaptive immune response. No genetic mechanisms known to cause constitutive PD-L1 expression were detected in these samples. Conclusions: Patients with advanced DM derive significant clinical benefit from PD-1/L1 inhibitors, likely related to the high mutational burden and a highly active adaptive immune response as the main mechanism of immune escape prior to therapy. Our results challenge the general conception that dense fibrous stroma around the malignant cells interferes with immune cell infiltration and efficacy of immunotherapy.
Read moreAbstract Introduction: Melanoma accounts for 5% of all skin cancers, yet is the most common cause of skin cancer-related deaths, and is the commonest lethal malignancy in young people (&lt;40yrs). Patients with BRAF mutant melanoma show survival benefit from MAPK pathway inhibition, although response rates vary. We conducted proteomic screening using mass spectrometry to detect cellular processes that might predict response to MEK inhibition more effectively than genotyping alone. Methods: Protein extracts from early passages of ten AJCC Stage III local lymph node metastatic melanoma cell lines with known MAPK mutational status (BRAFmut, NRASmut, MAPKwt,) were used. Further, 32 fresh frozen NRASmut or BRAFmut AJCC Stage IIIC metastatic melanoma (lymph node metastases) specimens were obtained from patients with varying survival. Lysed cells and tissue were digested with trypsin and analysed by LC-MS/MS using a TripleTOF 5600/6600 mass spectrometer. Data-independent acquisition (or SWATH-MS) was used to match multiplexed MS/MS spectra against a spectral ion library enabling relative protein quantification. Cell line viability in the presence of 2µM AZD6244 (MEKi) was determined using a PrestoBlue assay. Results: The SWATH-MS approach enabled quantification of ~2500 proteins across all 10 cell lines. Principal component analysis clearly segregated melanomas based on in vitro sensitivity to MEK inhibition, whereas genotype alone did not. In total, we show 57 proteins whose abundances correlate (r²&gt;0.75) with response to MEKi, revealing roles in cell pigmentation biosynthesis, lipid metabolism, adherence and inter-cell communication. Survival analysis demonstrated that patients whose cell lines were responsive to MEKi had lower mortality than patients whose cells were resistant to MEKi (survival &gt;7.5 years MEKi sensitive versus &lt;1.7 years MEKi resistant, p=0.01). Across the 32 tumour samples, ~2000 proteins were quantifiable. The proteome phenotype associated with MEK inhibition observed in patient cell lines was a dominant signal in fresh frozen stage III metastatic melanoma specimens, suggesting these patients may have profited from MAPK pathway inhibition had these drugs been available. In this patient cohort neither the genotype nor proteome phenotype correlated with survival. Conclusion: Proteomic profiling by mass spectrometry demonstrated a MEK inhibition phenotype in early passage cell lines and fresh frozen melanoma metastases which may provide utility for selecting patients to treat with MEK drugs. Citation Format: Christoph Krisp, Robert Parker, Dana Pascovici, James Wilmott, John F. Thompson, Graham J. Mann, Richard A. Scolyer, Nicholas K. Hayward, Mark P. Molloy. Proteome phenotype of stage III metastatic melanoma and response to MEK inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2206. doi:10.1158/1538-7445.AM2017-2206
Read more<b>Purpose</b>: 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. <b>Experimental Design</b>: 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). <b>Results</b>: 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 (<i>p </i>< 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. <b>Conclusions</b>: 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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