Melanoma Dataset Research Articles

Abstract 4034: DCLK1 is upregulated in melanoma and it is a novel predictive marker for survival and response

Abstract Melanoma is the deadliest form of all skin cancer, and the incidence of melanoma has been rising for the last 30 years. There are only a few reported biomarkers for prediction and prognosis for melanoma, therefore, there is an unmet medical need to identify predictive and prognostic biomarkers. Doublecortin-like kinase 1 (DCLK1) marks a population of tumor-initiating cells in colonic and pancreatic cancers, and it is overexpressed in many solid tumors. Our preliminary studies suggest that DCLK1 is overexpressed in serum samples of melanoma patients and it maybe a novel biomarker for melanoma. In this study we examined DCLK1 expression in melanoma tumor tissues and further evaluated whether its levels in serum could be quantitatively assessed in melanoma patients before and after therapy treatment. Immunohistochemistry (IHC) analysis was performed to detect DCLK1 in a tissue microarray with 60 tumor and normal adjacent tissues (NAT) from melanoma patients. ELISA assay were performed to detect DCLK1 levels in the serum samples of melanoma patients before and after treatment. Western blotting was also performed to detect DCLK1 protein in the serum samples of melanoma patients. Additionally, analysis of DCLK1 and correlative gene expressions was performed using TCGA Melanoma dataset. Here we report that the intensity of DCLK1 staining in melanoma tumor tissues are significantly increased compared to NAT. DCLK1 levels in the serum were elevated in melanoma patients (n=65) compared to healthy volunteers. Patients with higher levels of serum DCLK1 had reduced overall survival compared to patients with lower levels of serum DCLK1. We also compared DCLK1 levels in 10 patients for whom pre- and post- treatment samples were available. Among these patients, seven had elevated DCLK1 before treatment and had a significantly decrease (about 30-50%) in serum DCLK after treatment. Three patients had no change in DCLK1 levels after treatment. Interestingly, the patients with decrease level of DCLK1 post-treatment demonstrated significant clinical responses to therapy, however, the patients with no change in DCLK1 level post therapy showed no clinical improvement (p<0.002). Our study suggested that serum DCLK1 may be an independent prognostic factor, and treatment-induced reduction in serum DCLK1 was associated with excellent clinical response. These data taken together suggest that DCLK1 maybe a novel biomarker for melanoma for prognosis and response purposes. Citation Format: Dongfeng Qu, Nathaniel Weygant, Parthasarathy Chandrakesan, Kamille Pitts, Randal May, Adam Asch, Alexandra Ikeguchi, Courtney Houchen. DCLK1 is upregulated in melanoma and it is a novel predictive marker for survival and response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4034.

Consensus gene expression analysis to identify key hallmarks of cancer in malignant melanoma.

e21045 Background: Traditionally gene expression signatures (GES) are used individually to classify patients into subgroups. Signatures targeting the same biology are often developed independently and may not classify identically. We developed the claraT software tool that uses consensus between multiple published GES categorised by the Hallmarks of Cancer (Hanahan & Weinberg, 2011) to classify cancers. As metastatic melanoma represents poor prognostic disease (5-yr survival 15-20%), we applied claraT to the TCGA melanoma dataset to identify targetable biologies, validated in a cohort of melanoma patients treated with Ipilimumab. Methods: TCGA RNA-seq data ( n= 472) was analysed using the claraT platform including GES for immune ( n= 14), angiogenesis ( n= 9) and epithelial-mesenchymal transition (EMT) ( n= 12) Hallmarks. Samples were clustered for the combined and individual Hallmarks. Median progression-free (PFS) and overall-survival (OS) differences were analysed across identified subgroups. Analysis was validated in an Ipilimumab treated melanoma dataset ( n= 42) (Van Allen, 2015). Results: Clustering the combined Hallmarks identified 4 subgroups in the TCGA cohort: 1) Immune active, 2) Immune-EMT active, 3) EMT-Angiogenesis active, 4) All inactive. Groups 1&2 had significantly improved OS compared to Groups 3&4 (HR = 0.50, p< 0.0001). Clustering using single Hallmarks revealed that immune-positive tumours had significantly improved OS (HR = 0.53, p< 0.0001) compared to immune-negative tumours. Angiogenesis-negative tumours displayed improved PFS (HR = 0.73, p= 0.03) and OS (HR = 0.53, p <0.0001) compared to angiogenesis-negative tumours. Interestingly the EMT Hallmark was not found to be individually prognostic. When validated in the Ipilimumab treated dataset, patients classified as immune-positive had improved OS (HR = 0.357, p= 0.010) when compared to immune-negative. Similar trends were also observed for angiogenesis and EMT Hallmarks. Conclusions: This study demonstrates how simultaneous analysis of multiple GES ( n= 35 in this study) can identify robust biologies through consensus expression. This platform may have value in the identification of reliable biomarkers for clinical trials and could inform how combination therapies targeting key biologies may be used in cancer treatment.

Analysis of Chinese acral and mucosal melanoma patient genomic and neoantigen profiles in cancer vaccine development: A pilot study.

e14300 Background: Acral and mucosal melanoma are rare subtypes accounting for about 3% of all melanoma cases. The cutaneous melanoma genomic landscape is well defined; however, little is known about the acral and mucosal melanoma mutational spectrum. In this pilot study, we evaluated the genomic and neo-antigen profiles and tumor mutational burden (TMB) from acral and mucosal melanoma patients with the aim of designing personalized vaccines and longitudinally tracking patients’ clinical courses. Methods: Tumor whole exome sequencing and neo-antigen profiling of 5 acral and 3 mucosal melanoma patients at Shanghai Tenth Peoples Hospital, Tongji University, China between April 2018 and January 2019 was performed using YuceBio’s proprietary analytics platform. Watsonä for Genomics, an artificial intelligence decision-support system, was used for variant interpretation and annotation. A comparative analysis was performed on Chinese acral melanoma data with the published Caucasian acral cohort from the Translational Genomics Research Institute (TGen) and The Cancer Genome Atlas (TCGA) predominantly Caucasian cutaneous melanoma data set. Results: TMB in our acral/mucosal melanoma cohort was 2.26/Megabase (Mb) compared to over 20/Mb in published cutaneous melanoma studies. Tumor neo-antigen burden (TNAB) in our group was 1.03 neo-epitopes/Mb. Low TNAB levels were associated with low TMB levels in all tumors. Incidence of BRAF and NRAS mutant cases in our cohort was 0% (0/8) and 13% (1/8) respectively compared to 19% (5/27) and 7% (2/27) of the Caucasian acral population in the TGen dataset. Incidence of BRAF and NRAS mutations in the TCGA cutaneous melanoma dataset was 54% (237/440) and 28% (125/440), respectively. Conclusions: TMB was significantly lower in acral/mucosal than in cutaneous melanoma and may be a surrogate for TNAB. Detection of BRAF and NRAS mutations, the two most prevalent driver mutations in cutaneous melanoma, were significantly lower frequencies in both Chinese and Caucasian acral melanoma patients in this study, suggesting alternate cancer drivers may exist in this subtype. Strategies to address challenges of low TNAB in vaccine development are being explored.

High GILT expression and an active and intact MHC class II antigen presentation pathway are associated with improved survival in melanoma

Abstract The MHC class I antigen presentation pathway in melanoma cells has a well-established role in immune-mediated destruction of tumors. However, the clinical significance of the MHC class II antigen presentation pathway in melanoma cells is less clear. In antigen presenting cells, gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of multiple melanoma antigens. While not expressed in benign melanocytes of nevi, GILT and MHC class II expression is induced in malignant melanocytes in a portion of melanoma specimens. Analysis of The Cancer Genome Atlas (TCGA) cutaneous melanoma dataset showed that high GILT mRNA expression was associated with improved overall survival. Expression of IFN-γ, TNF-α, and IL-1β was positively associated with GILT expression in the TCGA melanoma specimens. These cytokines were capable of inducing GILT expression in melanoma cells in vitro. GILT protein expression in melanocytes was induced in halo nevi, which are nevi undergoing immune-mediated regression, and is consistent with the association of GILT expression with improved survival in melanoma. To explore potential mechanisms of GILT’s association with patient outcome, we analyzed the mRNA expression pattern in the MHC class II antigen presentation pathway. In contrast to healthy skin where the MHC class II pathway was nearly uniformly expressed and intact, there was substantial variation in the MHC class II pathway of the TCGA melanoma dataset. Both an active and intact MHC class II pathway were associated with improved overall survival in melanoma. These studies support a role for GILT and the MHC class II antigen presentation pathway in melanoma outcome.

BCAT1 and miR-2504: novel methylome signature distinguishes spindle/desmoplastic melanoma from superficial malignant peripheral nerve sheath tumor

Superficial/cutaneous malignant peripheral nerve sheath tumor is a rare soft tissue neoplasm that shares morphological, immunohistochemical, and molecular features with spindle/desmoplastic melanoma. We aimed to identify a methylome signature to distinguish these two entities. We analyzed 15 cases of spindle/desmoplastic melanoma and 15 cases of cutaneous malignant peripheral nerve sheath tumor in 23 men and 7 women. DNA from formalin-fixed, paraffin-embedded tissues was extracted and processed using the Illumina Infinium Methylation EPIC array interrogating 866,562 CpG sites. Using a home-grown informatics pipeline, we identified differentially methylated positions between the two entities. Functional network analysis for enrichment signatures was performed using DAVID tools. Identified differentially methylated positions were compared with the Cancer Genome Atlas's cutaneous melanoma dataset and a recently published malignant peripheral nerve sheath tumor dataset to assess the specificity of the identified signature. Unsupervised hierarchical clustering showed different patterns of methylation in cutaneous malignant peripheral nerve sheath tumor and spindle/desmoplastic melanoma. Two probes, cg20783223 and cg13332552, colocalized in the promoter region of BCAT1 and miR-2504. Pathway analysis highlighted enrichment in a subset of genes involved in breast and gastric cancer centered on BCAT1 and downstream activated genes in the mTOR pathway. Our study identifies BCAT1 as a novel methylome signature distinguishing spindle/desmoplastic melanoma from cutaneous malignant peripheral nerve sheath tumor.

An actionable axis linking NFATc2 to EZH2 controls the EMT-like program of melanoma cells

Discovery of new actionable targets and functional networks in melanoma is an urgent need as only a fraction of metastatic patients achieves durable clinical benefit by targeted therapy or immunotherapy approaches. Here we show that NFATc2 expression is associated with an EMT-like transcriptional program and with an invasive melanoma phenotype, as shown by analysis of melanoma cell lines at the mRNA and protein levels, interrogation of the TCGA melanoma dataset and characterization of melanoma lesions by immunohistochemistry. Gene silencing or pharmacological inhibition of NFATc2 downregulated EMT-related genes and AXL, and suppressed c-Myc, FOXM1, and EZH2. Targeting of c-Myc suppressed FOXM1 and EZH2, while targeting of FOXM1 suppressed EZH2. Inhibition of c-Myc, or FOXM1, or EZH2 downregulated EMT-related gene expression, upregulated MITF and suppressed migratory and invasive activity of neoplastic cells. Stable silencing of NFATc2 impaired melanoma cell proliferation in vitro and tumor growth in vivo in SCID mice. In NFATc2+ EZH2+ melanoma cell lines pharmacological co-targeting of NFATc2 and EZH2 exerted strong anti-proliferative and pro-apoptotic activity, irrespective of BRAF or NRAS mutations and of BRAF inhibitor resistance. These results provide preclinical evidence for a role of NFATc2 in shaping the EMT-like melanoma phenotype and reveal a targetable vulnerability associated with NFATc2 and EZH2 expression in melanoma cells belonging to different mutational subsets.

An accurate and powerful method for copy number variation detection.

Integration of multiple genetic sources for copy number variation detection (CNV) is a powerful approach to improve the identification of variants associated with complex traits. Although it has been shown that the widely used change point based methods can increase statistical power to identify variants, it remains challenging to effectively detect CNVs with weak signals due to the noisy nature of genotyping intensity data. We previously developed modSaRa, a normal mean-based model on a screening and ranking algorithm for copy number variation identification which presented desirable sensitivity with high computational efficiency. To boost statistical power for the identification of variants, here we present a novel improvement that integrates the relative allelic intensity with external information from empirical statistics with modeling, which we called modSaRa2. Simulation studies illustrated that modSaRa2 markedly improved both sensitivity and specificity over existing methods for analyzing array-based data. The improvement in weak CNV signal detection is the most substantial, while it also simultaneously improves stability when CNV size varies. The application of the new method to a whole genome melanoma dataset identified novel candidate melanoma risk associated deletions on chromosome bands 1p22.2 and duplications on 6p22, 6q25 and 19p13 regions, which may facilitate the understanding of the possible roles of germline copy number variants in the etiology of melanoma. http://c2s2.yale.edu/software/modSaRa2 or https://github.com/FeifeiXiaoUSC/modSaRa2. Supplementary data are available at Bioinformatics online.

A Leukocyte Infiltration Score Defined by a Gene Signature Predicts Melanoma Patient Prognosis.

Melanoma is the most aggressive type of skin cancer in the United States with an increasing incidence. Melanoma lesions often exhibit high immunogenicity, with infiltrating immune cells playing important roles in regression of tumors occurring spontaneously or caused by therapeutic treatment. Computational and experimental methods have been used to estimate the abundance of immune cells in tumors, but their applications are limited by the requirement of large gene sets or multiple antibodies. Although the prognostic role of immune cells has been appreciated, a systematic investigation of their association with clinical factors, genomic features, prognosis and treatment response in melanoma is still lacking. This study, identifies a 25-gene signature based on RNA-seq data from The Cancer Genome Atlas (TCGA)-Skin Cutaneous Melanoma (TCGA-SKCM) dataset. This signature was used to calculate sample-specific Leukocyte Infiltration Scores (LIS) in six independent melanoma microarray datasets and scores were found to vary substantially between different melanoma lesion sites and molecular subtypes. For metastatic melanoma, LIS was prognostic in all datasets with high LIS being associated with good survival. The current approach provided additional prognostic information over established clinical factors, including age, tumor stage, and gender. In addition, LIS was predictive of patient survival in stage III melanoma, and treatment efficacy of tumor-specific antigen vaccine. IMPLICATIONS: This study identifies a 25-gene signature that effectively estimates the level of immune cell infiltration in melanoma, which provides a robust biomarker for predicting patient prognosis.

The lncRNA RMEL3 protects immortalized cells from serum withdrawal-induced growth arrest and promotes melanoma cell proliferation and tumor growth.

RMEL3 is a recently identified lncRNA associated with BRAFV600E mutation and melanoma cell survival. Here, we demonstrate strong and moderate RMEL3 upregulation in BRAF and NRAS mutant melanoma cells, respectively, compared to melanocytes. High expression is also more frequent in cutaneous than in acral/mucosal melanomas, and analysis of an ICGC melanoma dataset showed that mutations in RMEL3 locus are preponderantly C>T substitutions at dipyrimidine sites including CC>TT, typical of UV signature. RMEL3 mutation does not correlate with RMEL3 levels, but does with poor patient survival, in TCGA melanoma dataset. Accordingly, RMEL3 lncRNA levels were significantly reduced in BRAFV600E melanoma cells upon treatment with BRAF or MEK inhibitors, supporting the notion that BRAF-MEK-ERK pathway plays a role to activate RMEL3 gene transcription. RMEL3 overexpression, in immortalized fibroblasts and melanoma cells, increased proliferation and survival under serum starvation, clonogenic ability, and xenografted melanoma tumor growth. Although future studies will be needed to elucidate the mechanistic activities of RMEL3, our data demonstrate that its overexpression bypasses the need of mitogen activation to sustain proliferation/survival of non-transformed cells and suggest an oncogenic role for RMEL3.

MMP7 sensitivity of mutant ECM proteins: An indicator of melanoma survival rates and T-cell infiltration

ObjectiveTo assess the potential impact of mutant ECM amino acids (AA) on melanoma-related matrix metalloproteinase-7 (MMP7) activity. Design and methodsWe applied a novel scripted algorithm, based on the MEROPS database, to reveal mutant-dependent sensitivity changes across the cancer genome atlas, melanoma dataset. ResultsThis approach revealed a strong bias in favor of mutant AA dependent protease sensitivity increases. Thus, melanoma specimens with relatively few mutations had only MMP7 mutant sensitive, ECM peptides. As mutations increased, melanoma specimens included mutant AA representing mostly increased sensitivity and a small but increasing number of mutant AA representing decreased MMP7 sensitivity. There was no detection of melanoma specimens with only decreases in MMP7 sensitivity. Furthermore, melanoma specimens with exclusively increased sensitivity and thereby only a few overall mutations represented reduced T-cell infiltrates and worse outcomes. ConclusionsOverall, the results indicated that changes in MMP7 sensitivity, attributable to mutant AA, have the potential of identifying patients with distinct survival outcomes as well as patients with cancer specimen immune activity.

A fast detection of fusion genes from paired-end RNA-seq data

BackgroundFusion genes are known to be drivers of many common cancers, so they are potential markers for diagnosis, prognosis or therapy response. The advent of paired-end RNA sequencing enhances our ability to discover fusion genes. While there are available methods, routine analyses of large number of samples are still limited due to high computational demands.ResultsWe develop FuSeq, a fast and accurate method to discover fusion genes based on quasi-mapping to quickly map the reads, extract initial candidates from split reads and fusion equivalence classes of mapped reads, and finally apply multiple filters and statistical tests to get the final candidates. We apply FuSeq to four validated datasets: breast cancer, melanoma and glioma datasets, and one spike-in dataset. The results reveal high sensitivity and specificity in all datasets, and compare well against other methods such as FusionMap, TRUP, TopHat-Fusion, SOAPfuse and JAFFA. In terms of computational time, FuSeq is two-fold faster than FusionMap and orders of magnitude faster than the other methods.ConclusionsWith this advantage of less computational demands, FuSeq makes it practical to investigate fusion genes in large numbers of samples. FuSeq is implemented in C++ and R, and available at https://github.com/nghiavtr/FuSeqfor non-commercial uses.

Analysis of Single-Cell RNA-Seq Identifies Cell-Cell Communication Associated with Tumor Characteristics

SUAMMARYTumor ecosystems are composed of multiple cell types that communicate by ligand-receptor interactions. Targeting ligand-receptor interactions (for instance, with immune checkpoint inhibitors) can provide significant benefits for patients. However, our knowledge of which interactions occur in a tumor and how these interactions affect outcome is still limited. We present an approach to characterize communication by ligand-receptor interactions across all cell types in a microenvironment using single-cell RNA sequencing. We apply this approach to identify and compare the ligand-receptor interactions present in six syngeneic mouse tumor models. To identify interactions potentially associated with outcome, we regress interactions against phenotypic measurements of tumor growth rate. In addition, we quantify ligand-receptor interactions between T cell subsets and their relation to immune infiltration using a publicly available human melanoma dataset. Overall, this approach provides a tool for studying cell-cell interactions, their variability across tumors, and their relationship to outcome.

Proportional Odds under Conway-Maxwell-Poisson Cure Rate Model and Associated Likelihood Inference

Cure rate models are useful while modelling lifetime data involving long time survivors. In this work, we discuss a flexible cure rate model by assuming the number of competing causes for the event of interest to follow the Conway-Maxwell Poisson distribution and the lifetimes of the non-cured individuals to follow a proportional odds model. The baseline distribution is considered to be either Weibull or log-logistic distribution. Under right censoring, we develop the maximum likelihood estimators using EM algorithm. Model discrimination among some well-known special cases are discussed under both likelihood- and information-based criteria. An extensive simulation study is carried out to examine the performance of the proposed model and the inferential methods. Finally, a cutaneous melanoma dataset is analyzed for illustrative purpose.

Abstract 5438: XPNPEP3: A novel transcriptional target of canonical Wnt/β-catenin signalling

Abstract Canonical Wnt/β-catenin signalling plays pivotal roles during embryonic development and adult tissue regeneration. Its aberrant activation however drives expression of a panoply of genes to facilitate colorectal tumorigenesis. Hence, it is imperative to delineate the complete β-catenin transcriptome. We performed genome-wide mRNA profiling of sporadic rectal cancer samples stratified by Wnt status. Results revealed significant up-regulation of XPNPEP3 transcript levels along with that of canonical Wnt targets AXIN2 and EPHB2 in Wnt+ samples. The differential expression of XPNPEP3 was further validated by quantitative PCR (Q-PCR) in additional samples. Nuclear stabilization of β-catenin achieved through LiCl treatment in three colorectal cancer (CRC) cell lines followed by Q-PCR and promoter-luciferase assays confirmed up-regulation of XPNPEP3. XPNPEP3 encodes X-prolyl aminopeptidase 3 which functions to remove the penultimate N-terminal Proline residue from nascent proteins and appears to play a role in ciliary function. Immunohistochemistry based expression analysis using a CRC tissue microarray revealed significant correlation between XPNPEP3 levels and β-catenin nuclear localization as well as increased XPNPEP3 expression in tumor compared to matched normal samples. Survival analysis of The Cancer Genome Atlas (TCGA) breast invasive carcinoma, skin cutaneous melanoma and lung adenocarcinoma data sets revealed poor survival among patients having perturbed XPNPEP3 expression. Characterization of possible oncogenic function of XPNPEP3 in CRC cells and in mice tumor xenografts is currently underway. Altogether, our results suggest XPNPEP3 to be a novel transcriptional target of Wnt/β-catenin signalling having a possible significance in CRC tumorigenesis. Citation Format: Raju Kumar, Ashmala Naz, Viswakalyan Kotapalli, Swarnalata Gowrishankar, Satish Rao, Jonathan R. Pollack, Murali Dharan Bashyam. XPNPEP3: A novel transcriptional target of canonical Wnt/β-catenin signalling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5438.

Abstract 3611: Serum DCLK1 levels are elevated in melanoma patients and it is a novel predictive marker for survival and response

Abstract Melanoma is the deadliest form of skin cancer, and the incidence of melanoma has been rising for the last 40 years. There is a paucity of readily available tools to predict for melanoma progression and response to therapy, leaving an unmet medical need to identify predictive and prognostic biomarkers. Doublecortin-like kinase 1 (DCLK1) is overexpressed and marks a population of tumor-initiating cells in colonic and pancreatic cancers. It regulates key oncogenes, pluripotency factors, angiogenic factors, and epithelial mesenchymal transition (EMT) related transcription factors. In this study, we evaluated whether DCLK1 can be detected in the bloodstream and if its levels in serum samples could be quantitatively assessed in melanoma patients. ELISA assay was performed to detect DCLK1 levels in the serum samples of melanoma patients before and after treatment with either targeted agents or immune checkpoint inhibitors. Western blotting was also performed to detect DCLK1 protein in the serum samples of melanoma patients. Immunohistochemistry (IHC) analysis was performed to detect DCLK1 in the same patients' tumor tissues. Additionally, analysis of DCLK1 and correlative gene expression profiles was performed using TCGA Melanoma dataset. Here we report that the intensity of DCLK1 staining in melanoma tumor tissues is increased compared to normal tissues. DCLK1 levels in the serum were elevated in melanoma patients (n=20) compared to healthy volunteers. Next, we compared DCLK1 levels in 10 patients for whom pre- and post- treatment samples were available. Among these patients, seven had elevated DCLK1 before treatment and had a significant decrease (about 30-50%) in serum DCLK after treatment. Three patients had no change in DCLK1 levels after treatment. The patients with a decrease in their level of DCLK1 post-treatment demonstrated radiologic response to therapy as well, while the patients with no change in DCLK1 level post therapy showed either no improvement or progression (p<0.002). These data suggest that reduction in serum DCLK1 levels following therapy for melanoma was associated with clinical response, and the absence of change in DCLK1 after therapy was associated with poor response. Analysis of the TCGA dataset demonstrated that melanoma patients with high levels of DCLK1 had worse overall survival (p<0.05) compared to the patients with low levels of DCLK1, suggesting that DCLK1 RNA levels could be used as a prognostic biomarker. These data taken together suggest that DCLK1 has potential utility as a novel predictive and on-treatment biomarker for melanoma prognosis and therapeutic response. Citation Format: Dongfeng Qu, Nathaniel Weygant, Parthasarathy Chandrakesan, Kamille Pitts, Randal May, Sripathi Sureban, Adam Asch, Alexandra Ikeguchi, Courtney Houchen. Serum DCLK1 levels are elevated in melanoma patients and it is a novel predictive marker for survival and response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3611.

Biological Validation of RNA Sequencing Data From Formalin-Fixed Paraffin-Embedded Primary Melanomas

Initiatives such as The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) have generated high-quality, multi-platform molecular data from thousands of frozen tumor samples. While these initiatives have provided invaluable insight into cancer biology, a tremendous potential resource remains largely untapped in formalin-fixed, paraffin-embedded (FFPE) samples that are more readily available, but which can present technical challenges due to crosslinking of fragile molecules such as RNA. We extracted RNA from FFPE primary melanomas and assessed two gene expression platforms -- genome-wide RNA sequencing (RNA-seq) and targeted NanoString -- for their ability to generate coherent biological signals. To do so, we generated an improved approach to quantifying gene expression pathways, in which we refine pathway scores through correlation-guided gene subsetting. We also make comparisons to the TCGA and other publicly available melanoma datasets. Comparison of the gene expression patterns to each other, to established biological modules, and to clinical and immunohistochemical data confirmed the fidelity of biological signals from both platforms using FFPE samples to known biology. Moreover, correlations with patient outcome data were consistent with previous frozen-tissue-based studies. FFPE samples from previously difficult-to-access cancer types - such as small primary melanomas - represents a valuable and previously unexploited source of analyte for RNA-seq and NanoString platforms. This work provides an important step towards the use of such platforms to unlock novel molecular underpinnings and inform future biologically-driven clinical decisions.

MicroRNA-365 Inhibits Cell Growth and Promotes Apoptosis in Melanoma by Targeting BCL2 and Cyclin D1 (CCND1).

BackgroundMicroRNA-365 (miR-365) is involved in the development of a variety of cancers. However, it remains largely unknown if and how miRNAs-365 plays a role in melanoma development.Material/MethodsIn this study, we overexpressed miR-365 in melanoma cell lines A375 and A2058, via transfection of miR-365 mimics oligos. We then investigated alterations in a series of cancer-related phenotypes, including cell viability, cell cycle, apoptosis, colony formation, and migration and invasion capacities. We also validated cyclin D1 (CCND1) and BCL2 apoptosis regulator (BCL2) as direct target genes of miR-365 by luciferase reporter assay and investigated their roles in miR-365 caused phenotypic changes. To get a more general view of miR-365’s biological functions, candidate target genes of miR-365 were retrieved via searching online databases, which were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses for potential biological functions. We then analyzed The Cancer Genome Atlas (TCGA) Skin Cutaneous Melanoma (SKCM) dataset for correlation between miR-365 level and clinicopathological features of patients, and for survival of patients with high and low miR-365 levels.ResultsWe found that miR-365 was downregulated in melanoma cells. Overexpression of miR-365 remarkably suppressed cell proliferation, induced cell cycle arrest and apoptosis, and compromised the migration and invasion capacities in A375 and A2058 cell lines. We also found that the phenotypic alterations by miR-365 were partially due to downregulation of CCND1 and BCL2 oncogenes. The bioinformatics analysis revealed that predicted targets of miR-365 were widely involved in transcriptional regulation and cancer-related signaling pathways. However, analysis of SKCM dataset failed to find differences in miR-365 level among melanoma patients at different clinicopathologic stages. The Kaplan-Meier analysis also failed to discover significant differences in overall survival and disease-free survival between patients with high and low miR-365 levels.ConclusionsOur findings suggested that miR-365 might be an important novel regulator for melanoma formation and development, however, the in vivo roles in melanoma developments need further investigation.

PolyClustR: defining communities of reconciled cancer subtypes with biological and prognostic significance

BackgroundTo ensure cancer patients are stratified towards treatments that are optimally beneficial, it is a priority to define robust molecular subtypes using clustering methods applied to high-dimensional biological data. If each of these methods produces different numbers of clusters for the same data, it is difficult to achieve an optimal solution. Here, we introduce “polyClustR”, a tool that reconciles clusters identified by different methods into subtype “communities” using a hypergeometric test or a measure of relative proportion of common samples.ResultsThe polyClustR pipeline was initially tested using a breast cancer dataset to demonstrate how results are compatible with and add to the understanding of this well-characterised cancer. Two uveal melanoma datasets were then utilised to identify and validate novel subtype communities with significant metastasis-free prognostic differences and associations with known chromosomal aberrations.ConclusionWe demonstrate the value of the polyClustR approach of applying multiple consensus clustering algorithms and systematically reconciling the results in identifying novel subtype communities of two cancer types, which nevertheless are compatible with established understanding of these diseases. An R implementation of the pipeline is available at: https://github.com/syspremed/polyClustR

Potential role of cyclin F mRNA expression in the survival of skin melanoma patients: Comprehensive analysis of the pathways altered due to cyclin F upregulation.

Cyclin F is a part of the Skp, Cullin, F-box containing ligase complex. The activity of cyclin F includes cell cycle control, centrosome duplication and response to DNA damage. The cyclin F expression pattern is very similar to cyclin A, but cyclin F is an orphan cyclin without its cyclin-dependent kinase partner. There is little evidence concerning the role of cyclin F in cancer. In the present study, for the first time, we present analysis from The Cancer Genome Atlas (TCGA) data in the context of expression of cyclin F mRNA in melanoma patients. Our original in silico analysis, not published elsewhere before, revealed that high expression of cyclin F in melanoma patients is associated with worse overall survival. Cyclin F and ribonucleotide reductase family member 2 (RRM2) compose a functional axis responsible for nucleotide metabolism. Impairment in this pathway may contribute to increased DNA damage repair and drug resistance. Additionally, we analyzed the expression of RRM2 mRNA and discovered that high expression of RRM2 is associated with worse overall survival. To shed more light on cyclin F overexpression in melanoma, we analyzed all protein data available in the TCGA melanoma dataset. It was found that in patients with upregulated cyclin F mRNA, we noted increased activity of pathways related to cell cycle and DNA damage repair. These data will support further in vitro and in vivo studies on the involvement of cyclin F in skin cutaneous melanoma.

Melanoma cases demonstrate increased carrier frequency of phenylketonuria/hyperphenylalanemia mutations

Identifying novel melanoma genetic risk factors informs screening and prevention efforts. Mutations in the phenylalanine hydroxylase gene (the causative gene in phenylketonuria) lead to reduced pigmentation in untreated phenylketonuria patients, and reduced pigmentation is associated with greater melanoma risk. Therefore, we sought to characterize the relationship between phenylketonuria carrier status and melanoma risk. Using National Newborn Screening Reports, we determined the United States phenylketonuria/hyperphenylalanemia carrier frequency in Caucasians to be 1.76%. We examined three publically available melanoma datasets for germline mutations in the phenylalanine hydroxylase gene associated with classic phenylketonuria and/or hyperphenylalanemia. Mutations were identified in 29/814 melanoma patients, with a carrier frequency of 3.56%. There was a twofold enrichment (p-value=3.4×10-5 ) compared to the Caucasian frequency of hyperphenylalanemia/phenylketonuria carriers. These data demonstrate a novel association between phenylalanine hydroxylase carrier status and melanoma risk. Further, functional investigation is warranted to determine the link between phenylalanine hydroxylase mutations and melanomagenesis.