A Digital mRNA Expression Signature to Classify Challenging Spitzoid Melanocytic Neoplasms

Spitzoid neoplasms are a challenging group of cutaneous melanocytic proliferations that occur in children or adolescents but can also arise in the elderly. They are characterized by epithelioid and/ or spindle-shaped melanocytes with a stromal background of variable amounts of lymphocytes, blood vessels and sclerosis. According to the WHO 2018 classification, Spitzoid melanocytic lesions are classified as benign Spitz nevi (SN), atypical Spitz tumors (AST) or malignant Spitz tumors (MST). The intermediate AST category represents a diagnostically challenging group since on purely histopathological grounds, their benign or malignant character remains unpredictable. This results in uncertainties in patient management and prognosis. The molecular properties of Spitzoid lesions, especially their transcriptomic landscape, remain poorly understood and genomic alterations in melanoma-associated oncogenes are typically absent. The aim of this study was to characterize the transcriptome of Spitzoid melanocytic neoplasms with digital mRNA expression profiling. Formalin-fixed-paraffin-embedded samples (including 27 SN, 10 AST and 14 MST) were analyzed using the RNA NanoString nCounter PanCancer Pathways Gene Expression panel. The number of significantly differentially expressed genes in SN vs. MST, SN vs. AST and AST vs. MST was 68, 167 and 18, respectively. Gene set enrichment analysis revealed an upregulation of pathways related to epithelial mesenchymal transition, immunomodulatory-, angiogenesis- as well as myogenesis associated processes in AST and MST. In addition, a specific gene expression signature of SN vs. MST was discovered based on the top-ranked six most informative gene expression markers: NRAS, NF1, BMP2, EIF2B4, IFNA17 and FZD9. The AST samples showed intermediate levels of the identified signature. This implies that the identified gene expression signature can potentially be used to distinguish high-grade from low-grade AST. This combined histopathological and transcriptomic methodology is promising for diagnostics of Spitzoid neoplasms and patient management in dermatological oncology in the future. mRNA expression of 27 Spitz nevi (SN), 10 atypical Spitz tumors (AST) and 14 malignant Spitz tumors (MST) were analyzed. Molecular profiling was done using the RNA NanoString nCounter Gene Expression Platform (No. of genes = 770). Gene expression counts were normalized based on total library size. As such, both the housekeeping and endogenous genes were used for normalization. The studied samples showed a mean library size of 307914 counts (median 292412; range 789054 (sample AST4) to 19042 (sample MST4)). All analyses were performed using R and Bioconductor packages. Marker Discovery and Validation of differentially expressed genes from the nCounter data was done using the limma and voom method in R. Three conditions were studied: (i) SN vs. MST, (ii) SN vs. AST, and (iii) AST vs. MST. The limma procedure borrows information across all genes, which makes the analysis robust even when the sample size is small (Ritchie, Phipson et al. 2015). The voom procedure was applied to non-parametrically estimate the mean-variance relationship of the log-transformed gene counts, which is important when dealing with expression count data