A digital mRNA expression signature to classify challenging Spitzoid melanocytic neoplasms

Abstract

Spitzoid neoplasms are a challenging group of cutaneous melanocytic proliferations. They are characterized by epithelioid and/or spindle-shaped melanocytes and 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 treatment 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 their transcriptome with digital mRNA expression profiling. Formalin-fixed paraffin-embedded samples (including 27 SN, 10 AST, and 14 MST) were analyzed using the NanoString nCounter PanCancer Pathways 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 upregulation of pathways related to epithelial-mesenchymal transition and immunomodulatory-, angiogenesis-, hormonal-, and myogenesis-associated processes in AST and MST. A molecular signature of SN vs. MST was discovered based on the top-ranked most informative genes: NRAS, NF1, BMP2, EIF2B4, IFNA17, and FZD9. The AST samples showed intermediate levels of the identified signature. This implies that the gene signature can potentially be used to distinguish high-grade from low-grade AST with a larger study cohort in the future. This combined histopathological and transcriptomic methodology is promising for prospective diagnostics of Spitzoid neoplasms and patient management in dermatological oncology.

Keywords: Spitz nevus; atypical Spitz nevus; gene expression profiling; mRNA; malignant Spitz tumor; molecular signature.

Fig. 1

Histomorphology of a prototypic SN, AST, and a MST. (A) Histology of a compound SN used in this study with a dome‐shaped configuration. H.E. staining, length of scale bar: 500 µm. (B) Illustration of an AST investigated in this study with a compound architecture. The lesion is slightly asymmetrical, and there is epidermal hyperplasia. H.E. staining, length of scale bar: 500 µm. (C) Morphology of a MST from the MST cohort. H.E. staining, length of scale bar: 500 µm. (D) Higher magnification of a SN depicted from (A) showing the two cell types of Spitzoid melanocytes with epithelioid Spitzoid morphology (encircled) with abundant eosinophilic ground‐glass cytoplasm containing vesicular nuclei with prominent nucleoli and spindle‐shaped Spitzoid melanocytes (asterisk). There are retraction clefts around the melanocytic cell nests. H.E. staining, length of scale bar: 100 µm. (E) Magnification of an AST from (B) showing nests of Spitzoid melanocytes with hyperchromatic nuclei and increase in N/C ratio. Spindle‐shaped melanocytes dominate in relation to epithelioid cells. A mitotic figure (encircled) is localized in the superficial area of the lesions at the epidermal junction, length of scale bar: 100 µm. (F) Higher magnification from of a MST depicted from (C) with frequent irregular mitotic figures (encircled), which are localized in the dermal depth of the lesion. There is strong cytonuclear atypia (asterisks) with irregular nuclear configuration, aberration of the nucleus‐to‐cytoplasm ratio, and hyperchromasia, length of scale bar: 100 µm.

Fig. 2

(A–C) Volcano plots identifying the differentially expressed genes in the three studied conditions: SN vs. MST (A), SN vs. AST (B), and AST vs. MST (C). The x‐axis shows the absolute log fold change (log 2FC), and the y‐axis represents the ‐log₁₀ of the P‐value, thereby indicating statistically significantly differentially expressed genes. Each dot in the scatter represents a gene, with significant genes colored in blue. There were 68 genes differentially expressed in SN in relation to MST (P‐value < 0.05), of which 27 had a higher mean expression level (positive log FC), and 41 genes had a lower mean expression level [negative log FC, (A)]. In the SN vs. AST group, there were 167 differentially expressed genes (B). Out of these, 36 genes showed a positive log FC and 131 had a negative log FC. In the AST vs. MST group, there were 18 differentially expressed genes with 10 transcripts showing a positive log FC and eight transcripts showing a negative log FC (C). The top five differentially expressed genes are labeled with gene names. The other differentially expressed gene names are provided in Table S2 in the supplemental material. (D–F) The identified top‐five‐ranked gene pathways are listed with corresponding –log₁₀ of the FDR Q‐value. Results of the Hallmark gene sets (including 50 gene sets), which were extracted from the MSigDB are shown for the three studied groups, that is, SN vs. MST (D), SN vs. AST (E), and AST vs. MST (F). Gray bars indicate upregulation, and black bars show downregulation of the gene pathways; for detailed characteristics of identified gene pathways, see also supplemental data, Table S4.

Fig. 3

(A) Coefficient path from LASSO regularization for classifying. The colored lines represent individual genes. The ordinate shows the value of the coefficients associated with each gene as a function of log λ. A 10‐fold CV identified the value for the tuning parameter that resulted in the lowest binomial deviance for classifying with a model of six top‐ranked gene expression markers. (B) Boxplots of signature levels (range 0–1) in the group of SN (left), AST (center), and MST (right). The AST group shows intermediate levels of the signature in between SN and MST. (C) Supervised heatmap of the top six validated most informative gene transcripts which were included in the signature. The samples are grouped by patients harboring a SN, an AST, or a MST; that is, Spitzoid melanoma. Each column represents a patient sample from the three groups of Spitzoid melanocytic neoplasms. The rows of the heatmap are the identified signature genes NRAS, EIF2B4, NF1, BMP2, FZD9, and IFNA17. The highest expression levels are shown in red staining, and lowest expression levels are shown in blue staining, yellow color represents intermediate expression (figure legend). The rows were clustered based on Euclidean distance and the complete linkage method.