Investigating the cell of origin and novel molecular targets in Merkel cell carcinoma: a historic misnomer

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with the poorest prognosis, posited to be derived from Merkel cells. Emerging evidence, however, suggests other potential origins for MCC including hematological lineages. Given the high fatality of MCCs and that many arise in the immune suppressed population, often precluding the use of immunotherapy, it is important to determine the cell of origin for these cancers to elucidate targetable pathways to enable novel treatment approaches. We utilized targeted and multi-omics approaches to explore expression patterns at both the protein and RNA level of MCCs. Western blotting, immunofluorescence, and immunohistochemistry were performed for two patient-derived MCC samples, one MCC cell line, and 92 FFPE samples, respectively, for numerous B-cell markers. Further, RNA sequencing was performed on 17 FFPE patient-derived MCC samples and evaluated by principal component analysis for differentially expressed genes between samples based on sex and MCPyV status. Finally, weighted gene correlation network analysis (WGCNA) and cell type enrichment analyses were employed to determine pathway and cell type enrichment, respectively. MCC patient-derived and cell line samples heterogeneously expressed B-cell and neuroendocrine markers including PAX5, TdT, IgA, CD19, CK20, and chromogranin A. Further, transcriptome analysis demonstrated differentially expressed genes based on sex and MCPyV status. MCPyV+ tumors notably demonstrated significant upregulation of genes involved in immune cell function and downregulation of processes related to neuronal activity. Finally, WGCNA highlighted enrichment for pathways involved in immune function including B-cell differentiation. Cell type enrichment analysis highlighted enrichment for multipotent stem cells, several immune cell types, and keratinocytes. Our findings together with previous results indicate that MCCs are not derived from Merkel cells and instead from multiple or divergent cell types, including those of B-cell lineage. Further, MCC cell of origin may depend on patient and tumour specific characteristics including sex and cell type/differentiation state of a cell infected by the MCPyV. Our work highlights the need for a more personalized approach to diagnosis/characterization and treatment of MCCs given the variability of potentially targetable pathways. Total RNA was quantified for 17 FFPE-preserved MCC samples, and its integrity was assessed on a PerkinElmer LabChip GXII (Massachusetts, USA). rRNA was depleted from 125 ng of total RNA using the QIAseq FastSelect platform (QIAGEN, Hilden, Germany). cDNA synthesis was achieved with the NEBNext RNA First Strand Synthesis and NEBNext Ultra Directional RNA Second Strand Synthesis Modules. The remaining steps of library preparation were carried out using and the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs, Massachusetts, USA). Adapters and PCR primers were purchased from New England BioLabs. Libraries were quantified using the KAPA Library Quanitification Complete Kit (Kapa Biosystems, Massachusetts, USA). Average size fragment was determined using a LabChip GXII (PerkinElmer) instrument (Massachusetts, USA). The libraries were normalized and pooled and then denatured in 0.05N NaOH and neutralized using HT1 buffer. The pool was loaded at 175pM on the Illumina NovaSeq S4 lane using the Xp protocol per the manufacturer’s recommendations. The run was performed for 2x100 cycles (paired-end mode). A phiX library was used as a control and mixed with libraries at 1% level. Base calling was performed using Real Time Analysis v3.4.4. Program bcl2fastq2 v2.20 was then used to demultiplex samples and generate FASTQ reads, which were subsequently aligned to the hg19 reference genome (Illumina, California, USA).