A pilot study of alternative TrkAIII splicing in Merkel cell carcinoma: a potential oncogenic mechanism and novel therapeutic target

Background: Merkel cell carcinomas (MCCs) are rare, aggressive, therapeutically-challenging skin tumours that are increasing in incidence and have poor survival rates. The majority are caused by genomic Merkel cell polyomavirus (MCPyV) integration and MCPyV T-antigen expression. Recently, a potential oncogenic role for the tropomyosin-related tyrosine kinase A receptor (TrkA) has been proposed in MCC. Alternative TrkAIII splicing is a TrkA oncogenic activation mechanism that can be promoted by SV40 large T-antigen, an analogue of MCPyV large T-antigen. In this pilot study, therefore, we have evaluated TrkAIII splicing as a novel potential oncogenic mechanism and therapeutic target in MCPyV positive MCC. Methods: Formalin-fixed paraffin-embedded MCC tissues, consisting of 10 stage IV, 1 stage IIIB, 1 stage IIB, 4 stage IIA and 2 stage I tumours, from patients diagnosed and treated from September 2006 to March, 2019, at the University of L'Aquila, L'Aquila, Italy, were compared to 3 primary basal cell carcinomas (BCCs), 3 primary squamous cell carcinomas (SCCs) and 2 normal skin samples by RT-PCR for MCPyV large T-antigen, small T-antigen, VP-1 expression and alternative TrkAIII splicing and by indirect IF for evidence of intracellular TrkA isoform expression and activation. Results: 9 of 10 Recurrent stage IV MCCs were from patients (P.1-3) treated with surgery plus loco-regional Melphalan chemotherapy and remaining MMCs, including 1 stage IV tumour, were from patients treated with surgery alone (P. 4-11). All MCPyV positive MCCs exhibiting MCPyV large T-antigen expression (17 of 18MCCs, 90%) exhibited alternative TrkAIII mRNA splicing (100%), which was exclusive in a significant number and predominant (> 50%) in all stage IV MCCs and the majority of stage 1-III MCCs. MCCs with higher TrkAIII to 18S rRNA expression ratios also exhibited strong or intermediate immunoreactivity to anti-TrkA antibodies, consistent with cytoplasmic TrkAIII expression and activation. In contrast, the MCPyV negative MCC, BCCs, SCCs and normal skin tissues all exhibited exclusive fully-spliced TrkA mRNA expression, associated with variable immunoreactivity for non-phosphorylated but not phosphorylated TrkA. Conclusions: MCPyV positive MCCs but not MCPyV negative MCC, BCCs and SCCs exhibit predominant alternative TrkAIII splicing, with evidence of intracellular TrkAIII activation. This establishes a new potential MCC subset, unveils a novel potential MCPyV oncogenic mechanism and identifies TrkAIII as a novel potential therapeutic target in MCPyV positive MCC.