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Merkel cell carcinomas (MCCs) are rare, aggressive, cutaneous neuroendocrine tumours, approximately 80% of which are caused by the genomic integration of Merkel cell polyomavirus (MCPyV). MCPyV-positive MCCs carry poor prognosis in approximately 70% of cases, highlighting the need for greater understanding of the oncogenic mechanisms involved in pathogenesis, progression and post-therapeutic relapse, and translation into novel therapeutic strategies. In a previous pilot study, we reported a potential relationship between MCPyV gene expression and oncogenic alternative Δ exon 6–7 TrkAIII splicing in formalin-fixed paraffin-embedded (FFPE) MCC tissues from a 12-patient cohort of >90% MCPyV-positive MCCs, diagnosed at San Salvatore Hospital, L'Aquila, Italy, characterising a new MCC subgroup and unveiling a novel potential MCPyV oncogenic mechanism and therapeutic target. This, however, could not be fully verified due to poor RNA quality and difficulty in protein extraction from FFPE tissues. Here, therefore, we extend our previous observations to confirm the relationship between MCPyV and oncogenic alternative Δ exon 6–7 TrkAIII splicing in fresh, nonfixed, MCPyV-positive MCC metastasis by detecting sequence-verified RT-PCR products, including full-length Δ exon 6–7 TrkAIII, and by Western blot detection of a 100 kDa TrkA protein isoform of identical size to 100 kDa Δ exon 6–7 TrkAIII expressed by stable transfected SH-SY5Y cells. We also report that in three MCC patients submitted for multidisciplinary treatment, including locoregional chemotherapy, MCPyV large T-antigen mRNA expression, Δ exon 6–7 TrkAIII mRNA expression and intracellular indirect immunofluorescence (IF) TrkA and phosphorylation protein isoform(s) immunoreactivity in FFPE tissues were not reduced in postchemotherapeutic-relapsed MCCs compared to pretherapeutic MCCs, extending the possible roles of this novel potential MCPyV oncogenic mechanism from MCC pathogenesis to post-therapeutic relapse and progression. Detection of alternative Δ exon 6–7 TrkAIII splicing in MCC, therefore, not only characterises a new MCPyV-positive MCC subgroup and unveils a novel potential MCPyV oncogenic mechanism but also identifies patients who may benefit from inhibitors of MCPyV T-antigen and/or TrkAIII expression or clinically approved Trk kinase inhibitors such as larotrectinib or entrectinib, which are known to inhibit activated TrkA oncogenes and to elicit durable responses in TrkA-fusion oncogene-driven cancers, supporting the call for a large-scale multicentre clinical study. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]