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

Abstract

BackgroundMerkel 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.MethodsFormalin-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.Results9 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.ConclusionsMCPyV 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.

Multidisciplinary Treatment, Including Locoregional Chemotherapy, for Merkel-Polyomavirus-Positive Merkel Cell Carcinomas: Perspectives for Patients Exhibiting Oncogenic Alternative Δ exon 6–7 TrkAIII Splicing of Neurotrophin Receptor Tropomyosin-Related Kinase A

“…Furthermore, analysis of potential promoters of alternative ∆ exon 6-7 TrkAIII splicing in NB cells identified the polyomavirus simian virus 40 (SV40) large T-antigen [30], raising the possibility that closely related polyomavirus MCPyV T-antigens may also promote alternative ∆ exon 6-7 TrkAIII splicing in MCPyV-positive MCCs. This hypothesis was supported in our pilot study [28], which detected a close relationship between MCPyV T-antigen expression and alternative ∆ exon 6-7 TrkAIII splicing in FFPE MCC tissues, characterising a new MCC subset and identifying a novel potential MCPyV oncogenic mechanism and therapeutic target (Figure 1). This, however, could not be fully verified due to the poor RNA quality and difficulty in protein extraction from FFPE tissue [28].…”

“…This hypothesis was supported in our pilot study [28], which detected a close relationship between MCPyV T-antigen expression and alternative ∆ exon 6-7 TrkAIII splicing in FFPE MCC tissues, characterising a new MCC subset and identifying a novel potential MCPyV oncogenic mechanism and therapeutic target (Figure 1). This, however, could not be fully verified due to the poor RNA quality and difficulty in protein extraction from FFPE tissue [28].…”

“…MCC diagnosis, however, continues to carry poor prognosis in spite of these advances, highlighting the need for a further understanding of oncogenic mechanisms involved in this rare tumour type and translation into novel therapeutic strategies. Within this context, in a recent pilot study of MCC FFPE tissues, we reported a novel potential MCPyV oncogenic mechanism, characterised by the promotion of oncogenic alternative ∆ exon 6-7 TrkAIII mRNA splicing of the tropomyosin-related tyrosine kinase receptor TrkA [28]. Oncogenic alternative ∆ exon 6-7 TrkAIII mRNA splicing was originally identified in advanced-stage primary human neuroblastomas (NBs), results in expression of the ∆ exon 6-7 TrkAIII oncoprotein that transforms NIH3T3 mouse embryo fibroblasts and exhibits oncogenic activity in a variety of in-vitro and in-vivo models [29,30].…”

A Prospective Study of Intraarterial Infusion Chemotherapy in Advanced Wild-Type BRAF Melanoma Patients

“…42 Finally, novel agnostic therapeutic agents, such as larotrectinib and entrectinib, that inhibit mutation and deletion-activated Trk oncogenes, elicit remarkable durable responses in a wide range of advanced stage Trkfusion oncogene-driven cancers, including melanoma. 43…”

Predictors of immunotherapy benefit in Merkel cell carcinoma

“…A small study ( n = 10) revealed that MCCP tumors may tend to display activating alternative splicing of NTRK1 mRNA. [ 23 ] Notably, none of the samples with NTRK1 SNVs in our cohort were MCCP. If TrkA activity is a key oncogenic pathway for MCCP tumors, we could speculate that NTRK1 alterations may confer an “MCCP-like” phenotype onto MCCN tumors by activating this pathway.…”