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

The aim of this study was to evaluate alternative TrkAIII splicing as a potential oncogenic mechanism and novel target in MCPyV positive MCC. Due to the rare nature of this tumour type, experiments were performed on a limited number of 18 FFPE MCC tissues from 11 patients, 3 individual BCCs and 3 individual SCCs from patients diagnosed and treated at the University of L’Aquila, L’Aquila, Italy from 2006 to 2019 and 2 normal skin samples, using appropriate RT-PCR-based and immunofluorescent (IF) techniques.

The 18 MCCs, 3 basal cell carcinomas (BCCs), 3 squamous cell carcinomas (SCCs) FFPE tissues were from a 17 patient cohort, comprised of 18 MCCs from 11 patients (7 females and 4 males, with a mean ± SD age of 72.06 ± 12.24 years), consisting of 4 sequential recurrent stage IV MCCs from patient 1 (P.1, i-iv) [27]; 3 contemporary recurrent stage IV MCCs from patient 2 (P.2, i-iii); 2 contemporary recurrent stage IV MCCs from patient 3 (P.3, i and ii); 1 primary stage I and 1 recurrent stage IV MCC from patient 4 (P.4, i and ii); 1 recurrent stage IIIB MCC from patient P.5; 4 stage IIA and 1 stage IIB primary MCCs from patients P.6-P.10; 1 primary stage 1 MCC from patient P.11; 3 primary stage 1 BCCs from patients P.12-P.14; 3 primary SCCs from patients P.15-P.17 and 2 normal skin samples (NS1 and NS2) (Tables 1 and 2). MCC diagnoses were confirmed by histopathological positivity for cytokeratin AE1/AE3 and CD56 and individual clinical data are presented in Table 1. Written consent was obtained from all patients and the study was approved by ASL (n.1) Ethics committee, Abruzzo, Italy [10/CE/2018: 19 July 2018 (n.1419)].

RNAs were purified from 50 μm MCC, SCC and BCC FFPE sections, using a total RNA purification kit for FFPE tissues, as directed (Thermo Fisher Scientific, CA), RNA purity and concentration was assessed using a nanodrop spectrophotometer, as directed (Thermo Fisher Scientific, CA) and purified RNAs reverse transcribed using a reverse transcription kit, as directed (Thermo Fischer Scientific, CA). Reverse transcription reactions (RTs) were then subjected to PCR using the following gene-specific primers: TrkA exon 5/6 splice-junction: 5′-CTGCAGTGTCATGGGCAA-3′ and 5′-CACATCCACCGAGGCATT-3′; TrkAIII exon 5–8 (covering the TrkAIII 5/8 splice junction): 5′-CTGCAGTGTCATGGGCAA-3′ and 5′-ACCAGTGGTGCATCTCCAC-3′; TrkA exons 3–8: 5′-AGTGGTCTCCCGTTTCGTGGCGCCA and 5′-ACCAGTGGTGCATCTCCAC-3′; GAPDH: 5′-CTGCACCACCAACTGCTTAG-3′ and 5′-GCAGTGATGGCATGGACTGT-3′; 18S rRNA: 5′-AAACGGCTACCACATCCACG-3′ and 5′-CCTCGAAAGAGTCCAGTATTG-3′; MCPyV VP1: 5′-CAACGAAAATTTGCCAGCTTA-3′ and 5’TTTAACAGAATATTGCCTCCCAC-3′; MCPyV small T-antigen: 5′-TGCCACCAGTCAAAACTTTC-3′ and 5′-AGCAAAAAAACTGTCTGACGTG-3′, and MCPyV large T-antigen: 5′-AAGGACCCATACCCAGAGGAAG-3′ and 5′-CCAACTCAAGATCCAGAAAGCC-3′. Primer sets were designed to generate 100 bp products to mitigate problems of RNA fragmentation in FFPE tissues [28] and facilitate comparative densitometric analysis. Optimum reverse transcription reaction (RT) dilutions for PCR were established by serial dilution in order to generated products within the linear-range. For TrkA, TrkAIII, MCPyV VP1, MCPyV small T-antigen and large T-antigen, RTs were undiluted, diluted 1:100 for GAPDH and 1:1,000 for 18S rRNA. RT-PCRs were performed in duplicate and repeated for each sample (n = 4). For densitometric analysis, 100 bp TrkA, TrkAIII, GAPDH, 18S rRNA, MCPyV VP1, MCPyV small T-antigen and MCPyV large T-antigen RT-PCR products from individual tissue samples were compared within the same 1% agarose gels, digitally photographed and analysed using Image J software (ImageJ bundled with Java 1.8.0_172) [29]. Inter-gel comparisons were made using common 18S rRNA RT-PCR product and DNA ladder standards.

FFPE 5 μm sections were de-paraffinized, re-hydrated and processed for antigen retrieval by incubation in 0.01 M sodium citrate buffer [pH 6.0] for 20 min at 98 °C. Sections were blocked in BSA/TX100 blocking solution, incubated overnight at 4 °C with polyclonal rabbit anti-human TrkA (C14, Santacruz, CA); polyclonal rabbit anti-human Y490-phopsphorylated TrkA (pY490-TrkA, Cell Signalling Technologies, CA) or mouse anti-human γ-tubulin (Santacruz, CA) primary antibodies, diluted 1:50 in blocking solution, washed then incubated with fluorochrome-conjugated secondary antibodies (Alexa Fluor, ThermoFisher Scientific, CA), diluted 1:1000 in blocking solution for 2 h at room temperature. Slides were then washed, mounted in VectaShield with DAPI (Vector Laboratories, CA) and visually scored with strong, intermediate, weak or negative immunoreactivity by scanning confocal microscopy (Leica TCS SP₅ II).

Densitometric data were analysed statistically by Student’s t-test, using the online t-test calculator at https://​www.​graphpad.​com/​quickcalcs/​ttest1.​cfm and statistical significances were associated with probabilities of ≤0.05.

RT-PCR detected MCPyV large T-antigen and small T-antigen expression in 17/18 (≈ 94%) MCCs, MCPyV VP-1 expression in 11/18 (≈ 61%) MCCs and trace level MCPyV large T-antigen expression in 1/3 BCCs. In contrast, MCPyV large T-antigen, small T-antigen and VP1 expression were not detected in 1 primary stage I MCC, 2/3 BCCs, 3/3 SCCs or 2 normal skin samples (Fig. 1a, Table 2). RT-PCR detected TrkAIII expression in all MCCs positive for MCPyV large T-antigen expression (100%), with predominant TrkAIII over fully spliced TrkA expression (> 50%) detected in the majority and exclusive TrkAIII mRNA expression in a significant number of MCCs. In contrast, MCPyV negative MCC, BCCs, SCCs and normal skin samples all exhibited exclusive expression of fully-spliced TrkA but not TrkAIII mRNA (Fig. 1a, Table 2). Densitometric analysis, revealed that MCPyV large T-antigen positive MCCs exhibited a significantly higher TrkAIII percentage of total TrkA expression corresponding to 80.92 ± 19.37% (mean ± SD) compared to 0.42 ± 0.397% in MCPyV negative MCC, BCCs, SCCs and normal skin (p < 0.0001, df = 24) (Fig. 1b and Table 2) that ranged from 76.6 ± 8.4% (n = 4) to 98.12 ± 4.5% (n = 4) in the 4 sequential recurrent stage IV MCCs (i-iv) from patient P.1; 68.1 ± 4.5% to 98.7 ± 6.1% in the 3 contemporary recurrent stage IV MCCs (i-iii) from patient P.2; 99.2 ± 8.4% (n = 4) to 99.6 ± 4.2% (n = 4) in the 2 contemporary stage IV recurrent MCCs (i and ii) from patient P.3; 87.6 ± 8.2% (n = 4) to 93.8 ± 8.6% (n = 4) in the primary stage I (i) and recurrent stage IV (ii) MCCs from patient P.4, and 41.5 ± 6.7% (n = 4) to 97.3 ± 6.4% (n = 4) in the remaining stage 1-III MCPyV positive MCCs from patients P.5–10 (Table 2). Consistent with this, MCPyV positive MCCs also exhibited a significantly lower 19.49 ± 19.71% (mean ± SD) TrkA percentage total TrkA expression compared to 99.48 ± 0.41% in MCPyV negative MCC, BCCs, SCCs and normal skin (P < 0.0001, df = 24) (Fig. 1b, Table 2). Furthermore, MCPyV positive MCCs exhibited a significantly lower TrkA:18S rRNA RT-PCR ratio of 0.063 ± 0.07 (mean ± SD) compared to 0.39 ± 0.37 in MCPyV negative MCC, BCCs, SCCs and normal skin (p = 0.0015, df = 24) and a significantly higher TrkAIII:18S rRNA ratio of 0.34 ± 0.25 (mean ± SD) compared to 0.014 ± 0.024 in MCPyV negative MCC, BCCs, SCCs and normal skin (p < 0.0009, df = 14) (Fig. 1b, Table 2). These results were also corroborated by comparing the 100 bp TrkAIII to the 300 bp TrkA RT-PCR product generated by the TrkA exon 5–8 primer set (Fig. 2a), despite the potential negative influence of RNA fragmentation in FFPE tissues on detection of fully spliced TrkA [28]. TrkAIII identity was confirmed in selected MCCs by direct PCR sequencing of the 300 bp fragment generated from the TrkA exon 3–8 primer set (Fig. 2b).

Using antibodies against TrkA and Y490 phosphorylated TrkA that also recognize TrkAIII [20], strong or intermediate immunoreactivity for both antibodies characterised MCPyV positive MCCs with high-level MCPyV large T-antigen expression (Fig. 3a), exclusive TrkAIII mRNA expression and relatively high TrkAIII to 18S rRNA RT-PCR ratios (Figs. 1a and 2a and Table 2), whereas weak immunoreactivity characterized MCPyV large T-antigen positive MCCs exhibiting low TrkAIII to 18S rRNA ratios (Figs. 1a, 2a and 3a and Table 2). The MCPyV negative stage I MCC (patient P.11) was negative for both TrkA and phosphorylated TrkA immunoreactivity (Fig. 3b, Table 2), despite low-level exclusive fully-spliced TrkA mRNA expression (Figs. 1a and 2a and Table 2). All recurrent stage IV MCCs (patients P.1–3) exhibited strong or intermediated immunoreactivity to both antibodies (Fig. 3a and Table 2). Strong TrkA immunoreactivity characterized normal skin epithelia exhibiting exclusive fully spliced TrkA mRNA expression and high TrkA:18S rRNA ratios, and also characterized the tumour component of 1 SCC, exhibiting exclusive TrkA mRNA expression. In contrast, scant immunoreactivity characterized 1/3 BCCs and 1/3 SCCs and the remaining BCCs (2/3) and SCC (1/3) were negative for TrkA immunoreactivity (Fig. 3b and Table 2). Immunoreactivity for phosphorylated TrkA was not detected in any of the MCPyV negative MCC, BCCs, SCCs or normal skin samples (Fig. 3b and Table 2). These data provide evidence for intracellular TrkAIII expression and activation in MCPyV positive MCCs and in particular stage IV tumours exhibiting exclusive TrkAIII mRNA expression, whereas fully spliced inactive TrkA expression characterised normal skin epithelia and the tumour component of some but not all MCPyV negative BCCs and SCCs. TrkA immunoreactivity also co-localized with centrosome γ-tubulin in a stage IV MCPyV positive MCC exhibiting exclusive TrkAIII mRNA expression (Fig. 4, Patient P. 1 i).