The Distinctive Mutational Spectra of Polyomavirus-Negative Merkel Cell Carcinoma

Abstract

Merkel cell carcinoma (MCC) is a rare but highly aggressive cutaneous neuroendocrine tumor. Merkel cell polyomavirus (MCPyV) may contribute to tumorigenesis in a subset of tumors via inhibition of tumor suppressors such as retinoblastoma (RB1) by mutated viral T antigens, but the molecular pathogenesis of MCPyV-negative MCC is largely unexplored. Through our MI-ONCOSEQ precision oncology study, we performed integrative sequencing on two cases of MCPyV-negative MCC, as well as a validation cohort of 14 additional MCC cases (n = 16). In addition to previously identified mutations in TP53, RB1, and PIK3CA, we discovered activating mutations of oncogenes, including HRAS and loss-of-function mutations in PRUNE2 and NOTCH family genes in MCPyV-negative MCC. MCPyV-negative tumors also displayed high overall mutation burden (10.09 ± 2.32 mutations/Mb) and were characterized by a prominent UV-signature pattern with C > T transitions comprising 85% of mutations. In contrast, mutation burden was low in MCPyV-positive tumors (0.40 ± 0.09 mutations/Mb) and lacked a UV signature. These findings suggest a potential ontologic dichotomy in MCC, characterized by either viral-dependent or UV-dependent tumorigenic pathways.

Figure 1. Next generation sequencing analysis of Merkel cell carcinoma index cases MO_1109 (A-D) and MO_1160 (E-H)

(A) Frozen section of biopsy for MO_1109 demonstrating a round cell malignancy (hematoxylin and eosin, 200×). (B) Copy number variations (CNVs) in MO_1109, including copy gain at MYCL1 and copy loss at RB1. (C) Somatic mutations in MO_1109 are dominated by C > T transitions. (D) Tandem substitutions in MO_1109 consist predominantly of CC > TT changes. (E) Frozen section of biopsy for MO_1160 demonstrating a round cell malignancy (hematoxylin and eosin, 200×). (F) CNVs in MO_1160, including copy loss at RB1 and TP53. (G) Somatic mutations in MO_1160 are dominated by C > T transitions. (H) Tandem substitutions in MO_1160 consist predominantly of CC > TT changes, consistent with UV-signature mutations.

Figure 2. Global mutation profiles of Merkel cell polyomavirus (MCPyV)-negative and MCPyV-positive Merkel cell carcinoma (MCC)

(A) Significantly higher somatic mutation rate in MCPyV-negative tumors. (B) C to T transitions are predominant in MCPyV-negative MCC, but not MCPyV-positive MCC. Asterisks indicate p < 0.0001. (C) Tandem substitutions are dominated by CC > TT changes (yellow) in melanoma and MCPyV-negative MCC. Other solid tumor types display predominantly CC > AA (red) or other changes (blue). For non-MCC tumors, profiles were generated from 10 randomly selected tumors of each type. (D) Trinucleotide mutation signatures demonstrate similar pattern of C to T transitions (red) in melanoma and a representative case of MCPyV-negative MCC. A randomly selected melanoma was used for comparison. Arrowheads indicate C > T transitions at dipyrimidine sites. CA: Carcinoma.

Figure 3. Mutational landscape of MCPyV-negative and MCPyV-positive tumors

Green: missense mutations, yellow: nonsense mutations, light blue: indels, purple: splice site mutations, red: copy gain (index cases only), dark blue: copy loss (index cases only).

Figure 4. Recurrent NOTCH family mutations in MCC

NOTCH1-4 mutations in MCC cluster in EGF and ankyrin repeat domains, similar to tumor types with inactivating NOTCH mutations. Mutated codons above the diagram indicate previously described mutations in cutaneous squamous cell carcinoma (Wang et al. 2011). EGF: Epidermal growth factor-like motifs. ANK: Ankyrin repeats. TAD: transactivation domain. RAM: RBP-Jκ–associated module. PEST: protein domain enriched in proline, glutamic acid, serine, and threonine residues. cSCC: Cutaneous squamous cell carcinoma. FPKM: fragments per kilobase of exon per million fragments mapped. CA: carcinoma.