Molecular analysis of a mammary analog secretory carcinoma in the upper lip: Novel search for genetic and epigenetic abnormalities in MASC

Abstract

Introduction: Mammary analog secretory carcinoma (MASC) is a newly described rare malignancy of the salivary glands characterized by an ETS variant 6 (ETV6)-neurotrophic tyrosine kinase receptor type 3 (NTRK3) fusion gene (EN fusion gene).

Presentation of case: We present a case of MASC derived from the left upper lip in a 61-year-old woman. ETV6 rearrangement was detected by fluorescence in situ hybridization (FISH). The presence of EN fusion transcripts was verified by reverse-transcriptase polymerase chain reaction (RT-PCR) and sequencing of the PCR products. Accordingly, this tumor was diagnosed as MASC. Moreover, we performed mutation analysis of the 50 known cancer-related genes using next-generation sequencing. No mutation of cancer-related genes was identified here. Subsequently, the methylation status in promoter region of tumor-suppressor genes, RASSF1A and RARB2, was examined. Both genes have been reported to be methylated in malignant salivary gland tumors, but they were found to be unmethylated.

Discussion: Recent studies have demonstrated that distinct types of malignant salivary gland carcinomas are driven by specific, highly recurrent genetic alterations. Detection of molecular abnormalities could be powerful diagnostic tools in the field of salivary gland tumors in near future.

Conclusion: We experienced a rare malignant salivary gland carcinoma, MASC. We diagnosed this tumor by molecular approach and subsequently tried to identify novel molecular abnormalities. Although no novel molecular alteration except for EN fusion gene was identified, this result might represent the favorable prognosis of patients with MASC.

Keywords: DNA methylation; ETV6-NTRK3 fusion gene; MASC; Mammary analog secretory carcinoma; Mutation; Salivary gland tumor.

Fig. 1

Clinical and histopathological findings. The arrowhead showed a soft and tender mass in the left upper lip (a). Microscopic findings are as follows (b–h). Hematoxylin-eosin staining showed that the tumor was composed of tubular structures or papillary architecture, with eosinophilic secretory fluid within the lumens of the tubules (b). Strong immunostaining was observed for the S-100 protein (c), vimentin (d) and EMA (e). Weak immunostaining was observed for α-SMA (f) and p63 (g). Strong immunoreactivity was observed for p53 (h).

Fig. 2

Molecular findings. (A) FISH analysis of ETV6 gene rearrangement using Vysis^®LSI^®ETV6 Break Apart Rearrangement Probe (Abbott Molecular/Vysis). The arrowheads show the cells which have ETV6 rearrangement. A yellow (red/green fusion) signal indicates an intact chromosome while separate red and green signals indicate an ETV6 gene break. (B) Expression of ETV6-NTRK3 fusion transcript in the MASC and noncancerous oral mucosa by RT-PCR. (1) DNA marker; (2) noncancerous oral mucosa; (3) cancerous lesion; (4) distilled water. (C) Validation of ETV6-NTRK3 fusion transcript by direct sequencing. Arrows show translocation breakpoint. (D) Methylation analysis of promoter CGIs of RASSF1A and RARB2 in MASC and non-cancerous specimens. M, primers specific to methylated DNA; Alu, primers that target the Alu repeat sequence were used as a control of the amount of bisulfite-treated DNA. Promoter CGIs of RASSF1A and RARB2 were unmethylated in both the non-cancerous specimen (1) and MASC (2). Fully methylated DNA was prepared by methylating genomic DNA using SssI-methylase (3). Fully unmethylated DNA was prepared by amplifying genomic DNA with ø29 DNA polymerase (4).