Comprehensive analysis of the MYB-NFIB gene fusion in salivary adenoid cystic carcinoma: Incidence, variability, and clinicopathologic significance

Abstract

Purpose: The objectives of this study were to determine the incidence of the MYB-NFIB fusion in salivary adenoid cystic carcinoma (ACC), to establish the clinicopathologic significance of the fusion, and to analyze the expression of MYB in ACCs in the context of the MYB-NFIB fusion.

Experimental design: We did an extensive analysis involving 123 cancers of the salivary gland, including primary and metastatic ACCs, and non-ACC salivary carcinomas. MYB-NFIB fusions were identified by reverse transcriptase-PCR (RT-PCR) and sequencing of the RT-PCR products, and confirmed by fluorescence in situ hybridization. MYB RNA expression was determined by quantitative RT-PCR and protein expression was analyzed by immunohistochemistry.

Results: The MYB-NFIB fusion was detected in 28% primary and 35% metastatic ACCs, but not in any of the non-ACC salivary carcinomas analyzed. Different exons in both the MYB and NFIB genes were involved in the fusions, resulting in expression of multiple chimeric variants. Notably, MYB was overexpressed in the vast majority of the ACCs, although MYB expression was significantly higher in tumors carrying the MYB-NFIB fusion. The presence of the MYB-NFIB fusion was significantly associated (P = 0.03) with patients older than 50 years of age. No correlation with other clinicopathologic markers, factors, and survival was found.

Conclusions: We conclude that the MYB-NFIB fusion characterizes a subset of ACCs and contributes to MYB overexpression. Additional mechanisms may be involved in MYB overexpression in ACCs lacking the MYB-NFIB fusion. These findings suggest that MYB may be a specific novel target for tumor intervention in patients with ACC.

Figure 1

Detection of MYB-NFIB fusion transcripts in ACCs. A, schematic representation of the MYB and NFIB genes. Arrows indicate primers used to identify MYB-NFIB fusion transcripts by RT-PCR. B, RT-PCR analysis of MYB-NFIB fusion transcripts using the indicated primers in ACCs and non-ACCs, including a myoepithelial carcinoma (#44), acinic cell carcinoma (#49), mucoepidermoid carcinoma (#50), salivary duct carcinoma (#53), Polymorphous low grade adenocarcinoma (#58), and epithelial-myoepithelial carcinoma (#61). Samples 62(M) and 63(M) represent metastatic ACCs. ACTB (beta actin) was used as internal control. C, identification of tumors expressing multiple MYB-NFIB fusion variants. RT-PCR using RNA from tumor #8, amplified with primers MYB1-NFIB1, showing two PCR products. D, sequencing profile generated from fusion transcripts shown in C, confirming MYB exon 15 linked to NFIB exon 12 (fusion 1) and exon 11 (fusion 2). MYB-NFIB fusion cDNA and predicted protein sequences are indicated. The nucleotide and protein sequence of MYB are based on NCBI database (accession number NM_001130173). NFIB sequences and exon numbers were obtained from accession number ENSG0000147862 for NFIB in the Ensembl database. E, FISH analysis using BAC clones containing MYB (green) and NFIB (red) genes in fusion-negative ACCs (top panels, samples #29, #33, #43) and fusion-positive ACCs (bottom panels, samples #6, #15, #22). Note overlap of the NFIB and MYB probes in the fusion-positive tumors but not in fusion-negative ACCs.

Figure 2

Expression of MYB transcripts in salivary gland tumors and normal salivary glands. A and B, relative expression of MYB using primers that amplify MYB transcripts containing coding sequences of exons 2-3 (A) or exons 15-16 (B). Relative expression of MYB represents fold expression compared to normal salivary gland. Red bars: fusion-positive ACCs; Blue bars: fusion-negative ACCs; Yellow bars: non-ACCs; N: normal salivary gland; ACC: Adenoid cystic carcinoma; MYC: Myoepithelial carcinoma; Aci: Acinic cell carcinoma; MEC: mucoepidermoid carcinoma; SDC: salivary duct carcinoma; PLGA: Polymorphous low grade adenocarcinoma; EMC: Epithelial-myoepithelial carcinoma. The asterisks in B denote tumors with MYB-NFIB fusions that occurred after exon 15 of MYB. C and D, cumulative expression of MYB exon 2-3 (C) or MYB exon 14-15 (D) in salivary gland tumors. Relative MYB expression for each tumor is represented with circles. White circles: normal salivary gland (N); Red circles: fusion-positive ACCs; Blue circles: fusion-negative ACCs; Yellow circles: non-ACCs. Horizontal bars represent the mean for each group.

Figure 3

Expression of MYB protein in salivary ACCs. A-C, immunostaining for MYB in MYB-NFIB fusion-positive ACCs. Each panel depicts a different fusion-positive ACC, showing strong staining in myoepithelial cells in the tumor shown in panel (A), ubiquitous expression in panel (B) and lack of staining in the ACC shown in (C). D-E, immunostaining for MYB in ACCs negative for the MYB-NFIB fusion, showing a representative tumor with high MYB expression (D) and another tumor that lacks MYB expression (E).