Recurrent RET Gene Rearrangements in Intraductal Carcinomas of Salivary Gland

Abstract

Intraductal carcinoma (IC) is the World Health Organization designation for lesions previously called low-grade cribriform cystadenocarcinoma. The relationship of IC to salivary duct carcinoma (SDC) is controversial, but currently these are considered distinct entities. It is hypothesized that IC and SDC should have different genomic signatures that may be identifiable by next-generation sequencing. A total of 23 ICs were identified: 14 pure IC and 9 invasive carcinomas with an intraductal component. Five invasive carcinomas were subjected to next-generation paired-end RNA sequencing. Data analysis was performed using FusionSeq and Mutation detection algorithms (MuTect and VarScan) for variant callers. Gene fusion candidates were validated by fluorescence in situ hybridization and reverse transcription polymerase chain reaction, and mutations by Sanger sequencing. Among the 9 invasive carcinomas, all except 1 were apocrine SDCs with an intraductal component. The remaining case showed typical intercalated duct type IC with invasive adenocarcinoma. The 14 pure ICs had typical intercalated duct features (2 showed hybrid intercalated/apocrine features). RNA sequencing predicted a NCOA4-RET fusion, confirmed by reverse transcription polymerase chain reaction, in the intercalated duct type IC invasive component. Six additional cases of pure IC showed RET rearrangement by fluorescence in situ hybridization (7/15=47%). No apocrine carcinomas showed RET rearrangement. RNA sequencing and Sanger sequencing identified PIK3CA (p.E545K/p.H1047R) and/or HRAS (p.Q61R) hotspot mutations in 6 of 8 (75%) apocrine carcinomas. In conclusion, 2 distinctive types of intraductal lesions are emerging based on molecular analysis. Classic intercalated type ICs commonly harbor fusions involving RET and rarely show widespread invasion. Apocrine intraductal lesions are typically associated with widespread invasion with no pure examples and show similar PIK3CA and HRAS mutations to SDC.

Fig. 1

Index case for sequencing of intraductal and invasive adenocarcinoma of intercalated duct type (case 1) (A–D). The intraductal component showed small bland cells forming solid and cribriform type nests with amphophilic cytoplasm (A). This component and all other intraductal carcinomas were S100 positive (Inset A). The invasive component showed cystic change with a fibrotic stroma and displayed prominent papillary tufts (B–C). The transition zone is seen in B. The papillary tufts floated in the spaces mimicking “spread through alveolar spaces” in pulmonary adenocarcinoma (D).

Fig. 2

Apocrine/salivary duct adenocarcinomas were always invasive with varying morphologies and showed focal intraductal components (A–D). Some cases showed typical features of salivary duct carcinoma with high-grade solid nests and comedonecrosis (A). A number of cases showed a prominent tubular morphology (B) and all cases had at least a focal intraductal component (arrow). A single case in a 38 year old showed a low to intermediate grade apocrine carcinoma not typical of conventional salivary duct carcinoma (C). However, all cases were androgen receptor (AR) positive (D) and considered in the salivary duct carcinoma spectrum.

Fig. 3

NCOA4-RET gene fusion in an invasive and intraductal adenocarcinoma with an intercalated duct phenotype (case #1). (A) Schematic representation of the NCOA4-RET fusion indicating the loci that are joined together; NCOA4 exon 6 being fused to RET exon 12 (left side of image). (B) RNA reads covering the fusion junction were isolated independent to FusionSeq analysis work flow, supporting the NCOA4-RET fusion candidate and confirmed by RT-PCR.

Fig. 4

Six additional pure intraductal carcinoma of the intercalated duct type showed RET rearrangement by FISH (A–D). Four of these cases showed a pure intraductal carcinoma of the intercalated duct type (A) and the other two showed a mixed intercalated duct type intraductal carcinoma with apocrine areas (B). The apocrine areas in case #2 showed prominent papillary architecture (top left of image). S100 was positive in all pure intraductal cases (not shown). FISH for RET shows break apart signals with one normal fused yellow signal and separate 5′ green and 3′ red signals indicating rearrangement of the gene (arrows) (case #2). Note the consistent distance of the split signals owing to the putative inversion in this case, which was the case in all RET rearranged tumours other than case #15 that showed a classic break-apart signal (not shown).

Fig. 5

The invasive apocrine/salivary duct carcinoma group showed common mutations in PIK3CA and HRAS. These included PIK3CA p.E545K (2 cases), PIK3CA p.H1047R (3 cases) and HRAS p.Q61R (3 cases). A total of 6/8 (75%) of these invasive carcinomas harbored mutations in one or both genes, suggesting that they are best classified as salivary duct carcinoma even when they show unusual morphology.