Intraductal carcinoma of the salivary gland with NCOA4-RET: expanding the morphologic spectrum and an algorithmic diagnostic approach

Abstract

After the publication of the 2017 World Health Organization Classification of Head and Neck Tumours, there has been increasing interest in the classification of newly categorized intraductal carcinomas. Intraductal carcinoma (IC) is an indolent tumor, typically arising in the parotid gland, with an intact myoepithelial layer and a cystic, papillary, often cribriform architecture. Early studies of IC identified a heterogeneous group of molecular alterations driving neoplasia, and recent studies have defined three primary morphological/immunohistochemical variants, subsequently linking these morphologic variants with defined molecular signatures. Although studies to date have pointed toward distinct molecular alterations after histological classification, this study used a novel approach, focusing primarily on six cases of IC with NCOA4-RET gene rearrangement as determined by next-generation sequencing and describing the spectrum of clinicopathologic findings within that molecularly-defined group, among them a unique association between the NCOA4-RET fusion and hybrid variant IC and the first case of IC arising in association with a pleomorphic adenoma. RET-rearranged IC show histological and immunohistochemical overlap with the more widely recognized secretory carcinoma, including low-grade morphology, a lumen-forming or microcystic growth pattern, and co-expression of S100, SOX10, and mammaglobin, findings undoubtedly leading to misdiagnosis. Typically regarded to have ETV6-NTRK3 fusions, secretory carcinomas may alternatively arise with RET fusions as well. Adding our cohort of six NCOA4-RET fusion-positive IC compared with four cases of secretory carcinoma with ETV6-RET fusions and a single case of fusion-negative IC with salivary duct carcinoma-like genetics, we propose a diagnostic algorithm that integrates histological elements, including atypia and invasiveness, and the likelihood of specific molecular alterations to increase diagnostic accuracy in what can be a very subtle diagnosis with important clinical implications.

Keywords: ETV6-RET; Intraductal carcinoma; NCOA4-RET; Salivary gland carcinoma; Secretory carcinoma.

Fig. 1

Schematic representation of RET gene rearrangements identified in salivary carcinoma. (A) NCOA4-RET isoforms include the fusion of exon 7 or exon 8 of NCOA4 (red) with exon 12 of RET (blue); (B) ETV6-RET isoforms include the fusion of exon 6 of ETV6 (red) with exon 10 or exon 12 of RET (blue). In the diagrams, the intron/exon structure of each fusion partner is depicted on the top panel, and the corresponding fusion transcripts are illustrated at the bottom. The images were generated using the Gene Structure Display Server 2.0 (http://gsds.cbi.pku.edu.cn) [18] and the following transcript identifiers: ETV6 (ENST00000396373.4), NCOA4 (ENST00000452682.1), and RET (ENST00000355710.3), obtained from the Ensembl GRCh37 release 100—April 2020 (http://grch37.ensembl.org/). The exons are displayed at the same scale for the three genes. To facilitate visualization, intron sizes were reduced to one-tenth of their original size for ETV6 and to half of their original size for NCOA4 and RET.

Fig. 2

Case 5, intercalated duct intraductal carcinoma arising in contiguity with a pleomorphic adenoma (A; 40×) with nuclear reactivity for PLAG1 by immunohistochemistry (A inset, 1000×) in the pleomorphic adenoma only, and typical morphological features including low-grade nuclei, eosinophilic cytoplasm, and circumscription by myoepithelial cells in the intraductal carcinoma (B; 400×). Tumor cells are positive by immunohistochemistry for S100 (C), SOX10 (D), and mammaglobin (E), and are negative for androgen receptor (E inset). Myoepithelial cells and rare tumor cells are highlighted by p63 (F).

Fig. 3

Case 2, intraductal carcinoma arising in an intraparotid lymph node (A; 20×), with intercalated duct morphology (3B; 400×) and a focal oncocytic component (C). The majority of the tumor has a preserved myoepithelial lining (3), except for one tumor nodule showing broad invasion (E), evidenced by loss of p63 on immunohistochemistry (F).

Fig. 4

Case 4, hybrid intraductal carcinoma with low (blue arrow) to intermediate (black arrow) grade cytologic atypia in the intercalated duct component (4A; 400×), as well as mucinous change in the intermediate-grade areas (4), confirmed by mucicarmine (not shown). The apocrine component is focal (C; 400×) but best visualized on immunohistochemistry, with nuclear reactivity for androgen receptor (D) and S100 loss (E), with intercalated duct nodule for comparison. CK5/6 highlights the intact myoepithelial layer around the minute apocrine glands (F) and is noticeably absent in a nearby focus of invasive intercalated duct component (arrow). Red circle: apocrine component.

Fig. 5

Case 6, hybrid intraductal carcinoma (A; 100×) with intercalated duct (red asterisk) and apocrine components (black asterisk). High-power examination shows intercalated duct component with cribriform architecture on the left and classic apocrine morphology on the right, including tumor cells containing abundant eosinophilic cytoplasm apical snouts and luminal decapitations (B; 400×). Immunohistochemistry demonstrates S100 (C) staining in the intercalated duct component and loss in the apocrine component, whereas HER2 was relatively increased in the apocrine component (D), showing stronger and more diffuse staining than in the intercalated duct component (D inset). Mammaglobin showed diffuse staining in both components (E). A large papillary proliferation (F; 200×) showed heterogeneous morphology (G; 400×) and immunophenotype, as demonstrated by S100 (G inset). In comparison, pure apocrine intraductal carcinomas (5; 400×) show higher grade cytomorphology and features of overt malignancy, including invasion (5 inset).

Fig. 6

Histological comparison of RET-translocated salivary gland carcinomas, including intraductal carcinoma (A; 400×) and secretory carcinoma (B; 400×), with both neoplasms showing mildly atypical round-to-ovoid nuclei, eosinophilic cytoplasm, and cribriform spaces containing eosinophilic secretions. Cytoplasmic vacuolation is seen in intercalated duct intraductal carcinoma (C) as well as RET-translocated secretory carcinoma (D). In intraductal carcinoma, vacuolated cells are often found at the luminal border of cystic spaces (E and F), where there is often mucinous metaplasia (F) and a layer of elongated, perpendicularly oriented “umbrella”-like cells with abundant, intensely staining eosinophilic cytoplasm (F, arrows). These cells do not stain with myoepithelial markers, such as p63 (F inset).

Fig. 7

Diagnostic flowchart for salivary gland malignancies with entirely or predominantly intraductal component. Instances where a diagnosis is favored over another is based on the preponderance of reported cases with the given feature, for example, molecular alteration. In advanced disease, a specific molecular driver must be identified for targeted therapy. Molecular alterations listed for rare variants of IC are listed in order of the respective variants. Confirmation of hybrid variant by RET FISH will be more easily appreciated with TRIM27-RET fusions than NCOA4-RET fusions, as the latter are intrachromosomal. FISH, fluorescence in situ hybridization; IC, intraductal carcinoma; Indel, insertion-deletion; NGS, next-generation sequencing; SC, secretory carcinoma; SNV, single-nucleotide variant. Red lines indicate diagnostic result. Dashed lines indicate supportive testing that is not necessary for confirmation.