Cytomorphologic Features of Intraductal Salivary Gland Carcinoma: A Multi-Institutional Study of 13 FNA Cases With Histologic, Molecular, and Clinical Correlations

Abstract

BACKGROUND:

Intraductal carcinoma of the salivary gland (IDC) is a rare cancer with potential actionable targets, including RET fusions. Histologic and molecular features of IDC were recently reported, but cytomorphologic data are limited. In the largest multi-institutional fine-needle aspiration (FNA) series, the authors describe the cytomorphologic features of 13 IDC cases with available clinical, radiologic, histopathologic, and molecular data.

METHODS:

The cases included 13 FNAs for 9 low-grade (LG) IDCs and 4 high-grade (HG) IDCs with corresponding histopathology and available molecular, imaging, and clinical data. Smears and liquid-based preparations available for 12 FNAs were semiquantitatively scored for key cytomorphologic findings and correlated with the corresponding resection.

RESULTS:

LG IDC FNAs showed a cellular, biphasic population of large, atypical ductal cells with mildly pleomorphic nuclei in a clean background and a minor population of small, uniform myoepithelial cells. In contrast, all HG IDC FNAs showed predominantly ductal cells with marked nuclear pleomorphism, coarse chromatin, and necrosis. With the Milan system, most LG and HG IDC FNAs were classified as either salivary gland neoplasms of uncertain malignant potential (54%) or malignant (31%). Immunohistochemistry showed ductal epithelial reactivity with mammaglobin, androgen receptor, and S100, whereas myoepithelial cells were positive for p63 and/or calponin. Among cases with next-generation sequencing, 4 LG IDCs showed NCOA4-RET gene fusions, whereas an HG IDC showed HRAS and PIK3CA mutations.

CONCLUSIONS:

The cytomorphology of IDC overlaps with other benign and malignant salivary gland neoplasms. Immunohistochemistry limits the differential diagnosis, but definitive classification requires molecular analysis. A diagnosis of IDC has potential implications for patient management.

INTRODUCTION

Intraductal carcinoma of the salivary gland (IDC) is an uncommon salivary gland malignancy. It was first described in 1983 by Chen,¹ and it has previously been called low-grade (LG) salivary duct carcinoma and LG cribriform cystadenocarcinoma. More recently, IDC, in its current iteration, was reported by Weinreb et al in 2006,² and their study seemed to better reflect the morphology and biological behavior of IDC and reduce confusion.³ It was described as such because of the presence of retained myoepithelial cells surrounding the proliferative surface epithelium. IDC was included for the first time in the 2017 edition of the World Health Organization Tumors of the Head and Neck⁴ as a unique diagnostic entity, and it has been described as analogous to atypical ductal hyperplasia or ductal carcinoma in situ of the breast. IDC is a biphasic tumor composed of ductal epithelial cells with intraductal or intracystic spread and exhibiting multiple architectural patterns, including papillary, solid, and cribriform patterns.⁴ The purely intraductal form of IDC is LG and has a well-defined myoepithelial component, but a high-grade (HG) form of IDC is also recognized.⁵

In part because of the recent application of molecular profiling to better characterize IDC, it has been the subject of several histologic studies.^5–9 IDC exists along a histologic, immunophenotypic, and molecular spectrum and, on the basis of these parameters, can be classified into 4 emerging subtypes: 1) an intercalated duct type with S100 and mammaglobin immunoreactivity and an NCOA4-RET gene fusion; 2) an oncocytic type, also immunoreactive for S100 and mammaglobin, showing unique molecular alterations, including TRIM33-RET gene fusions and BRAF V600E mutations; 3) an apocrine type expressing androgen receptor and gross cystic disease fluid protein 15 (GCDFP-15) with a complex mutational profile, including HRAS and PIK3CA mutations; and 4) a hybrid/mixed type, which has overlapping histologic and immunoprofiles with the other IDC subtypes but more often with TRIM27-RET gene fusions. An important aspect of accurate classification of IDC, either by fine-needle aspiration (FNA) or at the time of resection, is that some of the molecular changes associated with IDC represent potential therapeutic targets. In fact, these molecular changes are identifiable in both the ductal and myoepithelial components of the tumor, and this highlights the neoplastic nature of both populations with potential implications for the presumed “intraductal” nature of IDC.⁷

FNA paired with the Milan System for Reporting Salivary Gland Cytopathology (MSRSGC)¹⁰ represents the favored first step in the initial evaluation of salivary gland lesions. Although our understanding of the histologic and molecular features of IDC has significantly increased recently, data on the cytologic evaluation of IDC by FNA remain very limited and consist primarily of case reports.¹¹ In this multi-institutional study, we highlight key cytomorphologic features of IDC in the context of their histologic correlates and explore the idea of a stepwise approach to diagnosis using a combination of ancillary immunohistochemical stains and the potential for preoperative molecular testing in select cases.

MATERIALS AND METHODS

A retrospective search of the pathology archives at Massachusetts General Hospital (Boston, Massachusetts), University of Pennsylvania Hospital (Philadelphia, Pennsylvania), Beth Israel Deaconess Medical Center (Boston, Massachusetts), Newton-Wellesley Hospital (Boston, Massachusetts), Montefiore Medical Center (Bronx, New York), and Johns Hopkins Hospital (Baltimore, Maryland) identified 13 FNA specimens from 13 patients. The review of cases was approved by the respective collaborating hospital institutional review boards.

Data, including pathology reports, were evaluated for all patients. Radiologic imaging characteristics, including magnetic resonance imaging (MRI), computed tomography (CT), and/or ultrasound, were available for all IDC cases. Smear- or liquid-based preparation slides were available for review in 12 of the 13 FNA cases (9 LG IDC cases and 3 HG IDC cases). Preoperative cell block preparations were available for 44% of LG IDCs (4 of 9) and for 75% of HG IDCs (3 of 4). Adjunct molecular data were available in 9 cases (7 LG IDCs and 2 HG IDCs), including 4 cases with multiplex next-generation sequencing (NGS) only, 4 cases with fluorescence in situ hybridization (FISH) only, and 1 case with both NGS and FISH. Three of the histologic resections corresponding to FNAs have also been included in a separate study of histopathologic features only.¹²

Selected cytomorphologic features were scored semiquantitatively on a sliding scale by a senior cytopathologist at each respective institution. FNA specimens included air-dried Diff-Quik–stained smears, alcohol-fixed Papanicolaou-stained slides, and liquid-based preparations (SurePath [Becton Dickinson, East Rutherford, New Jersey] or ThinPrep [Hologic, Marlborough, Massachusetts]). FNA smears and liquid-based preparations were reviewed, and the following cytologic features were assessed: 1) cellularity (low, moderate, or high), 2) architecture (clusters, single cells, and papillary), 3) cell population (monomorphic or biphasic), 4) cell shape (oval, round, plasmacytoid, or stellate), 5) nuclear size (small, medium, or large), 6) nuclear pleomorphism (0-3), 7) presence of nucleoli, 8) cytoplasm quantity (scant, moderate, or abundant), 9) presence of cytoplasmic vacuoles, 10) cytoplasm color/consistency (delicate, dense, oncocytic, basophilic, or vacuolated), 11) chromatin quality (fine or coarse), 12) nuclear to cytoplasmic ratio (low, intermediate, or high), and 13) background findings (inflammation, necrosis, fibrosis, and stripped nuclei). If present, other unique cytologic features were noted. Representative hematoxylin-eosin–stained sections of the matched salivary gland resections were examined for cytologic-histologic correlation. Clinical parameters and radiologic imaging characteristics, when available, were obtained from the medical records. FNA diagnoses were classified with the MSRSGC criteria. All FNA Milan classifications were confirmed on review except for 1 case of LG IDC (case 2) that was initially classified at the time of diagnosis as nondiagnostic but was reclassified as atypia of undetermined significance upon rereview of the FNA smears.

RESULTS

Clinicopathologic and Radiologic Features of the IDC Cohort

Twelve IDC cases were localized to the parotid gland, and 1 was localized to the submandibular gland. Twelve FNAs were of primary tumors, and 1 case of LG IDC (case 4) was a tumor bed recurrence. In our IDC cohort (n = 13), 69% (n = 9) were LG, and 31% (n = 4) were HG (Table 1).

TABLE 1.

Clinicopathologic and Radiologic Parameters of the Intraductal Carcinoma FNA Cohort

Patient	Age, y	Gender	Sites Sampled on FNA	Size, cm	Imaging Findings	Surgical Management	Intraductal Carcinoma Histologic Subtype	Follow-Up Time, months	Recurrence or Metastasis (Yes or No)	Outcome	Additional Treatment
Low-grade intraductal carcinoma (n = 9)
1	53	M	Right parotid	1.9	US: complex cystic lesion MRI: complex, multiseptated cystic/solid mass centered in the superficial lobe	Right parotidectomy, right neck lymph node dissection	Intercalated duct	36	No	AWD	None
2	55	M	Left parotid	2.5	CT: well-circumscribed, partially cystic mass	Left parotidectomy with facial nerve preservation	Intercalated duct	3	No	AWD	Radiation for positive margin
3	44	F	Left parotid	1.4	MRI: T2 intermediate, T1 hypointense nodule	Left parotidectomy	Intercalated duct	57	No	AWD	Second surgery to remove residual disease
4	69	F	Right parotid bed	1.2	CT: heterogeneous mass with peripheral enhancement	Right parotidectomy and right neck dissection and re-excision of recurrence	Intercalated duct	12	Yes, recurrence	AWD	Second surgery for recurrence
5	57	M	Right parotid	1.3	CT: homogeneous isodense mass MRI: T1 and T2 hypointense mass	Right parotidectomy	Intercalated duct	144	No	AWD	None
6	41	M	Left parotid	1.5	MRI: solid and cystic mass with imaging features compatible with pleomorphic adenoma or another primary parotid tumor, including Warthin tumor	Left parotidectomy	Intercalated duct	6	No	NA	None
7	61	F	Right parotid	2	MRI: right parotid gland superficial mass, T1 isointense to muscle, T2 isointense to hypointense peripherally, and T2 hyperintense centrally. No definite evidence of associated restricted diffusion. This is only partially contained.	Right parotidectomy	Intercalated duct	43	No	AWD	None
8	38	F	Right parotid	1.3	US: round, anechoic simple cyst with possible thin internal septation or internal debris. No internal vascularity is present.	Right parotidectomy	Oncocytic	3	NA	AWD	NA
9	61	F	Left submandibular gland	2.4	MRI: lobulated, T2 hyperintense, mildly enhancing mass centered in the posterior left submandibular gland and extending posteroinferiorly into the submandibular space	Submandibular gland resection	Oncocytic	1	NA	AWD	NA
High-grade intraductal carcinoma (n = 4)
10	73	F	Left parotid, distal duct lesion	1.2	MRI: 1.3-cm mass involving the distal left parotid duct is suspect for neoplasm. Moderate dilatation of the parotid duct proximal to this. A 2.3-cm pathologic left level 1B lymph node is suspect for metastatic disease.	Left buccal and distal parotid dissection and left neck lymph node dissection	Outside case: slides unavailable for review	12	Yes, original tumor with metastasis to neck lymph nodes	AWD	Radiation therapy
11	77	F	Left parotid	1.8	MRI: multilobular, T2 hyperintense lesion	Left parotidectomy	Apocrine	20	No	AWD	Radiation therapy
12	59	M	Left parotid	0.9	Approximately 1-cm nodule within the inferior aspect of the superficial left parotid gland containing a focus of calcification	Left parotidectomy and left neck lymph node dissection	Apocrine	29	No	AWD	Radiation therapy
13	61	F	Right parotid	3.8	US: heterogeneous, hypoechoic, slightly lobulated solid mass with internal vascularity CT: heterogeneous, enhancing ill-defined neoplasm MRI: multicystic, partly enhancing mass with internal areas of hemorrhage	Right parotidectomy and right neck lymph node dissection	Apocrine	1	NA	NA	NA

Abbreviations: AWD, alive with disease; CT, computed tomography; F, female; FNA, fine-needle aspiration; M, male; MRI, magnetic resonance imaging; NA, not available; US, ultrasound.

LG IDC

The mean patient age was 53.2 years (range, 38-69 years) with a male to female ratio of 1:1.3 and an average tumor size of 1.7 cm (range, 1.2-2.5 cm). The radiologic imaging (n = 8; 89%) showed a range of features (Table 1 and Fig. 1). Both of the 2 cases with an ultrasound evaluation showed cystic changes. Among 3 cases with a CT evaluation, 1 was homogeneous, 1 was heterogeneous, and 1 was described as a well-circumscribed cystic mass. Lastly, 3 of the 6 cases with an MRI evaluation were described as either a T1 hypointense or isointense mass (cases 3, 5 and 7), 2 cases were described as a complex solid/cystic mass (cases 1 and 6), and 2 cases showed T2 hyperintensity (cases 7 and 9).

Figure 1.

Cytomorphologic features of low-grade intercalated-type IDC (case 5). (A) T1-weighted axial magnetic resonance imaging shows a heterogeneous, hypointense lesion at the junction of the superficial and deep lobes of the right parotid gland (white arrow). (B-F) Representative images demonstrate the biphasic nature of IDC with larger ductal cells (white arrows) and smaller myoepithelial cells (black arrows) in the low-grade IDC. Cytoplasmic features include delicate cytoplasm with round nuclei, fine chromatin, and small inconspicuous nucleoli ([B] Diff-Quik stain, original magnification ×100; [C] Papanicolaou stain, original magnification ×200; [D,E] Papanicolaou stain, original magnification, ×400; [F] SurePath preparation, original magnification × 200). IDC indicates intraductal carcinoma of the salivary gland.

Clinical follow-up was available for 7 of the 9 patients (78%); 2 had limited follow-up because of the recent diagnosis of LG IDC (cases 8 and 9; Table 1). The average follow-up time was 34 months. Four patients received no additional therapy after the initial surgery, whereas 2 patients required additional surgery: one for the removal of residual disease (case 3) and the other for a recurrence (case 4). One patient was treated with radiotherapy (6000 cGy) for a positive resection margin (case 2).

HG IDC

The mean patient age was 67.5 years (range, 59-77 years) with a male to female ratio of 1:3 and an average tumor size of 1.9 cm (range, 0.9-3.8 cm). All 4 cases had radiologic imaging and showed a range of features (Table 1, representative imaging in Fig. 5). An ultrasound and CT evaluation of 1 case (case 12) showed an ill-defined, heterogeneous, hypoechoic solid mass. An MRI evaluation was available for all 4 cases, and the finding was described as either a mass or a nodule, with 1 case showing partial enhancement and 1 case showing T2 hyperintensity.

Figure 5.

Cytomorphologic features of high-grade apocrine-type intraductal carcinoma (cases 11 and 13). (A) T2-weighted axial magnetic resonance imaging shows the multi-cystic, partly enhancing neoplasm with internal areas of hemorrhage. (B) The smears are highly cellular with a background showing necrotic debris (black arrow; Papanicolaou stain, original magnification ×100). (C) The Papanicolaou-stained slide highlights the biphasic nature of the intraductal carcinoma of the salivary gland with predominant larger ductal cells and fewer smaller myoepithelial cells (black arrows; Papanicolaou stain, original magnification ×200). (D) The cytoplasm of the larger ductal cells is abundant and granular (apocrine), and nuclei are pleomorphic (black arrow) in the ductal cells with prominent nucleoli (SurePath preparation, original magnification ×400). (E,F) Second example of a high-grade apocrine intraductal carcinoma (case 11) showing similar findings ([E] Diff-Quik, original magnification ×400; [F] Papanicolaou stain, original magnification ×100; black arrows in panel F highlight the biphasic population).

Clinical follow-up was available for all 4 patients with an average time to follow-up of 15.5 months. One patient had metastatic disease at the time of presentation to ipsilateral cervical lymph nodes (Table 1). All 3 patients who received adjuvant radiation therapy were alive without recurrence of the disease. One patient with a recent diagnosis of IDC (case 12) was scheduled for follow-up with radiation oncology.

Cytomorphologic Features of IDC, Milan System Classification, and Histologic Correlates

The cytomorphologic findings, Milan categorization, and immunohistochemical and molecular testing, when available, for each IDC in the cohort are detailed in Tables 2 and 3 and are highlighted next. An abbreviated summary of key cytomorphologic findings for the LG and HG IDC counterparts is presented in Table 4.

TABLE 2.

Cytomorphology, Molecular Profiles, and Histologic Features of the Intraductal Carcinoma Cohort

				Cytomorphologic Features
Case	Intraductal Carcinoma Histologic Subtype	FNA Preparation(s)	FNA MSRSGC Classification	Cellularity (Low, Moderate or High)	Architecture (eg, Groups, Papillary, Crowding, Single Cells)	Cell Population (Monophasic or Biphasic)	Cell Shape (eg, Plasmacytoid, Oval, Round, Stellate)	Nuclear Size (Small, Medium, or Large)	Nuclear Pleomorphism (0-3)	Nucleoli (Y or N)	Cytoplasm Amount (Scant, Moderate, or Abundant)	Cytoplasmic Vacuoles (Y or N)	Cytoplasm Color and Consistency	Chromati Quality (Fine or Coarse)	Nuclear to Cytoplasmic Ratio	Background Features	Stripped Nuclei	Necrosis	Inflammation	Fibrosis	Secretions	Additional Cytomorphologic Features
Low-grade intraductal carcinoma (n = 9)
1	Intercalated duct	DQ, Pap, TP, and Cellient cell block	SUMP	Moderate	Small groups and single cells	Biphasic	Round	Medium	2	Y	Moderate	Y	Vesicular/vacuolated	Fine	High	Cystic background with proteinaceous debris and macrophages	N	N	N	N	N	Small 3-dimensional group with scalloped borders
2	Intercalated duct	Pap and SP	Initially nondiagnostic reclassified as AUS on review	Low	Crowded groups and rare single cells	Monophasic–low cellularity–only atypical ductal cells seen	Round	Medium	1	Y, small	Moderate	N	Delicate, homogeneous	Fine	Intermediate	Histiocytes/cyst contents and blood	N	N	N	N	N	NA
3	Intercalated duct	TP	AUS	High	Crowded groups and single cells	Biphasic	Epithelioid/plasmacytoic	Variable (small cells and large cells)	1	Y, small	Moderate	N	Delicate	Fine	Intermediate (larger cells) to high (smaller cells)	Blood	Y	N	Y, scant lymphocytes	N	N	NA
4	Intercalated duct	Pap and SP	Malignant	High	Crowded groups and single cells	Biphasic, mostly ductal with few smaller myoepithelia cells	Epithelioid/plasmacytoic	Variable (small cells and large cells)	1	Y, small	Moderate	Y, rare	Delicate	Fine	Intermediate (larger cells) to high (smaller cells)	Blood	N	N	N	N	Y, scant	NA
5	Intercalated duct	DQ, Pap, and TP	SUMP	High	Crowded groups and single cells	Biphasic	Epithelioid/plasmacytoic	Variable (small cells and large cells)	1	Y, small	Moderate	Y, rare	Delicate	Fine	Intermediate (larger cells) to high (smaller cells)	Blood	N	N	N	Y	N	DQ stain showed granules, magenta secretion, fibrous fragments.
6	Intercalated duct	DQ, Pap, and TP	SUMP	Moderate	Predominantly large fragments, occasional small clusters, and rare individual cells	Biphasic, mostly smaller cells and separate fragment of larger cells with more cytoplasm	Mostly round, some oval	Variable (small cells and medium cells)	1	Y	Scant and moderate	N	Dense	Fine	Intermediate (larger cells) to high (smaller cells)	Blood, debris	Y	N	N	N	Y, scattered crystals and debris	Vacuoles, nuclear inclusions, matrix, larger cells with nucleoli, grooves, cribriform pattern
7	Intercalated duct	DQ and Pap	SUMP	High	Papillary	Biphasic, smaller cells and fragments of larger cells with more cytoplasm	Mostly round, some oval	Variable (small cells and medium cells)	2	Y	Scant and moderate	Y	Basophilic and dense	Fine	Intermediate	Blood, debris	Y	Y	N	Y	Y, abundant matrix and crystals	Very large papillary fragments, some with abundant vacuolated cytoplasm, cribriform pattern, naked nuclei, matrix, and crystals
8	Oncocytic	DQ, Pap, TP, and cell block	SUMP	High	Flat 2-dimensional sheets, rare crowded groups, and scattered single cells	Monophasic but heterogenous appearance	Round, oval, plasmacytoid	Medium	1	Y, subset	Abundant to moderate, oncocytic	Y, subset	Granular (oncocytic) occasionally vacuolated	Fine	Low	Blood	Y	N	N	N	Rare fragment in DQ slide only	Heterogeneous appearance
9	Oncocytic	TP and H & E-stained direct smears	SUMP	High	Predominantly crowded groups, occasional cribriform architecture	Biphasic, smaller basaloid cells and larger oncocytoid cells	Round, oval	Variable (small basaloid cells and large oncocytic cells)	Oncocytic cells: 2: basaloid cells: 1	Y (in subset of cells: not uniformly prominent)	Oncocytic cells: abundant; basaloid cells: scant	Y, subset	Pale basophilia, finely granular to finely vacuolated	Fine	Low (oncocytic cells); high (basaloid cells)	Fibrinous debris	N	N	N	N	Rare colloid-like proteinaceous-material	Heterogeneous appearance
High-grade intraductal carcinoma (n = 4)
10	Outside case: slides unavailable for review	DQ and cell block	Malignant	High	Small groups and single cells	Monophasic	Round	Large	3	Y	Moderate	N	Dense, and partly oncocytic	Coarse	High	Cellular debris	Y	Y	Y, lymphocytes	N	N	Focal features suggestive of squamous differentiation
11	Apocrine	DQ and Pap	SUMP	Moderate	Small to large clusters, intervening capillary network, and few single cells	Biphasic, predominantly polygonal cells, some with oncocytic cytoplasm, flanked by small, high N:C ratio, spindled to cuboidal darker cells	Plasmacytoid	Large	2	Y	Moderate	Y	Basophilic, vacuolated, and granular	Fine	Low	Necrotic	N	Y	N	N	N	Scattered, eosinophilic, granular cytoplasmic globules causing a signet ring–like appearance, prominent traversing capillaries
12	Apocrine	TP and cell block	Malignant	Moderate	Predominantly single cells, occasional small clusters, and macrophages	Biphasic, smaller cells and clusters of cells with more cytoplasm	Plasmacytoid, oval, round, stellate	Medium and large	3	Y	Moderate and abundant	Y	Basophilic and dense	Fine and coarse	Moderate to low	Blood, debris	Y	Y	N	N	Y, scant	Marked nuclear pleomorphism, necrosis, plasmacytoid cells, nuclear hyperchromasia
13	Apocrine	Pap, SP and cell block	Malignant	High	Sheets, single groups, and syncytial clusters	Biphasic, minimal	Polygonal	Large	2	Y, small and indistinct	Abundant eosinophilic cytoplasm	Y	Delicate, vacuolated	Fine	Low	Blood, necrotic debris	Y	Y	N	Y, minimal	N	Squamoid and oncocytic pattern, occasional mitotic figures

Abbreviations: AUS, atypia of undetermined significance; DQ, Diff-Quik; FNA, fine-needle aspiration; H & E, hematoxylin-eosin; MSRSGC, Milan System for Reporting Salivary Gland Cytopathology; N, no; NA, not available; N:C, nuclear to cytoplasmic; Pap, Papanicolaou; RBC, red blood cell; SP, SurePath; SUMP, salivary gland neoplasm of uncertain malignant potential; TP, ThinPrep; Y, yes.

TABLE 3.

Immunohistochemistry, Molecular Profiles, and Histologic Features of the Intraductal Carcinoma Cohort

Case	Intraductal Carcinoma Histologic Subtype	Immunohistochemistry Results of Cell Block	Immunohistochemistry Results of Surgical Resection	Molecular Results	Surgical Diagnosis
Low-grade intraductal carcinoma (n = 9)
1	Intercalated duct	Mucin stain negative	Positive for S100, p63 (rare), CK5/6 (rare), mucin stain (rare), Ki67 < 1%	Cytology cell block: Negative for ETV6 fusions by FISH	Low-grade cystadenocarcinoma AKA intraductal low-grade salivary duct carcinoma
2	Intercalated duct	NA	Positive for GATA-3, S100, mammaglobin, GCDFP-15 (patchy), p63/SMA/calponin (myoepithelial cells); negative for Her2, AR	Surgical: NCOA4-RET fusion	Low-grade intraductal carcinoma (no LVI, no PNI), positive margins
3	Intercalated duct	NA	Positive for mammaglobin, S100, p63 (myoepithelial cells), CK5/6 (focal); negative for AR	Surgical: NCOA4-RET fusion	Initially diagnosed as secretory carcinoma, reclassified as intraductal carcinoma, positive margins
4	Intercalated duct	NA	Positive for S100, SOX10, GATA-3; negative for p40, DOG1, TTF-1, c-Kit, MYB, AR, mammaglobin	Surgical: NCOA4-RET fusion	Initially diagnosed as polymorphous adenocarcinoma, reclassified as intraductal carcinoma
5	Intercalated duct	NA	Positive for calponin, SMA, p63 in myoepithelial cells supporting in situ	Surgical: NCOA4-RET fusion	Low-grade intraductal carcinoma
6	Intercalated duct	NA	Positive for S100, SOX10; focally positive for mammaglobin, DOG1; negative for SMA. p63, SMM-HC, and calponin highlight an intact myoepithelial layer around the tumor.	NA	Low-grade intraductal carcinoma (low-grade cribriform cystadenocarcinoma)
7	Intercalated duct	NA	Positive for mammaglobin and S100 in ductal cells; p63 highlights surrounding myoepithelial cells; negative for DOG1	NA	Intraductal carcinoma (cribriform cystadenocarcinoma)
8	Oncocytic	NA	Positive for (diffuse: S100, SOX10, mammaglobin; myoepithelial cells at periphery of cysts and tumor nodules: p40, calponin, SMA); negative for AR, DOG1, GCDFP15, HER2 (0−1+); low Ki-67 (1%-2%)	Negative for ETV6 and NR4A3 rearrangements by FISH	Low-grade salivary gland neoplasm, favor oncocytic low-grade intraductal carcinoma
9	Oncocytic	Positive for S100, p63 (rare cells); negative for TTF-1, thyroglobulin, PAX8, SOX10; equivocal MYB	Positive for S100, mammaglobin, SOX10; p63 positive in basal population around tumor nests; negative for DOG1, GCDFP15, AR	Negative for ETV6 rearrangement by FISH	Intraductal carcinoma, low-grade
High-grade intraductal carcinoma (n = 4)
10	Outside case: slides unavailable for review	Positive for CK7, MUC-1, CK8/18, CK7 (focal); negative for GATA-3, ER, CD10, p16, CEA, EGFR	Positive for CK7, CK20 (focal), p63/p40/CK5/6 rare cells, rare cells at the periphery of tumor positive for TTF-1, napsin A, and CDX2	Surgical: Negative for MAML2 fusions	Intraductal adenocarcinoma, high-grade, with focal invasion into duct wall and associated PNI, no LVI seen. Metastatic carcinoma with focal extranodal extension in 3/45 lymph nodes (lateral neck dissection).
11	Apocrine	NA	Positive for AR, mammaglobin (focal), HER-2 (2+, focal), p63 (stains basal layer); negative for mucicarmine	NA	Intraductal carcinoma, apocrine-type, high-grade (salivary duct carcinoma in situ), background tumor-associated lymphoid proliferation
12	Apocrine	Cytology (outside institution): positive for AE1/AE3, CK7; negative for CK20, TTF-1, napsin A, P63, CK5/6, CDX2, AR, calponin, SOX10	Positive for AR, Her2/Neu (2+). p63 highlights intact myoepithelial cells.	NA	High-grade intraductal carcinoma in association with a pleomorphic adenoma
13	Apocrine	Insufficient tumor cells for ancillary studies	Positive for GATA-3, mammaglobin, HER2 (3+), AR; p63 highlights myoepithelial cells; negative for SOX10, S100	Surgical: HRAS, PIK3CA mutations; HER2 FISH negative for amplification	Intraductal carcinoma of the parotid (3.8 cm) with focal infiltration of parenchyma and abundant comedo-type necrosis

Abbreviations: AKA, also known as; AR, androgen receptor; FISH, fluorescence in situ hybridization; GCDFP-15, gross cystic disease fluid protein 15; JHH, Johns Hopkins Hospital; LVI, lymphovascular invasion; NA, not available; PNI, perineural invasion; SMA, smooth muscle actin.

TABLE 4.

Summary of Cytomorphology in the Intraductal Carcinoma Cohort

	Tumor Type
Cytomorphologic Feature	Low-Grade Intraductal Carcinoma	High-Grade Intraductal Carcinoma
Cellularity	Moderate to high	Moderate to high
Architecture	Crowded groups and single cells	Crowded groups and single cells
Cell population (monomorphic or biphasic)	Mostly biphasic (larger ductal cells and smaller myoepithelial cells)	Often biphasic (larger ductal cells and smaller myoepithelial cells); can be monophasic (larger ductal cells only)
Cell shape	Epithelioid/plasmacytoid	Epithelioid/plasmacytoid
Nuclear features (shape, size, nucleoli, chromatin quality)	Round nuclei, fine chromatin, minimal pleomorphism, inconspicuous nucleoli	More often coarse chromatin, marked nuclear pleomorphism, prominent nucleoli
Cytoplasmic features	Delicate, rare vacuoles; can be oncocytic/granular	Dense to granular cytoplasm
Nuclear to cytoplasmic ratio	Intermediate (larger ductal cells) to high (smaller myoepithelial cells)	Intermediate (larger ductal cells) to high (smaller myoepithelial cells)
Background features	Mostly clean background, possibly cyst debris	Necrosis

For the LG IDCs (n = 9), under the MSRSGC, 67% (n = 6) were categorized as a salivary gland neoplasm of uncertain malignant potential (SUMP), 22% (n = 2) were categorized as atypia of undetermined significance, and 11% (n = 1) were categorized as malignant (Table 2). The FNA cellularity was moderate to high in 89% of the cases (n = 8) and low in 1 case (11%). Neoplastic cells were arranged either in crowded small groups or as single cells in 78% of the cases (n = 7) and less frequently in papillary or large crowded groups (22%; n = 2). Seventy-eight percent of the LG IDC FNA cases (n = 7) were biphasic and showed 2 readily identifiable cell populations, which consisted of a dominant population of larger ductal cells and a less prominent second population of either dispersed or loosely adherent smaller myoepithelial cells (Fig. 1). Two cases of LG IDC (cases 2 and 8), including an oncocytic subtype of IDC, showed a monotonous population of only large ductal cells without identifiable myoepithelial cells. The ductal cells in most cases (n = 7; 87.5%) were predominantly plasmacytoid to oval in shape with moderate amounts of delicate cytoplasm, round uniform nuclei, evenly distributed fine chromatin, and inconspicuous nucleoli with an overall intermediate nuclear to cytoplasmic ratio (Fig. 1). Two cases of the oncocytic subtype of LG IDC (cases 8 and 9) were remarkable for abundant granular oncocytic cytoplasm with a very low nuclear to cytoplasmic ratio (Fig. 2). Rare medium-sized cytoplasmic vacuoles were identified in a subset of ductal cells in 56% of the LG IDCs (n = 5; Fig. 3). In contrast to the larger ductal cells, the smaller myoepithelial component had scant delicate cytoplasm, a round nucleus, evenly distributed fine chromatin, small inconspicuous nucleoli, and an overall high nuclear to cytoplasmic ratio (Fig. 1).

Figure 2.

Cytomorphologic features and histologic appearance of low-grade oncocytic-type intraductal carcinoma (case 8). (A,B) The Diff-Quik– and Papanicolaou-stained slides show cellular groups with abundant oncocytic cytoplasm and centrally placed nuclei. Rare medium-sized cytoplasmic vacuoles are identifiable (black arrows in panel B). (C) Similar features can be noted on the ThinPrep preparation. (D-F) Corresponding histologic correlate and immunoprofile with S100 and mammaglobin (H & E and immunoperoxidase staining, original magnification ×200 for all images). Decorating myoepithelial cells were identified with smooth muscle actin and calponin (data not shown).

Figure 3.

Other interesting cytomorphologic features noted on fine-needle aspirates of intercalated-type intraductal carcinoma of the salivary gland with a corresponding histologic correlate: (A) proteinaceous secretions, (B) fibrosis, and (C) rare cytoplasmic vacuoles (Papanicolaou stain, original magnification ×100 for A-B, ×400 for C) and (D) their corresponding histologic features (larger arrows highlight the magenta secretion and fibrosis, and the smaller arrow highlights rare small vacuoles; case 2; H & E, original magnification ×100).

Fifty-six percent of the LG IDCs (n = 5) had debris or cyst contents in the background, whereas the other 44% had a background that either was clean or contained blood. Only 1 case (11%) of LG IDC showed evidence of background necrosis. Three LG IDCs and 1 HG IDC showed scant proteinaceous secretions, and 3 LG IDCs exhibited crystals or matrix-type material. Scant accompanying lymphocytes and fibrosis were seen in 22% (n = 2) and 11% (n = 1) of the FNA cases, respectively (Fig. 3 ).

On histologic resection, the majority of LG IDCs demonstrated the intercalated-duct type of histomorphology (Fig. 4) except for 2 cases of oncocytic IDC (cases 8 and 9). Histologically, the tumors consisted of well-demarcated tumor lobules outlined by small myoepithelial cells and separated by thin bands of fibrous stroma. The intraductal component was either solid or cribriform and was composed of uniform, histologically bland ductal cells with moderate amounts of eosinophilic cytoplasm and round nuclei (Fig. 4). The oncocytic IDC cases had abundant eosinophilic cytoplasm but otherwise showed morphologic features similar to those of the intercalated-duct subtype (Fig. 2).

Figure 4.

Histomorphologic features of low-grade intercalated-type intraductal adenocarcinoma (case 5). (A-D) The histologic image highlights the biphasic nature of the intraductal carcinoma of the salivary gland, with larger ductal cells containing moderate cytoplasm and round nuclei. These ductal cells are positive for mammaglobin and S100, whereas the myoepithelial cells are highlighted by p63 (H & E and immunoperoxidase staining, original magnification ×100 for all images).

Our HG IDC cohort comprised 4 cases, of which 3 (75%) were the apocrine subtype. Fifty percent (n = 2) were classified by the MSRSGC as malignant, and 50% (n = 2) were SUMP (Table 2). Overall, the HG cases exhibited marked nuclear atypia. The HG IDC FNAs were cellular with a predominance of large, atypical ductal cells in small crowded groups and single cells (Fig. 5). Unlike the LG IDC cases, the biphasic nature of the HG tumors was difficult to appreciate. Three of the HG IDC FNA cases (75%) exhibited a minor component of myoepithelial cells. The ductal cells contained a round nucleus, a prominent nucleolus, and dense, somewhat oncocytic cytoplasm, which imparted a squamoid appearance to 2 of the cases (cases 10 and 12). Occasional cytoplasmic vacuoles were present in 3 of the HG IDC cases (cases 10-12). All 4 HG IDC FNAs showed background necrosis (100%). No fibrosis was noted in any of the HG IDC FNAs.

The corresponding resected HG IDCs showed large, atypical ductal cells organized in crowded groups with pleomorphic nuclei and prominent nucleoli. One tumor (case 10) had squamoid features with focal invasion into the parotid duct wall, and the 2 apocrine-subtype cases (cases 11 and 13) showed abundant granular cytoplasm (Fig. 6). Necrosis was present in all 4 HG cases.

Figure 6.

Histologic example of high-grade apocrine-type intraductal carcinoma (case 11). (A-D) Histologic sections show an intraductal proliferation of large ductal cells with abundant granular (apocrine) cytoplasm, nuclear pleomorphism, and prominent nucleoli (the white arrow highlights myoepithelial cells; the black arrow highlights ductal cells). Necrosis is present. Immunophenotyping shows that the ductal cells are positive with AR, whereas p63 highlights the surrounding myoepithelial cells (H & E and immunoperoxidase staining, [A] original magnification ×200; [B] original magnification ×400; [C,D] original magnification ×100). AR indicates androgen receptor.

Ancillary Immunohistochemical and Molecular Profiling for IDC

Four of the FNA cohort cases had immunohistochemistry performed on both the cell block and surgical resection specimens, and 9 cases had immunohistochemistry only on the surgical resection. For the LG IDC cases with immunophenotyping, 100% (n = 8 of 8 LG IDCs stained with S100) were positive for S100, and 86% (n = 6 of 7 LG IDCs stained with mammaglobin) were positive for mammaglobin. Myoepithelial markers (p63, calponin, and smooth muscle actin), in the 7 cases for which they were performed, highlighted the surrounding basal myoepithelial layer in all LG IDCs tested, and this supported the intraductal nature of the lesions (Fig. 4). Among the HG IDCs, 75% (n = 3; cases 11-13) were positive for androgen receptor and Her2/Neu, and mammaglobin was positive in 2 cases (50%; cases 11 and 13). One HG IDC (case 10) did not show a specific immunoprofile, although S100, androgen receptor, and mammaglobin were not tested in that particular case. However, similarly to the LG IDCs, at least focal myoepithelial cells could be identified with p63 in all of the cases (100%; Fig. 6 and Table 3).

Nine IDC FNA cases had adjunct molecular data: 1 was performed on cell block material (case 1), and 8 were performed on surgical resections, including multiplex NGS and/or FISH (Table 3). FISH data were available for 5 patients (cases 1, 8, 9, 10, and 13). Case 1 was negative for an ETV6 rearrangement in the cytology cell block material, and this helped to exclude secretory carcinoma. Among surgical resections with FISH, cases 8 and 9 were negative for both ETV6 and NR4A3 rearrangements, and this excluded secretory carcinoma and acinic cell carcinoma; case 10 was negative for an MAML2 rearrangement in the histologic resections, and this excluded mucoepidermoid carcinoma (MEC). HER2 FISH for case 13 was negative for amplification. Molecular sequencing data were available only for the histologic resection for 4 LG IDC cases (cases 2-5) and 1 HG IDC case (case 13). All LG IDCs with available sequencing data demonstrated the characteristic NCOA4-RET gene fusion, whereas the single HG IDC case showed HRAS and PIK3CA mutations.

DISCUSSION

IDC is a rare salivary gland malignancy that has gained much attention in recent years through an improved understanding of its histomorphologic and molecular traits along with its most recent inclusion as a unique entity by the World Health Organization.^2–7,9,13 IDC demonstrates a spectrum of histological appearances from LG to HG, but a majority of IDCs are LG lesions associated with a good clinical prognosis.⁴ Although advances have been made in terms of the surgical pathology of IDC, the existing literature on the cytologic features of IDC is limited and is predominantly restricted to a few case reports as summarized in a recent systematic review by Palicelli.¹¹ As supported by our FNA cohort, IDCs are most common in adult patients in the sixth decade with an approximately equal gender distribution. Although 12 of our 13 cases occurred in the parotid gland, occasional cases have been reported in the minor salivary glands or the submandibular gland.¹

As shown in the current study and reported in the literature,¹¹ IDCs may be either LG or HG. LG IDCs are the most common and have bland cytologic features. Depending on the subtype of IDC, the cytoplasm can range from a dense squamoid type to being vacuolated. Oncocytic and apocrine subtypes, as may be hypothesized, have the most abundant cytoplasm with many vacuoles. The FNA background can be cystic and may have serous secretions, but background necrosis is more commonly seen in HG IDC cases. Because of the range of LG cytomorphologic features, which overlap with several other primary salivary gland tumors, most FNA cases of IDC, on the basis of cytomorphology alone, would be classified as SUMP in the MSRSGC. In contrast, HG forms of IDC, distinguished by their marked ductal cell nuclear atypia, hyperchromasia, and background necrosis, are usually classified as either suspicious for malignancy or malignant.

Although not previously described in other FNA reports of IDC,¹¹ a biphasic cellular pattern was very common, and this can be used to help limit the differential cytologic diagnosis (Table 5). The second population of cells consists of small, bland myoepithelial cells present either as single cells in the background or along the periphery of ductal epithelial clusters. An important caveat is that the myoepithelial population, though often present in both LG and HG IDCs, may be less conspicuous in the HG forms of IDC or if the population is not sampled, as in case 2 of our cohort, which had low cellularity. Thus, the absence of the biphasic pattern does not necessarily preclude the diagnosis of IDC, but if it is present, it would lend support to the diagnosis.

TABLE 5.

Cytomorphologic and Molecular Features of Key Entities in the Differential Diagnosis for Intraductal Carcinoma

Differential Diagnoses	Salient Cytomorphologic Features	Molecular Features	Intraductal Carcinoma Subtype	IHC Testing
				S-100	GATA-3	Mammaglobin	p63	p40	Other Unique IHC
Intraductal carcinoma	Two cell populations: smaller myoepithelial cells and larger atypical ductal cells	Intercalated duct variant: NCOA4-RET, less common STRN-ALK, TUT1-ETV5, KIAA1217-RET	Intercalated duct variant	+	+	+	+ (myoepithelial cells)	+ (myoepithelial cells)	MUC4 negative
	Moderate cytoplasm, very rare small vacuoles	Apocrine variant: complex, multiple mutations (HRAS, PIK3CA, TP53 among others)	Apocrine variant	−	+	+			AR+, Her2+
	Epithelioid/plasmacytoid with round nucleus	Oncocytic variant: TRIM33-RET, BRAF V600E, less common NCOA4-RET	Oncocytic variant	+	NA	+			AR−
	Fine chromatin and small nucleoli	Mixed hybrid variant: TRIM27-RET, less common NCOA4-RET	Mixed hybrid variant	+ (intercalated-duct-like areas)	NA	NA			AR+ in apocrine areas
Secretory carcinoma	Single population of intermediate-sized cells Micro/macrovacuolated cytoplasm in most cells Round nuclei and small nucleoli	ETV6-NTRK3, ETV6-RET, VIM-RET fusions		+	+	+	−	−	MUC4+ Phosphorylated STAT5+
Salivary duct carcinoma	Abundant eosinophilic cytoplasm Marked nuclear pleomorphism with prominent nucleoli and coarse chromatin Background necrosis	Complex, multiple mutations (HRAS, PIK3CA, TP53 among others)		−	+	Variable	−	−	AR+, Her2+
Epithelial-myoepithelial carcinoma	Two cell populations: smaller myoepithelial cells and larger atypical ductal cells Epithelioid/plasmacytoid with round nucleus Fine chromatin and small nucleoli	RAS mutations		+	Variable	−	+ (myoepithelial cells)	+ (myoepithelial cells)	Keratin 7 highlights ductal cells.
Acinic cell carcinoma with TALP	Oncocytic cells with vacuolated cytoplasm in 3-dimensional clusters Stripped-naked nuclei Admixed lymphocytes	t(4;9) and HTN3-MSANTD3 translocation		−	Variable	−	−	−	DOG1+, SOX10+ NR4A3+ PAS-D highlights zymogen granules.
Mucoepidermoid carcinoma with TALP	Mucinous background Glandular mucinous cells and squamoid cells Muciphages	MAML2 translocation		Variable	Variable	Variable	+ (diffuse in epidermoid cells)	+ (diffuse in epidermoid cells)	Mucicarmine may highlight mucin-containing glandular cells.
Polymorphous adenocarcinoma (especially in minor salivary gland)	Scant cytoplasm, high nuclear to cytoplasmic ratio Vesicular chromatin, small nucleoli, and nuclear grooves Stromal/matrix globules	PRKD1 mutations or rearrangements		+	Variable	+	+ (diffuse)	−	NA
Oncocytoma	Bland-appearing oncocytes	NA		NA	+	NA	−	−	NA

Abbreviations: AR, androgen receptor; IHC, immunohistochemistry; NA, not applicable or not available; TALP, tumor-associated lymphoid proliferation.

A definitive FNA diagnosis of IDC on FNA cytomorphology alone is not possible because of the cytomorphologic overlap with other benign and malignant salivary gland neoplasms. Although our FNA cohort has revealed cytomorphologic features that affirm or might suggest the diagnosis of IDC, ancillary studies are essential for a definitive diagnosis. The 2 most common entities in the differential diagnosis are epithelial-myoepithelial carcinoma and secretory carcinoma. Similar to epithelial-myoepithelial carcinoma, cases of IDC show a biphasic pattern; however, while the ductal cell is the dominant cell type in IDCs, the myoepithelial cell either dominates or is present in equal proportions in epithelial-myoepithelial carcinomas. Both IDC and secretory carcinoma contain LG cells with moderate cytoplasm, cytoplasmic vacuoles, background secretions, and similar immunohistochemical features, but secretory carcinoma lacks the often conspicuous biphasic pattern of IDC.¹⁴ In addition, cytoplasmic vacuolization is much more pronounced in secretory carcinoma, whereas vacuoles are sparse in IDC. Importantly, if only the large ductal population were sampled from an IDC, it would be difficult to distinguish it from secretory carcinoma without ancillary studies.

MEC also presents in the cytologic differential diagnosis of IDC, as considered in 1 of the cases in our cohort (case 1). In the systematic review by Palicelli,¹¹ the diagnosis of MEC carcinoma was suggested in 2 cases. Although MEC contains a population of p63-positive epidermoid and intermediate cells, which can mimic the appearance of ductal cells, it lacks myoepithelial cells. Most MEC cases show a mucinous background, which was not present in our cases. Mucinous features have been rarely reported in IDCs as either entrapped native mucinous cells or mucinous metaplasia.¹¹

The differential diagnosis for the oncocytic and apocrine subtypes of IDC includes oncocytoid/squamoid salivary gland neoplasms such as secretory carcinoma, MEC, oncocytoma, Warthin tumor, and acinic cell carcinoma. Cyst debris associated with oncocytic ductal cells can be seen in some IDC FNA cases. Although not observed in our IDC cohort, tumor-associated lymphocytes have been described in some IDC cases.^11,15 An awareness of the spectrum of cytologic appearances of IDC is, therefore, useful to avoid misinterpreting an IDC as a Warthin tumor or oncocytoma. For cases in which IDC is a diagnostic possibility, ancillary studies can be used to avoid a misdiagnosis.

Finally, with respect to HG IDCs (Figs. 5 and 6), the limited HG cases in our FNA cohort suggest that FNA will at least be suggestive if not diagnostic of an HG tumor, and this could be critical for clinical management. The specific classification, however, can be exceptionally challenging, and the differential will include other HG neoplasms such as salivary duct carcinoma, HG MEC, and salivary gland carcinoma with HG transformation. Ancillary studies, including immunohistochemistry and possibly molecular analysis, are needed to limit the differential diagnosis and provide a specific classification; however, aside from being an HG carcinoma, this would not necessarily affect the management.

With regard to a definitive FNA classification of IDC, ancillary studies, including both immunohistochemistry and especially molecular analysis on cell block specimens, could be very useful, if not essential.¹⁶ LG IDC usually demonstrates an immunohistochemical profile akin to neoplastic proliferations of breast origin, namely atypical ductal hyperplasia and LG ductal carcinoma in situ, and as such is strongly and diffusely positive for mammaglobin, GATA-3, and S100, with p63 highlighting the subpopulation of myoepithelial cells.^5–7 This immunoprofile can be used to exclude entities such as epithelial-myoepithelial carcinoma, MEC, and acinic cell carcinoma from consideration. However, there is significant overlap between the immunoprofiles of LG IDC and secretory carcinoma, which is also diffusely positive for S100, mammaglobin, and GATA-3.^4,6,17 In general, secretory carcinoma would lack the p63-positive subpopulation of myoepithelial cells present in most cases of LG IDC.¹⁴ Most recently, a few studies have highlighted that MUC4 and phosphorylated STAT5 are sensitive and specific immunohistochemical surrogates for secretory carcinoma, and they are negative in other mimics, including IDC.^18,19 Polymorphous adenocarcinoma may have a similar immunoprofile overlapping with IDC; however, this is most often in the minor salivary glands, whereas IDC is most often localized to the parotid gland.^4,20 In addition, polymorphous adenocarcinoma often displays an unusual and distinct p63+/p40− immunoprofile. In contrast to LG IDC, 67% of HG IDCs express androgen receptor and Her2/Neu. Among the 2 HG IDC cases tested for GATA-3, 50% showed expression; however, the sampling was limited in 1 case (case 10). As expected, this staining pattern overlaps with that of salivary duct carcinoma and is, therefore, not entirely specific.²¹

Despite being an expensive and technologically demanding ancillary test, molecular profiling, particularly when combined with the cytomorphology and immunoprofile, provides a highly effective tool that can be applied to cytology to improve the accuracy of the FNA diagnosis. In addition, it can also provide preoperative information about potential actionable targets that can maximize therapeutic efficacy while minimizing side effects. With regard to IDC, 1 such oncogene target is RET proto-oncogene.²² As shown in our cohort of IDC cases, alterations in RET have recently been shown to be the most characteristic feature of many IDCs, where certain RET fusions, depending on the specific histologic subtype, are overrepresented.^5,6,23,24

Specific molecular changes identified in IDC include NCOA4-RET (most common in the intercalated-duct type), TRIM27-RET (often in hybrid/mixed IDC), TRIM33-RET (often in oncocytic IDC), BRAF V600E mutations (often in oncocytic IDC), complex molecular alterations of HRAS and PIK3CA (apocrine IDC), and rarely STRN-ALK fusion (rare cases of IDC).^5,6,9,23,24 In our cohort, all LG IDCs with available molecular sequencing data demonstrated NCOA4-RET fusions corresponding to the intercalated-duct type of morphology on histology. A single HG IDC FNA case with molecular sequencing data showed HRAS and PIK3CA alterations, which are consistent with an apocrine-type IDC (Table 3). In view of the range of molecular features that can be identified in cases of IDC, molecular profiling using a platform such as NGS is favored. An important caveat is that FISH analysis has diagnostic limitations for the identification of IDC, particularly because the NCOA4-RET fusion often produces a false-negative FISH result.^5,9,24

Because of the prominence of RET fusions in a majority of IDCs, RET inhibitors are gaining attention as potential therapeutic tools for the treatment of patients with aggressive or recurrent IDC.²⁵ In fact, Bishop et al⁷ have shown evidence that the RET alteration is present in both the ductal and myoepithelial cell populations, and this highlights the neoplastic nature of the entire lesion and raises the possibility that this may represent an invasive carcinoma, in which case these targeted therapeutics may become even more relevant for treatment. Recent clinical trials in patients with various RET fusion–positive cancers using RET inhibitors, including selpercatinib and pralsetinib, have demonstrated therapeutic responses with rates ranging from 56% to 63%.^26–28

Currently, rationales for molecular analysis in selected salivary neoplasias detected by FNA are useful when one is considering preoperative neoadjuvant therapy, in cases in which surgical planning would be either altered or deferred on the basis of the result, or in cases in which surgery is not an option because of the patient’s clinical status. Recent studies with kinase fusion–related thyroid carcinomas^29,30 provide a useful template to apply in the context of cytologic and small biopsy specimens of kinase-fusion or kinase-mutated salivary gland malignancies such as IDC when the result would potentially affect the clinical management (Fig. 7).

Figure 7.

Proposed algorithm for selecting salivary gland FNA cases for preoperative molecular testing. On the basis of the cytomorphology of the smear preparations from oncocytoid/squamoid salivary gland neoplasms, if a biphasic or monophasic population of round, large, ductal-type cells with delicate vacuolated or oncocytoid/squamoid cytoplasm is noted, then the differential is broad and includes intraductal carcinoma, secretory carcinoma, mucoepidermoid carcinoma, and epithelial-myoepithelial carcinoma. If background necrosis, marked nuclear pleomorphism, and/or coarse chromatin is noted, then the possibility of a high-grade component can be suggested. In either circumstance, it is worthwhile to collect a dedicated FNA pass for a cell block preparation for subsequent ancillary testing with IHC with select markers to narrow down the differential. If the IHC is definitive, then molecular testing to identify actionable targets can be considered if the patient is a suboptimal surgical candidate or if the patient needs neoadjuvant therapy. Conversely, if the IHC is inconclusive and if the surgical management can be affected by the diagnosis, molecular testing should be performed; this not only may help to identify the unique genetic alterations but also might provide actionable therapeutic targets. FNA indicates fine-needle aspiration; IHC, immunohistochemistry.

Our multi-institutional FNA series highlights several key cytomorphologic features of rarely encountered IDCs, most importantly the biphasic pattern characteristic of LG IDCs, which include a predominant population of larger ductal cells and a minor population of smaller p63-positive myoepithelial cells. Additional features may include background necrosis, marked nuclear pleomorphism, and coarse chromatin, which might suggest an HG variant of IDC. Being aware of the spectrum of cytomorphologic features of IDC is important for providing the most inclusive differential diagnosis, particularly for LG salivary gland tumors. In such cases, adequate specimens should be obtained for adjunct immunohistochemical and molecular profiling because a definitive preoperative diagnosis of carcinoma could significantly influence the surgical management. For most cases, a salivary gland FNA with an indeterminate interpretation may be sufficient as a preoperative screen, but in cases in which the salivary gland FNA is a tissue end point, more precise classification may be needed.