pitfalls in the biopsy diagnosis of intraoral minor salivary gland...

Pitfalls in the Biopsy Diagnosis of Intraoral Minor SalivaryGland Neoplasms: Diagnostic Considerations and

Recommended Approach

Andrew T. Turk, MD* and Bruce M. Wenig, MDw

Abstract: Among the more common types of intraoral minor sali-vary gland neoplasms are pleomorphic adenoma, basal cell ade-noma, polymorphous low-grade adenocarcinoma, and adenoidcystic carcinoma. These minor salivary gland neoplasms sharesimilar morphologic features and to a large extent immunohis-tochemical findings. Differentiation between these benign andmalignant neoplasms is often predicated on the presence or absenceof invasion. As such, in the presence of limited tissue sampling thattypifies the initial testing modalities, including fine needle aspira-tion biopsy and/or incisional biopsy, it often is not possible todifferentiate a benign from malignant minor salivary gland neo-plasm. The diagnostic difficulties arise from the absence in needleor incisional biopsy of the tumor’s periphery to determine whetherinfiltrative growth is or is not present. In this manuscript we discusslimitations and considerations associated with evaluation of inci-sional biopsies of intraoral minor salivary gland tumors. We offer adiagnostic approach to evaluating these biopsies, and suggestdiagnostic terminology for biopsy specimens in which distinctionbetween benignancy and malignancy is not feasible. The patholo-gist’s approach to this distinction is critical, as treatment of benignneoplasms is generally conservative, whereas malignant lesions maywarrant more aggressive management.

Key Words: intraoral minor salivary gland neoplasms, incisional

biopsy, pleomorphic adenoma, basal cell adenoma, polymorphous

low-grade adenocarcinoma, adenoid cystic carcinoma

(Adv Anat Pathol 2014;21:1–11)

The 2005 World Health Organization classification systemrecognizes 24 malignant and 10 benign salivary gland

epithelial tumors.1 Since 2005, additional entities have beenidentified and/or more clearly defined, including (but notlimited to) mammary analog secretory carcinoma2 and cri-briform adenocarcinoma of the tongue.3 With rare exceptions,any neoplasm that occurs in major salivary glands may alsoarise in minor salivary glands, which are located throughoutthe mucosa of the upper aerodigestive tract. The oral cavityrepresents one of the most common locations of minor salivarygland tumors. Frequently encountered intraoral minor salivarygland neoplasms include benign tumors such as pleomorphicadenoma and monomorphic adenoma, basal cell type (also

known as basal cell adenoma), and malignant lesions such asmucoepidermoid carcinoma, adenoid cystic carcinoma, andpolymorphous low-grade adenocarcinoma.

The initial diagnostic modality for a patient with anintraoral mass is fine-needle aspiration biopsy or incisionalbiopsy. The utility of these techniques is constrained bylimitations related to tissue sampling. In addition, sim-ilarities between the growth patterns, cytomorphology, andimmunoreactivity of common intraoral minor salivarygland tumors (benign and malignant) further complicatesthe interpretation of fine-needle aspiration biopsy andincisional biopsy specimens. Consequently, distinctionbetween benign and malignant intraoral minor salivarygland lesions can be difficult, especially given limited biopsymaterial. Mucoepidermoid carcinoma represents a poten-tial exception to this problem. This tumor is characterizedby cellular proliferation that includes mucocytes, epi-dermoid cells, and intermediate cells (Fig. 1). As no othersalivary gland neoplasm consists of these cellular compo-nents, identification of these cell types permits a diagnosisof mucoepidermoid carcinoma even in the context of lim-ited sampling. Other lesions such as pleomorphic adenoma,basal cell adenoma, adenoid cystic carcinoma, and poly-morphous low-grade adenocarcinoma, however, share lightmicroscopic and immunohistochemical (IHC) features suchthat distinction between benign and malignant lesions oftendepends upon identification of invasive growth. Biopsysampling of intraoral minor salivary gland tumors fre-quently consists of lesional material without surroundingtissues, and therefore does not facilitate assessment ofinvasive growth (Fig. 2). Within this context, a specificdiagnosis rendered on the basis of fine-needle aspirationbiopsy or incisional biopsy may result in a discordantdiagnosis after complete surgical resection.

RESULTS

Shared Findings Among Minor Salivary GlandNeoplasms

Aside from mucoepidermoid carcinoma, the mostcommon intraoral minor salivary gland neoplasms listedabove show overlapping light microscopic and IHC fea-tures. Any of these lesions may show circumscriptionwithout encapsulation, multiple growth patterns, blandcytomorphology, combination of epithelial and myoepi-thelial cell differentiation, absent to minimal increase inmitotic activity, and to a large extent similar IHC reactivity.

Circumscription, Encapsulation, and InvasiveGrowth

All minor salivary gland neoplasms, whether benign ormalignant, are unencapsulated. Accordingly, the presence orabsence of a capsule does not differentiate benign and

From the *Department of Pathology and Cell Biology, ColumbiaUniversity; and wDepartment of Diagnostic Pathology andLaboratory Medicine, Beth Israel Medical Center, St Luke’s andRoosevelt Hospitals, New York, NY.

The authors have no funding or conflicts of interest to disclose.Reprints: Bruce M. Wenig, MD, Department of Diagnostic Pathology

and Laboratory Medicine, Beth Israel Medical Center, St Luke’sand Roosevelt Hospitals, New York, NY 10003 (e-mail: [email protected]).

All figures can be viewed online in color at http://www.anatomicpathology.com.

Copyright r 2013 by Lippincott Williams & Wilkins

REVIEW ARTICLE

Adv Anat Pathol � Volume 21, Number 1, January 2014 www.anatomicpathology.com | 1

mailto:[email protected]

mailto:[email protected]

http://www.anatomicpathology.com

http://www.anatomicpathology.com

malignant minor salivary gland tumors. Both benign andmalignant intraoral minor salivary gland neoplasms may becircumscribed (Fig. 3), but malignant lesions show invasivegrowth, including invasion into non-neoplastic seromucousglands (Fig. 4A) and/or soft tissues (Fig. 4B), perineuralinvasion (PNI) (Fig. 4C), and lymph-vascular invasion (LVI).Metastatic disease would certainly confer a diagnosis ofmalignancy, but herein we focus on biopsies of intraoralminor salivary gland lesions without clinically evident nodalmetastasis. It should be noted that extension of tumor to (andeven involvement of) the surface (squamous) epithelium does

not represent a criterion for malignancy (Fig. 5). If biopsysampling shows unequivocal evidence of infiltrative growth,then a definitive diagnosis of carcinoma can be rendered.However, such findings are uncommon in biopsies ofintraoral minor salivary gland neoplasms, which frequentlyshow only lesional tissue, and lack sufficient sampling ofsurrounding tissues for assessment of invasive growth.

Growth PatternsSalivary gland neoplasms of both major and minor sali-

vary gland origin often show >1 growth pattern. In other

FIGURE 2. Incisional biopsy of intraoral minor salivary gland neoplasms often yield limited tissue especially the absence of tumorborders to evaluate for invasive growth. A and B, Incisional biopsy shows a cellular neoplasm comprised of tubules with isomorphicbasaloid-appearing nuclei without features allowing for determining whether it is benign or malignant. C, After complete excisionshowing circumscription without invasion, a diagnosis of basal cell adenoma can be rendered. D and E, Incisional biopsy of anotherintraoral minor salivary gland neoplasm showing a cellular neoplasm comprised of tubules with isomorphic basaloid-appearing nucleiwithout features allowing for determining whether it is benign or malignant. F, After complete excision, perineural invasion is presentallowing for a diagnosis of carcinoma, in this case polymorphous low-grade adenocarcinoma.

FIGURE 3. All minor salivary gland neoplasms are unencapsu-lated and often even malignant neoplasms such as poly-morphous low-grade adenocarcinoma appears circumscribed inits superficial aspect.

FIGURE 1. Mucoepidermoid carcinoma. This tumor is comprisedof an admixture of cells types including mucocytes, epidermoidcells, and intermediate cells. The identification of these cell typesonly seen in mucoepidermoid carcinoma is diagnostic.

Turk and Wenig Adv Anat Pathol � Volume 21, Number 1, January 2014

2 | www.anatomicpathology.com r 2013 Lippincott Williams & Wilkins

words, polymorphic architecture in an intraoral minor salivarygland neoplasm is not equivalent to any specific diagnosis suchas polymorphous low-grade adenocarcinoma. Pleomorphicadenoma, monomorphic adenoma, and adenoid cystic carci-noma may show a combination of growth patterns includingtubular-ductular, solid, (micro)cystic, cribriform, trabecular,nodular, lattice-like, fascicular, and/or papillary. Accordingly,no single growth pattern or combination of growth patternsdefines any specific neoplasm or distinguishes between minorsalivary gland tumors. It should also be noted that significantmorphologic variability may exist within a single tumor, andbetween different examples of a single diagnostic entity. Forinstance, basal cell adenoma and basal cell adenocarcinomaoccur as tubular, trabecular, solid, cribriform, and mem-branous variants; adenoid cystic carcinoma shows tubular,cribriform, and solid patterns; polymorphous low-grade ade-nocarcinoma exhibits tubular, cribriform, solid, and papillarygrowth. Hybrid adenoid cystic carcinoma, in which adenoidcystic carcinoma coexists with another histologic subtype ofsalivary gland carcinoma, by definition shows different archi-tectural features in different areas. This heterogeneity withinspecific entities frequently complicates the interpretation ofsalivary gland biopsies, especially incisional biopsies that pro-vide limited material.

Cribriform growth is stereotypically associated withadenoid cystic carcinoma (Fig. 6A), and is not generally con-sidered characteristic of pleomorphic adenoma or basal celladenoma. Cribriform growth may suggest a diagnosis of

adenoid cystic carcinoma, but is not pathognomonic of thislesion. This finding may also be present in other intraoralminor salivary gland neoplasms including polymorphous low-grade adenocarcinoma (Fig. 6B), as well as pleomorphic ade-noma (Fig. 6C) and basal cell adenoma (Fig. 6D).

Various other associations exist between certain tumorsand specific architectural features. Nuclear palisading ofbasaloid cells along the stromal interface is often seen in basalcell adenoma, although this feature is not specifically diag-nostic of basal cell adenoma. In polymorphous low-gradeadenocarcinoma, swirling or whorling patterns (Fig. 7A), oftenat the lesion’s periphery, are characteristic (although notpathognomonic). However, given their localization at theperipheral aspects, these arrangements may not be evident inlimited incisional biopsy specimens. Polymorphous low-gradeadenocarcinoma may also show single-file formations oftumor cells (Fig. 7B) (akin to lobular carcinoma of the breast),although this feature is not consistently seen and is absent inmany cases. Another characteristic although not patho-gnomonic finding associated with polymorphous low-gradeadenocarcinoma is the incorporation of non-neoplastic sero-mucous glands (Fig. 7C). Conversely, pleomorphic adenoma,basal cell adenoma, and adenoid cystic carcinoma generallycause architectural effacement such that residual seromucousglands cannot be identified within the tumor. Althoughenvelopment of residual glands (individually or in clusters)warrants suspicion of polymorphous low-grade adenocarci-noma, this feature does not represent unequivocal evidence ofpolymorphous low-grade adenocarcinoma. Diagnosis of pol-ymorphous low-grade adenocarcinoma (ie, diagnosis of minorsalivary gland carcinoma) requires identification of PNI, LVI,and/or invasive growth.

Despite purportedly specific growth patterns, pleo-morphic adenoma, basal cell adenoma, polymorphous low-grade adenocarcinoma, and adenoid cystic carcinoma sharetoo much architectural overlap to permit definitive diag-nosis based on any single growth pattern, or combinationof architectural features.

CytomorphologyThe extent of cytomorphologic similarity between

pleomorphic adenoma, basal cell adenoma, polymorphouslow-grade adenocarcinoma, and adenoid cystic carcinomaseverely hinders definitive diagnosis based on limited tissuesampling. These tumors consist of cells with limited nuclearpleomorphism, absent to low mitotic activity, and absenceof necrosis. Certain cytologic features may suggest a specificdiagnosis, such as adenoid cystic carcinoma characteristi-cally contains abundant myoepithelial (abluminal) cells

FIGURE 4. The presence of infiltration confers a diagnosis of malignancy. Polymorphous low-grade adenocarcinoma showing invasionof (A) seromucous glands and (B) skeletal muscle; perineural invasion in adenoid cystic carcinoma (C).

FIGURE 5. Pleomorphic adenoma showing lesional cellsextended to and even involvement of the surface (squamous)epithelium. This finding is not diagnostic for malignancy.

Adv Anat Pathol � Volume 21, Number 1, January 2014 Intraoral Minor Salivary Gland Neoplasms

r 2013 Lippincott Williams & Wilkins www.anatomicpathology.com | 3

with increased nuclear-to-cytoplasmic ratio, and basaloid(hyperchromatic) angulated nuclei without identifiable nucleoli(Fig. 8). Similar nuclear features, however, can be present inbasal cell adenoma and even in pleomorphic adenoma. Incontrast, basaloid hyperchromatic nuclei are not generallyevident in polymorphous low-grade adenocarcinoma, whichtypically consists of cells with vesicular chromatin and incon-spicuous small nucleoli (Fig. 8). Squamous differentiation (eg,squamous eddies) with or without keratinization is relativelycommon basal cell adenoma, particularly the membranousvariant (Fig. 9). Squamous metaplasia also occurs in pleo-morphic adenoma and polymorphous low-grade adenocarci-noma (see below under the Metaplasia section), but this phe-nomenon is highly unusual in adenoid cystic carcinoma.Beyond associations such as these, other cytologic features areless specific. Clear cells and oncocytic cells, for instance, arepresent in a wide variety of benign and malignant lesions,including pleomorphic adenoma, monomorphic adenoma,adenoid cystic carcinoma, and polymorphous low-gradeadenocarcinoma.

Ductal Cells (Luminal Cells)Ductal cells are columnar, cuboidal, or flat appearing.

Normal and neoplastic ductal cells form tubules, ductules,and glands; tumors derived from these cells may show

cystic or even diffuse/solid architecture. Such growth pat-terns may be seen within a single neoplasm, with or withoutany single predominant pattern. Duct lumina may containmucinous material that is diastase resistant, periodic acidSchiff (DPAS) positive, and weakly mucicarmine positive.Intracytoplasmic mucicarmine positivity is typically absent.Metaplastic changes of ductal cells may manifest as squ-amous cells, oncocytic cells, clear cells, sebaceous cells, and/or goblet cells, which would contain intracytoplasmicmucin-positive material (see below under the Metaplasiasection). Although myoepithelial cells are generally pre-dominant in basal cell adenoma and adenoid cystic carci-noma, these tumors may display small ductular or tubularstructures lined by luminal cells with round-to-oval nucleiand eosinophilic cytoplasm (although these cells aresometimes difficult to identify by light microscopy)(Fig. 10A). In basal cell adenoma, ductal (luminal) cells aremost conspicuous in the tubular subtype (Fig. 10B).

Myoepithelial Cells (Abluminal Cells)Myoepithelial cells can be present in all tumors types

discussed in this manuscript, visible by light microscopy and/orIHC (see below), although myoepithelial cells are not charac-teristic of polymorphous low-grade adenocarcinoma. Con-sequently, the presence of myoepithelial cells does not

FIGURE 6. Cribriform growth in salivary gland tumors. A, Although characteristically seen in adenoid cystic carcinoma, cribriformgrowth pattern can be seen in other salivary gland tumors including: (B) polymorphous low-grade adenocarcinoma; (C) basal celladenoma; and (D) pleomorphic adenoma.



unequivocally distinguish between these tumors. Neoplastic(modified) myoepithelial cells may display various morphologyby light microscopy, including plasmacytoid (Fig. 11A) andspindle shaped (so-called “hyaline cells”) (Fig. 11B);

FIGURE 8. Comparison of the nuclear features between adenoidcystic carcinoma and polymorphous low-grade adenocarcinoma.A, In adenoid cystic carcinoma, the predominant cell type is themyoepithelial (abluminal) cells comprised of isomorphic, basa-loid (hyperchromatic), and angulated nuclei without identifiablenucleoli. B, Polymorphous low-grade adenocarcinoma alsocomprised of isomorphic nuclei but in contrast show vesicular-appearing nuclei with small identifiable nucleoli.

FIGURE 7. Polymorphous low-grade adenocarcinomas show characteristic but not pathognomonic growth patterns including: (A)swirling/whorling cellular arrangements at the lesion’s periphery; (B) single cell file formation (akin to lobular carcinoma of the breast;and (C) incorporation or envelopment (rather than effacement) of residual non-neoplastic seromucous glands.

FIGURE 9. The presence of squamous eddies (with and withoutkeratinization) is a feature that can be seen in basal cell adenoma.



myoepithelial cells may also appear as epithelioid, cuboidal,stellate, or clear cells. Plasmacytoid myoepithelial cells are ovalshaped with eccentrically positioned round-to-ovoid nuclei andeosinophilic cytoplasm. These cells lack the perinuclear clearzone (so-called hof, representing the Golgi apparatus) of lym-phocyte-derived plasma cells. Plasmacytoid myoepithelial cellsoften occur in pleomorphic adenoma (and myoepithelioma),and accordingly may be informative in limited tissue sampling,although they are not pathognomonic of pleomorphicadenoma.

The presence of myoepithelial cells in polymorphouslow-grade adenocarcinoma is controversial. Some authorsconsider myoepithelial cells to be an integral cell compo-nent in addition to ductal cells in polymorphous low-gradeadenocarcinoma4–6; other authors feel that myoepithelialcells are limited or even absent in polymorphous low-gradeadenocarcinoma.7 For any given case of polymorphous

low-grade adenocarcinoma, IHC staining (see below) formyoepithelial differentiation may not be substantially dif-ferent from the pleomorphic adenoma, basal cell adenoma,and adenoid cystic carcinoma to allow for differentiation.

Tumor StromaFeatures of the extracellular matrix/stroma produced by

salivary gland tumors are sometimes diagnostically useful.Chondromyxoid stroma represents a definitive histologiccomponent of pleomorphic adenoma; even in limited quan-tity, chondromyxoid stroma facilitates distinction of pleo-morphic adenoma from monomorphic adenoma. Pleomor-phic adenoma with scant but identifiable extracellular stromawarrants a diagnosis of cellular pleomorphic adenoma. Thechondromyxoid stroma derives from neoplastic (modified)myoepithelial cells, and often includes myoepithelialcells “streaming” through the matrix (Fig. 12A). The

FIGURE 10. A, In adenoid cystic carcinoma, small ductular or tubular structures lined by luminal cells with round-to-oval nuclei andeosinophilic cytoplasm are seen (although these cells are sometimes difficult to identify by light microscopy) among the predominantmyoepithelial (abluminal) cell type. B, Ductular or tubular structures are readily apparent in this basal cell adenoma, tubular subtype.

FIGURE 11. The light microscopic features of myoepithelial cells in pleomorphic adenoma include: (A) plasmacytoid and (B) spindleshaped (hyaline cells).



chondromyxoid stroma of pleomorphic adenoma may appearsimilar to the slate blue-gray mucohyaline matrix sometimesseen in polymorphous low-grade adenocarcinoma (Fig. 12B).Adenoid cystic carcinoma shows pseudocysts with basophilic-appearing and eosinophilic-appearing material (Fig. 12C) andhyaline eosinophilic pseudocysts (Fig. 12D). Other thanchondromyxoid, the stroma of pleomorphic adenoma mayalso appear myxoid, chondroid, hyaline, and (less often)osseous. Because of limitations of tissue sampling, the chon-dromyxoid stroma of pleomorphic adenoma may not bepresent in an incisional biopsy specimen. Consequently, theabsence of chondromyxoid stroma does not preclude adiagnosis of pleomorphic adenoma, nor should lack ofchondromyxoid stroma prompt a diagnosis of monomorphicadenoma. Hyalinized (collagenized) stroma can be seen inbasal cell adenoma, particularly the membranous subtype(Fig. 13A), as well as in pleomorphic adenoma and adenoidcystic carcinoma. Basal cell adenoma sometimes shows small“droplets” of matrix within nests of neoplastic cells(Fig. 13B). Stromal features are potentially useful within theappropriate context, but definitive diagnosis of salivary glandtumors generally depends upon additional considerations.

MetaplasiaPleomorphic adenoma, monomorphic adenoma, and

polymorphous low-grade adenocarcinoma may show meta-plasia which may occur spontaneously or more oftenfollow trauma such as a fine-needle aspiration biopsy orincisional biopsy. Metaplastic alterations include squamousmetaplasia in the form of cells with keratinization andintercellular bridges. Other metaplastic cell types mayinclude mucocytes (goblet cells) or oncocytes, particularlyin pleomorphic adenoma. In contrast to other tumor types,adenoid cystic carcinoma rarely displays squamous meta-plasia, even after biopsy. Therefore, metaplasia may beinformative, particularly in ruling out adenoid cystic car-cinoma as the identification of squamous metaplasiapotentially excludes adenoid cystic carcinoma, therebynarrowing the differential diagnosis.

Mitotic ActivityPleomorphic adenoma, basal cell adenoma, poly-

morphous low-grade adenocarcinoma, and adenoid cysticcarcinoma are frequently devoid of mitotic activity by lightmicroscopy, although scattered mitotic figures may be

FIGURE 12. Pleomorphic adenoma. A, Classic admixture of tubular/ductular differentiation and chondromyxoid stroma, the lattercontaining myoepithelial cells. B, Chondromyxoid-predominant pleomorphic adenoma showing copious chondromyxoid stroma withassociated myoepithelial cells and identifiable but limited presence of ductular/tubular differentiation. C, Polymorphous low-gradeadenocarcinoma with associated blue-gray–appearing stroma similar in appearance to the chondromyxoid stromal component inpleomorphic adenoma. D, Adenoid cystic carcinoma showing basophilic and focally slightly eosinophilic material within thepseudocysts.



evident in benign and malignant intraoral minor salivarygland tumors. Malignant lesions sometimes show markedincrease in mitotic activity with atypical mitoses, butmitotic figures do not distinguish malignant lesions fromtheir benign counterparts, and even atypical mitoses do notabsolutely confer a malignant diagnosis. In addition, Ki-67(MIB-1) IHC does not definitively distinguish adenoidcystic carcinoma and/or polymorphous low-grade adeno-carcinoma from pleomorphic adenoma or basal cell ade-noma. Skalova et al8 reported a significantly higher Ki-67labeling index in adenoid cystic carcinoma compared withpolymorphous low-grade adenocarcinoma, suggesting theutility of Ki-67 IHC in the distinction between these tumortypes. However, although Schwarz et al9 showed somewhathigher overall Ki-67 labeling indices in adenoid cystic car-cinoma relative to polymorphous low-grade adenocarci-noma, these authors reported overlapping indices of r5%in most cases of adenoid cystic carcinoma and poly-morphous low-grade adenocarcinoma in their cohort. Weagree with these authors that Ki-67 IHC is of limited valuein the distinction of these tumors.

Miscellaneous FindingsOther miscellaneous features may provide evidence

supporting particular diagnoses, but are not independentlydiagnostic of any specific entity. Many salivary glandtumors contain crystals and psammomatoid concretions.Tyrosine-rich crystalloids are perhaps most characteristic ofpleomorphic adenoma, but may also be present in poly-morphous low-grade adenocarcinoma and other tumors.Psammomatoid concretions can be present in a variety ofsalivary gland neoplasms including (but not limited to)polymorphous low-grade adenocarcinoma, pleomorphicadenoma, and monomorphic adenoma.

HistochemistryThere are no significant differences in the histo-

chemical staining of pleomorphic adenoma, monomorphicadenoma, polymorphous low-grade adenocarcinoma, andadenoid cystic carcinoma, which would facilitate distinctionbetween these tumors. All of these lesions lack intra-cytoplasmic mucin, and show negative intracytoplasmic

staining with mucicarmine. Conversely, any of these lesionsmay contain extracellular (eg, intraluminal) mucicarmine-positive material. Any of these lesions may also showintracytoplasmic glycogen (ie, positive intracytoplasmicstaining with DPAS).

ImmunohistochemistryAs previously discussed, the lesions considered in this

manuscript consist of admixture of epithelial cells andmyoepithelial cells, although myoepithelial differentiationaccording to some authors occurs much less frequently inpolymorphous low-grade adenocarcinoma. Given their epi-thelial and myoepithelial components, these neoplasms shareoverlapping IHC features, and IHC does not generallyfacilitate distinction between these lesions. IHC markers thatstain the epithelial component include pancytokeratin, low–molecular weight cytokeratins (eg, CK7, CK19, andCAM5.2), epithelial membrane antigen, and carcinoem-bryonic antigen. The myoepithelial component shows pos-itive staining with pancytokeratins, high–molecular weightcytokeratin, vimentin, p63, calponin, muscle-specific andsmooth muscle actin (SMA), S100 protein, and glial fibrillaryacidic protein. In general, the diagnosis of an intraoral minorsalivary gland tumor should not depend upon the IHCfindings alone as too often IHC does not allow differ-entiation among such tumors. IHC staining of salivary glandtumors can show significant variability, even within a singlelesion, and any specific entity may not demonstrate the IHCprofile as described in the literature. Arguably, the only IHCstain that might prove beneficial in limited biopsies is S100protein in the attempt to identify neurotropism, a findingthat would confer a diagnosis of carcinoma. However, theabsence of PNI in limited tissue sampling does not exclude adiagnosis of carcinoma.

c-Kit (CD117) IHC has been investigated as a poten-tial marker of adenoid cystic carcinoma, particularly interms of distinguishing this lesion from polymorphous low-grade adenocarcinoma.9–13 Schwarz et al9 posited that dif-ferences in c-Kit staining represent the most striking andreproducible distinction between adenoid cystic carcinomaand polymorphous low-grade adenocarcinoma, suggestingthe potential utility of c-Kit in the evaluation of small

FIGURE 13. Basal cell adenoma. A, Membranous subtype showing islands of basaloid cells surrounded by thick extracellular hyalinized(basement membrane) material. B, Small “droplets” of membranous matrix material within nests of basaloid neoplastic cells.



biopsies. Beltran et al14 reported statistically significantdifferences between adenoid cystic carcinoma and poly-morphous low-grade adenocarcinoma in terms of IHCstaining patterns using c-Kit, SMA, and Ki-67, with sig-nificantly stronger expression of these markers in adenoidcystic carcinoma compared with polymorphous low-gradeadenocarcinoma. Similarly, Epivatianos et al15 found dif-ferences between adenoid cystic carcinoma and poly-morphous low-grade adenocarcinoma in terms of c-Kit andSMA IHC. Among other findings, these authors reportedpositive a-SMA staining in 83% of adenoid cystic carci-noma cases (with staining in >50% of lesional cells), andpositive c-Kit staining in only 11% of polymorphous low-grade adenocarcinoma cases (with staining in <50% oflesional cells). These authors also reported differentexpression patterns of a-SMA and c-Kit within the tubularstructures of adenoid cystic carcinoma versus those ofpolymorphous low-grade adenocarcinoma. These studiessupport the potential utility of IHC as an adjunctive con-sideration in the distinction between adenoid cystic carci-noma and polymorphous low-grade adenocarcinoma.

Although c-Kit (and SMA) staining of adenoid cysticcarcinoma may be more consistently positive and strongcompared with polymorphous low-grade adenocarcinoma,IHC of salivary gland tumors should be interpreted withcircumspection. Various authors have noted that c-Kitexpression is not unique to adenoid cystic carcinoma, asthis marker sometimes stains polymorphous low-gradeadenocarcinoma and basal cell adenoma.13,16 Our experi-ence is similar to these authors, as we frequently observec-Kit expression in various salivary gland tumors includingpolymorphous low-grade adenocarcinoma, pleomorphicadenoma, and basal cell adenoma. Especially in equivocalcases, c-Kit does not provide definitive distinction betweenadenoid cystic carcinoma and polymorphous low-gradeadenocarcinoma, or between malignant and benignneoplasms.13

Molecular GeneticsAssociations have been demonstrated between several

salivary gland neoplasms and specific molecular geneticmarkers (Table 1), including adenoid cystic carcinoma andfusion involving MYB-NFIB17–25; low-grade/intermediate-grade mucoepidermoid carcinoma and CRTC1-MAML226–29;mammary analog secretory carcinoma and ETV6-NTRK32,30,31; and hyalinizing clear cell carcinoma andEWSR1-ATF1.32–35 Rearrangement involving the PLAG1 andHMGA2 oncogenes occur specifically in pleomorphic ade-noma.36–40 Molecular markers of polymorphous low-gradeadenocarcinoma remain to be characterized. Persson et al22

investigated occurrence of the MYB-NFIB fusion (associatedwith adenoid cystic carcinoma) in polymorphous low-gradeadenocarcinoma; of the 9 cases analyzed, 1 case demonstratedthe MYB-NFIB fusion, illustrating the difficulties associatedwith diagnosis of these morphologically similar lesions. Theseauthors posit that the genome of polymorphous low-gradeadenocarcinoma is genetically stable and contains relativelyfew copy-number alterations, consistent with the clinicalbehavior of polymorphous low-grade adenocarcinoma as anindolent low-grade carcinoma with low metastatic potential.Molecular genetic evaluation potentially offers significantutility in the diagnosis and differential diagnosis of intraoralminor salivary gland tumors. Most laboratories, however, arenot presently equipped to perform molecular analysis of these

lesions, and pathologists must rely on the more conventionaldiagnostic methods of light microscopy and IHC.

Recommendations for the Diagnosis of IntraoralMinor Salivary Gland Neoplasms

Distinction between benign and malignant salivarygland lesions generally depends upon identification ofmalignant features. High-grade cytomorphologic featuresincluding marked nuclear pleomorphism, increased mitoticactivity with atypical mitoses, and tumor necrosis (ofindividual cells and/or confluent foci) facilitate a diagnosisof malignancy. In typical cases of pleomorphic adenoma,basal cell adenoma, polymorphous low-grade adenocarci-noma, and adenoid cystic carcinoma, however, limitedbiopsies lack overtly malignant cytomorphologic features.Consequently, the diagnosis of malignancy in these speci-mens relies upon other findings. The presence of metastaticdisease would certainly be diagnostic for a malignant neo-plasm, but most intraoral minor salivary gland carcinomas,including polymorphous low-grade adenocarcinoma andadenoid cystic carcinoma (as well as low-grade mucoepi-dermoid carcinoma), are infrequently associated withmetastatic disease at the time of diagnosis.

Other than metastasis, definitive features of malignancyin minor salivary gland neoplasms are predicated on infil-trative growth. Infiltrative growth includes invasion into non-neoplastic seromucous glands, soft tissues and/or bone, PNI,and LVI. By definition, invasive growth occurs at a lesion’speriphery. As previously discussed, specific aspects of atumor’s growth, such as envelopment of residual seromucousglands by polymorphous low-grade adenocarcinoma, mayfactor into the differential diagnosis of a given lesion. Other-wise, incisional biopsies of intraoral minor salivary glandtumors generally provide limited sampling of the lesion’speriphery, and therefore do not permit assessment of invasivegrowth. The considerable histopathologic and IHC overlapbetween the various intraoral minor salivary gland neoplasmscomplicates the interpretation of incisional biopsies. Whenevaluating a limited biopsy that shows neoplastic proliferationof intraoral minor salivary gland origin, the surgical patholo-gist must then consider the appropriate diagnostic terminologyand potential recommendations regarding management.

In this scenario, pathologists are advised to renderrelatively broad diagnosis while providing as much infor-mation as feasible regarding the differential diagnosis. Inthe context of an intraoral minor salivary gland neoplasmthat lacks overtly malignant features, but does not permitexclusion of malignancy, appropriate diagnostic terminol-ogy would resemble “Minor salivary gland neoplasm, notfurther specified,” with recommendation of conservative

TABLE 1. Salivary Gland Neoplasms With Known ChromosomalTranslocations/Gene Fusions

Neoplasm Chromosomal Translocation Gene Fusion

PA Rearrangement of 8q12Rearrangement of 12q13-15

PLAG1HMGA2

MEC t (11;19)(q21;p13) CRTC1-MAML2AdCC t (6;9)(q22-23;p23-24) MYB-NFIBHCCC t (12;22)(q13;q12) EWSR1-ATF1MASC t (12;15)(p13q25) ETV6-NTRK3

AdCC indicates adenoid cystic carcinoma; HCCC, hyalinizing clear cellcarcinoma; MASC, mammary analogue secretory carcinoma; MEC,mucoepidermoid carcinoma; PA, pleomorphic adenoma.



but complete surgical excision with tumor-free margins. Onthe basis of limited material, a more specific diagnosiswhether benign or malignant, may be discordant with thediagnosis resulting from a subsequent resection specimen.A lesion diagnosed as pleomorphic adenoma or basal celladenoma based on an incisional biopsy may show invasivegrowth (eg, PNI) after complete resection (Fig. 2). Con-versely, a limited biopsy showing cytologic features ofpolymorphous low-grade adenocarcinoma or adenoidcystic carcinoma may lead to a resection specimen thatlacks invasive growth, thereby warranting a benign diag-nosis. As an important caveat, among salivary glandlesions, some malignancies can be diagnosed in a circum-scribed to encapsulated lesion without invasive growth.Examples of such an occurrence include mucoepidermoidcarcinoma and acinic cell adenocarcinoma in which the celltypes for these respective neoplasms are diagnostic as thereare no benign neoplasms with the constituent cells diag-nostic for either of these tumor types. Such encapsulated ornoninvasive malignant neoplasms arise more frequently inmajor salivary glands, and are less of a concern in the dif-ferential diagnosis of intraoral minor salivary gland tumors.

CONCLUSIONSWe have attempted to demonstrate the spectrum of

pathologic findings shared by intraoral benign and malig-nant minor salivary gland neoplasms, such that limitedbiopsies of these lesions are frequently insufficient fordefinitive diagnosis. This generalization applies to incisionalbiopsies and fine-needle aspiration biopsy. Our clinicalcolleagues may believe that biopsy material should facilitatedistinction between benign and malignant neoplasms.Unfortunately, in our experience, this expectation is fre-quently unrealistic due to sampling issues. The prudentdiagnostic approach regarding incisional biopsy specimensentails terminology such as “Minor salivary gland neo-plasm, not further specified,” confirming the presence of aneoplastic proliferation that necessitates complete surgicalresection with clear margins. After resection, evaluation ofthe excised specimen facilitates assessment of invasivegrowth, and establishment of a definitive diagnosis. Anintraoral minor salivary gland neoplasm lacking invasivegrowth is benign (eg, pleomorphic adenoma or basal celladenoma per the focus of this manuscript), and completeresection of benign tumors is curative. Identification ofinvasive growth warrants a malignant diagnosis (eg, poly-morphous low-grade adenocarcinoma or adenoid cysticcarcinoma per the focus of this manuscript). In the caseof polymorphous low-grade adenocarcinoma, completeresection with tumor-free margins is considered curative,and additional therapy (eg, radiotherapy) is generally notindicated. The potential complications of radiotherapy inthe setting of polymorphous low-grade adenocarcinomamay include osteoradionecrosis representing a far greaterclinical problem for the patient than any untoward com-plications due to the neoplasm, such as recurrence yearsafter resection. Adenoid cystic carcinoma, in contrast,necessitates adjuvant therapy, specifically radiation, inaddition to complete surgical resection.

REFERENCES

1. Barnes L, Eveson JW, Reichert P, et al. World HealthOrganization classification of tumours. Pathology and Genetics

of Head and Neck Tumours. Lyon, France: IARC Press;2005:210.

2. Skalova A, Vanecek T, Sima R, et al. Mammary analoguesecretory carcinoma of salivary glands, containing the ETV6-NTRK3 fusion gene: a hitherto undescribed salivary glandtumor entity. Am J Surg Pathol. 2010;34:599–608.

3. Michal M, Skalova A, Simpson RH, et al. Cribriformadenocarcinoma of the tongue: a hitherto unrecognized typeof adenocarcinoma characteristically occurring in the tongue.Histopathology. 1999;35:495–501.

4. Fonseca I, Felix A, Aores J. Cell proliferation in salivary glandadenocarcinomas with myoepithelial participation. A study of78 cases. Virchows Arch. 1997;430:227–232.

5. Batsakis JG, el-Naggar AK. Myoepithelium in salivary andmammary neoplasms is host-friendly. Adv Anat Pathol. 1999;6:218–226.

6. Edwards PC, Bhuiya T, Kelsch RD. Assessment of p63expression in the salivary gland neoplasms adenoid cysticcarcinoma, polymorphous low-grade adenocarcinoma, basalcell adenoma and canalicular adenoma. Oral Surg Oral MedOral Pathol Oral Radiol Endod. 2004;97:613–619.

7. Araujo V, Sousa S, Jaeger M, et al. Characterization of thecellular component of polymorphous low-grade adenocarci-noma by immunohistochemistry and electron microscopy. OralOncol. 1999;35:164–172.

8. Skalova A, Simpson RHW, Lehtonen H, et al. Assessment ofproliferative activity using the MIB1 antibody helps todistinguish polymorphous low grade adenocarcinoma fromadenoid cystic carcinoma of salivary glands. Pathol Res Pract.1997;193:695–703.

9. Schwarz S, Muller M, Ettl T, et al. Morphological hetero-geneity of oral salivary gland carcinomas: a clinicopathologicstudy of 41 cases with long term follow-up emphasizing theoverlapping spectrum of adenoid cystic carcinoma andpolymorphous low-grade adenocarcinoma. Int J Clin ExpPathol. 2011;4:336–348.

10. Jeng YM, Lin CY, Hsu HC. Expression of the c-kit protein isassociated with certain subtypes of salivary gland carcinoma.Cancer Lett. 2000;154:107–111.

11. Penner CR, Folpe AL, Budnick SD. C-kit expressiondistinguishes salivary gland adenoid cystic carcinoma frompolymorphous low-grade adenocarcinoma. Mod Pathol. 2002;15:687–691.

12. Mino M, Pilch BZ, Faquin WC. Expression of KIT (CD117)in neoplasms of the head and neck: an ancillary markerfor adenoid cystic carcinoma. Mod Pathol. 2003;16:1224–1231.

13. Edwards PC, Bhuiya T, Kelsch RD. C-kit expression in thesalivary gland neoplasms adenoid cystic carcinoma, polymor-phous low-grade adenocarcinoma, and monomorphicadenoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod.2003;95:586–593.

14. Beltran D, Faquin WC, Gallagher G, et al. Selectiveimmunohistochemical comparison of polymorphous low-gradeadenocarcinoma and adenoid cystic carcinoma. J OralMaxillofac Surg. 2006;64:415–423.

15. Epivatianos A, Poulopoulos A, Dimitrakopoulos I, et al.Application of alpha-smooth muscle actin and c-kit in thedifferential diagnosis of adenoid cystic carcinoma from poly-morphous low-grade adenocarcinoma.Oral Oncol. 2007;43:67–76.

16. Andreadis D, Epivatianos A, Poulopoulos A, et al. Detectionof C-KIT (CD117) molecule in benign and malignant salivarygland tumours. Oral Oncol. 2006;42:57–65.

17. Persson M, Andren Y, Mark J, et al. Recurrent fusion of MYBand NFIB transcription factor genes in carcinomas of thebreast and head and neck. Proc Natl Acad Sci USA.2009;106:18740–18744.

18. Mitani Y, Li J, Rao PH, et al. Comprehensive analysis of theMYB-NFIB gene fusion in salivary adenoid cystic carcinoma:Incidence, variability, and clinicopathologic significance. ClinCancer Res. 2010;16:4722–4731.

19. Bhaijee F, Pepper DJ, Pitman KT, et al. New developments inthe molecular pathogenesis of head and neck tumors: a review



of tumor-specific fusion oncogenes in mucoepidermoid carci-

noma, adenoid cystic carcinoma, and NUT midline carcinoma.

Ann Diagn Pathol. 2011;15:69–77.20. Brill LB II, Kanner WA, Fehr A, et al. Analysis of MYB

expression and MYB-NFIB gene fusions in adenoid cystic

carcinoma and other salivary neoplasms. Mod Pathol. 2011;24:

1169–1176.21. Bell D, Roberts D, Karpowicz M, et al. Clinical significance of

Myb protein and downstream target genes in salivary adenoid

cystic carcinoma. Cancer Biol Ther. 2011;12:569–573.22. Persson F, Fehr A, Sundelin K, et al. Studies of genomic

imbalances and the MYB-NFIB gene fusion in polymorphous

low-grade adenocarcinoma of the head and neck. Int J Oncol.

2012;40:80–84.23. Mitani Y, Rao PH, Futreal PA, et al. Novel chromosomal

rearrangements and break points at the t(6;9) in salivary

adenoid cystic carcinoma: association with MYB-NFIB

chimeric fusion, MYB expression, and clinical outcome. Clin

Cancer Res. 2011;17:7003–7014.24. Persson M, Andren Y, Moskaluk CA, et al. Clinically

significant copy number alterations and complex rearrange-

ments of MYB and NFIB in head and neck adenoid cystic

carcinoma. Genes Chromosomes Cancer. 2012;51:805–817.25. Moskaluk CA. Adenoid cystic carcinoma: clinical and

molecular features. Head Neck Pathol. 2013;7:17–22.26. Tonon G, Modi S, Wu L, et al. t(11;19)(q21;p13) translocation

in mucoepidermoid carcinoma creates a novel fusion product

that disrupts a Notch signaling pathway. Nat Genet.

2003;33:208–213.27. Martins C, Cavaco B, Tonon G, et al. A study of MECT1-

MAML2 in mucoepidermoid carcinoma and Warthin’s tumor

of salivary glands. J Mol Diagn. 2004;6:205–210.28. Behboudi A, Enlund F, Winnes M, et al. Molecular

classification of mucoepidermoid carcinomas-prognostic sig-

nificance of the MECT1-MAML2 fusion oncogene. Genes

Chromosomes Cancer. 2006;45:470–481.29. Seethala RR, Dacic S, Cieply K, et al. A reappraisal of the

MECT1/MAML2 translocation in salivary mucoepidermoid

carcinomas. Am J Surg Pathol. 2010;34:1106–1121.

30. Fehr A, Loning T, Stenman G. Mammary analogue secretorycarcinoma of the salivary glands with ETV6-NTRK3 genefusion. Am J Surg Pathol. 2011;35:1600–1602.

31. Bishop JA. Unmasking MASC: bringing to light the uniquemorphologic, immunohistochemical and genetic features of thenewly recognized mammary analogue secretory carcinoma ofsalivary glands. Head Neck Pathol. 2013;7:35–39.

32. Antonescu CR, Katabi N, Zhang L, et al. EWSR1-ATF1 fusionis a novel and consistent finding in hyalinizing clear-cell carci-noma of salivary gland. Genes Chromosomes Cancer. 2011;50:559–570.

33. Shah AA, LeGallo RD, van Zante A, et al. EWSR1 geneticrearrangements in salivary gland tumors: a specific and verycommon feature of hyalinizing clear cell carcinoma. Am J SurgPathol. 2013;37:571–578.

34. Tanguay J, Weinreb I. What the EWSR1-ATF1 fusion hastaught us about hyalinizing clear cell carcinoma. Head NeckPathol. 2013;7:28–34.

35. Thway K, Fisher C. Tumors with EWSR1-CREB1 andEWSR1-ATF1 fusions: the current status. Am J Surg Pathol.2012;36:e1–e11.

36. Matsuyama A, Hisaoka M, Nagao Y, et al. Aberrant PLAG1expression in pleomorphic adenomas of the salivary gland: amolecular genetic and immunohistochemical study. VirchowsArch. 2011;458:583–592.

37. Kas K, Voz ML, Roijer E, et al. Promoter swapping betweenthe genes for a novel zinc finger protein and beta-catenin inpleomorphic adenomas with t(3;8)(p21;q12) translocations.Nat Genet. 1997;15:170–174.

38. Kas K, Roijer E, Voz M, et al. A 2-Mb YAC contig andphysical map covering the chromosome 8q12 breakpointcluster region in pleomorphic adenomas of the salivary glands.Genomics. 1997;43:349–358.

39. Voz ML, Astrom AK, Kas K, et al. The recurrent trans-location t(5;8)(p13;q12) in pleomorphic adenomas results inupregulation of PLAG1 gene expression under control of theLIFR promoter. Oncogene. 1998;16:1409–1416.

40. Geurts JM, Schoenmakers EF, Roijer E, et al. van de Ven WJ.Identification of NFIB as recurrent translocation partnergene of HMGIC in pleomorphic adenomas. Oncogene.1998;16:865–872.



pitfalls in the biopsy diagnosis of intraoral minor salivary gland...

Documents