Pitfalls in the Biopsy Diagnosis of Intraoral Minor Salivary Gland Neoplasms: Diagnostic Considerations and Recommended Approach

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1 REVIEW ARTICLE Pitfalls in the Biopsy Diagnosis of Intraoral Minor Salivary Gland Neoplasms: Diagnostic Considerations and Recommended Approach Andrew T. Turk, MD* and Bruce M. Wenig, MDw Abstract: Among the more common types of intraoral minor salivary gland neoplasms are pleomorphic adenoma, basal cell adenoma, polymorphous low-grade adenocarcinoma, and adenoid cystic carcinoma. These minor salivary gland neoplasms share similar morphologic features and to a large extent immunohistochemical findings. Differentiation between these benign and malignant neoplasms is often predicated on the presence or absence of invasion. As such, in the presence of limited tissue sampling that typifies the initial testing modalities, including fine needle aspiration biopsy and/or incisional biopsy, it often is not possible to differentiate a benign from malignant minor salivary gland neoplasm. The diagnostic difficulties arise from the absence in needle or incisional biopsy of the tumor s periphery to determine whether infiltrative growth is or is not present. In this manuscript we discuss limitations and considerations associated with evaluation of incisional biopsies of intraoral minor salivary gland tumors. We offer a diagnostic approach to evaluating these biopsies, and suggest diagnostic terminology for biopsy specimens in which distinction between benignancy and malignancy is not feasible. The pathologist s approach to this distinction is critical, as treatment of benign neoplasms is generally conservative, whereas malignant lesions may warrant 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 system recognizes 24 malignant and 10 benign salivary gland epithelial tumors. 1 Since 2005, additional entities have been identified and/or more clearly defined, including (but not limited to) mammary analog secretory carcinoma 2 and cribriform adenocarcinoma of the tongue. 3 With rare exceptions, any neoplasm that occurs in major salivary glands may also arise in minor salivary glands, which are located throughout the mucosa of the upper aerodigestive tract. The oral cavity represents one of the most common locations of minor salivary gland tumors. Frequently encountered intraoral minor salivary gland neoplasms include benign tumors such as pleomorphic adenoma and monomorphic adenoma, basal cell type (also From the *Department of Pathology and Cell Biology, Columbia University; and wdepartment of Diagnostic Pathology and Laboratory Medicine, Beth Israel Medical Center, St Luke s and Roosevelt 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 s and Roosevelt Hospitals, New York, NY ( bwenig@ chpnet.org). All figures can be viewed online in color at pathology.com. Copyright r 2013 by Lippincott Williams & Wilkins known as basal cell adenoma), and malignant lesions such as mucoepidermoid carcinoma, adenoid cystic carcinoma, and polymorphous low-grade adenocarcinoma. The initial diagnostic modality for a patient with an intraoral mass is fine-needle aspiration biopsy or incisional biopsy. The utility of these techniques is constrained by limitations related to tissue sampling. In addition, similarities between the growth patterns, cytomorphology, and immunoreactivity of common intraoral minor salivary gland tumors (benign and malignant) further complicates the interpretation of fine-needle aspiration biopsy and incisional biopsy specimens. Consequently, distinction between benign and malignant intraoral minor salivary gland lesions can be difficult, especially given limited biopsy material. Mucoepidermoid carcinoma represents a potential exception to this problem. This tumor is characterized by cellular proliferation that includes mucocytes, epidermoid cells, and intermediate cells (Fig. 1). As no other salivary gland neoplasm consists of these cellular components, identification of these cell types permits a diagnosis of mucoepidermoid carcinoma even in the context of limited sampling. Other lesions such as pleomorphic adenoma, basal cell adenoma, adenoid cystic carcinoma, and polymorphous low-grade adenocarcinoma, however, share light microscopic and immunohistochemical (IHC) features such that distinction between benign and malignant lesions often depends upon identification of invasive growth. Biopsy sampling of intraoral minor salivary gland tumors frequently consists of lesional material without surrounding tissues, and therefore does not facilitate assessment of invasive growth (Fig. 2). Within this context, a specific diagnosis rendered on the basis of fine-needle aspiration biopsy or incisional biopsy may result in a discordant diagnosis after complete surgical resection. RESULTS Shared Findings Among Minor Salivary Gland Neoplasms Aside from mucoepidermoid carcinoma, the most common intraoral minor salivary gland neoplasms listed above show overlapping light microscopic and IHC features. Any of these lesions may show circumscription without encapsulation, multiple growth patterns, bland cytomorphology, combination of epithelial and myoepithelial cell differentiation, absent to minimal increase in mitotic activity, and to a large extent similar IHC reactivity. Circumscription, Encapsulation, and Invasive Growth All minor salivary gland neoplasms, whether benign or malignant, are unencapsulated. Accordingly, the presence or absence of a capsule does not differentiate benign and Adv Anat Pathol Volume 21, Number 1, January

2 Turk and Wenig Adv Anat Pathol Volume 21, Number 1, January 2014 FIGURE 1. Mucoepidermoid carcinoma. This tumor is comprised of an admixture of cells types including mucocytes, epidermoid cells, and intermediate cells. The identification of these cell types only seen in mucoepidermoid carcinoma is diagnostic. malignant minor salivary gland tumors. Both benign and malignant intraoral minor salivary gland neoplasms may be circumscribed (Fig. 3), but malignant lesions show invasive growth, including invasion into non-neoplastic seromucous glands (Fig. 4A) and/or soft tissues (Fig. 4B), perineural invasion (PNI) (Fig. 4C), and lymph-vascular invasion (LVI). Metastatic disease would certainly confer a diagnosis of malignancy, but herein we focus on biopsies of intraoral minor salivary gland lesions without clinically evident nodal metastasis. It should be noted that extension of tumor to (and even involvement of) the surface (squamous) epithelium does FIGURE 3. All minor salivary gland neoplasms are unencapsulated and often even malignant neoplasms such as polymorphous low-grade adenocarcinoma appears circumscribed in its superficial aspect. not represent a criterion for malignancy (Fig. 5). If biopsy sampling shows unequivocal evidence of infiltrative growth, then a definitive diagnosis of carcinoma can be rendered. However, such findings are uncommon in biopsies of intraoral minor salivary gland neoplasms, which frequently show only lesional tissue, and lack sufficient sampling of surrounding tissues for assessment of invasive growth. Growth Patterns Salivary gland neoplasms of both major and minor salivary 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 tumor borders to evaluate for invasive growth. A and B, Incisional biopsy shows a cellular neoplasm comprised of tubules with isomorphic basaloid-appearing nuclei without features allowing for determining whether it is benign or malignant. C, After complete excision showing circumscription without invasion, a diagnosis of basal cell adenoma can be rendered. D and E, Incisional biopsy of another intraoral minor salivary gland neoplasm showing a cellular neoplasm comprised of tubules with isomorphic basaloid-appearing nuclei without features allowing for determining whether it is benign or malignant. F, After complete excision, perineural invasion is present allowing for a diagnosis of carcinoma, in this case polymorphous low-grade adenocarcinoma. 2 r 2013 Lippincott Williams & Wilkins

3 Adv Anat Pathol Volume 21, Number 1, January 2014 Intraoral Minor Salivary Gland Neoplasms FIGURE 4. The presence of infiltration confers a diagnosis of malignancy. Polymorphous low-grade adenocarcinoma showing invasion of (A) seromucous glands and (B) skeletal muscle; perineural invasion in adenoid cystic carcinoma (C). words, polymorphic architecture in an intraoral minor salivary gland neoplasm is not equivalent to any specific diagnosis such as polymorphous low-grade adenocarcinoma. Pleomorphic adenoma, monomorphic adenoma, and adenoid cystic carcinoma may show a combination of growth patterns including tubular-ductular, solid, (micro)cystic, cribriform, trabecular, nodular, lattice-like, fascicular, and/or papillary. Accordingly, no single growth pattern or combination of growth patterns defines any specific neoplasm or distinguishes between minor salivary gland tumors. It should also be noted that significant morphologic variability may exist within a single tumor, and between different examples of a single diagnostic entity. For instance, basal cell adenoma and basal cell adenocarcinoma occur as tubular, trabecular, solid, cribriform, and membranous variants; adenoid cystic carcinoma shows tubular, cribriform, and solid patterns; polymorphous low-grade adenocarcinoma exhibits tubular, cribriform, solid, and papillary growth. Hybrid adenoid cystic carcinoma, in which adenoid cystic carcinoma coexists with another histologic subtype of salivary gland carcinoma, by definition shows different architectural features in different areas. This heterogeneity within specific entities frequently complicates the interpretation of salivary gland biopsies, especially incisional biopsies that provide limited material. Cribriform growth is stereotypically associated with adenoid cystic carcinoma (Fig. 6A), and is not generally considered characteristic of pleomorphic adenoma or basal cell adenoma. Cribriform growth may suggest a diagnosis of FIGURE 5. Pleomorphic adenoma showing lesional cells extended to and even involvement of the surface (squamous) epithelium. This finding is not diagnostic for malignancy. adenoid cystic carcinoma, but is not pathognomonic of this lesion. This finding may also be present in other intraoral minor salivary gland neoplasms including polymorphous lowgrade adenocarcinoma (Fig. 6B), as well as pleomorphic adenoma (Fig. 6C) and basal cell adenoma (Fig. 6D). Various other associations exist between certain tumors and specific architectural features. Nuclear palisading of basaloid cells along the stromal interface is often seen in basal cell adenoma, although this feature is not specifically diagnostic of basal cell adenoma. In polymorphous low-grade adenocarcinoma, swirling or whorling patterns (Fig. 7A), often at the lesion s periphery, are characteristic (although not pathognomonic). However, given their localization at the peripheral aspects, these arrangements may not be evident in limited incisional biopsy specimens. Polymorphous low-grade adenocarcinoma may also show single-file formations of tumor cells (Fig. 7B) (akin to lobular carcinoma of the breast), although this feature is not consistently seen and is absent in many cases. Another characteristic although not pathognomonic finding associated with polymorphous low-grade adenocarcinoma is the incorporation of non-neoplastic seromucous glands (Fig. 7C). Conversely, pleomorphic adenoma, basal cell adenoma, and adenoid cystic carcinoma generally cause architectural effacement such that residual seromucous glands cannot be identified within the tumor. Although envelopment of residual glands (individually or in clusters) warrants suspicion of polymorphous low-grade adenocarcinoma, this feature does not represent unequivocal evidence of polymorphous low-grade adenocarcinoma. Diagnosis of polymorphous low-grade adenocarcinoma (ie, diagnosis of minor salivary gland carcinoma) requires identification of PNI, LVI, and/or invasive growth. Despite purportedly specific growth patterns, pleomorphic adenoma, basal cell adenoma, polymorphous lowgrade adenocarcinoma, and adenoid cystic carcinoma share too much architectural overlap to permit definitive diagnosis based on any single growth pattern, or combination of architectural features. Cytomorphology The extent of cytomorphologic similarity between pleomorphic adenoma, basal cell adenoma, polymorphous low-grade adenocarcinoma, and adenoid cystic carcinoma severely hinders definitive diagnosis based on limited tissue sampling. These tumors consist of cells with limited nuclear pleomorphism, absent to low mitotic activity, and absence of necrosis. Certain cytologic features may suggest a specific diagnosis, such as adenoid cystic carcinoma characteristically contains abundant myoepithelial (abluminal) cells r 2013 Lippincott Williams & Wilkins 3

4 Turk and Wenig Adv Anat Pathol Volume 21, Number 1, January 2014 FIGURE 6. Cribriform growth in salivary gland tumors. A, Although characteristically seen in adenoid cystic carcinoma, cribriform growth pattern can be seen in other salivary gland tumors including: (B) polymorphous low-grade adenocarcinoma; (C) basal cell adenoma; and (D) pleomorphic adenoma. with increased nuclear-to-cytoplasmic ratio, and basaloid (hyperchromatic) angulated nuclei without identifiable nucleoli (Fig. 8). Similar nuclear features, however, can be present in basal cell adenoma and even in pleomorphic adenoma. In contrast, basaloid hyperchromatic nuclei are not generally evident in polymorphous low-grade adenocarcinoma, which typically consists of cells with vesicular chromatin and inconspicuous small nucleoli (Fig. 8). Squamous differentiation (eg, squamous eddies) with or without keratinization is relatively common basal cell adenoma, particularly the membranous variant (Fig. 9). Squamous metaplasia also occurs in pleomorphic adenoma and polymorphous low-grade adenocarcinoma (see below under the Metaplasia section), but this phenomenon is highly unusual in adenoid cystic carcinoma. Beyond associations such as these, other cytologic features are less specific. Clear cells and oncocytic cells, for instance, are present in a wide variety of benign and malignant lesions, including pleomorphic adenoma, monomorphic adenoma, adenoid cystic carcinoma, and polymorphous low-grade adenocarcinoma. 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 patterns may be seen within a single neoplasm, with or without any single predominant pattern. Duct lumina may contain mucinous material that is diastase resistant, periodic acid Schiff (DPAS) positive, and weakly mucicarmine positive. Intracytoplasmic mucicarmine positivity is typically absent. Metaplastic changes of ductal cells may manifest as squamous cells, oncocytic cells, clear cells, sebaceous cells, and/ or goblet cells, which would contain intracytoplasmic mucin-positive material (see below under the Metaplasia section). Although myoepithelial cells are generally predominant in basal cell adenoma and adenoid cystic carcinoma, these tumors may display small ductular or tubular structures lined by luminal cells with round-to-oval nuclei and eosinophilic cytoplasm (although these cells are sometimes difficult to identify by light microscopy) (Fig. 10A). In basal cell adenoma, ductal (luminal) cells are most 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/or IHC (see below), although myoepithelial cells are not characteristic of polymorphous low-grade adenocarcinoma. Consequently, the presence of myoepithelial cells does not 4 r 2013 Lippincott Williams & Wilkins

5 Adv Anat Pathol Volume 21, Number 1, January 2014 Intraoral Minor Salivary Gland Neoplasms 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. unequivocally distinguish between these tumors. Neoplastic (modified) myoepithelial cells may display various morphology by light microscopy, including plasmacytoid (Fig. 11A) and spindle shaped (so-called hyaline cells ) (Fig. 11B); FIGURE 8. Comparison of the nuclear features between adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma. A, In adenoid cystic carcinoma, the predominant cell type is the myoepithelial (abluminal) cells comprised of isomorphic, basaloid (hyperchromatic), and angulated nuclei without identifiable nucleoli. B, Polymorphous low-grade adenocarcinoma also comprised of isomorphic nuclei but in contrast show vesicularappearing nuclei with small identifiable nucleoli. FIGURE 9. The presence of squamous eddies (with and without keratinization) is a feature that can be seen in basal cell adenoma. r 2013 Lippincott Williams & Wilkins 5

6 Turk and Wenig Adv Anat Pathol Volume 21, Number 1, January 2014 FIGURE 10. A, In adenoid cystic carcinoma, small ductular or tubular structures lined by luminal cells with round-to-oval nuclei and eosinophilic cytoplasm are seen (although these cells are sometimes difficult to identify by light microscopy) among the predominant myoepithelial (abluminal) cell type. B, Ductular or tubular structures are readily apparent in this basal cell adenoma, tubular subtype. myoepithelial cells may also appear as epithelioid, cuboidal, stellate, or clear cells. Plasmacytoid myoepithelial cells are oval shaped with eccentrically positioned round-to-ovoid nuclei and eosinophilic cytoplasm. These cells lack the perinuclear clear zone (so-called hof, representing the Golgi apparatus) of lymphocyte-derived plasma cells. Plasmacytoid myoepithelial cells often occur in pleomorphic adenoma (and myoepithelioma), and accordingly may be informative in limited tissue sampling, although they are not pathognomonic of pleomorphic adenoma. The presence of myoepithelial cells in polymorphous low-grade adenocarcinoma is controversial. Some authors consider myoepithelial cells to be an integral cell component in addition to ductal cells in polymorphous low-grade adenocarcinoma 4 6 ; other authors feel that myoepithelial cells are limited or even absent in polymorphous low-grade adenocarcinoma. 7 For any given case of polymorphous low-grade adenocarcinoma, IHC staining (see below) for myoepithelial differentiation may not be substantially different from the pleomorphic adenoma, basal cell adenoma, and adenoid cystic carcinoma to allow for differentiation. Tumor Stroma Features of the extracellular matrix/stroma produced by salivary gland tumors are sometimes diagnostically useful. Chondromyxoid stroma represents a definitive histologic component of pleomorphic adenoma; even in limited quantity, chondromyxoid stroma facilitates distinction of pleomorphic adenoma from monomorphic adenoma. Pleomorphic adenoma with scant but identifiable extracellular stroma warrants a diagnosis of cellular pleomorphic adenoma. The chondromyxoid stroma derives from neoplastic (modified) myoepithelial cells, and often includes myoepithelial cells streaming through the matrix (Fig. 12A). The FIGURE 11. The light microscopic features of myoepithelial cells in pleomorphic adenoma include: (A) plasmacytoid and (B) spindle shaped (hyaline cells). 6 r 2013 Lippincott Williams & Wilkins

7 Adv Anat Pathol Volume 21, Number 1, January 2014 Intraoral Minor Salivary Gland Neoplasms FIGURE 12. Pleomorphic adenoma. A, Classic admixture of tubular/ductular differentiation and chondromyxoid stroma, the latter containing myoepithelial cells. B, Chondromyxoid-predominant pleomorphic adenoma showing copious chondromyxoid stroma with associated myoepithelial cells and identifiable but limited presence of ductular/tubular differentiation. C, Polymorphous low-grade adenocarcinoma with associated blue-gray appearing stroma similar in appearance to the chondromyxoid stromal component in pleomorphic adenoma. D, Adenoid cystic carcinoma showing basophilic and focally slightly eosinophilic material within the pseudocysts. chondromyxoid stroma of pleomorphic adenoma may appear similar to the slate blue-gray mucohyaline matrix sometimes seen in polymorphous low-grade adenocarcinoma (Fig. 12B). Adenoid cystic carcinoma shows pseudocysts with basophilicappearing and eosinophilic-appearing material (Fig. 12C) and hyaline eosinophilic pseudocysts (Fig. 12D). Other than chondromyxoid, the stroma of pleomorphic adenoma may also appear myxoid, chondroid, hyaline, and (less often) osseous. Because of limitations of tissue sampling, the chondromyxoid stroma of pleomorphic adenoma may not be present in an incisional biopsy specimen. Consequently, the absence of chondromyxoid stroma does not preclude a diagnosis of pleomorphic adenoma, nor should lack of chondromyxoid stroma prompt a diagnosis of monomorphic adenoma. Hyalinized (collagenized) stroma can be seen in basal cell adenoma, particularly the membranous subtype (Fig. 13A), as well as in pleomorphic adenoma and adenoid cystic carcinoma. Basal cell adenoma sometimes shows small droplets of matrix within nests of neoplastic cells (Fig. 13B). Stromal features are potentially useful within the appropriate context, but definitive diagnosis of salivary gland tumors generally depends upon additional considerations. Metaplasia Pleomorphic adenoma, monomorphic adenoma, and polymorphous low-grade adenocarcinoma may show metaplasia which may occur spontaneously or more often follow trauma such as a fine-needle aspiration biopsy or incisional biopsy. Metaplastic alterations include squamous metaplasia in the form of cells with keratinization and intercellular bridges. Other metaplastic cell types may include mucocytes (goblet cells) or oncocytes, particularly in pleomorphic adenoma. In contrast to other tumor types, adenoid cystic carcinoma rarely displays squamous metaplasia, even after biopsy. Therefore, metaplasia may be informative, particularly in ruling out adenoid cystic carcinoma as the identification of squamous metaplasia potentially excludes adenoid cystic carcinoma, thereby narrowing the differential diagnosis. Mitotic Activity Pleomorphic adenoma, basal cell adenoma, polymorphous low-grade adenocarcinoma, and adenoid cystic carcinoma are frequently devoid of mitotic activity by light microscopy, although scattered mitotic figures may be r 2013 Lippincott Williams & Wilkins 7

8 Turk and Wenig Adv Anat Pathol Volume 21, Number 1, January 2014 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. evident in benign and malignant intraoral minor salivary gland tumors. Malignant lesions sometimes show marked increase in mitotic activity with atypical mitoses, but mitotic figures do not distinguish malignant lesions from their benign counterparts, and even atypical mitoses do not absolutely confer a malignant diagnosis. In addition, Ki-67 (MIB-1) IHC does not definitively distinguish adenoid cystic carcinoma and/or polymorphous low-grade adenocarcinoma from pleomorphic adenoma or basal cell adenoma. Ska lova et al 8 reported a significantly higher Ki-67 labeling index in adenoid cystic carcinoma compared with polymorphous low-grade adenocarcinoma, suggesting the utility of Ki-67 IHC in the distinction between these tumor types. However, although Schwarz et al 9 showed somewhat higher overall Ki-67 labeling indices in adenoid cystic carcinoma relative to polymorphous low-grade adenocarcinoma, these authors reported overlapping indices of r5% in most cases of adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma in their cohort. We agree with these authors that Ki-67 IHC is of limited value in the distinction of these tumors. Miscellaneous Findings Other miscellaneous features may provide evidence supporting particular diagnoses, but are not independently diagnostic of any specific entity. Many salivary gland tumors contain crystals and psammomatoid concretions. Tyrosine-rich crystalloids are perhaps most characteristic of pleomorphic adenoma, but may also be present in polymorphous low-grade adenocarcinoma and other tumors. Psammomatoid concretions can be present in a variety of salivary gland neoplasms including (but not limited to) polymorphous low-grade adenocarcinoma, pleomorphic adenoma, and monomorphic adenoma. Histochemistry There are no significant differences in the histochemical staining of pleomorphic adenoma, monomorphic adenoma, polymorphous low-grade adenocarcinoma, and adenoid cystic carcinoma, which would facilitate distinction between these tumors. All of these lesions lack intracytoplasmic mucin, and show negative intracytoplasmic staining with mucicarmine. Conversely, any of these lesions may contain extracellular (eg, intraluminal) mucicarminepositive material. Any of these lesions may also show intracytoplasmic glycogen (ie, positive intracytoplasmic staining with DPAS). Immunohistochemistry As previously discussed, the lesions considered in this manuscript consist of admixture of epithelial cells and myoepithelial cells, although myoepithelial differentiation according to some authors occurs much less frequently in polymorphous low-grade adenocarcinoma. Given their epithelial and myoepithelial components, these neoplasms share overlapping IHC features, and IHC does not generally facilitate distinction between these lesions. IHC markers that stain the epithelial component include pancytokeratin, low molecular weight cytokeratins (eg, CK7, CK19, and CAM5.2), epithelial membrane antigen, and carcinoembryonic antigen. The myoepithelial component shows positive staining with pancytokeratins, high molecular weight cytokeratin, vimentin, p63, calponin, muscle-specific and smooth muscle actin (SMA), S100 protein, and glial fibrillary acidic protein. In general, the diagnosis of an intraoral minor salivary gland tumor should not depend upon the IHC findings alone as too often IHC does not allow differentiation among such tumors. IHC staining of salivary gland tumors can show significant variability, even within a single lesion, and any specific entity may not demonstrate the IHC profile as described in the literature. Arguably, the only IHC stain that might prove beneficial in limited biopsies is S100 protein in the attempt to identify neurotropism, a finding that would confer a diagnosis of carcinoma. However, the absence of PNI in limited tissue sampling does not exclude a diagnosis of carcinoma. c-kit (CD117) IHC has been investigated as a potential marker of adenoid cystic carcinoma, particularly in terms of distinguishing this lesion from polymorphous lowgrade adenocarcinoma Schwarz et al 9 posited that differences in c-kit staining represent the most striking and reproducible distinction between adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma, suggesting the potential utility of c-kit in the evaluation of small 8 r 2013 Lippincott Williams & Wilkins

9 Adv Anat Pathol Volume 21, Number 1, January 2014 Intraoral Minor Salivary Gland Neoplasms biopsies. Beltran et al 14 reported statistically significant differences between adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma in terms of IHC staining patterns using c-kit, SMA, and Ki-67, with significantly stronger expression of these markers in adenoid cystic carcinoma compared with polymorphous low-grade adenocarcinoma. Similarly, Epivatianos et al 15 found differences between adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma in terms of c-kit and SMA IHC. Among other findings, these authors reported positive a-sma staining in 83% of adenoid cystic carcinoma cases (with staining in >50% of lesional cells), and positive c-kit staining in only 11% of polymorphous lowgrade adenocarcinoma cases (with staining in <50% of lesional cells). These authors also reported different expression patterns of a-sma and c-kit within the tubular structures of adenoid cystic carcinoma versus those of polymorphous low-grade adenocarcinoma. These studies support the potential utility of IHC as an adjunctive consideration in the distinction between adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma. Although c-kit (and SMA) staining of adenoid cystic carcinoma may be more consistently positive and strong compared with polymorphous low-grade adenocarcinoma, IHC of salivary gland tumors should be interpreted with circumspection. Various authors have noted that c-kit expression is not unique to adenoid cystic carcinoma, as this marker sometimes stains polymorphous low-grade adenocarcinoma and basal cell adenoma. 13,16 Our experience is similar to these authors, as we frequently observe c-kit expression in various salivary gland tumors including polymorphous low-grade adenocarcinoma, pleomorphic adenoma, and basal cell adenoma. Especially in equivocal cases, c-kit does not provide definitive distinction between adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma, or between malignant and benign neoplasms. 13 Molecular Genetics Associations have been demonstrated between several salivary gland neoplasms and specific molecular genetic markers (Table 1), including adenoid cystic carcinoma and fusion involving MYB-NFIB ; low-grade/intermediategrade mucoepidermoid carcinoma and CRTC1-MAML ; mammary analog secretory carcinoma and ETV6- NTRK3 2,30,31 ; and hyalinizing clear cell carcinoma and EWSR1-ATF Rearrangement involving the PLAG1 and HMGA2 oncogenes occur specifically in pleomorphic adenoma Molecular markers of polymorphous low-grade adenocarcinoma remain to be characterized. Persson et al 22 investigated occurrence of the MYB-NFIB fusion (associated with adenoid cystic carcinoma) in polymorphous low-grade adenocarcinoma; of the 9 cases analyzed, 1 case demonstrated the MYB-NFIB fusion, illustrating the difficulties associated with diagnosis of these morphologically similar lesions. These authors posit that the genome of polymorphous low-grade adenocarcinoma is genetically stable and contains relatively few copy-number alterations, consistent with the clinical behavior of polymorphous low-grade adenocarcinoma as an indolent low-grade carcinoma with low metastatic potential. Molecular genetic evaluation potentially offers significant utility in the diagnosis and differential diagnosis of intraoral minor salivary gland tumors. Most laboratories, however, are not presently equipped to perform molecular analysis of these TABLE 1. Salivary Gland Neoplasms With Known Chromosomal Translocations/Gene Fusions Neoplasm Chromosomal Translocation Gene Fusion PA Rearrangement of 8q12 Rearrangement of 12q13-15 PLAG1 HMGA2 MEC t (11;19)(q21;p13) CRTC1-MAML2 AdCC t (6;9)(q22-23;p23-24) MYB-NFIB HCCC t (12;22)(q13;q12) EWSR1-ATF1 MASC t (12;15)(p13q25) ETV6-NTRK3 AdCC indicates adenoid cystic carcinoma; HCCC, hyalinizing clear cell carcinoma; MASC, mammary analogue secretory carcinoma; MEC, mucoepidermoid carcinoma; PA, pleomorphic adenoma. lesions, and pathologists must rely on the more conventional diagnostic methods of light microscopy and IHC. Recommendations for the Diagnosis of Intraoral Minor Salivary Gland Neoplasms Distinction between benign and malignant salivary gland lesions generally depends upon identification of malignant features. High-grade cytomorphologic features including marked nuclear pleomorphism, increased mitotic activity with atypical mitoses, and tumor necrosis (of individual cells and/or confluent foci) facilitate a diagnosis of malignancy. In typical cases of pleomorphic adenoma, basal cell adenoma, polymorphous low-grade adenocarcinoma, and adenoid cystic carcinoma, however, limited biopsies lack overtly malignant cytomorphologic features. Consequently, the diagnosis of malignancy in these specimens relies upon other findings. The presence of metastatic disease would certainly be diagnostic for a malignant neoplasm, but most intraoral minor salivary gland carcinomas, including polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma (as well as low-grade mucoepidermoid carcinoma), are infrequently associated with metastatic disease at the time of diagnosis. Other than metastasis, definitive features of malignancy in minor salivary gland neoplasms are predicated on infiltrative growth. Infiltrative growth includes invasion into nonneoplastic seromucous glands, soft tissues and/or bone, PNI, and LVI. By definition, invasive growth occurs at a lesion s periphery. As previously discussed, specific aspects of a tumor s growth, such as envelopment of residual seromucous glands by polymorphous low-grade adenocarcinoma, may factor into the differential diagnosis of a given lesion. Otherwise, incisional biopsies of intraoral minor salivary gland tumors generally provide limited sampling of the lesion s periphery, and therefore do not permit assessment of invasive growth. The considerable histopathologic and IHC overlap between the various intraoral minor salivary gland neoplasms complicates the interpretation of incisional biopsies. When evaluating a limited biopsy that shows neoplastic proliferation of intraoral minor salivary gland origin, the surgical pathologist must then consider the appropriate diagnostic terminology and potential recommendations regarding management. In this scenario, pathologists are advised to render relatively broad diagnosis while providing as much information as feasible regarding the differential diagnosis. In the context of an intraoral minor salivary gland neoplasm that lacks overtly malignant features, but does not permit exclusion of malignancy, appropriate diagnostic terminology would resemble Minor salivary gland neoplasm, not further specified, with recommendation of conservative r 2013 Lippincott Williams & Wilkins 9

10 Turk and Wenig Adv Anat Pathol Volume 21, Number 1, January 2014 but complete surgical excision with tumor-free margins. On the basis of limited material, a more specific diagnosis whether benign or malignant, may be discordant with the diagnosis resulting from a subsequent resection specimen. A lesion diagnosed as pleomorphic adenoma or basal cell adenoma based on an incisional biopsy may show invasive growth (eg, PNI) after complete resection (Fig. 2). Conversely, a limited biopsy showing cytologic features of polymorphous low-grade adenocarcinoma or adenoid cystic carcinoma may lead to a resection specimen that lacks invasive growth, thereby warranting a benign diagnosis. As an important caveat, among salivary gland lesions, some malignancies can be diagnosed in a circumscribed to encapsulated lesion without invasive growth. Examples of such an occurrence include mucoepidermoid carcinoma and acinic cell adenocarcinoma in which the cell types for these respective neoplasms are diagnostic as there are no benign neoplasms with the constituent cells diagnostic for either of these tumor types. Such encapsulated or noninvasive malignant neoplasms arise more frequently in major salivary glands, and are less of a concern in the differential diagnosis of intraoral minor salivary gland tumors. CONCLUSIONS We have attempted to demonstrate the spectrum of pathologic findings shared by intraoral benign and malignant minor salivary gland neoplasms, such that limited biopsies of these lesions are frequently insufficient for definitive diagnosis. This generalization applies to incisional biopsies and fine-needle aspiration biopsy. Our clinical colleagues may believe that biopsy material should facilitate distinction between benign and malignant neoplasms. Unfortunately, in our experience, this expectation is frequently unrealistic due to sampling issues. The prudent diagnostic approach regarding incisional biopsy specimens entails terminology such as Minor salivary gland neoplasm, not further specified, confirming the presence of a neoplastic proliferation that necessitates complete surgical resection with clear margins. After resection, evaluation of the excised specimen facilitates assessment of invasive growth, and establishment of a definitive diagnosis. An intraoral minor salivary gland neoplasm lacking invasive growth is benign (eg, pleomorphic adenoma or basal cell adenoma per the focus of this manuscript), and complete resection of benign tumors is curative. Identification of invasive growth warrants a malignant diagnosis (eg, polymorphous low-grade adenocarcinoma or adenoid cystic carcinoma per the focus of this manuscript). In the case of polymorphous low-grade adenocarcinoma, complete resection with tumor-free margins is considered curative, and additional therapy (eg, radiotherapy) is generally not indicated. The potential complications of radiotherapy in the setting of polymorphous low-grade adenocarcinoma may include osteoradionecrosis representing a far greater clinical problem for the patient than any untoward complications due to the neoplasm, such as recurrence years after resection. Adenoid cystic carcinoma, in contrast, necessitates adjuvant therapy, specifically radiation, in addition to complete surgical resection. REFERENCES 1. Barnes L, Eveson JW, Reichert P, et al. World Health Organization classification of tumours. Pathology and Genetics of Head and Neck Tumours. Lyon, France: IARC Press; 2005: Skálova A, Vanecek T, Sima R, et al. Mammary analogue secretory carcinoma of salivary glands, containing the ETV6- NTRK3 fusion gene: a hitherto undescribed salivary gland tumor entity. 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Assessment of proliferative activity using the MIB1 antibody helps to distinguish polymorphous low grade adenocarcinoma from adenoid cystic carcinoma of salivary glands. Pathol Res Pract. 1997;193: Schwarz S, Mu ller M, Ettl T, et al. Morphological heterogeneity of oral salivary gland carcinomas: a clinicopathologic study of 41 cases with long term follow-up emphasizing the overlapping spectrum of adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma. Int J Clin Exp Pathol. 2011;4: Jeng YM, Lin CY, Hsu HC. Expression of the c-kit protein is associated with certain subtypes of salivary gland carcinoma. Cancer Lett. 2000;154: Penner CR, Folpe AL, Budnick SD. C-kit expression distinguishes salivary gland adenoid cystic carcinoma from polymorphous low-grade adenocarcinoma. Mod Pathol. 2002; 15: Mino M, Pilch BZ, Faquin WC. Expression of KIT (CD117) in neoplasms of the head and neck: an ancillary marker for adenoid cystic carcinoma. 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11 Adv Anat Pathol Volume 21, Number 1, January 2014 Intraoral Minor Salivary Gland Neoplasms of tumor-specific fusion oncogenes in mucoepidermoid carcinoma, adenoid cystic carcinoma, and NUT midline carcinoma. Ann Diagn Pathol. 2011;15: 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: 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: 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: 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: Persson M, Andrén Y, Moskaluk CA, et al. Clinically significant copy number alterations and complex rearrangements of MYB and NFIB in head and neck adenoid cystic carcinoma. Genes Chromosomes Cancer. 2012;51: Moskaluk CA. Adenoid cystic carcinoma: clinical and molecular features. Head Neck Pathol. 2013;7: 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: 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: Behboudi A, Enlund F, Winnes M, et al. Molecular classification of mucoepidermoid carcinomas-prognostic significance of the MECT1-MAML2 fusion oncogene. Genes Chromosomes Cancer. 2006;45: 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: Fehr A, Lo ning T, Stenman G. Mammary analogue secretory carcinoma of the salivary glands with ETV6-NTRK3 gene fusion. Am J Surg Pathol. 2011;35: Bishop JA. Unmasking MASC: bringing to light the unique morphologic, immunohistochemical and genetic features of the newly recognized mammary analogue secretory carcinoma of salivary glands. Head Neck Pathol. 2013;7: Antonescu CR, Katabi N, Zhang L, et al. EWSR1-ATF1 fusion is a novel and consistent finding in hyalinizing clear-cell carcinoma of salivary gland. Genes Chromosomes Cancer. 2011;50: Shah AA, LeGallo RD, van Zante A, et al. EWSR1 genetic rearrangements in salivary gland tumors: a specific and very common feature of hyalinizing clear cell carcinoma. Am J Surg Pathol. 2013;37: Tanguay J, Weinreb I. What the EWSR1-ATF1 fusion has taught us about hyalinizing clear cell carcinoma. Head Neck Pathol. 2013;7: Thway K, Fisher C. Tumors with EWSR1-CREB1 and EWSR1-ATF1 fusions: the current status. Am J Surg Pathol. 2012;36:e1 e Matsuyama A, Hisaoka M, Nagao Y, et al. Aberrant PLAG1 expression in pleomorphic adenomas of the salivary gland: a molecular genetic and immunohistochemical study. Virchows Arch. 2011;458: Kas K, Voz ML, Ro ijer E, et al. Promoter swapping between the genes for a novel zinc finger protein and beta-catenin in pleomorphic adenomas with t(3;8)(p21;q12) translocations. Nat Genet. 1997;15: Kas K, Ro ijer E, Voz M, et al. A 2-Mb YAC contig and physical map covering the chromosome 8q12 breakpoint cluster region in pleomorphic adenomas of the salivary glands. Genomics. 1997;43: Voz ML, Astro m AK, Kas K, et al. The recurrent translocation t(5;8)(p13;q12) in pleomorphic adenomas results in upregulation of PLAG1 gene expression under control of the LIFR promoter. Oncogene. 1998;16: Geurts JM, Schoenmakers EF, Ro ijer E, et al. van de Ven WJ. Identification of NFIB as recurrent translocation partner gene of HMGIC in pleomorphic adenomas. Oncogene. 1998;16: r 2013 Lippincott Williams & Wilkins 11