Salivary gland tumours: diagnostic challenges and an update on the latest WHO classification

Open AccessPublished:February 28, 2020DOI:https://doi.org/10.1016/j.mpdhp.2020.01.001

      Abstract

      Salivary gland tumours are one of the most difficult areas of diagnostic pathology, with significant morphological diversity and many overlapping features. The latest WHO classification has attempted to simplify the classification but there are still more than 30 tumours for the pathologist to grapple with. These include two new entities – secretory carcinoma and sclerosing polycystic adenosis – and a number of name changes. Most controversial is the removal of “low grade” from polymorphous low-grade adenocarcinoma and the inclusion of intraductal carcinoma as a unifying entity. There are also more nuanced changes in categorisation or terminology that may influence the way a diagnostic report is written. Despite advances in immunohistochemistry and molecular pathology, the WHO still use histomorphology as the primary basis for classification. However, morphological similarities can make diagnosis difficult without the assistance of ancillary techniques. In this short review we describe these changes in the latest WHO classification, discuss particular areas of diagnostic difficulty, and suggest some useful antibodies that can be used to assist diagnosis.

      Keywords

      Introduction and overview

      Primary epithelial salivary gland tumours (SGT) are a morphologically diverse group of neoplasms, which may present considerable diagnostic challenges to the pathologist, and management conundra to surgeons and oncologists. SGT are rare, with only about 720 cases per year in the United Kingdom. The overall incidence of benign and malignant tumours is less than 5 per 100,000 head of population per year. Since about 80% of all tumours are benign it can be appreciated that salivary gland malignancies are very rare with reported incidences of only 1.2–1.3 cases per 100,000 and representing only around 3% of all cancers of the head and neck.,
      • Lin H.H.
      • Limesand K.H.
      • Ann D.K.
      Current state of knowledge on salivary gland cancers.
      As a general rule patients are over the age of 40 with males and females equally affected. However, it is important to note that some of the more common tumours, especially pleomorphic adenoma (PA), show a predominance for females with a M:F ratio of about 1:1.4.
      • Valstar M.H.
      • de Ridder M.
      • van den Broek E.C.
      • et al.
      Salivary gland pleomorphic adenoma in The Netherlands: a nationwide observational study of primary tumor incidence, malignant transformation, recurrence, and risk factors for recurrence.
      Around 80% of SGT are benign and 65% of these are PA which are by far the most common of all SGT, comprising about 55% of major gland lesions and 50% of minor gland lesions.
      • Valstar M.H.
      • de Ridder M.
      • van den Broek E.C.
      • et al.
      Salivary gland pleomorphic adenoma in The Netherlands: a nationwide observational study of primary tumor incidence, malignant transformation, recurrence, and risk factors for recurrence.
      ,
      • Speight P.M.
      • Barrett A.W.
      Salivary gland tumours.
      Tumours in the parotid gland account for approximately 70% of SGT, the submandibular gland accounts for around 10% and the sublingual gland less than 1%, thus the minor glands are affected by about 20%. Although tumours are less common in the minor glands about 50% are malignant, compared to only about 20% in the major glands. Of note, tumours in the sublingual gland are almost always malignant. Of the 70% of all tumours encountered in the parotid gland, 50–60% are PA, 20–30% are Warthin tumours and about 10% are mucoepidermoid carcinomas (MEC).
      • Lin H.H.
      • Limesand K.H.
      • Ann D.K.
      Current state of knowledge on salivary gland cancers.
      • Valstar M.H.
      • de Ridder M.
      • van den Broek E.C.
      • et al.
      Salivary gland pleomorphic adenoma in The Netherlands: a nationwide observational study of primary tumor incidence, malignant transformation, recurrence, and risk factors for recurrence.
      • Speight P.M.
      • Barrett A.W.
      Salivary gland tumours.
      There is a dauntingly extensive literature on this topic, but for the diagnostic pathologist there are a number of excellent current histopathology textbooks
      • Hellquist H.B.
      • Skalova A.
      Histopathology of the salivary glands.
      ,
      which, as well as the latest AFIP fascicle
      • Ellis G.L.
      • Auclair P.L.
      Tumors of the salivary glands. AFIP atlas of tumor pathology.
      and current WHO classification,
      World Health organization classification of head and neck tumours.
      may provide succinct guidance.
      In this article we aim to update our previous review
      • Khurrram S.A.
      • Barrett A.W.
      • Speight P.M.
      Diagnostic difficulties in lesions of the minor salivary glands.
      of the main changes in the latest WHO classification of salivary gland tumours and address some areas of interest, dispute and difficulty.

      Changes in the classification of salivary gland tumours

      In 1952, the World Health Organization (WHO) initiated a programme to produce an internationally acceptable classification system for all human tumours. The process has been reviewed by Sobin,
      • Sobin L.H.
      The World Health Organization's programme for the histopathological definition and classification of tumours.
      but the overall aim was to produce classifications that would use a uniform nomenclature to facilitate global communication. Number 7 in the series was published in 1972 and was the first edition of the WHO classification of salivary gland tumours.
      • Thackray A.C.
      • Sobin L.H.
      Histological typing of salivary gland tumours. International histological typing of tumours. No 7.
      This first classification listed only 10 primary epithelial SGT using terminology which, with three exceptions, current pathologists would not recognize. By the time of the second edition in 1991
      • Seifert G.
      • Sobin L.H.
      Histological typing of salivary gland tumours. World Health organization international histological classification of tumours.
      the number of entities had risen significantly to 39. These classifications were based almost exclusively on histomorphology and were essentially a simple list of lesions ordered by frequency of occurrence. Such a classification system has been criticized, especially by surgical oncologists, for being too complicated and for a lack of precision or applicability to modern oncological practice. In a previous review, we discussed at length the benefits or otherwise of “lumping” or “splitting” SGT
      • Speight P.M.
      • Barrett A.W.
      Salivary gland tumours.
      and suggested that these comprehensive classifications had developed because of the wide morphological diversity of the tumours, and were needed to ensure accurate diagnosis and correct categorisation which in turn would guide treatment. We believe that this is still the case, and indeed subsequent editions of the WHO classification have maintained this approach. In the 2005 classification
      • Eveson J.W.
      • Auclair P.L.
      • Gnepp D.R.
      • El-Naggar A.K.
      Tumours of the salivary glands.
      the number of entities remained virtually stable at 37, and in the latest (2017) classification has been slightly reduced to 33.
      World Health organization classification of head and neck tumours.
      The major changes in the classification are summarized in Table 1 and are discussed below. The reason for the reduction was explained by El-Naggar in the introduction to the 2017 WHO classification.
      World Health organization classification of head and neck tumours.
      There was an overall desire to make the classification less complex and more streamlined. Therefore, some particularly rare malignant entities were grouped together as “adenocarcinoma NOS” and the two variants of ductal papilloma (intraductal papilloma, inverted ductal papilloma) were merged. Despite several candidates, only two new entities were included, as they had thoroughly documented novel phenotypes and had become universally recognized. Also, a major principle of the WHO's international approach has been maintained – that findings based on sophisticated techniques should not be used as a basis for classification, because these techniques are not globally available.
      • Sobin L.H.
      The World Health Organization's programme for the histopathological definition and classification of tumours.
      Thus the current classification is still largely based on basic histomorphology and requires proficiency in routine diagnostic microscopy. Nevertheless, immunohistochemical findings are still described, and where appropriate, molecular changes are included because they enable more accurate diagnosis of some SGT. Some of these will be discussed below and are shown in Table 2, but it should be noted that, at the present time, use of these adjunctive techniques is rarely essential for diagnosis or management, although when present they may define a particular entity. For example, two carcinomas can be defined by specific gene translocations (secretory carcinoma and clear cell carcinoma), while a number of other tumours have molecular abnormalities that can assist diagnosis but may not be consistently found (MEC, adenoid cystic carcinoma, cribriform adenocarcinoma of minor salivary glands, PA and sclerosing polycystic “adenoma”). As our understanding of these molecular changes increases, we anticipate that genomic alterations will become more important as diagnostic biomarkers and that future classifications will become more reliant on molecular changes, especially as many of these provide specific targets for new therapies.
      Table 1Key changes in the 2017 WHO classification of salivary gland tumours
      World Health organization classification of head and neck tumours.
      Key changesExplanatory notes
      New entities
      Secretory carcinomaFirst described in 2010.
      • Skálová 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.
      Formerly known as mammary analogue secretory carcinoma (MASC)
      Sclerosing polycystic adenosisFirst described in 1996.
      • Smith B.C.
      • Ellis G.L.
      • Slater L.J.
      • Foss R.D.
      Sclerosing polycystic adenosis of major salivary glands. A clinicopathologic analysis of nine cases.
      There is controversy over its status as a neoplasm
      New names
      Polymorphous adenocarcinomaFormerly polymorphous low-grade adenocarcinoma
      Intraductal carcinomaFormerly low grade cribriform cystadenocarcinoma, low grade salivary duct carcinoma, salivary duct carcinoma in situ
      Poorly differentiated carcinomaSingle category includes undifferentiated carcinoma, large and small cell neuroendocrine carcinoma
      Clarifications, changes
      Adenocarcinoma NOSDefinition broadened to include rare entities, including: cystadenocarcinoma, mucinous (cyst)adenocarcinoma, papillary cystadenocarcinoma
      CystadenocarcinomaCystadenocarcinoma is removed as a separate entity (see above)
      Mucinous adenocarcinomaMucinous adenocarcinoma is removed as a separate entity (see above)
      Metastasising pleomorphic adenomaMoved from malignant category to a variant of benign pleomorphic adenoma
      Carcinoma ex-pleomorphic adenomaClarifications on diagnostic terminology: should explicitly state the histological type of malignant component.

      Definition of minimally invasive changed from 1.5 mm to “<4–6 mm”
      Sialadenoma papilliferumGiven its own category. No longer a “ductal papilloma”
      Ductal papillomaA single name for two variants: inverted ductal papilloma and intraductal papilloma
      LymphadenomaA single category replacing sebaceous and non-sebaceous lymphadenomas. Sebaceous-type is regarded as a simple variant
      Non-neoplastic epithelial lesionsNew category, includes sclerosing polycystic adenosis, nodular oncocytic hyperplasia, lymphoepithelial sialadenitis, intercalated duct hyperplasia
      Table 2Antibodies that are useful in the diagnosis of salivary gland tumours
      AntibodyTargetDiagnostic utility
      Cytokeratins
      CK7Type II keratin. Mainly non-keratinising simple epitheliaVirtually all SGT are positive for CK7. Useful to confirm a salivary origin for unusual tumours and metastases. If a tumour is CK7 negative, first exclude an alternative diagnosis to SGT
      CK20Type I keratin. Mainly GI epitheliumVirtually all SGT are negative for CK20. Useful to exclude salivary origin for unusual tumours and metastases. CK7/CK20 phenotypes define many tumour types.
      CK14Myoepithelial cellsNot entirely specific but marks myoepithelial cells in most tumours. Useful for abluminal cells in EMC.
      AE1/AE3Duct cellsStains most SGT. Useful to identify duct (luminal cells) especially in EMC.
      Myoepithelial markers
      SMASmooth muscle actinReliable marker of mature myoepithelial cells. Note that myoepithelial cells in PA (including plasmacytoid cells) are largely negative. All myoepithelial markers are useful in EMC and for demonstrating peripheral cells around tumour islands in intraductal (“in situ”) carcinomas.
      CalponinBasic smooth muscle proteinReliable myoepithelial cell marker. Stains plasmacytoid cells in PA, so useful to use SMA and calponin together.
      p63Transcription factorReliable myoepithelial and basal cell marker. Useful for EMC and stains peripheral cells in AdCC, BUT not specific. Positive in other tumours, in particular PAC (and CAMSG) shows strong diffuse nuclear staining (and are p40-negative). AcCC and SC are negative. p63 is also a good marker for tumours of squamous origin.
      p40An isoform of p63Has very similar staining pattern as p63, BUT PAC and CAMSG are negative. p63-positive/p40-negative phenotype is useful for diagnosis of PAC. Note that 26% of matrix-rich PA may also be p40-negative.
      SOX-10Transcription factorPositive in most cells derived from neural crest, but useful in PA and has a similar distribution to DOG-1 in AcCC.
      S100Family of S100 proteinsTraditionally a myoepithelial marker but lacks specificity and little utility for myoepithelial cells. Useful for diagnosis of SC and PAC where it is strong and diffusely positive in 100% of tumour cells. AcCC and AdCC are negative or weak and patchy.
      Cell cycle markers
      Ki67/MIB-1Cell cycle marker (G1/G2/S/M)Useful as indicator of malignancy and aggression. High expression associated with high grade lesions, BUT malignant SGT have notoriously low expression – e.g. very low in cribriform AdCC and MEC.
      MCM2Cell cycle marker (G1/G2/S)Shown to always be >10% in AdCC but <10% in PA and PAC. Useful in small biopsies where there is a suspicion of AdCC.
      Other markers
      DOG-1Luminal aspect of acini and small ductsMost useful for differentiation of AcCC from SC. AcCC is positive, SC is negative. Occasionally positive in ducts in a variety of tumours including PA and AdCC. Occasionally stains myoepithelial cells.
      CD117c-KIT (a tyrosine kinase)Not absolutely specific, but positivity suggests AdCC (80%+ of cases are positive)
      PLAG1PLAG1 proteinUseful for diagnosis of PA, with positive nuclear staining in about 95%. Rarely seen in PAC and negative in AdCC.
      GFAPGlial fibrillary acidic proteinUseful marker for PA. It is almost always positive, especially in myxoid areas. Rarely seen in any other SGT.
      Pan-TrkTropomyosin receptor kinaseNew antibody that targets tumours with NTRK fusion proteins. Useful for diagnosis of SC.
      MammaglobinMember of the uteroglobin family of glycoproteinsMost useful for differentiation of AcCC from SC; AcCC is negative, SC is strongly positive.
      MybMyb proteinPositive in AdCC with the MYB-NFIB fusion. Positive in about 65% of cases.
      Androgen receptorTranscription factorNuclear expression in 70% of salivary duct carcinomas.
      HER2Epidermal growth factor receptorPositive in 25–30% of salivary duct carcinomas, but also in high-grade intraductal carcinoma.
      AcCC, acinic cell carcinoma; AdCC, adenoid cystic carcinoma; CAMSG, cribriform adenocarcinoma of minor salivary gland; EMC, epithelial-myoepithelial carcinoma; MEC, mucoepidermoid carcinoma; PA, pleomorphic adenoma; PAC, polymorphous adenocarcinoma; SC, secretory carcinoma.

      New entities

       Secretory carcinoma

      It has long been recognized that, occasionally, a tumour that otherwise resembles acinic cell carcinoma (AcCC) does not show the typical PAS-positive intracellular granules that defines acinic differentiation. These were often referred to as “granule poor” AcCC or relegated to the adenocarcinoma NOS category. In 2010 Skalova et al.
      • Skálová 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.
      recognized that these neoplasms were similar to secretory carcinoma of the breast and also shared the specific ETV6-NTRK3 gene fusion. They called this tumour mammary analogue secretory carcinoma (MASC) and it quickly became established as a new entity with more than 250 cases now reported. It is included in the WHO classification, under a new simpler name, as secretory carcinoma (SC).
      World Health organization classification of head and neck tumours.
      SC is found most often in the parotid gland (70% of cases) with most of the remainder arising in the buccal mucosa, lips and palate. Few cases have been reported in the submandibular or sublingual glands. Males appear to be slightly more often affected than females and there is a wide age range, with a number of cases reported in children. Nodal metastases have been reported in up to 25% of cases, but overall this is an indolent tumour with survival exceeding 95%.
      Histologically, SC shows features similar to AcCC with a lobular growth pattern and microcystic, solid, follicular and papillary cystic features. However, SC lacks the sheets of basophilic granular acinic cells that typify AcCC and the papillary cystic pattern may be more common in SC. In our experience, PAS (with diastase) staining is the single most useful diagnostic aid.
      • Khurrram S.A.
      • Barrett A.W.
      • Speight P.M.
      Diagnostic difficulties in lesions of the minor salivary glands.
      In AcCC the acinic cells contain many PAS-positive granules, but the pale eosinophil cells in SC are negative. However, in SC PAS does show a very characteristic globular staining within the cystic and intercellular spaces. It was long felt that demonstration of the ETV6-NTRK3 gene rearrangement (by FISH) was essential for diagnosis, but studies have now shown that there is a characteristic immunohistochemical phenotype
      • Khurram S.A.
      • Sultan-Khan J.
      • Atkey N.
      • Speight P.M.
      Cytogenetic and immunohistochemical characterization of mammary analogue secretory carcinoma of salivary glands.
      that is almost diagnostic and easily distinguishes SC from AcCC. SC shows strong, diffuse positivity for S100 and mammaglobin, but is negative for DOG-1. Conversely AcCC show consistent luminal positivity for DOG-1 and SOX-10 but are negative or only patchily positive for S100 and mammaglobin. More recently, a new antibody has become available to tropomyosin receptor kinase (pan-TRK) and this has been shown to be highly specific for the diagnosis of SC.
      • Xu B.
      • Haroon Al Rasheed M.R.
      • Antonescu C.R.
      • et al.
      Pan-Trk immunohistochemistry is a sensitive and specific ancillary tool in diagnosing secretory carcinoma of salivary gland and detecting ETV6-NTRK3 fusion.
      In the field of precision medicine, TRK inhibitors (e.g. larotrectinib, entrectinib) have now been approved for the clinical treatment of tumours with NTRK fusions regardless of their histology or site – a new tumour-agnostic approach to therapy.
      • Yan L.
      • Zhang W.
      Precision medicine becomes reality-tumor type-agnostic therapy.
      This will include SC and the results of ongoing clinical trials may prove this to be an effective therapy.

       Sclerosing polycystic adenosis

      Although only about 60 cases of this controversial lesion have been reported, sclerosing polycystic adenosis has been included as a new entity. It was first described as an unusual inflammatory or reactive disorder
      • Smith B.C.
      • Ellis G.L.
      • Slater L.J.
      • Foss R.D.
      Sclerosing polycystic adenosis of major salivary glands. A clinicopathologic analysis of nine cases.
      that resembles fibrocystic disease of the breast. It is almost exclusively found in the parotid gland, with only occasional cases reported in the submandibular gland or minor glands. The lesions are characterized by lobular areas of densely collagenous fibrous tissue with multiple, cystically dilated ducts, often with a cribriform or papillary cystic pattern. Apocrine secretion, mucous cells, sebaceous cells and squamous metaplasia may be seen as well as a chronic inflammatory cell infiltrate. There is significant controversy over the nature of this lesion, since some authorities regard the changes as neoplastic. There is good evidence for this since lesions have been shown to be monoclonal in nature and more recently it has been shown that sclerosing polycystic adenosis shows genetic alterations in the PI3K pathway and PTEN mutations.
      • Bishop J.A.
      • Gagan J.
      • Baumhoer D.
      • et al.
      Sclerosing polycystic "adenosis" of salivary glands: a neoplasm characterized by PI3K pathway alterations more correctly named sclerosing polycystic adenoma.
      This suggests that these lesions are neoplasms. Intriguingly, although the WHO classify it as a non-neoplastic epithelial lesion they give “sclerosing polycystic adenoma” as a synonym.
      World Health organization classification of head and neck tumours.

      New names

       Polymorphous adenocarcinoma

      Polymorphous low grade adenocarcinoma was first named in 1984 as a low grade and indolent tumour, often found on the palate. The lesion is widely infiltrative and often exhibits a cribriform pattern similar to adenoid cystic carcinoma (AdCC), with which it is often confused – hence the term “low grade” was included in the name to clearly distinguish its behaviour from that of AdCC. Previously we have noted that the behaviour of this lesion can be unpredictable and some do not behave in a low grade manner and we suggested the term “low grade” should be removed from the name.
      • Speight P.M.
      • Barrett A.W.
      Salivary gland tumours.
      Although overall survival is better than 95%, individual lesions can be unpredictable and lesions may recur and metastasise. Recurrence rates of 5–33% have been reported and about 10% overall show lymph node metastases.
      • Vander Poorten V.
      • Triantafyllou A.
      • Skálová A.
      • et al.
      Polymorphous adenocarcinoma of the salivary glands: reappraisal and update.
      In effect the behaviour of the polymorphous “low grade” adenocarcinoma was similar to MEC, yet even in 2005 the WHO kept “low grade” in the name – the only tumour to have its grade defined in the name. In 2017 “low grade” has been dropped and the new term for this tumour is polymorphous adenocarcinoma (PAC). Removing the term “low grade” will help reduce inappropriate conservative management and ensure that the lesion is managed in the same way as other malignant SGT, based on clinical stage and a careful consideration of the histological features. Over 90% of PAC are found in the minor salivary glands and it is the second most common intra-oral salivary malignancy (after MEC), comprising about 40% of the total. It most often occurs on the palate and females are affected almost twice as often as males.
      • Vander Poorten V.
      • Triantafyllou A.
      • Skálová A.
      • et al.
      Polymorphous adenocarcinoma of the salivary glands: reappraisal and update.
      Histologically PAC shows a range of diverse morphological features and diagnosis is based on recognition of these polymorphous features, which may only be apparent in a good-sized biopsy or excision specimen. The features include a lobular pattern, widespread infiltration, single cell filing and a characteristic perineural infiltration with a whorling or targetoid pattern (see below). A cribriform pattern is a common feature, which, along with nerve involvement, is the main reason for misdiagnosis as AdCC (Figure 1). Many lesions also show a papillary cystic morphology and it has been reported that these may be more aggressive with a higher incidence of lymph node metastases.
      • Vander Poorten V.
      • Triantafyllou A.
      • Skálová A.
      • et al.
      Polymorphous adenocarcinoma of the salivary glands: reappraisal and update.
      In 1999 a typically cribriform or papillary cystic variant of PAC was reported in the tongue and was given the name cribriform adenocarcinoma of the tongue ("CAT").
      • Michal M.
      • Skálová A.
      • Simpson R.H.
      • et al.
      Cribriform adenocarcinoma of the tongue: a hitherto unrecognized type of adenocarcinoma characteristically occurring in the tongue.
      Subsequently lesions were reported at other intra-oral sites and the tumour is now called cribriform adenocarcinoma of minor salivary glands (CAMSG). This lesion was considered to be more aggressive than PAC and many regard it as a new entity.
      • Vander Poorten V.
      • Triantafyllou A.
      • Skálová A.
      • et al.
      Polymorphous adenocarcinoma of the salivary glands: reappraisal and update.
      ,
      • Michal M.
      • Skálová A.
      • Simpson R.H.
      • et al.
      Cribriform adenocarcinoma of the tongue: a hitherto unrecognized type of adenocarcinoma characteristically occurring in the tongue.
      Its distinction from PAC is supported by the finding that over 80% of cases harbour rearranged PRKD1-3 genes compared to only 10% of cases of PAC.
      • Weinreb I.
      • Zhang L.
      • Tirunagari L.M.
      • et al.
      Novel PRKD gene rearrangements and variant fusions in cribriform adenocarcinoma of salivary gland origin.
      Conversely, more than 70% of PAC show a specific PRKD (E710D) mutation which is only rarely seen in CAMSG.
      • Vander Poorten V.
      • Triantafyllou A.
      • Skálová A.
      • et al.
      Polymorphous adenocarcinoma of the salivary glands: reappraisal and update.
      ,
      • Weinreb I.
      • Zhang L.
      • Tirunagari L.M.
      • et al.
      Novel PRKD gene rearrangements and variant fusions in cribriform adenocarcinoma of salivary gland origin.
      Controversially, the WHO specialist panel did not include CAMSG as a new entity but rather considered it to be a variant within the morphological spectrum of PAC (this is well reviewed and critiqued by Vander Poorten et al.
      • Vander Poorten V.
      • Triantafyllou A.
      • Skálová A.
      • et al.
      Polymorphous adenocarcinoma of the salivary glands: reappraisal and update.
      ). With some justification this is based on the fact that they share similar morphology and immunophenotype, but also appear to involve genetic changes within the same gene family.
      Figure 1
      Figure 1Four different tumours show a similar cribriform morphology, suggesting that simple “pattern-matching” is not appropriate in the diagnosis of SGT. a) Polymorphous adenocarcinoma, b) canalicular adenoma, c) pleomorphic adenoma, d) adenoid cystic carcinoma.

       Intraductal carcinoma

      Intraductal carcinoma is an exceedingly rare new entity that has developed from the concept that some salivary malignancies are confined within ductal structures, or can be considered as “in situ”, similar to in situ ductal lesions in the breast. Over the years a number of lesions have been described that have been suggested to have an in situ morphology. The names for these lesions have included low-grade salivary duct carcinoma, salivary duct carcinoma in situ and low grade cribriform cystadenocarcinoma (reviewed by Kuo et al.
      • Kuo Y.J.
      • Weinreb I.
      • Perez-Ordonez B.
      Low-grade salivary duct carcinoma or low-grade intraductal carcinoma? Review of the literature.
      ). A common feature of these tumours is that the intraductal component is surrounded by an intact layer of myoepithelial cells that can be demonstrated with a myoepithelial marker (e.g. calponin, p63, CK14 or smooth muscle actin – see Table 2). The concept of an in situ lesion was first described by Delgado et al.
      • Delgado R.
      • Klimstra D.S.
      • Albores-Saavedra J.
      Low grade salivary duct carcinoma. A distinctive variant with a low grade histology and a predominant intraductal growth pattern.
      who observed that not all salivary duct carcinomas were high grade or aggressive. They reported 10 cases that had bland cytological features and predominantly intraductal growth and used the term low-grade salivary duct carcinoma. The lesion was included in the 2005 WHO classification but was called low grade cribriform cystadenocarcinoma. However, it is apparent that similar lesions may show high grade cytological features, while at the same time having an intraductal morphology.
      • Kuo Y.J.
      • Weinreb I.
      • Perez-Ordonez B.
      Low-grade salivary duct carcinoma or low-grade intraductal carcinoma? Review of the literature.
      These lesions have been called salivary duct carcinoma in situ but did not have a place in the 2005 classification.
      The new 2017 classification provides a unifying solution by incorporating all these lesions into one category of intraductal carcinoma (Table 1). These lesions are found almost exclusively in the parotid gland and usually present as asymptomatic well demarcated swellings. Histologically they show a range of features but are mostly composed of multiple duct-like cystic spaces with cribriform or papillary morphology. Cytological atypia, necrosis and mitoses indicate a high grade lesion. The WHO description is not helpful in suggesting diagnostic criteria and does not discuss the usefulness of immunohistochemistry. However, it is implicit that the diagnosis can only be made if an intact circumferential layer of myoepithelial cells is immunohistochemically demonstrable around the tumour islands. There is also confusion over the relationship between high grade intraductal carcinoma and salivary duct carcinoma. These may show similar immunophenotypes and any evidence of invasion must indicate the latter diagnosis. In our view more research is needed to properly characterize these lesions. The WHO criteria are not clear, no indication is given of diagnostic criteria and the images suggest areas of invasive tumour that are clearly not “intraductal”. The statement that “The significance of local invasion by these lesions is uncertain” is also unhelpful. The immunohistochemistry and differential diagnosis of these lesions have been well discussed by Kuo et al.
      • Kuo Y.J.
      • Weinreb I.
      • Perez-Ordonez B.
      Low-grade salivary duct carcinoma or low-grade intraductal carcinoma? Review of the literature.

       Poorly differentiated carcinoma

      Poorly differentiated carcinoma is not a new name but appears as a new category that includes several very rare lesions, which can only be diagnosed after exclusion of metastases or other primary tumours. The category includes undifferentiated carcinoma and large and small cell neuroendocrine carcinomas.

      Clarifications and changes

      As well as changes in categorisation of some tumours, as discussed above and shown in Table 1, the text in the 2017 classification includes a number of subtle changes, some of which may change our perception of a lesion. Two changes in particular need to be considered: the re-categorisation of metastasising PA, and the terminology to be used when reporting carcinoma ex-PA.
      In previous classifications metastasising PA has been categorized as a separate entity under malignant salivary gland tumours. In the 2005 classification
      • Eveson J.W.
      • Auclair P.L.
      • Gnepp D.R.
      • El-Naggar A.K.
      Tumours of the salivary glands.
      the lesion was described in a whole section, which drew attention to its poor prognosis with up to 40% of patients dying of disease. In the latest classification
      World Health organization classification of head and neck tumours.
      metastasising PA merits only a single short paragraph describing its benign histological features and stating only that the prognosis is “generally good”. This change may be misleading and may result in inappropriate management of these lesions.
      With regards to carcinoma ex-PA the new edition explicitly states that it should not be considered as a standalone diagnosis. Whereas in previous editions, it was implicit that carcinoma ex-PA was a diagnostic term, in 2017 it clearly states that the histological type of the malignant component must be stated in the diagnostic report. In most cases the malignant component is a high grade adenocarcinoma NOS or salivary duct carcinoma, but sometimes a low grade lesion such as epithelial-myoepithelial carcinoma may be encountered. Another important change in the 2017 classification is the definition of minimal invasion. Carcinoma ex-PA is divided into three prognostic categories: intracapsular, minimally invasive and widely invasive. The significance of this is that intracapsular and minimally invasive lesions have a similarly good prognosis, implying that it is important to be able to clearly define a cut-off between a minimally invasive lesion and a widely invasive lesion. In the 2005 edition it was clearly stated that tumour that had invaded further than 1.5 mm from the capsule was widely invasive with an associated poor prognosis. However, this appeared to be arbitrary and discussion of prognosis included data for cut-off points of 8 mm, 6 mm and 5 mm. In 2017, minimally invasive is defined as “<4–6 mm extension beyond the PA border”, but further states that this is a preliminary threshold and that further research and validation are needed.
      Unfortunately, this is little help to the diagnostic pathologist and it remains important to consider each case on its merits. The key feature to define an invasive lesion must be histological evidence of destructive infiltration into soft tissues beyond the margins of the tumour. This judgement is made more difficult by the fact that PA, especially in minor glands, may have a poorly defined capsule and that recurrent lesions are often multinodular.

      Areas of diagnostic difficulty

       Core, punch and small biopsies

      As was emphasised in our previous articles,
      • Speight P.M.
      • Barrett A.W.
      Salivary gland tumours.
      ,
      • Khurrram S.A.
      • Barrett A.W.
      • Speight P.M.
      Diagnostic difficulties in lesions of the minor salivary glands.
      for most SGT a diagnosis can be made on H&E-stained sections but this depends on adequate histological material so that the full range of morphological and cytological features can be appreciated. Often however, the diagnostic pathologist is faced with a small biopsy and core and punch biopsies are now encountered frequently, so misdiagnoses on first biopsy are increasingly a potential hazard. Figure 1 shows small areas from a range of tumours that all have a similar cribriform morphology and illustrates how pattern-matching can be inappropriate and, with a lack of attention to detail or a lack of experience, may lead to a misdiagnosis. In particular situations immunohistochemistry is helpful or even essential to establish a definitive diagnosis and Table 2 summarizes antibodies that are particularly useful in specific applications. Molecular pathology is also increasingly being applied to the classification and diagnosis of SGT and some of these are mentioned. Molecular pathology is covered in detail in a sister article in this issue.
      • Moutassim K.
      Salivary gland tumours: update on molecular diagnostics.
      Particular care is needed when examining incisional biopsies from the palate, which is the site where most minor gland tumours will arise, but swellings caused by adenomas and adenocarcinomas are often clinically indistinguishable. However, 50% will be benign and 50% malignant. One of the most difficult problems for the diagnostic pathologist is to differentiate between the three most common lesions encountered at this site, namely PA (and its malignant variants), PAC and AdCC, all of which show variable and often similar features, the cribriform pattern being the most obvious (Figure 1). “Hyaline” or plasmacytoid cells (Figure 2) are almost pathognomonic of palatal PA, so are a most useful feature, and single cell filing and classic perineural whorling usually indicate a PAC (Figure 3).
      Figure 2
      Figure 2“Hyaline” or plasmacytoid cells in a palatal PA. These are rare in other types of SGT and are almost pathognomonic for PA.
      Figure 3
      Figure 3Polymorphous adenocarcinoma showing characteristic a) single cell filing and b) perineural whorls.
      Distinguishing benign from malignant is further complicated by the fact that benign SGT, including PA, basal cell adenoma, canalicular adenoma, Warthin tumour, cystadenoma and oncocytic lesions may be multifocal or lack a capsule or both. If not managed carefully, PA has the potential to recur, but is the only benign SGT to do so with any frequency and developments in surgical management (particularly extracapsular dissection of parotid tumours) have virtually eliminated this problem. Intravascular PA, which may be genuine or artefactual, is a rare but well documented phenomenon,
      • Skalova A.
      • Altemani A.
      • Di Palma S.
      • et al.
      Pleomorphic adenoma of the salivary glands with intravascular tumor deposits: a diagnostic pitfall.
      but has no adverse prognostic significance. By contrast, malignant tumours may be well circumscribed, and even encapsulated, when small or early stage (Figure 4), or are cytologically bland (thus accounting for the limitations of cytopathology in SGT). Hence, although the key determining factor in establishing the malignant nature of a SGT is the histological demonstration of an infiltrative margin, care is needed and a good working knowledge of SGT histomorphology is essential.
      Figure 4
      Figure 4A small (<2 cm) tumour in the parotid gland is surrounded by a thick fibrous capsule and appears benign. However, the morphology is that of a typical adenoid cystic carcinoma.
      Figure 5 shows a quite typical core biopsy, which provides little diagnostic information. However, at one end of the sample there are tubules and evidence of a cribriform morphology (Figure 5b), and at the other a solid, more basaloid component composed of small, hyperchromatic cells with a high nuclear:cytoplasmic ratio (Figure 5c). Such a morphological combination is typical of AdCC. The tumour arose in the submandibular gland and clinically there was paralysis of the marginal mandibular branch of the facial nerve; neural involvement is a characteristic feature of AdCC and this information assisted in making the diagnosis in this case. The presence of necrosis also suggests a malignant neoplasm. The features of the SGT in Figure 6 suggest a solid AdCC, but elsewhere the morphology was that of a PAC, with the diagnosis confirmed with immunohistochemistry.
      Figure 5
      Figure 5a) A small core biopsy from a submandibular gland tumour. Very little tumour tissue is present making diagnosis difficult. At one end, b) there are tubules and small cribriform areas, and at the other c) a solid, basaloid component. These feature support the diagnosis of adenoid cystic carcinoma.
      Figure 6
      Figure 6Polymorphous adenocarcinoma showing comedo-like necrosis.
      In the past immunohistochemistry (especially the widespread use of S100) has been more of a hindrance than a help when it comes to SGT differential diagnosis, but now there are several very useful, and more specific, markers that can aid diagnosis (Table 2). Figure 7 shows a core biopsy of a deep lobe tumour of the parotid. Morphologically the variable pattern of epithelium in a loosely fibrous matrix, which may be myxoid, mucoid or chondroid, is consistent with a PA, but positive expression of GFAP and SOX-10 further supports the diagnosis. The discovery that PAC are negative for p40,
      • Rooper L.
      • Sharma R.
      • Bishop J.A.
      Polymorphous low grade adenocarcinoma has a consistent p63+/p40− immunophenotype that helps distinguish it from adenoid cystic carcinoma and cellular pleomorphic adenoma.
      whilst retaining expression of p63 (Figure 8a, 8b) has almost revolutionized diagnosis in small biopsies when PA, PAC and AdCC form the principal differential diagnosis, since both PA (and carcinoma ex-PA) and AdCC are consistently p63 and p40-positive (Figure 8c, 8d). Although up to 26% of PA may also be p40-negative, these are tumours with a prominent chondromyxoid matrix. Such PA tend to occur in the major salivary glands, so the effectiveness of this unusual phenotype is less likely to be reduced with PA of the minor salivary glands. The cellular component of occasional PA may be negative for both p63 and p40, but virtually 100% of AdCC are positive for both.
      • Rooper L.
      • Sharma R.
      • Bishop J.A.
      Polymorphous low grade adenocarcinoma has a consistent p63+/p40− immunophenotype that helps distinguish it from adenoid cystic carcinoma and cellular pleomorphic adenoma.
      Most AdCC are also positive for CD117. Proliferation markers can also distinguish between PA, PAC and AdCC. AdCC have a high proliferative index and Ki-67 (Mib-1) uniformly stains more than >10% of cells in this neoplasm. By contrast PA and PAC consistently stain less than 10%. Even clearer is minichromosome maintenance protein-2 (MCM-2), which Vargas and coworkers showed was expressed by 30% of cells in AdCC but in only 5% and 7% respectively in PAC and PA.
      • Vargas P.A.
      • Cheng Y.
      • Barrett A.W.
      • Craig G.T.
      • Speight P.M.
      Expression of Mcm-2, Ki-67 and geminin in benign and malignant salivary gland tumours.
      Immunohistochemistry for the transcription factors PLAG1 (on 8q12) and HMGA (on 12q14-15), which appear to be consistently and specifically expressed by PA, is as yet not widely available.
      Figure 7
      Figure 7A fragmented core biopsy of a deep lobe tumour of the parotid. a) H&E, b) GFAP shows diffuse nuclear positivity in the myxoid component, with c) SOX-10 expression in both this and the tumour islands. The myxoid areas and the immunohistochemistry confirm a diagnosis of pleomorphic adenoma.
      Figure 8
      Figure 8Polymorphous adenocarcinoma shows a characteristic and almost diagnostic phenotype, i.e. a) strong nuclear positivity for p63 and b) uniform negativity for p40. By contrast, c) carcinoma ex-pleomorphic adenoma and d) adenoid cystic carcinoma show strong positive staining for p40.
      In other SGT, the diffuse nuclear expression of androgen receptor by 70% of salivary duct carcinomas helps exclude a breast metastasis, as does lack of expression of oestrogen and progesterone receptors. About 25–30% of salivary duct carcinomas are also positive for HER-2 (ERBB2). The consistent CK7-positive/CK20-negative profile of SGT may help to confirm a salivary origin for the lesion, and also assist in excluding a metastasis. The application of DOG-1, SOX-10 and mammaglobin in the discrimination between SC and AcCC has been discussed above.

       Clear cell tumours

      Figure 9a shows a portion of an extensive tumour on the palate, comprising sheets of clear cells. The differential diagnosis must include clear cell carcinoma of minor salivary gland origin, odontogenic clear cell carcinoma, a metastasis (particularly renal cell carcinoma) and a clear cell variant of MEC. Clear cell carcinoma is much more common intra-orally than in the major salivary glands, and although an odontogenic clear cell carcinoma can only occur within the bones of the jaws it may present as a soft tissue swelling with or without ulceration. In the major salivary glands, the differential diagnosis is more straightforward, comprising principally MEC, a metastasis and the benign candidates - clear cell oncocytoma and oncocytic hyperplasia, all of which may contain sheets of clear cells as the main component. To this list could be added epithelial-myoepithelial carcinoma and other tumours which may contain clear cells such as PA and AcCC, but in these clear cells are rarely predominant.
      Figure 9
      Figure 9a) A palatal tumour shows sheets of monomorphic clear cells. b) Elsewhere in the same tumour there are areas of intermediate cells and cystic structures lined by intermediate and mucous cells. The diagnosis was mucoepidermoid carcinoma with extensive clear cell change.
      Of these options MEC is by far the most likely diagnosis, indeed most studies suggest it is the most common malignant SGT, representing approximately 10% of all SGT and 25% of salivary malignancies. Although most common in the parotid gland, MEC is frequently encountered in the oral cavity in the palate, cheeks, lips, tongue and retromolar region, and is the most common salivary malignancy in children. When a clear cell lesion is encountered in the oral cavity, an MEC should always be considered first, and excluded by careful examination of multiple blocks.
      Histological diagnosis of MEC is based on the identification of an admixture of epidermoid (squamous) and mucous cells, with solid areas containing cytologically bland, so-called “intermediate cells”, and on this basis the tumour in Figure 9a was diagnosed as a MEC since further examination revealed sheets of intermediate cells and cystic structures lined by intermediate and mucous cells (Figure 9b). The proximity of minor mucous salivary glands, “tatty” histological appearance and tumour-associated lymphoid infiltrate are other helpful features, although they are more commonly found in parotid than intra-oral neoplasms. Low grade, extensively cystic MECs comprise over 70% of cases so diagnosis is not often a problem. However, especially in an incisional biopsy, some MEC may appear almost unicystic and resemble a mucous cyst or, if arising in a site such as the retromolar minor glands, a glandular odontogenic cyst if the tumour has infiltrated bone despite low grade histology.
      • Barrett A.W.
      • Abdullakutty A.
      • Norris P.M.
      • et al.
      Molecular diagnostics in the differential diagnosis of glandular odontogenic cyst and mucoepidermoid carcinoma.
      MEC does not have a characteristic immunohistochemical profile, but most are positive for the “glandular” cytokeratins (CK) CK7, CK8, CK18 and CK19, and the characteristic t(11; 19)(q21; p13) translocation and CRTC1-MAML2 gene fusion can also be a useful marker in difficult cases and in small indeterminate biopsies, but does require access to molecular methods. MAML2 alterations cannot be relied on for grading or prediction of outcome, and conventional histological schemes are preferable.
      • Brandwein M.S.
      • Ivanov K.
      • Wallace D.I.
      • et al.
      Mucoepidermoid carcinoma. A clinicopathological study of 80 patients with special reference to histological grading.
      Clear cell carcinoma lacks mucous, epidermoid and/or intermediate cells, and many have a strikingly hyalinized stroma that divides the otherwise solid sheets of clear cells. There is usually diffuse expression of p63, but apart from the characteristic EWSR1 rearrangement the distinction between MEC and clear cell carcinoma is morphological. Differentiating clear cell carcinoma from an odontogenic clear cell carcinoma may therefore be particularly difficult, since both have an EWSR1 rearrangement. However, odontogenic carcinomas may lack expression of CK7, CK8 and CK18, although CK19 is usually positive. Radiology may also help in establishing the intra-bony origin of a central odontogenic neoplasm. Clearly the patient's history will be important in considering whether the clear cell tumour is a metastasis, classically from the kidney, and positive immunohistochemistry for vimentin, PAX-8 and CD10, and negative CK7 staining, will help to confirm the diagnosis.

      Summary and conclusions

      Salivary gland tumours are one of the most difficult areas of diagnostic pathology, with significant morphological diversity and many overlapping features. The problems are compounded by the number of different tumours. Despite efforts to simplify the classification, the latest WHO classification still includes more than 30 entities. There are two new entities – secretory carcinoma (formerly known as mammary analogue secretory carcinoma) and sclerosing polycystic adenosis and several important name changes. The former polymorphous low grade adenocarcinoma has lost its designation as “low grade” and should be called polymorphous adenocarcinoma, which properly reflects its unpredictable behaviour. The WHO still emphasise that classifications are based primarily on histomorphological features, but the morphological diversity and overlapping features of SGT can make diagnosis difficult. In particular pathologists are often faced with small biopsies from the palate, or core biopsies from major glands. “Pattern-matching” is potentially dangerous, inappropriate and can lead to confusion, especially between PA, PAC and AdCC. While attention to detail and to subtle cytological features is essential, immunohistochemistry can be helpful. A range of antibodies are now available that, if used carefully, can be almost diagnostic or can help differentiate between the tumour types.
      • The new WHO classification includes a number of changes, some of which are significant while others are nuanced, but may influence how diagnostic reports are written.
      • Secretory carcinoma (formerly called mammary analogue secretory carcinoma) is a new entity that must be differentiated especially from acinic cell carcinoma. Secretory carcinoma is strongly positive for S100 and mammaglobin and negative for DOG-1 and SOX-10. Conversely, acinic cell carcinoma is positive for DOG-1 and SOX-10, and negative (or weak and patchy) for S100 and mammaglobin. They also have different patterns of PAS staining. Secretory carcinoma does not contain intracytoplasmic PAS-positive granules.
      • The term “low grade” is removed from polymorphous low grade adenocarcinoma.
      • An area of particular diagnostic difficulty is differentiating between pleomorphic adenoma, polymorphous adenocarcinoma and adenoid cystic carcinoma in small biopsies.
      • Attention to morphological detail is important, but immunohistochemistry, especially with antibodies to p63, p40, GFAP, Ki-67, MCM2 and CD117, may be helpful in distinguishing these three tumours.
      • Polymorphous adenocarcinoma shows a characteristic p63-positive/p40-negative immunophenotype.
      • When reporting carcinoma ex-pleomorphic adenoma, the specific tumour type of the malignant component should be named.
      • The definition of widely invasive in carcinoma ex-pleomorphic adenoma remains uncertain but a cut-off of >4–6 mm is suggested.

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