Epithelial-myoepithelial carcinoma of salivaryglands

R H W Simpson, T J Clarke, P T L Sarsfield, P G C Gluckman

AbstractFour cases of epithelial-myoepithelialcarcinoma of the salivary glands arose as

painless masses in patients over 60 years

old, three in the parotid and one in thesubmandibular gland. Histologically, allthe tumours were composed of smallducts with a- double cell liningsurrounded by a basement membrane.The inner cells were epithelial and theouter cells myoepithelial, the latterusually possessing clear cytoplasm.There was a variable degree ofintervening hyalinised stroma. All thetumours were partly encapsulated, butalso displayed local invasiveness. One ofthe tumours also showed areas ofdedifferentiation when it later recurredand metastasised. The other three were

apparently cured by initial excision, withadjuvant radiotherapy in one instance.In the past this tumour has beendescribed as clear cell adenoma, and itwas only recently that its true malignantnature, albeit low grade, was recognised.Reports of epithelial-myoepithelial

carcinoma are still relatively few, withonly one case described from Britain. Itis recommended that this histologicallydistinct neoplasm deserves widerrecognition.

PostgraduateMedical School,University of Exeter,Barrack Road, Exeter,Devon EX2 5DWR H W SimpsonArea Department ofPathology, ExeterT J ClarkeP T L SarsfieldDepartment of Ear,Nose and ThroatSurgery, Royal Devonand Exeter HospitalP G C GluckmanCorrespondence to:Dr R H W SimpsonAccepted for publication31 October 1990

The 1972 WHO classification of salivary glandtumours' lists adenocarcinoma, but does notattempt further subdivision. It has sincebecome increasingly clear, however, that thereis considerable variation within this group,both in histological appearance and in clinicalbehaviour. One distinctive subtype was firstdescribed as epithelial-myoepithelial carcin-oma (Epithelial-myoepitheliales Schaltstfick-carcinom) by Donath in 1972,2 althoughseveral similar tumours had previously beendocumented under other names.'3 Thisneoplasm was illustrated in the WHOclassification' and the AFIP Tumor Fascicle'as clear cell monomorphic adenoma, but thiswas before its truly malignant nature had beenrealised. By 1987, a review of publishedfindings had identified 35 cases,8 and a 1989study from Germany and Sweden added a

further 21.9 The entity was not specificallyreferred to either in a 1976 Scottish survey of643 salivary tumours,'0 or among the 2410cases of the British Salivary Gland TumourPanel reported in 1985." We have found onlyone case from the United Kingdom described

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as epithelial-myoepithelial carcinoma,12although the parotid clear cell adenomareported by Corridan in 1956' was almostcertainly another.The purpose of this study was to present

four new cases of epithelial-myoepithelialcarcinoma and to document the clinical,histopathological, and immunohistochemicalfindings, and thus permit wider rccognition ofthis distinctive tumour.

MethodsThe Area Department of Pathology in Exeterserves a population of about 300 000 in EastDevon. The surgical pathology archives con-

tained 212 parotid tumours between 1978 andJune 1990, of which three were classified on

review as epithelial-myoepithelial carcinoma.One was found among 25 submandibular neo-

plasms, but none among 43 minor salivarygland tumours. Paraffin wax embedded blockswere available from all four cases. Sectionswere prepared in the conventional manner andstained with haematoxylin and eosin, periodicacid Schiff (PAS) (before and after diastasedigestion) alcian blue at pH 2-5, Masson'strichrome, Weigert's stain for elastin with a van

Giesen counterstain, and Gordon and Sweet'sreticulin. A panel of commercially availableantibodies (table 1) was applied to sectionsusing the streptavidin-biotin technique.Appropriate positive and negative controlswere used. Immunoperoxidase staining was

assessed semiquantitatively, with 0 as

negative, ± as equivocal, and +, + +, and+ + + indicating different intensities ofpositive reaction. "F" indicated that thepattern was focal. Where mitotic counts are

given, the area ofa high power field is 0 16 mm2.

ResultsCLINICAL FINDINGSThese are summarised in table 2. All thepatients were British and white, and there wereno common factors in their medical or

occupational histories. All presented with a

painless salivary gland mass. None hadenlarged lymph nodes at presentation, andthere was no evidence ofnerve symptoms. Theoverlying skin appeared normal in each case.

General clinical and radiological examinationexcluded the possibility that any was a

metastasis. Surgical excision was consideredcomplete in each patient, and case 1 received a

course of postoperative radiotherapy. One ofthe patients (case 3) developed a local

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recurrence 18 months later, which wascompletely excised. At the same time she wasnoted to have an enlarged supraclavicularlymph node, which was not biopsied. Twopatients (cases 1 and 2) died of presumedcerebrovascular disease, and case 4 was lost atsea in a boating accident. None had any clinicalevidence of persistent tumour. Post mortemexaminations were not performed.

PATHOLOGICAL FINDINGSMacroscopically, all the tumours were wellcircumscribed masses with a grey cut surface.Haemorrhagic areas were noted in case 3.

Microscopically, each tumour was composedof small ducts, some of which contained smallquantities of intraluminal inspissated mucin.The lining consisted of two layers: the innercomprised small cuboidal or low columnar cellswith generally eosinophilic cytoplasm, and wassurrounded by an outer mantle of cells, almostall of which had clear cytoplasm. The nuclei inboth cell types were single with evenlydispersed chromatin, and rare almost in-conspicuous nucleoli. Glycogen could beshown in both layers, especially the outer, butno intracellular mucin was seen. The outerlayer was surrounded by prominent, PASpositive basement membrane material, and theintervening stroma were composed of almostacellular collagen, with neither elastin noralcian blue positive mucin.The relative prominence of the stroma and

both cell types varied not only from case tocase, but also within each individual tumour.Three basic morphological patterns could berecognised, as well as transitional areas. In thefirst, or classic pattern (fig 1) the two layerslining the ducts were easily discernable, as were

the surrounding basement membranes. In thesecond, or clear cell predominant pattern (fig 2)

sheets of cells resembling the outer layer of theclassic pattern were divided into alveolar struc-tures by thin strands of stroma. An inner layerof cells was present, but was often difficult toidentify. Zones with this pattern were observedin all four tumours, and were particularlyprominent in case 1. The third, or scleroticpattern (figs 3 and 4) consisted of abundanthyalinised stroma separating relatively sparse

double-layered ducts. There was no myxoid orchondroid change, as seen in a pleomorphicadenoma: Small cyst formation (cases 3 and 4),scanty microcalcification (cases 2 and 4), andfocal necrosis (case 4) wereuncommon findings.Although the tumours were well circum-

scribed and partly encapsulated, each showedinvasion into surrounding tissue includingblood vessels and nerves (fig 5). There was no

pleomorphism, and mitotic figures were rare

(fewer than 3 per 20 high power fields). Anexception was the recurrent tumour in case 3which showed areas of dedifferentiation withpleomorphic spindle and polygonal clear cells(fig 6), prominent haemorrhage, and necrosisand numerous mitotic figures (36 per 20 highpower fields).

IMMUNOHISTOCHEMISTRYThe findings are summarised in table 1. In allcases there was a noticeable difference in thereactions of the two ductal cell layers: the innercells showed luminal staining with HMFG 1

and 2 antibody, and cytoplasmic positivity withlow molecular weight cytokeratins (AE1 andCAM 5.2); the outer layer stained almostexclusively with S100 protein (fig 7) indicatingmyoepithelial differentiation. Myosin (ICN,dilution 1 in 10) was applied to cases 3 and 4and was completely negative in each case. Thisbiphasic staining reaction was best seen in theclassic pattern, but was preserved even in the

Table 1 Immunohistochemicalfindings

Case I Case 2 Case 3 Case 4

Inner Outer Inner Outer Inner Outer Inner OuterMarker Source Dilution layer layer layer layer layer layer layer layer

AEI ICN 1:200 + + + F+ ++ +0++ 0 + ++ 0AE3 ICN 1:800 + 0 0 0 0 0 F + 0CAM 5 2 Becton-Dickinson 1:8 + + + + F+ 0 + + + 0 + 0Keratin Dako 1:1500 + + + + +± 0 + O + +0CHMFG 1 & 2 Oxoid 1:600 + ++ 0 F+ 0 + ++ 0 + + OCEA Dako 1:400 + + F+ 0 0 + + + +S100 Dako 1:1500 F+ + + + + + + + F+ + +0+ + +Vimentin Dako 1:15 F+ + + 0 F+ + + 0 F+Desmin Dako 1:50 C C C O C C C C

Table 2 Clinicalfindings

Age atCase No Sex diagnosis Clinical presentation Size (cm) Initial treatment Outcome

1 M 62 Left parotid swelling many years; 5 x 4 x 3 5 Superficial parotidectomy DOC 8 yr 7 m NEDrecent growth over 2 months;at operation firm, lobulated tumoursuperficial lobe

2 F 69 Left parotid nodule 6 weeks 3-5 x 3 x 2 Total parotidectomy DOC4m NED3 F 103 Left submandibular mass I year; 6 x 5 x 3 Excision Recurrence 1 yr 6 m;

rapid growth 2 months supra-clavicular lymph node;alive 2 yr 6 m

4 M 71 Left parotid mass 10 years; 3-5 x 25 Superficial parotidectomy DOC 2 yrNEDrecent growth 3 months

DOC: died of other causes NED: no evidence of disease

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Salivary epithelial-myoepithelial carcinoma

:; t''-,'Figure I Case 4: classic pattern illustrating ducts lined Figure 4 Case 2: transition between clear cellby inner low columnar cells and an outer mantle of clear predominant pattern on the left and sclerotic pattern oncells surrounded by a basement membrane (PAS the right, with thickened basement membranes (PASdiastase). diastase).

Figure 2 Case 1: clear cell predominant pattern with Figure 5 Case 4: invasion of a nerve by tumour. Theclear cell-filled alveolar structures, some of which contain outer cell layer also shows positive staining (SIOOsmall lumina (haematoxylin and eosin). protein).

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Figure 3 Case 4: sclerotic area, showing relativelysparse double lined ducts separated by hyalinised stroma(haematoxylin and eosin).

clear cell predominant areas (fig 8), where theinner ductal cells were often difficult to identify.The dedifferentiated areas in the recurrenttumour in case 3 showed a patchy reaction withcytokeratins, HMFG 1 and 2 and CEA, butnegative staining for S100 protein and all othermarkers.

Figure 6 Case 3: recurrence showing a solid mass of cellswith increased pleomorphism and mitotic activity(haematoxylin and eosin).

DiscussionEpithelial-myoepithelial carcinoma is a raretumour accounting for slightly fewer than 1%of salivary gland neoplasms.9 Our four casesamong 280 salivary tumours (1 -4%) indicated abroadly similar relative incidence.

Previous studies have shown that it is some-

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what more prevalent in women, and that themean age at presentation is about 60 years, witha range of 27 to 103, the latter being our case 3.Most tumours arise in the major salivaryglands, especially the parotid, but cases havebeen described in the minor glands of themouth, and even in the maxillary sinus.8 Thehistory is typically that of a mass enlarging over

several months or even years. Pain and facialnerve palsy have been reported on occasion,'3but were not noted in our cases.

The tumours are solitary and range in sizefrom 2 to 8 cm maximal diameter. The micro-scopic appearances, as in our cases, encompassclassic, sclerotic, and clear cell predominantpatterns, and any particular tumour may showone or more of these zones. A constant featureis the double layer ductal lining of inner smallepithelial cells and outer clear myoepithelialcells. Immunohistochemistry can help toidentify the two layers when they are

morphologically indistinct. We found AE1 andS100 protein to be the best combination ofmarkers for this. Electron microscopicalexamination has confirmed the inner epithelialand outer myoepithelial differentiation.'4 15

Although the tumours are locally invasive, theydo not exhibit nuclear pleomorphism or a highmitotic rate, an exception being the recurrenttumour in our case 3 which resembled a

malignant spindle cell myoepithelioma.'6The morphological features of epithelial-

myoepithelial carcinoma are generally quitedistinct from other salivary tumours. Mono-

morphic or pleomorphic adenomas do notinvade locally, and the latter often containabundant alcian blue positive stromal mucin.Salivary duct adenocarcinoma8 is aggressiveand resembles ductal carcinoma of the breast.Terminal duct carcinoma8 (or polymorphouslow grade adenocarcinoma) is a locally invasivetumour arising almost always in minor salivaryglands, and is characterised by cytologicaluniformity and histological diversity; single-lined ducts often with intraluminal papillae aretypical, but a biphasic ductal lining is not afeature.The clear cell predominant pattern of

epithelial-myoepithelial carcinoma, however,can be confused with other clear cell tumours.Monomorphic clear cell carcinoma is found inminor salivary glands and lacks S100 positivemyoepithelial differentiation.'7 The clear cellsof mucoepidermoid carcinoma contain neutralepithelial mucin (PAS positive), and squamousdifferentiation is also, by definition, a feature ofthese tumours. Sebaceous carcinoma is com-posed of "foamy," lipid-rich clear cells, andsometimes intracytoplasmic mucin." Asalivary gland oncocytoma may be composedlargely of clear cells, although scattered typicaloncocytes are usually present to betray its truenature. The clear cells often have faintly gran-ular cytoplasm with some diastase resistantPAS positive granules, and numerous mito-chondria are seen on electron microscopicalexamination.'9 The clear cells in some aciniccell carcinomas are generally considered torepresent a fixation artefact, and it is notthought that a true clear cell variant exists202' asa careful search will show foci with the charac-teristic secretory granules of normal acinarcells. A parotid mass may be the first manifesta-tion of metastatic carcinoma, particularly fromthe kidney, and this can also occur up to nineyears after nephrectomy.22 The immuno-histochemical reactions of renal carcinoma arevariable, but they will not show the biphasicpattern of epithelial-myoepithelial carcinoma.Nevertheless, exclusion of this possibility insome instances may still require the use ofimaging techniques such as abdominal ultra-sound examination and computed tomographyscanning.The histogenesis of epithelial-myoepithelial

carcinoma is uncertain. It has been suggestedthat there is bidirectional differentiation from astem cell to form myoepithelial and inter-calated ductal epithelial cells.'5 Our observa-tions highlight the constant and intimateassociation of these two cell types, but do notfurther elucidate the histogenesis.Although originally thought to be benign, it

is now apparent that epithelial-myoepithelialcarcinoma is a genuine low grade malignancy.In a review of 35 cases8 37% had recurrencesand 17% lymph node metastases. Threepatients developed distant metastatic spreadand died nine months, 10 years, and 27 yearsafter initial diagnosis.

Information on the effectiveness of differenttreatments is scanty. In one series of ninepatients" seven were initially treated bysurgical excision alone and five later developed

Figure 7 Case 3: mainlybasal positive staining ofthe outer cell layer (S100protein).

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one or more recurrence. In contrast, bothpatients treated with surgical excisionplus immediate postoperative radiotherapyremained tumour free after nine years. In thepresent study all four patients were treatedwith surgical excision, including the one wholater had a recurrence. Of the other three, onlyone had received a course of radiation shortlyafter surgery.

The prognostic value of other factors is alsonot established. The histological differentiation(cellular anaplasia and mitotic activity) is saidnot to matter,9 but tumour size may. In one

series9 the recurrence rate was higher intumours larger than 4 cm. In another'5 none ofthe tumours smaller than 3 cm in maximumdiameter recurred, while all the larger ones did.Among our cases the recurrence happened inthe largest tumour, but in case 1, a carcinoma of5 x 4 x 3 5 cm failed to recur in over eight anda half years (initial treatment included radio-therapy).

In conclusion, epithelial-myoepithelialcarcinoma of salivary glands is a low grademalignancy with a distinct histologicalappearance. It deserves wider recognition.

We are most grateful to Messrs P Beasley, A J Knox, andT T Irvin, and Drs A H Ridler, M J W Slot, and J A G Bensonfor additional clinical information on their patients. We alsothank all the technical staffof the Histopathology Department inExeter, especially Brian Moores, Farid Moshtael, and DerekBoustred. We are also most grateful to Louise Parkhouse forsecretarial help.

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