Clin Pathol 1995;48:164-167

Malignant myoepithelioma (myoepithelialcarcinoma) of the breast:. a detailed cytokeratinstudy

S R Lakhani, M J O'Hare, P Monaghan, J Winehouse, J-C Gazet, J P Sloane

AbstractAims-To study the expression of a range

of cytokeratins by malignant myo-

epithelioma of the breast.Methods-Immunophenotyping was car-

ried out using a panel of antibodies on

paraffin wax embedded and frozenmaterial using immunohistochemistryand double-labelled immunofluorescence.Electron microscopy was also performed.Results-The tumour cells were positivefor CAM 5 2, actin, vimentin, and cyto-keratin 14 and negative for cytokeratins18 and 19. Electron microscopy showedwell formed desmosomes and hemi-desmosomes together with pinocytic ves-icles, plentiful rough endoplasmic re-

ticulum and 6 nM microfilaments withfocal densities.Conclusions-The pattern of cytokeratinexpression provides further evidence thattumours with a specific myoepithelialphenotype occur rarely in the breast.(J7 Clin Pathol 1995;48:164-167)

Keywords: Malignant myoepithelioma, cytokeratin,breast.

Most breast neoplasms have epithelial cell char-acteristics but a few are composed partly or

purely of cells exhibiting myoepithelial differ-entiation. Tavassoli2 recognised three maintypes of myoepithelial proliferations in thebreast: (i) myoepitheliosis, (ii) adeno-myoepithelioma with a potential for local

Department ofHistopathology,The Royal MarsdenHospital,Down's Road, Sutton,Surrey SM2 5PTS R LakhaniJ P Sloane

Department ofSurgeryJ WinehouseJ-C Gazet

Institute of CancerResearchM J O'HareP Monaghan

Correspondence to:Dr S R Lakhani.

Accepted for publication1 July 1994

recurrence but not metastasis, and (iii) myo-

epithelial carcinoma. Most of the cases, as inother series, were classified as adeno-myoepitheliomas, which are generally of lowgrade malignancy. Myoepithelial carcinomas,however, usually give rise to metastases,34 al-though one purely intraductal case has beendescribed.5 These lesions are extremely rare

spindle cell tumours requiring a combinationofmorphology, electron microscopy and/or im-munohistological investigations to distinguishthem from sarcomas and spindle cell car-

cinomas. The antibodies most commonly usedare CAM 5-2, epithelial membrane antigen(EMA), smooth muscle actin, S100 protein,and glial fibrillary acidic protein (GFAP).These antibodies are not specific for my-

oepithelial cells but in combination, positivefindings indicate myoepithelial differentiationwith a reasonable degree of confidence.At least 20 different keratins have been dis-

tinguished in human epithelia,6 with differentcombinations in different cell types. The myo-

epithelial cells of the breast express keratins 5,14, and 17 and the luminal cells keratins 7, 8,18, and 19.7 8Although staining for keratins 14and 17 has been observed in normal luminalcells and some carcinomas,8 it is generally focaland weak compared with the uniform stainingobserved in myoepithelial cells. The difficultywith detailed keratin phenotyping is the re-

quirement for fresh tissue as many of the anti-bodies do not bind to formalin fixed, paraffinwax embedded sections. In this study we haveundertaken immunophenotyping with a panel

Antibodies used and reactivity with tumour samples

Antibodies Source Antigen Reactivity with tumour*

Immunohistochemistry**CAM 5-2 Becton-Dickinson CK8,18,19 + +ICR-2 Dr C Dean, ICR Epithelial membrane antigen +Anti-smooth muscle actin (1A4) Sigma a-Actin + + +Anti-vimentin (MV1) Dako Vimentin + + +Anti-SI00 Dako S100 protein +Anti-desmin EuroDiagnostics Desmin

Double immunofluorescencetVimentin/LP34 Labsystems/EBL Vimentin/CK5,8,18 + + + + +LE61/LL002 EBIJEBL CK18/CK14 -+ +LP2K/RCK107 EBL/FR CK19/CK14 -+ +LL002/RCK107 EBL/FR CK14/CK14 ++/++1A4/LL002 Sigma/EBL oa-Actin/CK14 ++ +/++LL002/MV1 EBLtDako CK14/Vimentin + +/+ + +MV1/lA4 Dako/Sigma Vimentin/ot-actin + + +/+ +RCK105/IC7 FR/FR CK7/CK13 /

Key to scoring system: + + +, >75% tumour cells positive; + +, 25-75% tumour cells positive; +, <25% tumour cells positive;-, all tumour cells negative.* In all combinations where there was positivity for both antigens, which reflected extensive co-expression by the tumour cellsand not separate cell populations (see fig 2).** Undertaken on formalin fixed, paraffin wax embedded sections.tUndertaken on acetone fixed, frozen sections; in all combinations the primary antibodies were of different IgG subclasses,permitting specific visualisation by subclass specific RITC or FITC conjugated secondary antibodies.EBL, E B Lane; FR, F Ramaekers.Source: Becton-Dickinson, Oxford, UK; Sigma, Poole, Dorset, UK; EuroDiagnostics, Stratton, Bude, Cornwall, UK; Labsystems,Basingstoke, UK; Dako, High Wycombe, UK.

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Malignant myoepithelioma of the breast

of monospecific and polyspecific antikeratinantibodies to determine whether a tumourdiagnosed as myoepithelial carcinoma bymorphology, immunohistology, and electronmicroscopy exhibited a keratin profile con-sistent with myoepithelial differentiation.

Figure 1 Low power view of a haematoxylin and eosinstained section of the lesion from the axilla showing aspindle cell tumour with only mild pleomorphism andoccasional mitotic figures.

04

Figure 2 Double indirect immunofluorescence staining of tumour frozen sections showingantigens visualised with RITC on the left and FITC on the right in the same field. A antB show staining for CK14 and CK18, respectively; C and D CK14 together with smoothmuscle actin; E and F CK14 and vimentin; and G and H smooth muscle actin andvimentin staining. Tumour fascicles staining for CK14 (but not CK18), smooth muscleactin and vimentin can be seen, interspersed with cytokeratin negativelvimentin positivecells (F and G) that probably represent normal stromal cells (original magnificationx 270).

MethodsCLINICAL DATAIn April 1987 a 67 year old woman presentedwith a mass located in the upper outer quadrantof the right breast. Wide local excision andlevel I axillary dissection were performed. Shepresented again in June 1991 with another massin the lower inner quadrant of the same breast.Macroscopic examination showed a cyst-likestructure with a smooth grey lining. Staginginvestigations were again negative and a widelocal excision was carried out. In March 1993a mass in the ipsilateral axilla was excised. Alimited skeletal survey at this time revealedmetastatic disease in the skull, ribs, lumbarspine, and sternum. She received tamoxifen(20 mg mane) but with no discernible clinicaleffect. She developed hypercalcaemia and diedin June 1993.At necropsy, there was residual tumour in

the right breast and metastases in the left breast,lymph nodes, bones, myocardium, peri-cardium, kidneys, adrenal glands, liver, spleen,and lungs.Immunohistochemistry was carried out on

paraffin wax embedded tissue from all threesurgical specimens using the alkaline phos-phatase-antialkaline phosphatase method with

« Fast Red TR as the substrate. The antibodiesused were as shown in the table. Frozen tissuewas available only from the mass in the axilla;

i acetone fixed frozen sections from this speci-men were stained with a combination ofmono-specific (except LP34) antikeratin monoclonalantibodies and visualised using narrow-bandpass green and red filters and subclass specificsecond antibody fluorochrome (RITC andFITC) conjugates (Southern Biotechnology viaEuropath, Bude, UK).For ultrastructural analysis, formalin fixed

tissue obtained from the axillary mass was used.It was post-fixed in 1% (w/v) osmium tetroxidein 0 1 M phosphate buffer for two hours, de-hydrated in ethanol and embedded in Epon/Araldite. Representative areas of the tumour

, were selected from toluidine blue stained 1 gimsections. Ultrathin sections were cut with adiamond knife on a Leica (Milton Keynes, UK)OMU4 ultramicrotome, collected on copper

; grids, contrasted with uranyl acetate and leadcitrate (Leica Ultrostainer), and viewed in a

i Philips CM1O at 80 Kv.

ResultsHISTOLOGICAL FINDINGS

d All surgical specimens, two from the breast andone from the axilla, showed similar features.The mass from the axilla consisted ofmetastaticdeposits in lymph nodes. The tumour wascomposed of uniform spindle cells with only amild degree of nuclear pleomorphism (fig 1)

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Lakhani, O'Hare, Monaghan, Winehouse, Gazet, Sloane

and exhibited moderate numbers of mitoses(five per 10 high power fields). In some areasthe cells were plump and epithelioid, and ap-peared to line cleft-like spaces. Foci of necrosiswere also seen. The original specimen from1987 showed some glandular structures butthey were present only focally, did not exhibitcytological or structural atypia, and did notshow any evidence oftransition with the spindlecell component. They were thus interpreted asentrapped rather than neoplastic structures.There was no evidence of glandular differ-entiation or in situ tumour in any of the speci-mens. Sections of all the organs involved atnecropsy revealed tumour of identical ap-pearance to that seen in the surgical specimens.

IMMUNOHISTOCHEMISTRY/IMMUNOFLUORESCENCEImmunohistology using conventional markersshowed that the tumour cells were CAM 5-2,actin and vimentin positive. The immuno-histochemical profile ofthe tumour is presentedin the table. Staining with a polyspecific anti-cytokeratin antibody (LP34) showed that atleast 75% of the cells in the lesion were keratinpositive. All the cells that were keratin positivewere also vimentin positive when frozen tumour

sections were simultaneously stained withLP34 and an antibody to vimentin.Three staining patterns were seen using

double-labelling immunofluorescence: (i) ap-proximately two thirds of the tumour cells,organised largely in discrete fascicles, werecytokeratin (CK) 14, smooth muscle actin andvimentin positive (figs 2A, 2D, and 2F). Thesecells were also CK18 and CK19 negative. (ii)The areas with pattern A merged at their edgeswith less obviously fascicular areas of smoothmuscle actin and vimentin positive but CK14negative cells. These cells accounted for aboutone quarter of the tumour cells (fig 2C). (iii)The remainder of the cells were vimentin pos-itive and smooth muscle actin and CK14 neg-ative. Some of these were interspersed betweenthe cytokeratin positive cell bundles and werealmost certainly stromal fibroblasts (figs 2Eand 2F). Others appeared to be contiguouswith cells showing pattern B and could havebeen either fibroblasts or tumour cells that hadlost both actin and CK14 expression. Theyrepresented the least numerically significantcomponent. Hence, over half the cells in thismetastatic lesion showed a specific myo-epithelial phenotype with concurrent CK14,smooth muscle actin and vimentin positivestaining and CK1 8 and CK1 9 negative stainingin the same cells.

ELECTRON MICROSCOPYThe tumour cells were spindle shaped (fig 3A)and joined by well formed desmosomes. Thecytoplasm showed plentiful rough endoplasmicreticulum and zones of 6 nM microfilamentswith focal densities near the plasma membrane.Keratin filaments were present in many cells(fig 3B). Basement membrane-like material wasassociated with occasional tumour cells whichexhibited focal membrane thickening and oc-casional hemidesmosomes, sometimes in as-sociation with pinocytotic vesicles (fig 3C).

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Figure 3 A: Low power electron micrograph of spindle shaped tumour cells (originalmagnification x 5200). B: Electron micrograph showing two tumour cells with peripheralareas of myofilaments with focal densities (arrows). The tumour cells are joined by a

desmosome. Keratin filaments are present in the cell cytoplasm (original magnificationx 19 600). C: High power electron micrograph of the membrane of a tumour cell showinghemidesmosomes and numerous pinocytotic vesicles (arrows). The cell cytoplasm alsocontains myofilaments (F) (original magnification x 48 000).

DiscussionMyoepithelial carcinomas are difficult to diag-nose. Unlike adenomyoepitheliomas which ex-hibit distinctive combined patterns of epithelialand myoepithelial differentiation, they are com-posed purely of spindle cells and consequentlyvirtually impossible to differentiate fromspindle cell carcinomas and sarcomas on mor-phological examination. It is essential to beable to recognise myoepithelial carcinomas inorder to learn more about their natural historyand response to treatment. It was interestingto note that tamoxifen had no effect on thetumour in this patient.To date, immunohistochemistry has been

limited to antibodies against smooth muscleactin, S100 protein and GFAP. However, noneof these markers on their own are entirelyspecific for myoepithelial cells or tumours de-rived from them. Actin may be expressed ontumours exhibiting myofibroblastic or smoothmuscle differentiation, and GFAP and S100on tumours of nerve sheath origin. Combinedstaining with these antibodies and epithelial

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markers, however, provides adequate evidenceof myoepithelial differentiation for routinediagnostic purposes. Electron microscopy mayprovide further evidence of myoepithelialdifferentiation.

Detailed keratin profiles have not previouslybeen reported presumably because of the needfor frozen material, the infrequency with whichthe relevant antibodies are used in routine diag-nostic practice and the complexity of the stain-ing procedures. In the present case half of thetumour cells in the lesion showed a very specificmyoepithelial phenotype-that is, coexpressionof CK14, smooth muscle actin and vimentinin the same cells, which were negative for CK7,13, 18, and 19. These findings provide furtherdefinitive evidence of the truly myoepithelialnature of a small proportion of malignant tu-mours in the breast.

We are grateful to Professors E B Lane and F Ramaekers forthe gift of anticytokeratin antibodies.

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