J Clin Pathol 1990;43:905-908

Spherical connective tissue inclusions in epithelialhyperplasia of the breast ("collagenousspherulosis")

C A Wells, C W Wells, P Yeomans, M Vifia, S Jordan, A J d'Ardenne

AbstractPartial myoepithelial differentiation iscommon in simple epithelial hyperplasia(epitheliosis) of the breast but functionalmyoepithelial differentiation withbasement membrane production isexceedingly rare. A peculiar change ofhyaline globules within benign epithelialhyperplasia has been recognised beforeas "collagenous spherulosis" and type IVcollagen has been shown by immuno-histochemistry. Another seven cases aredescribed which show the presence oflaminin and collagens IV and III withinthe proliferation. Electron micro-scopical examination of two cases usingmaterial retrieved from the wax blockshowed varying degrees of myoepithelialdifferentiation of the cells immediatelysurrounding the spherules and basallamina material, including maturecollagen fibrils in one case. The degree ofmyoepithelial differentiation of the cellssurrounding the spherules seemed tocorrelate with the differing types andamounts of extracellular matrix in thespherule.Histopathologists should be aware of

this rare change as it may be misinter-preted as in situ carcinoma.

Department ofHistopathology, StBartholomew'sHospital, LondonC A WellsCW WellsS JordanA J d'ArdenneNuffield Departmentof Pathology, JohnRadcliffe Hospital,OxfordP YeomansM VifiaCorrespondence to:Dr C A WellsNuffield Department ofPathology, John RadcliffeHospital Phase II, HeadleyWay, Headington, OxfordOX3 8DUAccepted for publication2 July 1990

In 1987 Clement, Young, and Azzopardi des-cribed a peculiar change within the lumina ofbreast acini and ductules which they desig-nated "collagenous spherulosis".l Thischange had been noted in referral materialwhere it had occasionally been confused withmalignancy, especially adenoid cystic carcin-oma, and in archival material. The authors ofthis paper were able to show that the hyalinematerial within the intraluminal space wasrich in collagen by conventional histochemis-try, and in a subsequent letter they were ableto show one component of basement mem-brane by immunocytochemistry.2 We presenta further seven cases with immunohisto-chemical results showing the varying propor-tions of basal lamina proteins and interstitialcollagens in the spherules. In two cases thiswas confirmed by ultrastructural examinationof tissue taken from the paraffin wax blocks.

MethodsCases of "collagenous spherulosis" were iden-tified in tissue sections from the files of theJohn Radcliffe Hospital by reviewing 934

biopsy specimens diagnosed as fibrocysticchange or one of its synonyms. One casereferred for opinion to CAW was alsoincluded (courtesy of Dr W Harrison, NorthMiddlesex Hospital).

Blocks were recut, and where sufficientmaterial was available, sections were stainedby the immunoperoxidase technique, withappropriate controls, for laminin (Gibco Ltd),type IV collagen (Euro-diagnostics Ltd), typeI and type III collagen (Bio-Nuclear ServicesLtd), S-100 protein (Dako Ltd), and actin(Amersham Diagnostics Ltd).

Laminin, collagen, types I, III, and IVimmunostaining were performed using theavidin-biotin complex method with 25 min-utes in pronase type 24 (Sigma Ltd) for diges-tion. Actin immunostaining was performed bythe avidin-biotin complex method using 10minutes of trypsin digestion. S100 immuno-staining was performed by the peroxidase-antiperoxidase method using 10 minutes oftrypsin digestion.

In two cases the abnormal area could beidentified from the paraffin wax block and aportion of the lesion was retrieved for electronmicroscopical examination. This area wasexcised from the block, dewaxed in xylene,and rehydrated through graded alcohol into0-IM cacodylate buffer (pH 7 4). This wasthen fixed in 3% gluteraldehyde, post-fixed in1% osmium tetroxide, dehydrated andembedded in TAAB premix resin. Semi-thin(0 5 gum) sections were cut and stained withtoluidine blue. Ultrathin sections (70 nm)were cut, stained with uranyl acetate and leadcitrate, and viewed in a JEOL 100 SX trans-mission electron microscope.

Case follow up was performed as describedpreviously.3

Results"Collagenous spherulosis" was found in six ofthe 934 biopsy specimens reviewed, anincidence of 0 64%, and a further referred casewas studied. The age range of patients fromwhom biopsy specimens had been taken was 26to 47 years (mean 40 4 years). Follow up wasavailable on four, one of whom was registeredas having breast carcinoma with the OxfordCancer Registry in 1974, having had a benignbiopsy showing "collagenous spherulosis",moderate simple hyperplasia (epitheliosis),papillary apocrine metaplasia, and a radial scarin 1965. The carcinoma was of poorly differen-tiated ductal type with lymph node metastases.Three other patients are alive with no evidence

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Figure 1 "Collagenous spherulosis" showing amorphousrounded hyaline material in an intraluminal locationwithin a ductule. Note the occasional epithelial lumina(haematoxylin and eosin).

of breast carcinoma 30, 27, and 23 years aftertheir benign breast biopsy. No follow-up infor-mation is available for the other two patients.

HISTOPATHOLOGY"Collagenous spherulosis" was present as anincidental finding in all cases apart from thereferral case where it was associated with aradial scar and was an integral part of theabnormality. The appearances on haematoxy-lin and eosin staining were as describedpreviously,' and showed discrete hyaline orfibrillar material within a sieve-like intra-acinar or intraductular proliferation (fig 1),accompanied by a dimorphic population ofcells. The cells immediately surrounding thespherules showed some spindling reminiscentof myoepithelial differentiation while cells withpure epithelial appearances, surroundinglumina often containing secretion, were locatedaway from the spherules. The change wasintimately associated with a radial scar in threeof the seven cases, with an area of nodularsclerosing adenosis in one, and in three caseswas found incidently in small ductules or acinarunits not associated with another lesion.Fibrocystic changes were noted elsewhere inthe tissue in all cases.

IMMUNOHISTOCHEMISTRYLaminin staining showed a characteristicappearance in all the lesions in which further

Figure 2 Laminin immunoreactivity showingcharacteristic circumferential positivity and "spider-like"processes extending to a central core (lamininimmunoperoxidase).

Figure 3 Sf00 immunoreactivity showing positive cellssurrounding the amorphous hyaline material and negativecells surrounding true epithelial lumina (S100immunoperoxidase).

sections were available for study (all but onecase). This was present in a linear form aroundthe edge of the spherules similar to that seen inadenoid cystic carcinoma.45 In five of the sixcases there was also a spider-like distributionwithin the spherules themselves (fig 2). TypeIV collagen staining was performed on caseswhere material was still available and thestaining pattern was similar to the laminindistribution, and as described previously.2S100 staining showed that the cellsimmediately surrounding the spherules wereuniformly positive with negative cells surroun-ding occasional epithelial lumina (fig 3). Actinstaining was only possible on the referral casedue to technical problems, but it showed faintstaining of the same cells as in the S100immunostain. Immunostaining for types I andIII collagen showed that occasional spherulescontained small amounts of immunoreactivity.This was most conspicuous in the case withabsent basal lamina proteins in the centre of thespherules.

ELECTRON MICROSCOPYPreservation of the tissue was remarkably goodconsidering that the tissue had been embeddedin paraffin wax for some 30 years, althoughfixation was suboptimal.At low magnification the lesion appeared as a

number of spheres surrounded by proliferatedcells (fig 4). The proliferation was all contained

Figure 4 Low power electron micrograph showing thespherules within a ductule.Insert: high power view of collagen fibresfrom the core ofa spherule showing characteristic striations.

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Collagenous spherulosis of the breast

Figure 5 Myoepithelial cell showing cytoplasmic Figure 7 Spherule from the second case studiedfilaments, dense bodies, and hemidesmosomes attaching demonstrating the lack of a collagen core and radiatingthe cell to the basement membrane surrounding the basement membrane strands.collagen core of a spherule.

within ductules or acinar spaces.In the first case studied, at higher magnifica-

tion, the spherules were shown to consist of amature collagen core (fig 4, insert) surroundedby an intact basement membrane layer (fig 5).

Subtending the basement membrane werecells showing the full spectrum of myoepi-thelial differentiation, with hemidesmosomesattached to the basement membrane andparallel arrays of microfilaments with densebodies (fig 5). One or two true epithelial luminawith microvilli and surrounded by epithelialcells joined by desmosomes and tight junctions(fig 6 and insert) were identified within theproliferation.

In the second case the core of the spherulewas completely devoid of mature collagenfibres and seemed to contain a central basementmembrane core and radiating strands of re-duplicated basement membrane extendingfrom the centre to the peripheral rim. Betweenthe strands the matrix was electron-lucent (fig7). The cells subtending the spherules showedhemidesmosomes, again attached to the base-ment membrane material, but microfilamentsand dense bodies were not generally seen.These appearances mirrored the immuno-

histological findings, the first case studiedbeing the case with no radiating strands on thelaminin immunostaining and with strongpositive immunoreactivity for collagens I andIII. The second case was more typical of thegroup as a whole.

Figure 6 Epithelial lumen with microvilli and mucinsecretion in an area of "collagenous spherulosis". Insert:tight junction at high powerfrom the epithelial cells seenin the main figure.

DiscussionThe finding of laminin and type IV collagen inthe spherules within "collagenous spherulosis"suggests that the spherules contain basementmembrane material, which confirms the find-ings of the previous study.'2 This would sug-gest that the lesion is an extreme example ofmyoepithelial differentiation in epithelialhyperplasia of the breast. Additional supportfor this can be seen in the results with S 100 andactin staining.' Although S100 protein does notmark myoepithelial cells exclusively"6 as wasoriginally thought,7 the combination of weakactin staining with this suggests myoepithelialdifferentiation.6 The electron microscopicappearances also strongly support this.

"Collagenous spherulosis" seems to be alittle heterogeneous in that some cases show amature collagen core to the spherules while inmost cases the spherules contain reduplicatedbasal lamina only, without mature collagen.This suggests that in some examples there is amore "mature" type of spherule with acollagenous centre surrounded by basementmembrane. The electron microscopical resultssugggest that the degree of collagen formationwithin the spherule is related to the degree ofmyoepithelial differentiation of the cellssurrounding the spherule, and suggests thatthis change occurs due to production ofbasement membrane material and, in somecases, mature collagen by the myoepithelialcomponent of the proliferation.

Myoepithelial cell production of interstitialcollagen fibres is often seen in benign salivarytumours such as plemorphic adenomata. Therewere no vessels or fibroblasts within any of thespherules, indicating that these were notpapillary cores.The lesion bears a similarity to adenoid

cystic carcinoma45 in its light and electronmicroscopic appearances and the immunocyto-chemical staining. Both lesions contain twotypes of lumina, one surrounded by cells show-ing epithelial differentiation and containingepithelial mucin, the other surrounded by cellsshowing myoepithelial differentiation and con-taining basement membrane material. Inter-stitial collagen fibres, while numerous in thespherules of the first example of "collagenousspherulosis" at electron microscopy, are rarely

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found in the pseudocysts of adenoid cysticcarcinoma,5 but the electron microscopicappearances of the second case with re-duplicated basal lamina were very similar tothose of adenoid cystic carcinoma.

Interestingly, the lesion often seems to berelated to radial scars, and this was true of theprevious study and our own. Its detection,therefore, may well increase in frequency as alarger number of radial scars are removed as aresult of breast screening programmes.

In common with the previous authors' weemphasise the benign nature of this lesion andalert practising histopathologists to the entityso as to avoid the misdiagnosis of this lesion aseither adenoid cystic carcinoma or cribriformcarcinoma of either in situ8 or invasive types.9This may be a special problem when the lesionis associated with a radial scar which may showpseudo-infiltration. The recognition of the lowpower appearance of intra-acinar proliferationaround a hyaline core with pseudo-infiltrationis crucial to the diagnosis. This contrasts withthe haphazard arrangement of in situ carcin-oma and infiltrating tubules within the baso-philic desmoplastic stroma of a tubular carcin-oma.

Basal lamina immunohistology can be help-ful in these cases, provided it is interpretedwith care.9

We thank Dr W Harrison and Mr J M Beaugie for kindpermission to report one oftheir cases; DrW Gullick for the kindprovision of antibodies; Mr J Hopwood for photographicassistance; Dr R Holloway, Dr B Don, Mrs Jane Orr and MrsAudrey Tutt for help with patient tracing, and the many GeneralPractitioners in Oxfordshire who helped with follow up infor-mation on women in their practices.This work was partially supported by a research grant fromOxford Regional Health Authority (No 88/17).AJd'A is supported, in part, by the Imperial Cancer ResearchFund.

1 Clement PB, Young RH, Azzopardi JG. Collagenousspherulosis of the breast. Am J Surg Pathol 1987;11:411-17.

2 Clement PB. Collagenous spherulosis. Am J Surg Pathol1987;11:907.

3 Vina M, Wells CA. Clear cell metaplasia of the breast: alesion showing eccrine differentiation. Histopathol1989;15:85-92.

4 d'Ardenne AJ, Kirkpatrick P, Wells CA, Davis JD. Lamininand fibronectin in adenoid cystic carcinomas. J ClinPathol 1986;39:138-44.

5 Wells CA, Nicol SM, Ferguson DJP. Adenoid cysticcarcinoma of the breast-a case with proven lymph nodemetastasis. Histopathol 1986;10:415-24.

6 Gillett CE, Bobrow LG, Millis RR. S100 protein in humanmammary tissue-Immunoreactivity in breast carcinomaincluding Paget's disease of the nipple, and value as amarker of myoepithelial cells. J Pathol 1990;160:19-24.

7 Egan MJ, Newman J, Crocker J, Collard M. Immunohisto-chemical localisation of S100 protein in benign andmalignant conditions of the breast. Arch Pathol Lab Med1987;111:28-31.

8 Page DL, Anderson TJ, Rogers LW. Cribriform andmicropapillary carcinoma in-situ. In: Diagnostic histo-pathology of the breast. London: Churchill Livingstone,1987:163-74.

9 Page DL, Anderson TJ, Sukamoto G. Invasive cribriformcarcinoma. In: Diagnostic histopathology of the breast.London: Churchill Livingstone, 1987:227-35.

10 d'Ardenne AJ. Use of basement membrane markers intumour diagnosis. J Clin Pathol 1989;42:449-57.

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