Pathology International

2006;

56

: 211–216 doi:10.1111/j.1440-1827.2006.01948.x

Blackwell Publishing AsiaMelbourne, AustraliaPINPathology International1320-54632006 Japanese Society of PathologyApril 2006564211216Case Report

Myoepithelial carcinoma of breastB. Han

et al

.

Correspondence: Bo Han, MD, Department of Pathology, WakayamaMedical University, Kimiidera 811-1, Wakayama 641-8509, Japan.Email: hanbo@wakayama-med.ac.jp

Received 3 August 2005. Accepted for publication 11 December2005.

Case Report

Myoepithelial carcinoma arising in an adenomyoepithelioma of the breast: Case report with immunohistochemical and mutational analysis

Bo Han, Ichiro Mori, Misa Nakamura, Xiaojuan Wang, Takashi Ozaki, Yasushi Nakamura and Kennichi Kakudo

Department of Pathology, Wakayama Medical University, Kimiidera, Wakayama, Japan

Adenomyoepithelioma (AME) of the breast is an uncommontumor characterized by biphasic proliferation of both epi-thelial and myoepithelial cells. In rare instances, the epithe-lial, the myoepithelial or both components of an AME maybecome malignant. Described herein is the case of a 69-year-old woman who presented with myoepithelial carci-noma of the breast in an AME. Malignancy of myoepithelialcomponent (MEC) was evidenced by the presence of cyto-logical atypia, high mitotic rate, necrosis and local invasion.Immunohistochemical study demonstrated strong expres-sion of P53 and phosphorylated extracellular signal-regulated kinase 1/2 in MEC. Laser capture microdissectiontechnique and mutational analysis further revealed pointmutation of the

p53

gene (T

Æ

G transversion at codon 270)in this population, but not in glandular epithelial cells oradjacent normal ductal epithelium. No mutations in exons1 and 2 of the K-, H-, and N-

ras

genes were identified in anyof the neoplastic component. To the authors’ knowledgethis is the first report of a mutation in the

p53

gene in amalignant AME of the breast.

Key words:

adenomyoepithelioma, breast, mutation, myoepi-thelial carcinoma,

p53

Adenomyoepithelioma (AME) of the breast is an uncommontumor characterized by biphasic proliferation of both epithelialand myoepithelial cells. It was first recognized by Hamperl in1970,

1

and approximately 140 cases of AME of the breasthave been reported in English-language literature.

2–24

Theage of the patients described ranges from 24 to 86 years,with a mean of 57 years. All cases except two occurred inwomen.

Although most of the AME described have been consid-ered to be benign, they can progress to a malignant stateand give rise to metastases in rare instances. Malignanttransformation may involve epithelial, myoepithelial or bothcellular elements.

12

According to the most recent WorldHealth Organization (WHO) classification, malignant AME ofthe breast includes epithelial carcinoma, myoepithelialcarcinoma, biphasic epithelial and myoepithelial carcinoma,sarcoma and carcinosarcoma (Table 1).

25

Because of rarityof malignant AME, the majority of previously reported caseshave been in the form of isolated case reports or small seriesdescribing mainly the clinicopathological features. To the bestof our knowledge, there has been no report analyzing molec-ular genetic changes or aberrantly active signaling pathwaysof malignant AME to date. We herein present histological andimmunohistochemical findings of a rare case of myoepithelialcarcinoma of the breast in an AME. We also report the resultsof our investigation of the

p53

gene mutation on this tumorwith a laser capture microdissection technique.

CLINICAL SUMMARY

A 69-year-old woman presented to Kokuho Susami Hospitalof Wakayama in May 2004 with a palpable mass in the upper,outer region of her right breast. There was no family historyof breast disease. Physical examination revealed a2.5

×

2 cm, movable and painless mass with firm consis-tency. Neither the axillary nor supraclavicular lymph nodeswere palpable. Mammography showed relatively well-circumscribed tumor with focally ill-defined border. No micro-calcification was identified in the tumor mass. The tumor wastreated by complete local excision and there was no evidenceof metastatic involvement of five dissected lymph nodes.The patient’s postoperative course was uneventful, and norecurrence or metastasis was identified after 9 months offollow up.

212 B. Han

et al

.

MATERIALS AND METHODS

Immunohistochemical analysis

Four

µ

m-thick, 10% buffered formalin-fixed and paraffin-embedded specimens from the breast tumor were used forimmunohistochemical analysis. Immunohistochemical stain-ing was performed using the standardized labeled streptavi-din biotin (LSAB) kit (DakoCytomation, Carpinteria, CA,USA) according to the manufacturer’s instructions. For visu-alization, 3,3-diaminobenzidine tetrahydrochloride was usedas chromogen and methyl green was used as a counterstain.The primary antibodies and pretreatment procedures usedfor antigen retrieval are listed in Table 2.

Microdissection and DNA extraction

Laser capture microdissection (LCM) procedures have beendescribed previously.

26

Briefly, six serial 10

µ

m-thick histolog-ical sections were cut and deparaffinized. After HE staining,the myoepithelial component (MEC) and glandular epithelialcomponent (GEC; 5–7 foci in each element, approximately100–2000 cells), were carefully microdissected, respectively,using the AS-LCM system (Leica Microsystems, Germany).Before microdissection, MEC and GEC were confirmed bycomparing them with the serial sections stained with S-100protein and epithelial membrane antigen (EMA). Adjacentnormal ductal epithelium was also dissected (

>

5 mm next tothe neoplastic area) for DNA extraction. Microdissected tis-sues were digested overnight at 56

°

C in 50

µ

L proteinase Ksolution.

26

For inactivation of proteinase K, the samples werecentrifuged and incubated at 95

°

C for 10 min. Aliquot of thelysates were stored at

−

20

°

C until used for polymerase chainreaction (PCR).

Mutational analysis

PCR amplification was performed in a total volume of 50

µ

Lof reaction mixture including 5

µ

L lysates in a thermal cycler(Gene Amp PCR System 9700, Perkin-Elmer, Norwalk, CT,USA) for 40 cycles. The primers for exons 4–9 of

p53

andexons 1–2 of the K-, H-, and N-

ras

genes for PCR weredescribed previously.

27,28

Table 2

Immunohistochemical panel results

Antibodies to Source Clone Pretreatment Dilution GEC MEC

α

-SMA Sigma, Saint Louis, MO, USA 1A4 None 1:400 –

+

Bcl-2 Novocastra, Newcastle upon Tyne, UK bcl-2/100/D5 Microwave 1:80 – –CK 7 and 8 Becton Dickinson, Mountain View, CA, USA CAM5.2 Trypsin 1:10

+ +

Calcitonin DakoCytomation, Carpinteria, CA, USA None 1:200 – –CD34 DakoCytomation, Carpinteria, CA, USA QBEnd10 Microwave 1:100 –

+

, FocalCK14 Novocastra, Newcastle upon Tyne, UK LL002 Microwave 1:80 –

+

CK19 DakoCytomation, Carpinteria, CA, USA RCK108 Microwave 1:100

+

–c-Kit IBL, Fujioka, JAPAN K963 Microwave 1:20 – –Desmin DakoCytomation, Carpinteria, CA, USA DER-11 Microwave 1:100 – +EMA DakoCytomation, Carpinteria, CA, USA E29 Microwave 1:500 + –ER DakoCytomation, Carpinteria, CA, USA 1D5 Microwave 1:100 – –GFAP DakoCytomation, Carpinteria, CA, USA Polyclonal None 1:100 – +HER2/neu DakoCytomation, Carpinteria, CA, USA Autoclave Prediluted – –Maspin Novocastra, Newcastle upon Tyne, UK EAW24 Microwave 1:50 + +Ki-67 DakoCytomation, Carpinteria, CA, USA MIB-1 Microwave 1:50 – +, 60–70%p53 DakoCytomation, Carpinteria, CA, USA DO7 Microwave 1:100 – +, 50–70%p63 DakoCytomation, Carpinteria, CA, USA 4A4 Autoclave 1:400 – +p-Akt Cell Signaling Technology, Beverly, MA, USA Polyclonal Autoclave 1:100 – –p-ERK 1/2 Cell Signaling Technology, Beverly, MA, USA Polyclonal Autoclave 1:200 – +PgR DakoCytomation, Carpinteria, CA, USA PgR636 Microwave 1:100 – –S-100 protein DakoCytomation, Carpinteria, CA, USA Polyclonal None 1:400 – +

α

-SMA,

α

-smooth muscle actin; CK, cytokeratin; EMA, epithelial membrane antigen; ER, estrogen receptor; GEC, glandular epithelial component;GFAP, glial fibrillary acidic protein; MEC, myoepithelial component; p-Akt, phosphorylated Akt; p-ERK1/2, phosphorylated extracellular signal-regulatedkinase 1/2; PgR, progesterone receptor.

Autoclave, treated in Citra Plus solution, pH 6.0 (Biogenex, San Ramon, CA, USA) in an autoclave at 121

°

C for 10 min.Microwave, heated in 10 mmol/L citrate buffer, pH 6.0 at 100

°

C for 3 min, five times, using a microwave oven.

Table 1

World Health Organization classfication of myoepitheliallesions of the breast

25

1. Myoepitheliosisa. Intraductalb. Periductal

2. Adenomyoepithelial adenosis3. Adenomyoepithelioma

a. Benignb. With malignant changes (specify the subtype)• Myoepithelial carcinoma arising in an adenomyoepithelioma• Epithelial carcinoma arising in an adenomyoepithelioma• Malignant epithelial and myoepithelial components• Sarcoma arising in adenomyoepithelioma• Carcinosarcoma arising in adenomyoepithelioma

4. Malignant myoepithelioma (ME carcinoma)

Myoepithelial carcinoma of breast 213

The amplified PCR products were then purified using theQIAquick PCR purification Kit (Qiagen, Valencia, CA, USA),and electrophoresed on 2% agarose gel. Amplified DNAfragments were sequenced using a DNA Sequencing Kit(Bigdye Terminator v3.0 cycleSequencing Ready Reaction;Applied biosystems, Foster City, CA, USA) and subjected todirect sequencing using an autosequencer model 310(Applied Biosystems, Model 310) in both directions.

PATHOLOGICAL FINDINGS

Gross and histological findings

Grossly, the presented tumor was elastic, hard, and mea-sured 25

×

21

×

15 mm. The cut surface was firm and gray–yellow in color (Fig. 1). It appeared solid and relatively well-circumscribed but with a focal ill-defined margin adjacent tosurrounding tissue. Several small cystic areas were alsonoted. No apparent hemorrhage or necrosis was identified.

Histologically, the tumor was composed of a predominantlyspindle cell proliferation surrounding scattered glandular epi-thelial lined spaces (Fig. 2a). The inner GEC had abundantpinkish cytoplasm and oval nuclei with delicate chromatinand inconspicuous nucleoli. No apparent apocrine changewas identified (Fig. 2b). In contrast, the conspicuous spindlecells showed clear or eosinophilic cytoplasm. They prolifer-ated in broad sheets, strands or in a multinodular fashion,

and were separated by hyalinized colleagnous stroma withvarying thickness. All of these features suggested a spindle-cell type of AME described by Tavassoli.

2

However, at highmagnification, foci of cellular atypia are present in MEC,demonstrating enlarged pleomorphic nuclei and easily rec-ognizable mitoses (Fig. 2c). These proliferating spindle cellswere mainly arranged in cords or nodules, in which areas ofnecrosis were also seen (Fig. 2d). Mitotic figures werenumerous throughout the MEC of the tumor, and up to 17mitoses were counted in 10 high-power fields (HPF; Fig. 2c).Furthermore, invasive irregular margins were present insome regions. At these areas, fibrous or adipose tissueswere invaded by less well-defined nests and cords of MEC(Fig. 2e). Therefore, these features were suggestive of amore aggressive phenotype for MEC in this rare tumor.

Immunohistochemical findings

Immunohistochemical results are listed in Table 2. Biphasicepithelial and myoepithelial character of the tumor was con-firmed by immunohistochemistry. That is, the inner GEC wasstrongly positive for epithelial membrane antigen (EMA) andcytokeratin (CK) 19; whereas the outer MEC was diffuselypositive for S-100 protein,

α

-smooth muscle actin (

α

-SMA),glial fibrillary acidic protein (GFAP), p63 and CK14. BothMEC and GEC were consistently negative for estrogenreceptor (ER), progesterone receptor (PgR) and HER2/neu.The Ki-67 labeling index in MEC was 50–70%. In contrast, itwas

<

5% in GEC.On the basis of the histological and immunohistochemical

analysis, we considered that ‘myoepithelial carcinoma arisingin adenomyoepithelioma’ might be an appropriate histologi-cal diagnosis. Notably, in this tumor strong immunohis-tochemical staining of p53 protein (

>

50%; Fig. 3) wasidentified only in MEC, but not in GEC. In addition, usingphosphorylated extracellular signal-regulated kinase (ERK)1/2 (Thr202/Tyr204) and phosphorylated Akt (Ser473) anti-bodies, we investigated activation status of ERK-regulatedcell proliferation and Akt-regulated survival pathway. Therewas diffuse reactivity for phosphorylated ERK1/2 in MEC, butnegatively in GEC (Fig. 4). No neoplastic component waspositive for phosphorylated Akt.

Detection of

p53

and

ras

mutations

Direct sequencing revealed somatic mutation at codon 270of exon 8 (T

→

G transversion) in MEC, namely myoepithelialcarcinoma component (Fig. 5). No mutation of the

p53

genewas observed in GEC or adjacent normal ductal epithelium.No mutations in exons 1 and 2 of the K-, H-, and N-

ras

geneswere identified in any of the neoplastic component.

Figure 1

Gross appearance of the tumor. The cut surface is gray-yellow, solid but with several small cystic areas, measuring25

×

21

×

15 mm.

214 B. Han

et al

.

a

b

c

d

e

Figure 2

Histological findings. (

a

) Biphasic character of the tumor:solid proliferation of myoepithelial component (MEC) and scatteredglandular epithelial component (GEC) (HE). (

b

) GEC has blandnuclei and rich pinkish cytoplasm. No apparent apocrine change wasidentified (HE). (

c

) Areas of cellular pleomorphism and abundantmitotic figures (arrows) are apparent in MEC (HE). (

d

) Presence ofnecrotic area in MEC (HE). (

e

) Local invasion of surrounding adiposetissue by MEC (HE).

Figure 3

Immunohistochemical staining of P53. The myoepithelialcomponent is strongly positive for P53, whereas the glandular epi-thelial component is negative for P53 (immunohistostaining withmethyl green background staining).

Figure 4

Immunohistochemical staining of phosphorylated extra-cellular signal-regulated kinase 1/2 (p-ERK1/2). The myoepithelialcomponent is strongly positive for p-ERK1/2, whereas the glandularepithelial component is negative for p-ERK1/2. Note that positivestaining was in the nuclei of tumor cells (immunohistostaining withmethyl green background staining).

Figure 5

Sequences in codon 270 of the

p53

gene. T

→

G transver-sion mutation (arrow) is detected in the myoepithelial component(MEC) but not in the glandular epithelial component (GEC) or adja-cent normal tissue. N, normal ductal epithelium; M, MEC; G, GEC.

Myoepithelial carcinoma of breast 215

DISCUSSION

So far, 18 cases of carcinoma arising in an AME have beenreported in the literature.

2,4,7–12

Histologically, malignancyarising in an AME appears to adopt two patterns: either asan area of obvious malignancy arising in an otherwise typicalAME, or as giving an overall appearance of an AME but seenon close examination to contain cellular atypia and increasedmitotic activity throughout the tumor.

10

Loose

et al

. havedescribed guidelines for the identification of malignancy inthe second group and the present case might fall into thiscategory.

5

It has been suggested that the term ‘malignantAME’ can also be applied to tumors that have not yet metas-tasized but which are locally invasive and have high mitoticactivity and marked cytological atypia.

12

In the present case,although the background lesion was adenomyoepithelioma-tous in appearance, the presence of local invasion, highmitotic activity, cytological atypia and necrosis of MEC mayindicate malignancy in the absence of metastasis.

To date, only a small number of reports focusing on molec-ular analysis of malignant AME have been published. Trojani

et al

. described a malignant AME with lung metastases andidentified a DNA diploid epithelial population and DNA tetra-ploid myoepithelial cells.

3

In another report, Jones

et al

. per-formed comparative genomic hybridization (CGH) analysison a malignant AME case.

18

They found that both GEC andMEC of primary tumor acquired loss of heterozygosity (LOH)at 11q23-q24 and 16q22-q23, whereas the liver metastasisshowing exclusively myoepithelial differentiation had an addi-tional LOH at 12q24. In the present study, for the first time,we detected a somatic mutation of the

p53

gene at codon270 (TTT

Arg

→

TGT

Cys

) in myoepithelial carcinoma of thebreast in an AME. This mutation is not within the ‘hotspot’codons, but has been reported in colorectal cancer, glioblas-toma and common types of breast cancer.

29

We highlight thisfinding because myoepithelial cells seem to be resistant totransformation, and myoepithelial carcinomas of the breastexhibit few genetic alterations.

30,31

In addition, in the presenttumor, it is suggested that

p53

gene mutation might beinvolved in the malignant transformation of MEC. Recentlysome investigators have speculated that the evolution ofmalignant AME seems to begin with adenosis with or withoutmyoepithelial hyperplasia, proceeds to benign AME, andends in a malignant state.

12

However, not much is knownabout molecular change during this neoplastic progression.It has been reported that LOH in 17p, where the

p53

geneis localized, occurs in myoepithelial carcinoma of thebreast.

32

Angele

et al

. have also reported that p53 protein isconsistently negative in benign myoepithelial lesions butoverexpressed in 44.4% (8/18) of malignant myoepithelialtumors of the breast.

33

However, further molecular analysisof the

p53

gene was not provided in that study. Because noother investigations of

p53

gene mutations in malignant AME

have been reported except that for the present case, morestudies are needed to test a possible role of

p53

gene muta-tion in the carcinogenesis of myoepithelial carcinoma in AME.

Assuming that aberrantly signaling pathways of cellulargrowth, survival and apoptosis contribute to breast tumorprogression, we also investigated two kinases that areinvolved in these important cellular events: ERK1/2 and Akt.To our knowledge, there has been no report relating tomolecular pathway in myoepithelial tumors of the breast. Inthe present study, we identified exclusive overexpression ofphosphorylated ERK1/2 in malignant MEC. Because acti-vated ERK1/2 plays a pivotal role in cell proliferation andsurvival,

34

its overexpression, as well as high Ki-67 labelingindex, might reflect a proliferative state of MEC in this tumor.It is known that in many types of cancer, mutated forms ofthe

ras

gene are responsible for ERK1/2 activation; but inbreast cancer activating ras mutations are rare.

35

Instead,

ras

genes may be overexpressed and this process correlateswith carcinogenesis and cancer progression.

35,36

Consistentwith previous findings, we failed to identify K-, H-, or N-

ras

genes mutations in each neoplastic component. Thus oneexplanation was that overexpression of phosphrylated ERK1/2 in MEC might be due to overexpression of

ras

protein. Incontrast, some other unknown genes might modulate over-expression of ERK1/2. However, the mutated

p53

gene doesnot appear to play a major role in this regard because of theabsence of

ras

gene mutation.In conclusion, for the first time, we have identified

p53

mutation in a rare case of malignant AME of the breast.Because the present study is based on a single case report,molecular analysis of more cases of malignant AME isneeded to further define the genetic profile and carcinogen-esis of this rare category of breast carcinoma.

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