CASE REPORT / CAS CLINIQUE

Assessment of hormonal receptors and Her-2/neu status in breast cancerusing cell block: a case study

Évaluation de l’état des récepteurs hormonaux et du HER2/neu dans les cas de cancer du seinau moyen d’un échantillon de cellules : étude de cas

J. Nyagol · V. Kisato · W. Ochuk · M. Wakio

Received: 6 February 2013; Accepted: 27 June 2013© Springer-Verlag France 2013

Abstract Objective: To report the expressions of estrogenreceptor (ER), PR (progesterone receptor), and human epi-dermal growth factor receptor type 2 (Her-2/neu) in a breastcancer patient using cell block prepared from fine needleaspirate (FNA).Study design: FNAwas obtained from the subject and cyto-morphological diagnosis of stage II ductal carcinoma doneon hematoxylin and eosin stain. This was followed withpreparation of cell blocks from a portion of the aspirateand subsequent immunocytochemical technique, usingheat-induced epitope retrieval (HIER). Specific monoclonalantibodies were used to determine the expressions of ER,PR, and Her-2/neu receptors. Positive and negative controlswere included in the procedure at the same time the testswere done for the receptor assessments. For each receptorassessed, cells were counted from ten representative high-power fields and an average of cells that stained positivefor respective markers, out of the total number of cellsobserved, was calculated. The data was produced in tripli-cate by three independent pathologists, from which morethan 95% concordance was obtained.Results: In this reported case, the steroid receptors werescored as: ER-positive (ER+) and PR-positive (PR+). Her-2/neu was positive (3+), implying a strong complete mem-brane staining in more than 10% of the tumor cells.Conclusion: The successful use of FNA and immunocyto-chemistry (ICC) following HIER for the assessment of hor-monal and Her-2/neu receptor status in breast cancer is herereported to have been done for the first time in our setting.The results must, however, be interpreted with caution usingthe counted cells captured in representative high-powerfields assessed.

Keywords Breast cancer · Fine needle aspirate · Hormonalreceptors · Her-2/neu · Endocrine therapy · Herceptin

Résumé Objectif : Rendre compte de l’expression des récep-teurs ER, PR et HER2/neu chez une patiente atteinte d’uncancer du sein en utilisant un échantillon de cellules préparépar une ponction à l’aiguille fine (PAF).Étude : La ponction a été effectuée sur le sujet et un diag-nostic cytomorphologique de carcinome canalaire en phaseII a été établi après coloration à l’hématoxyline et à l’éosine.Ensuite, un groupe de cellules issues de la ponction a étépréparé pour être analysé par démasquage des épitopesinduit par la chaleur (DEIC), une technique d’immunocy-tochimie. Des anticorps monoclonaux spécifiques ont étéutilisés afin de déterminer l’expression des récepteurs ER,PR et HER2/neu. Des contrôles positifs et négatifs ont étéinclus dans la procédure parallèlement aux tests d’évalu-ation des récepteurs. Pour chaque récepteur évalué, lescellules ont été comptées sur la base de dix champs àfort grossissement représentatifs, et une moyenne des cel-lules qui ont subi une coloration positive par leurs mar-queurs respectifs a été calculée par rapport au nombretotal de cellules observées. Les données ont été produitesen trois exemplaires par trois pathologistes indépendants,chez lesquels une concordance de plus de 95 % a étéobtenue.Résultats : Dans le cas rapporté ici, les récepteurs des stér-oïdes ont été notés comme suit : ER-positif (+++) et PR-positif (++). HER2/neu était positif (3+), ce qui impliqueune coloration forte et complète de la membrane de plus de10 % des cellules tumorales.Conclusion : L’usage réussi de la PAF et de la techniqued’immunocytochimie DEIC pour l’évaluation de l’état desrécepteurs hormonaux et HER2/neu dans le cancer dusein est rapporté ici comme étant une première dans notreétablissement. Cependant, ces résultats (obtenus par compt-age des cellules capturées dans les champs représentatifs

J. Nyagol (*) · V. Kisato · W. Ochuk · M. WakioDepartment of Human Pathology, University of Nairobi (UoN),P.O Box 19676-00202, KNH, Nairobi, Kenyae-mail : Joshua.nyagol@uonbi.ac.ke

J. Afr. Cancer (2013) 5:180-184DOI 10.1007/s12558-013-0279-4

à fort grossissement analysés) doivent être interprétésavec prudence.

Mots clés Cancer du sein · Ponction à l’aiguille fine ·Récepteurs hormonaux · HER2/neu · Thérapieendocrinienne · Herceptine

Introduction

Breast cancer is by far now the leading cause of cancer deathamong females in economically developing countries, a shiftfrom the previous decade during which the most commoncause of cancer death was cervical cancer. Although overallbreast cancer incidence rate in the developing world is halfthat seen in developed world, the mortality rates from thedisease are generally similar. However, breast cancer sur-vival tends to be poorer in developing countries, most likelybecause of a combination of a late stage at diagnosis andlimited access to timely and standard treatment. One of thechallenges in treating the disease is addressing the biologicalheterogeneity evident in the existence of several histologicand molecular subtypes [1–4].

The response of breast cancer patients to endocrine ther-apy is currently guided by the expression of two steroid hor-mone receptors (HR): estrogen receptor-α (ER-α) and pro-gesterone receptor (PR). Expression of PR, in fact, has beenreported to confer good prognosis to breast cancer patients[5]. Another molecular marker that is increasingly beingexamined in breast cancer for therapeutic potential as wellas a prognostic indicator is the human epidermal growth fac-tor receptor type 2 (Her-2/neu) oncoprotein [6–8]. Her-2/neugene encodes a membrane receptor protein in the epidermalgrowth factor receptor family, and has been documented inmany studies to be amplified and overexpressed in approxi-mately 20–25% of human breast cancers. This is in tandemwith our previous study of the receptor expression in cohortof Kenyan women with breast cancer [9–11].

Overexpression of Her-2/neu protein on the cell surfaceand/or gene amplification at the nuclear level predicts theresponse to anti-Her-2/neu immunotherapy with Herceptin/Trastuzumab. This alteration is associated with shorterdisease-free and overall survival in women with either lymphnode-negative or lymph node-positive breast cancer. Besides,Her-2/neu alteration is also associated with lack of respon-siveness to Tamoxifen anti-estrogen therapy, as well as alteredresponsiveness to a variety of cytotoxic chemotherapy regi-mens [12–14]. However, both as a single agent and in combi-nation with conventional chemotherapy, clinical trials of anti-Her-2/neu therapy with monoclonal antibody, trastuzumab,have demonstrated an improved response rate or improved

overall survival of women with Her-2/neu-positive metastaticbreast cancers [15–17].

Since the standards of care in breast cancer require knowl-edge of tumor features, and HR as well as Her-2/neu status,the assessment of HRs and HER-2/neu by immunohis-tochemistry and even chromogenic in situ hybridization(CISH) procedure is necessary to select patients who arecandidates for hormonal and anti-HER-2 therapy (Trastuzu-mab/Herceptin). The evaluation of these parameters is gen-erally carried out in biopsy from primary tumors and so far, itis not clear if reassessment in metastatic lesions might havean impact on patients’management [18]. Equally, the assess-ment of breast cancer receptors from cell blocks have notbeen a common practice even in advanced laboratories dueto inherent limitations in tumor staging and inter-observervariations in reporting immunostains.

Here, we document immunocytochemistry results obtainedfrom receptor status evaluation from FNA of a multi-parouspatient who presented with grade II ductal carcinoma in theupper outer quadrant of the left breast.

Materials and methods

After obtaining institutional and informed consent from theconcerned subject, routine hematoxylin and eosin from FNAdone on left breast of a 37-year-old patient with previouslydiagnosed grade II ductal carcinoma was done for confirma-tion of the previous diagnosis and immunocytochemistry test.Two slide smears were prepared for the hematoxylin/eosin,while a portion of remaining aspirate in the needle was pre-served in a bottle containing normal saline and taken to thelaboratory for processing and cell block preparation. In brief,10–20 ml of the aspirate was centrifuged and supernatant dis-carded. Two drops of pooled plasma were added, followedwith gentle shaking. Thereafter, two drops of thromboplastinwere added and mixed well to activate clotting factor, fol-lowed with two drops of calcium ion. The mixture was leftto stand for 5 min, followed with transfer of the clot to moist-ened filter paper number 1. The clot was wrapped well, put ina cassette, and then fixed in buffered formalin for 4 h. Thesample was then processed as routine, from which cell blockwas prepared the following day.

Immunocytochemistry for ER, PR,and Her-2/neu analyses

Immunocytochemistry (ICC) assay was performed on 4 μmsections cut from the cell blocks and float mounted on silane-coated slides. The standard strepavidin-biotin-peroxidase wasused. In brief, the technique involved heat-induced epitoperetrieval in 0.1 M boiling citrate buffer (pH 6.0) in microwave

J. Afr. Cancer (2013) 5:180-184 181

for 15 min at 750 W, quenching endogenous peroxidase with0.03% hydrogen peroxide in methanol, followed with block-ing of non-specific protein binding sites with normal horseserum, and then binding with respective primary antibodies.The sections were incubated for 1 h at room temperature withER (clone NCL-ER-alpha, Novocastra), PR (clone 1A6,Novocastra), and Her-2/neu (clone CB11, Novocastra) spe-cific antibodies. Streptavidin-biotin-peroxidase complex wasapplied, followed by diaminobenzidine (DAB) chromogenstaining. The slides were then counter-stained with hematoxy-lin and eosin, dehydrated, and mounted. For each run of thetests, positive and negative controls were included. The ERand PR were considered positive when distinct nuclear stainswere observed in an average of more than 15% of the totaltumor cells counted from ten high-power fields. Her-2/neustain was evaluated according to HercepTest system (four-tier scale—DAKO). A strong complete membrane stainingin an average of more than 10% of tumor cells was considered3+ positive, while a weak to moderate complete membranestaining in an average of more than 10% of the tumor cellswas scored at 2+ positive. The slides were reviewed by twoindependent pathologists from which 98% concordance wasachieved. In case of discordance, the slides were simulta-neously reviewed by the two Pathologists to achieve aconsensus.

Results

Clinical Data of the Patient

The case reported here is of a pre-menopausal, multipa-rous (two children) woman of 37 years, with no familyhistory of breast cancer. Although multifocal, tumor loca-tion was noted at the upper outer quadrant of the leftbreast, from which FNA was initially done on a growinglump adjacent to another, and diagnosis made of grade IIductal carcinoma.

Cytological Features

On repeat FNA for confirmation of previous diagnosis, thetypical ductal cells were observed (Fig. 1.a) (Table 1).

Hormonal Receptors and Her-2/neu Status

ER was reported in this case as positive (+++), while PR waspositive (++) guided by the average number of cells that werepositive out of the ten high-power fields evaluated for estro-gen and progesterone receptors (Figs 1.b and c, respectively).On the other hand, Her-2/neu was scored as positive (3+),implying that complete membrane staining was observed in

Fig. 1 Hematoxylin/eosin and receptors staining.

Hematoxylin and eosin shows a cluster of ductal cells from FNA, Fig. 1.a. Both ER and PR shows strong nuclear staining (Figs 1.b and

1.c, respectively), while the score of Her-2/neu is at 3+, indicating a complete membrane staining in more than 10% of the tumor cells

observed at high-power field (Fig. 1.d)

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more than 10% of the tumor cells at high-power field in tendifferent regions assessed (Fig. 1.d and Table 2).

Discussion

Breast cancer is emerging to be the most common canceramong Kenyan women in the reproductive age group, withalmost a thousand new cases reported per year at the healthinstitutions in Nairobi alone. Therefore, including the assess-ment of both steroid and Her-2/neu receptor status, a routinediagnostic tool in breast cancer, will reflect positive clinicaland therapeutic outcome in the economically developingcountries, Kenya inclusive, similar to what has beenobserved in the affluent nations [19,20]. With the currentlimited standard care for metastatic breast cancer, character-ization of individual patient’s tumor is of value in treatmentdecisions.

The determination of ER and PR status has become stan-dard practice in the evaluation of patients with invasivebreast cancer, having important prognostic and therapeuticimplications [21]. Breast cancer patients whose tumorsexpress estrogen receptor-α (ER-α) may be offered endo-crine or anti-estrogen therapy in addition to or in place ofconventional chemotherapies. Currently, the most widelyused anti-estrogen is the triphenylethylene Tamoxifen(TAM), which functions as a partial antagonist by competingwith estrogen for binding to ER. TAM is known to induce astatistically significant improvement in the overall survivalrate from breast cancer, and approximately 70% of all ER-positive (ER+)/PR-positive (PR+) breast cancers willrespond to TAM [22,23].

Equally, Her-2/neu has been documented to be overex-pressed in about 20–30% of ductal carcinoma of the breast.These cancers tend to grow faster, spread more rapidly, recurmore often, and have a poorer prognosis than other breast

cancers. Assessment of HER-2/neu is therefore importantto evaluate the response to Herceptin (Trastuzumab) therapyfor primary and metastatic breast cancers [24,25].

Regardless of tumor subtype, the development of endo-crine resistance is a pervasive clinical problem. One thirdof ER+/ PR+ breast tumors treated with TAM do notrespond to initial treatment, and on average, 70% are stillat risk of relapse in the future. Although details of themechanisms continue to be unclear, a number of mechan-isms have been proposed to confer anti-estrogen resistancein ER+ breast cancer, including overexpression of Her-2/neu [26,27].

It is in this context that we used ICC technique to demon-strate the expression of hormonal receptors and Her-2/neu incell block prepared from FNA, being a success instead ofusing solid tumor sample from a biopsy. Although stagingof the tumor and evaluation of invasiveness cannot beobtained from cytology specimen, use of FNA can easilypredict the benefit of anti-ER and/or anti-Her-2/neu targetedtherapy in breast cancer patients.

Conflict of interest: the authors have no conflicts of interestto declare.

References

1. Ahmedin J, Freddie B, Melissa MC, et al (2011) Global cancerstatistics. CA Cancer J Clin 61:69–90

2. Ferlay J, Shin HR, Bray F, et al (2010) Estimates of worldwideburden of cancer. In: GLOBOCAN 2008. Int J Cancer 127(12):2893–917

3. Harford JB, Otero IV, Anderson BO, et al (2011) Problem solv-ing for breast health care delivery in low and middle resourcecountries (LMCs): consensus statement from the Breast HealthGlobal Initiative. Breast 20(Suppl 2):S20–S9

Table 1 Total count and average of ER and PR positive cells.

Positive cells out of the total number counted in each high-power field (F) Total (%)

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10

ER 7/8 12/14 13/13 12/15 12/15 10/11 8/8 11/12 10/14 10/10 105/120 (87.5)

PR 10/15 10/12 9/14 11/16 6/10 9/13 8/12 10/11 12/15 12/13 97/131 (74)

Table 2 Total count and average of Her-2/neu positive (3+) cells.

Positive (3+) cells out of the total number counted in each high-power field (F) Average (%)

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10

Her-2/neu 12/12 11/13 10/10 8/10 11/12 12/12 10/11 11/11 9/10 10/10 104/111 (94)

J. Afr. Cancer (2013) 5:180-184 183

4. Rodrigues AD, Bustamante-Teixeira MT (2011) Breast cancerand cervical cancer mortality trends in a medium-sized city inSouthern Brazil, 1980–2006. Cad Saude Publica 27(2):241–8

5. Anderson BO, Yip CH, Smith RA, et al (2008) Guideline imple-mentation for breast healthcare in low-income and middle-income countries: overview of the Breast Health Global InitiativeGlobal Summit 2007. Cancer 113(Suppl 8):2221–43

6. Ghosh R, Narasanna A, Wang SE, et al (2011) Trastuzumab haspreferential activity against breast cancers driven by HER2 homo-dimers. Cancer Res 71(5):1871–82

7. Konecny G, Pauletti G, Pegram M, et al (2003) Quantitativeassociation between HER-2/neu and steroid hormone receptorsin hormone receptor-positive primary breast cancer. Natl CancerInst 95(2):142–53

8. James R, Thriveni K, Krishnamoorthy L, et al (2011) Clinicaloutcome of adjuvant endocrine treatment according to Her-2/neustatus in breast cancer. Indian J Med Res 133(1):70–5

9. Nyagol J, Nyong’o A, Byakika B, et al (2006) Routine assess-ment of hormonal receptor and Her-2/neu status underscores theneed for more therapeutic targets in Kenyan women with breastcancer. Anal Quant Cytol Histol 28(2):97–103

10. Sauter G, Lee J, Bartlett JM, et al (2009) Guidelines for humangrowth factor receptor 2 testing: biologic and methodologic con-siderations. J Clin Oncol 27(8):1323–33

11. Mayr D, Heim S, Weyrauch K, et al (2009) Chromogenic in situhybridization for Her-2/neu-oncogene in breast cancer: compari-son of a new dual-color chromogenic in situ hybridization withimmunohistochemistry and fluorescence in situ hybridization.Histopathology 55(6):716–23

12. Suzuki Y, Saito Y, Terao M, et al (2010) Trastuzumab and che-motherapy after the treatment failure of lapatinib for HER2-positive metastatic breast cancer. Tokai J Exp Clin Med 35(4):148–51

13. Piccat-Gebhart MJ (2011) New developments in hormonereceptor-positive disease. Oncologist 16(Suppl 1):40–50

14. Higgins MJ, Baselga J (2011) Breast cancer in 2010: novel tar-gets and therapies for a personalized approach. Nat Rev ClinOncol 8(2):65–6

15. Xiao C, Gong Y, Han EY, et al (2011) Stability of HER2-positivestatus in breast carcinoma: a comparison between primary andpaired metastatic tumors with regard to the possible impact of

intervening trastuzumab treatment. Ann Oncol Jan 14. [Epubahead of print]

16. Nielsen DL, Andersson M, Kamby C (2009) HER2-targeted ther-apy in breast cancer. Monoclonal antibodies and tyrosine kinaseinhibitors. Cancer Treat Rev 35(2):121–36

17. Dean-Colomb W, Esteva FJ (2008) Her2-positive breast cancer:herceptin and beyond. Eur J Cancer 44(18):2806–12

18. Guarneri V, Giovannelli S, Ficarra G, et al (2008) Comparison ofHER-2 and hormone receptor expression in primary breast can-cers and asynchronous paired metastases: impact on patient man-agement. Oncologist 13(8):838–44

19. Lawrence LS, Walter W, Amy S, Felicia MK (2010). Breast can-cer in developing countries: opportunities for improved treatment.J Oncol: doi:10.1155/2010/595167

20. Stark A, Kleer CG, Martin I, et al (2010) African ancestry andhigher prevalence of triple-negative breast cancer: findings froman international study. Cancer 116(21):4926–32

21. James R, Thriveni K, Krishnamoorthy L, et al (2011) Clinicaloutcome of adjuvant endocrine treatment according to Her-2/neustatus in breast cancer. Indian J Med Res 133(1):70–5

22. Gianni L, Dafni U, Gelber RD, et al (2011) Treatment with tras-tuzumab for 1 year after adjuvant chemotherapy in patients withHER2-positive early breast cancer: a 4-year follow-up of a ran-domized controlled trial. Lancet Oncol 12(3):236–44

23. Konecny G, Pauletti G, Pegram M, et al (2003) Quantitativeassociation between HER-2/neu and steroid hormone receptorsin hormone receptor-positive primary breast cancer. Natl CancerInst 95(2):142–53

24. Garrett JT, Arteaga CL (2011) Resistance to HER2-directed anti-bodies and tyrosine kinase inhibitors: mechanisms and clinicalimplications. Cancer Biol Ther 11(9):793-800

25. Emens LA (2005) Trastuzumab: targeted therapy for the manage-ment of HER-2/neu-overexpressing metastatic breast cancer. AmJ Ther 12(3):243–53

26. Sunami E, Shinozaki M, Sim MS, et al (2008) Estrogen receptorand HER2/neu status affect epigenetic differences of tumor-related genes in primary breast tumors. Breast Cancer Res 10(3):R46

27. Buzdar AU (2009) Role of biologic therapy and chemotherapy inhormone receptor- and HER2-positive breast cancer. Ann Oncol20(6):993–9

184 J. Afr. Cancer (2013) 5:180-184