ORIGINAL ARTICLES

Estrogen and ProgesteroneHormone Receptor Status inBreast Carcinoma:Comparison of Immunocytochemistry andImmunohistochemistrySvetlana Tafjord, M.D., Per J. Bøhler, M.D., Bjørn Risberg, M.D., Ph.D., andEmina Torlakovic, M.D.*

We evaluated the correlation between histologic and cytologicspecimens in the determination of estrogen receptor (ER) andprogesterone receptor (PR) status in breast carcinoma and inves-tigated the causes of clinically significant discrepancies. We ana-lyzed 70 immunoassays for ER and 60 for PR from 71 patients withbreast carcinoma. Concordance between cytology and histologywas 89% for ER and 63% for PR using scores from pathologyreports. Concordance between cytology and histology was 98% forER and 91% for PR using consensus scores (obtained after re-evaluation by the team of pathologists). Thirty of 130 (23%) testshad clinically relevant discrepancies, 53% of which were causedby wrong interpretation of cytologic findings, 10% by wronginterpretation of histologic findings, 17% by sampling error and20% were not available for reevaluation. Wrong interpretation ofthe results for ER and PR status in cytology was a far morefrequent cause of clinically relevant discrepancies than samplingerrors. The use of strict criteria is recommended. Diagn. Cyto-pathol. 2002;26:137–141; DOI 10.1002/dc.10079© 2002 Wiley-Liss, Inc.

Key Words: immunohistochemistry; immunocytochemistry; corre-lation; estrogen receptor; progesterone receptor; breast carcinoma

With the advent of newer detection methods, improvedantibodies, and better antigen retrieval methods, immuno-histochemistry has become a reliable means of detectionand quantitation of estrogen receptors (ERs) and progester-one receptors (PRs) in formalin-fixed/paraffin-embeddedtissue.1–9 Fine-needle aspiration biopsy (FNA) offers a suit-able alternative for hormone receptor analysis for patientswith inoperable or metastatic lesions and in cases selectedfor preoperative chemotherapy or radiation.10,11 The ER/PRstatus is particularly important in the treatment of patients

with breast cancer. Moreover, the absence of ERs in suchtumors has been associated with early recurrence indepen-dent of other known prognostic factors.10,12 Assessment ofERs and PRs also may be useful as a diagnostic adjunct inthe evaluation of patients with metastatic tumors of un-known origin.13 In our study, we compared the results of ERand PR immunoassays performed on FNA specimens andcorresponding histology specimens, to find out whetherthere were any significant discrepancies between these twoprocedures and, if so, to identify their causes.

Materials and MethodsMaterialsAll materials were collected from the archives of the De-partment of Pathology, Norwegian Radium Hospital, Oslo,Norway. Included were biopsy specimens of primary andmetastatic breast carcinoma from December 1998 to Feb-ruary 2000 for which FNA cytologic material with ER andPR status was also available. Of the 71 patients, 70 werefemale and one was male; their average age was 61 yr.There were 61 invasive ductal carcinomas, 4 invasive lob-ular carcinomas, 1 mucinous carcinoma, and 5 metastatictumors (4 from invasive ductal carcinoma and 1 from inva-sive lobular carcinoma). No selection was made with re-spect to tumor size or grade, lymph node status, or patient’ssex or age.

Specimens were fixed in 4% neutral formalin up to 48 hr.All samples were embedded in paraffin and stained byhematoxylin and eosin. FNA was done by ultrasound guid-ance or by free hand using an FNA device with a 23-gaugeneedle. The aspirate was smeared onto silane-coated slidesand immediately put into an acetone/formalin fixative (1 l ofacetone/formalin: 150 mg of Na2HPO4, 1,200 mg ofKH2PO4, 300 ml of distillated H2O, 450 ml of acetone, and250 ml of 37–40% formaldehyde at pH 6.6).

Department of Pathology, Norwegian Radium Hospital, Oslo, Norway*Correspondence to: Emina Emilia Torlakovic, M.D., Department of

Pathology, Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.Received 31 July 2001; Accepted 19 November 2001

© 2002 WILEY-LISS, INC. Diagnostic Cytopathology, Vol 26, No 3 137

ImmunohistochemistryParaffin blocks were cut at 4 �m. Heat-induced epitoperetrieval was performed in EDTA buffer at pH 8.9 bycooking in an Electrolux microwave oven (1,000 W) for 20min (4 � 5 min). Anti-ER (diluted 1:20; clone 6F11;NovoCastra, Newcastle upon Tyne, U.K.) and anti-PR (di-lution 1:20, clone 1A6; Novo Castra) antibodies were used,incubated for 30 min at room temperature. The EnVisionmethod (Dako, Glostrup, Denmark) was employed for therest of the procedure, according to the manufacturer’s in-structions. The slides were counterstained by hematoxylin.

ImmunocytochemistryAll materials used for ER and PR determination were FNAspecimens, which were fixed in acetone/formalin. The slideswere fixed immediately after aspirated material wassmeared on the slide. After 2 hr of fixation, the slides weretransferred to phosphate-buffered saline for several hoursbefore staining of the slides. Antigen retrieval was effectedin citrate buffer at pH 6.8 in an Electrolux microwave oven(1,000 W) for 20 min. Anti-ER and anti-PR primary anti-bodies (same as earlier) were used, incubated for 30 min atroom temperature. The EnVision method (Dako) was usedfor the rest of the procedures, according to the manufactur-er’s instructions. The slides were counterstained byhematoxylin.

Scoring of ER and PR PositivitySimilarly to other studies, the cutoff value for the test to beconsidered positive for either ER or PR was detection ofimmunostaining in at least 10% of tumor nuclei.1,3,5 Tumorswith positivity in 10–50% of nuclei were interpreted asweakly positive, and those with positivity in more than 50%were regarded as strongly positive. Two sets of scores wererecorded. The first set of scores was obtained from theoriginal pathology reports, and the second set of scores(consensus scores) was generated during review of all avail-able material by the team of pathologists. “Clinically rele-vant discrepancy” refers to differences in scoring, whichcould cause different treatment decisions. “Clinically irrel-evant discrepancy” refers to differences in scoring thatshould not cause different treatment decisions but that couldbe of technical importance because of noticeable differencein the intensity of staining.

ResultsScores From Histology and Cytology ReportsPerfect correlation between scores obtained by immunohis-tochemistry and immunocytochemistry was found in 46 of70 (64%) ER scores and 27 of 60 (45%) PR scores. Whenonly clinically relevant discrepancies were included in thecalculation, the correlation increased to 89% for ER testsand 63% for PR tests. These results are summarized in

Table I and Table II. Of 71 cases included in the study, 8 ERand 22 PR tests had discrepant scores that were clinicallyrelevant.

Consensus ScoresOf 70 ER tests, 5 were not available for review, and 2 weretechnically suboptimal. Of 60 PR tests, 7 were not availablefor review, and 8 were technically suboptimal. In 2 of 70(3%) ER cases and 8 of 60 (13%) PR cases, it was notpossible to rescore slides, because they were judged to betechnically suboptimal as the result of extensive air-dryingartifacts (about 70% of cases) or a very small number ofnuclei (about 30% of cases). The much higher percentage ofsuboptimal slides for the PR test stemmed from the labora-tory’s policy of choosing better smears for ER tests.

In toto, 63 ER and 45 PR tests were rescored by the teamof pathologists. Fifty-four breast carcinomas were found tobe true positive for ER, and 34 were found to be truepositive for PR (by immunohistochemistry, immunocyto-chemistry, or both). The sensitivity of histology was 98%for ER and 94% for PR, while the sensitivity of cytologywas 93% for ER and 71% for PR. Results of reevaluationare shown in Table III and Table IV. Perfect correlation forER expression was found in 60 of 63 (95%) cases. Perfectcorrelation for PR expression was found in 37 of 45 cases(82%). When only clinically relevant discrepancies wereincluded in the calculation, the correlation increased to 98%for ER tests and 91% for PR tests.

DiscrepanciesThe causes of discrepancies are shown in Table V and TableVI. Altogether, 30 of 130 (23%) tests were judged to have

Table I. Comparison of Scoring of Positivity for Estrogen Receptor inHistologic and Cytologic Samples From Pathology and CytologyReportsa

IHC (histology)

ICC (cytology)

Negative �10% 10–50% �50% Total

Negative 7 3 3 — 13�10% 2 — — 2 410–50% 1 — 2 2 5�50% — 2 9 37 48Total 10 5 14 41 70aIHC, immunohistochemistry; ICC, immunocytochemistry.

Table II. Comparison of Scoring of Positivity for ProgesteroneReceptor in Histologic and Cytologic Samples From Pathologyand Cytology Reportsa

IHC (histology)

ICC (cytology)

Negative �10% 10–50% �50% Total

Negative 10 1 3 1 15�10% 5 2 1 — 710–50% 5 4 2 3 15�50% 4 3 2 13 25Total 25 10 8 17 60aIHC, immunohistochemistry; ICC, immunocytochemistry.

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clinically relevant discrepancies, and an additional 27(21%) had clinically irrelevant discrepancies in scoring.Clinically relevant discrepancies were caused by wronginterpretation on the part of the histopathologist in 3 of 30(10%) cases, by wrong interpretation by the cytopathologistin 16 of 30 (53%), and by sampling error in 5 of 30 (17%).The rest, 6 of 30 (20%), were not available for reevaluation.

The great majority of the cases incorrectly interpreted bythe cytopathologist were due to acceptance of technicallysuboptimal slides for scoring. Most of these cases hadsignificant air-drying artifact, many of which caused eitherfalse-negative test results or false-low scores (Fig. C-1).Occasionally, the cytopathologist also accepted slides withvery few cells (less than 100 nuclei), which were deemednonrepresentative at consensus scoring. In 3 of 30 (10%)

clinically relevant discrepancies, the cytologic preparationgave higher scores than the histologic preparation owing tosampling error. Additional paraffin blocks were tested inthose cases, and areas with corresponding levels of ER orPR expression were found.

Twenty-seven patients had the above-described 30 clini-cally relevant discrepancies. In 6 of these 27 patients (22%),false-positive results were reported owing to wrong inter-pretation. The tumors from these patients were found to benegative for both ERs and PRs on consensus scoring. Therewere 17 of 60 (28%) clinically relevant false-negative PRtests in cytology. Only 2 of those 17 (12%) were due tosampling error. This means that overall only 2 of 60 (3%)false-negative PR tests were attributed to sampling error.Similarly, only 2 of 60 (3%) PR and 1 of 70 (1%) ERfalse-negative test results in histology were caused by sam-pling error. There were no false-negative ER test results incytology that resulted from sampling error, but all 3 false-negative ER tests were due to wrong interpretation oncytology.

DiscussionRailo and co-workers found that needle-core biopsy (NC)and FNA biopsy could be used for preoperative assessmentof hormonal status in breast carcinoma.1 They compared ERand PR scores in NC and FNA specimens with those ob-tained from surgical specimens. The results of the compar-ison of FNA and surgical specimens were not very good.The kappa value obtained was only 0.3 for ER scores and0.4 for PR scores. These researchers also did not separate

Table III. Comparison of Scoring of Positivity for ER in Histologic and Cytologic Samples From Consensus Score After Reevaluationof ER Scoresa

IHC (histology)

ICC (cytology)

Negative �10% 10–50% �50% TS NA Total

Negative 11 2 — — — 1 14�10% — — 1 1 — — 210–50% — — 4 1 2 7�50% — — — 44 1 1 46TS — — — — — — —NA — — — — — 1 1Total 11 2 5 45 2 5 70aER, estrogen receptor; IHC, immunohistochemistry; ICC, immunocytochemistry; TS, technically suboptimal; NA, not available.

Table IV. Comparison of Scoring of Positivity for PR in Histologic and Cytologic Samples From Consensus Score After Reevaluation of PR Scoresa

IHC (histology)

ICC (cytology)

Negative �10% 10–50% �50% TS NA Total

Negative 14 — — 1 — — 15�10% 2 3 1 — 1 — 710–50% — 1 5 — 2 1 9�50% 1 — 2 15 5 — 23TS — — — — — — —NA — — — — — 6 6Total 17 4 8 16 8 7 60aPR, progesterone receptor; IHC, immunohistochemistry; ICC, immunocytochemistry; TS, technically suboptimal; NA, not available.

Table V. Causes of Clinically Relevant Discrepanciesa

Causes

Clinically relevantdiscrepancies

Total

H�/C� H�/C�

ER PR ER PR

Sampling error — 2 1 2 5Wrong interpretation by

histopathologist — 2 — 1 3Wrong interpretation by

cytopathologist 3 9 3 1 16NA — 4 1 1 6Total 3 17 5 5 30aH, histology; C, cytology; ER, estrogen receptor; PR, progesterone recep-tor; NA, not available.

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Fig. C-1. A: Histologic specimen shows uniformly strong expression of ER throughout the tumor. B: In the cytologic preparation, only the central partof the specimen correlates with findings on histology. C,D: Gradual loss of reactivity for anti-ER is seen towards the periphery of the slide. This findingis consistent with drying artefact. If drying artefact is not recognized, the scoring of positivity will be falsely low.

Table VI. Causes of Clinically Irrelevant Discrepanciesa

Causes

Clinically irrelevant discrepancies

Total

H�/C � 10% C�/H � 10%H10–50%/C � 50%

H � 50%/C10–50%

ER PR ER PR ER PR ER PR

Sampling error — 1 — — — — — 1 2Wrong interpretation by histopathologist — — 1 3 — 1 1 — 6Wrong interpretation by cytopathologist 3 — — 1 — 1 6 1 12NA — — 1 1 2 1 2 — 7Total 3 1 2 5 2 3 9 2 27aH, histology; C, cytology; ER, estrogen receptor; PR, progesterone receptor; NA, not available.

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140 Diagnostic Cytopathology, Vol 26, No 3

clinically relevant from clinically irrelevant discrepancies.We have analyzed two sets of scores: initial scores from thepatients’ reports and consensus scores obtained by the teamof pathologists who reevaluated the results.

It was previously suggested that discrepancies betweencytology and histology could be ascribed mainly to tissueheterogeneity, alteration of hormone receptors during thestorage period or during the assay, methodologic pitfalls,use of different antibodies for immunocytochemical andimmunohistochemical assays, and failure of antigen re-trieval by microwave irradiation.5 In our study, tissue het-erogeneity (different tissue sampling) was the cause of 5 of30 (17%) clinically relevant discrepancies between cytologyand histology, seen in only 5 of 130 (4%) tests in toto. Withcytology, there is a potentially serious concern about false-negative ER and PR tests, which may result from differentor inadequate tissue sampling. We found that only 3% offalse-negative PR tests and no false-negative ER tests inFNA preparations were due to sampling error.

The most important cause of clinically relevant discrep-ancies in our study was not the technical aspects of immu-nostaining or tissue selection for the test but overly enthu-siastic scoring by cytopathologists. The cytopathologistsaccepted technically suboptimal slides as valid for scoring.These included slides with less than 100 nuclei and air-driedspecimens. The FNA material in our study was unselectedand representative of daily practice, which also includesmaterials obtained by residents. One hundred nuclei hasbeen used as a cutoff point for scoring of steroid receptorexpression in breast carcinoma in the past and is recom-mended by several groups.1,2,7 In one study, only specimenswith more than 200 nuclei were acceptable, and, in theother, 500 cells were evaluated for each specimen.3,5

Our study shows that a recommendation to use at least100 nuclei for evaluation should be followed. The concor-dance between cytology and histology was much better forconsensus scores than for scores from the reports. Theconsensus scores were superior to the scores from the re-ports because specimens with a small number of nuclei werenot accepted for evaluation and areas with air-drying arti-facts were not included in the scoring. The concordance ofthe scores obtained from the reports, however, compareswell with the previously published results of several groups,and the concordance did not appear to differ significantlybetween studies that used 100 or 500 nuclei for scoring.1,5

Proper attention to specimen collection, handling, andprocessing is mandatory for the determination of ER and PRstatus.4 This is particularly true for cytologic specimens.The use of various fixatives for cytologic preparations wasstudied by Hudock et al.2 Even though slides can be storeddried before staining, we believe that they should not be driedbefore fixation. Air-drying is known to cause deterioration ofERs and PRs.6 ThinPrep specimens also were used success-

fully for ER/PR determination, but this method includes fixa-tion of the cells before they are exposed to drying.14,15

Despite relatively strict technical standards for successfulER/PR determination in histology and cytology, our resultsshowed that, ultimately, it was the interpretation of theresults that caused most clinically relevant discrepancies.Immunocytochemical study of FNA specimens was foundto be a good test for the determination of ER and PR statusin breast carcinoma, with a sensitivity of 93% for ER and71% for PR. False-positive results for ER and PR status inbreast carcinoma should not occur in FNA specimens ifstrict guidelines for interpretation are followed. We believethat there is no need for the confirmation of positive resultsusing histologic specimens.

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