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Indian Journal of Cancer, Vol. 47, No. 2, April-June, 2010, pp. 148-150 Original Article Estrogen and progesterone hormone receptor status in breast carcinoma: Comparison of immunocytochemistry and immunohistochemistry Radhika K, Prayaga AK Department of Pathology, Nizam's Institute of Medical Sciences, Hyderabad, India Correspondence Address: Dr. K Radhika, Department of Pathology, Nizam's Institute of Medical Sciences, Hyderabad, India kotturadhika@yahoo.com Code Number: cn10038 PMID: 20448377 DOI: 10.4103/0019-509X.63006 Abstract Context : Estrogen receptors (ER) and progesterone receptors (PR) play a significant role in the prognosis of breast cancer. For preoperative chemotherapy in locally advanced lesions, trucut biopsy is used to localize the ER and PR receptors by immunohistochemistry. Immunocytochemistry can be a better alternative to immunohistochemistry as it better fixes cells. Keywords: Breast cancer, Estrogen receptors: estrogen, Progesterone receptors: progesterone, immunocytochemistry, immunohistochemistry Introduction The introduction of immunocytochemistry opened the gateway to a new era of pathology, in which immunolocalization of specific cellular antigens has moved from being a purely scientific research tool to becoming an invaluable diagnostic adjunct in most pathology laboratories. [1] Some tumors, notably carcinoma of the breast and prostate, are often responsive to hormones, a property that has been exploited by the use of endocrine surgery and, medically, using drug therapy to influence hormone levels or inhibit the effects of hormones on tumor cells. Trucut biopsy is currently used to determine the locally advanced lesions for a preoperative chemotherapy. Formalin may destroy some epitopes in paraffin-embedded tissues and hence immunocytochemistry can be a better alternative as it results in more adequate fixation of cells. The present study was carried out to evaluate the degree of correlation between immunocytochemical (ICC) and immunohistochemical (IHC) determination of estrogen receptors (ERs) and progesterone receptors (PRs) in breast cancer. Materials and Methods Fine needle aspiration cytology (FNAC) of 100 primary breast cancers immunostained for ER and PR during a period of 1 year 7 months, i.e. January 2006-July 2007, formed the material of the study. Papanicolaou-stained slides were taken from the period of January 2006 to April 2007, destained, fixed in cold acetone and submitted for ICC markers ER and PR. Successive FNAC smears were straightaway fixed in cold acetone and submitted for ER and PR. The cases were included from May 2007 to July 2007, a 3-month period. For immunocytochemistry, the technique adopted was peroxidase, antiperoxidase. Monoclonal antibodies were used from manufacturer Biogenex. ERiAb No. 272M-ASR, PR: mouse monoclonal antibody (clone; PR 88). The principle adopted was biotin-streptavidin IHC detection system in combination with antigen retrieval pretreatment with 0.9 M Tris HCl buffer in a decloaking chamber at 120 o C. The scoring was performed according to the Allred Score System, [2],[5] which includes proportion score and intensity score to get the total score. The numbers of calculated cells were 100. The proportion score is calculated by the number of positively stained tumor cells. The intensity score represents the staining intensity of positive tumor cells. Statistics Sensitivity and specificity were calculated for ER and PR. ER showed 33% sensitivity, 75% specificity, 67% +ve predictive value, 43% -ve predictive value and 50% concordance. PR showed 25% sensitivity, 33% specificity, 33% +ve predictive value, 25% -ve predictive value and 29% concordance. Results A total of 100 cases were studied on FNAC smears with immunocytochemistry and compared with immunohistochemistry for estrogen and progesterone receptors. The numbers of cases suitable for study were 76 among the total 100 cases. Of these, 24 (confirmed) expressed hormone receptors and 24 cases were labeled as invalid because of the improper staining. After considering both tests of the 24 positive cases, both ER and PR were positive in five cases, ER was positive in only three, PR in one and both were negative in nine cases. Among individual receptor analysis, the total number of ER-positive cases were 20 (27%) [Figure - 1] and negative cases were 56 (73%). In the present study, cases comparable on immunocytochemistry vs. immunohistochemistry were 24 [Table - 1]. False-negative cases in ICC were nine and in IHC were three. But, false-negative cases were not due to the use of destained smears. In unstained smears, eight (40%) were ER positive and 30 (55%) were ER negative. In destained smears, 12 (60%) were ER positive and 25 cases were (45%) ER negative. More number of ER-positive cases were observed in destained smears while more number of ER-negative cases were observed in unstained smears. Total number of PR +ve cases were 10 (27%) [Figure - 2], destained six (60%) and unstained four (40%). Total number of PR -ve cases were 27 (73%), destained 10 (37%) and unstained 17 (63%). Differences between groups were analyzed and correlations were assessed by the Spearman Rank Correlation test. Sensitivity and specificity were calculated for ER and PR. ER showed 33% sensitivity, 75% specificity, 67% positive predictive value, 43% negative predictive value and 50% concordance. PR showed 25% sensitivity, 33% specificity, 33% positive predictive value, 25% negative predictive value and 29% concordance [Table - 2]. Discussion In the present study, among 76 valid cases, total number of ER-positive cases was 20 (27%) and negative cases was 55 (73%). Total number of PR-positive cases was 10 (27%) and negative cases was 27 (73%). Our sensitivity of 33% for ER was much lower than that reported by Nizzoli et al. [3] Immunocytochemistry is a well-accepted technique to determine the ER and PR status in the treatment of breast cancer. Several studies are available in the literature. The crucial steps involved are fixation and antigen retrieval in a pressure cooker in immunocytochemistry as well as immunohistochemistry. The fixatives used in the present study were 4% buffered formalin, cold acetone, ether alcohol and destained Papanicolaou smears. Antigen retrieval was performed in 0.9 M Tris-HCl buffer in a decloaking chamber at 120°C. A mixture of acetone and methanol was used by Malaviya et al. for fixation of their cytology smears, all of which were freshly made, and they used sodium citrate retrieval as compared to Tris buffer in our study, which could be the reason for the lower positivity rate documented by us. It is known that antigen retrieval is the most crucial step in documentation of ER/PR receptors in breast cancer. This derogatory step can be removed in immunocytochemistry. Although a superior antigen retrieval solution like ethylenediammine tetraacetic acid (EDTA) is preferred, sodium citrate is used in most of the Indian laboratories because EDTA is expensive. Many Indian authors have suggested using a pressure cooker for heating compared to a microwave on account of financial constraints, although microwave heating is proved to give better results in the west. Lowering the cut-off points for reporting hormone receptors will help in reducing the false-negatives for hormone receptors in breast cancers. PR serves as a check of ER testing and helps in quality control of the ER antigen. The concordance between cytology and histology was 84% for ER and 90% for PR in the study by Malaviya et al.[6] Both the positivity rates and the staining intensity scores were higher for cytochemistry than for histochemistry. We agree with Shet et al.[7] that participation in an external quality assurance scheme for immunocytochemistry, including an audit of the percentage-positive tumors in a laboratory against the accepted norms, annually is needed. Hormone receptor expression in breast cancers in India is lower, but 32% positivity reported by us may be due to a play of technical factors. Suboptimal assays can be the etiological factors for erroneous results before considering the genetic differences. Although antigen retrieval is not a problem in immunocytochemistry, the present study could not give expected results. The chief causes for lower results in this study are use of improper antigen retrieval techniques. Participation in external quality assessment schemes for immunocytochemistry will help laboratories. The crucial question that remains unanswered in the literature is if a case is immunocytochemistry-positive and immunohistochemistry-negative, should the patient receive benefit of hormonal therapy? Studies in the future should seek to answer these questions. References
Copyright 2010 - Indian Journal of Cancer The following images related to this document are available:Photo images[cn10038t3.jpg] [cn10038t1.jpg] [cn10038t4.jpg] [cn10038t2.jpg] [cn10038f1.jpg] |
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