Potential Value of Hormone Receptor Assay in Carcinoma In Situ of Breast

Transcription

1 Potential Value of Hormone Receptor Assay in Carcinoma In Situ of Breast ROBERT BARNES, M.D. AND SHAHLA MASOOD, M.D. The estrogen receptor (ER) expression of invasive breast cancer has been extensively studied both biochemically and with specific monoclonal antibodies against ER. Relatively few studies have attempted to characterize ER pattern in breast carcinoma in situ (CIS) and in other premalignant lesions. In the current study, the authors investigated the pattern of ER expression in 62 cases of breast CIS, 30 of which had a component of invasive cancer, and 36 cases of atypical hyperplasia. Paraffin sections of formalin-fixed breast tissue underwent enzyme pretreatment to expose nuclear antigenic sites as previously described. Breast tissues then underwent estrogen immunocytochemical assay using specific monoclonal antibodies (Abbott Laboratory, Chicago, IL). The cases were evaluated for heterogeneity, intensity of staining, and percentage of positive cells. An attempt was made to study the relation between the pattern of ER expression, nuclear pleomorphism, and type of CIS. The results of ER immunocytochemical assay showed positive nuclear staining for ER in 75% of the CIS, 73% of CIS with invasive cancer, and 100% of atypical hyperplasias. ER expression in CIS agreed with that in the invasive carcinoma in 29 of 30 cases. This study also suggests that comedocarcinoma has a higher incidence of negative ER expression than the other types of CIS, particularly when it is associated with significant nuclear pleomorphism. There was no significant difference in ER tumor heterogeneity between premalignant and malignant lesions. (Key words: Estrogen receptor; Carcinoma in situ; Atypical hyperplasia; Breast cancer) Am J Clin Pathol 1990;94: THE DEVELOPMENT and use of estrogen receptor (ER) assays has led to significant improvements in the therapy ofbreast cancer. Characterizing a carcinoma of the breast by ER status can aid in predicting the aggressiveness of the tumor The presence of ER is associated with favorable cell kinetics and prognosis for the patient.' 01 Those patients with positive tumors, amenable to hormone therapy, may be treated for prolonged periods with endocrine therapy and avoid the undesirable side effects associated with cytotoxic chemotherapy. 19 The profound influence estrogen receptors may have on the progression and outcome ofbreast carcinoma make it a compelling factor to assess in premalignant lesions. Received December 1, 199; received revised manuscript and accepted for publication February 23, This paper was presented at the ASCP/CAP Fall Meeting, Washington, D.C., October 2-November 3, 199, and the 12th Annual San Antonio Breast Cancer Symposium, San Antonio, Texas, December -9, 199. Address reprint requests to Dr. Masood: Department of Pathology, University Hospital of Jacksonville, 655 W. th Street, Jacksonville, Florida Department of Pathology, University of Florida Health Science Center, Jacksonville Division, Jacksonville, Florida Extensive studies have provided information regarding ER expression in invasive breast cancers 24 " 6 "; however, relatively few reports have attempted to characterize ER expression in preinvasive breast lesions such as those described by Dupont and Page. 3 Their epidemiologic studies indicate an elevated risk of invasive breast cancer associated with atypical hyperplasia (moderately increased risk: four to five times normal) and carcinoma in situ (high risk: eight to ten times normal). In the recent study, we examined the patterns and degrees of ER positivity in breast carcinoma in situ without an invasive component (CIS), CIS with infiltrating breast cancer (CIS/CA), and atypical hyperplasia (AH) to discern whether there were any distinguishing differences in ER expression between preinvasive lesions (CIS and AH) and CIS/CA. We studied ER expression using monoclonal antibodies, which not only have a high degree of concordance with traditional, cytosolic steroid binding assays 6914 but also offer some unique advantages. The antibody is highly specific and sensitive 5 for nuclear ER and allows ER detection without interference by estrogen, antiestrogens, or other steroid-binding proteins. 15 The use of monoclonal antibodies is ideal on fine-needle aspiration biopsy or when breast lesions are too small for conventional estrogen assays. 1 U2 It provides a further advantage in that the histologic characteristics, as well as the degree of heterogeneity of estrogen receptor positivity, can be simultaneously evaluated. 19 Because no sophisticated equipment is needed and the technique is relatively easy, immunohistochemistry is adaptable to the community hospital." Materials and Methods Paraffin blocks from 62 cases of breast CIS (30 cases with a component of infiltrating carcinoma) and 36 cases of atypical hyperplasia were obtained from the files of the surgical pathology department of University Hospital of Jacksonville, Florida. The reagents used in the immunoperoxidase procedure (including blocking reagents from primary antibody negative control, bridging antibody, peroxidase-antiperoxi- 533

2 534 BARNES AND MASOOD A.J.C.P. November 1990 dase complex [PAP] substrate, and substrate buffer) were obtained from commercially available kits (ICA monoclonal; Abbott Laboratories Chicago, IL). The slides were prepared from paraffin blocks using a method previously described. 2,20 The tissue was cut in 5-fim sections and placed on adhesive slides provided in the ICA kit. They were deparaffinized with toluene and dehydrated through gradient alcohols. Appropriate phosphate-buffered saline (PBS) rinses were performed. All incubations were at room temperature. Protease was applied to each slide and allowed to incubate for 9 minutes. The slides were rinsed, and normal horse serum was used as a blocking agent before primary antibody, bridging antibody, and PAP solutions. The slides were incubated for 10 minutes, and then the primary antibody was applied and incubated overnight. After incubation the bridging antibody was applied and incubated for two hours. The PAP complex was applied and incubated for two hours. The chromogen, diaminobenzidine (DAB), was applied and allowed to react for 10 minutes. Finally, 2% (w/v) osmium tetroxide was used to enhance the DAB reaction, and the slides were counterstained with methyl green. Specimens with a minimum of 20% malignant cells staining positively were considered ER positive. 7 All ERpositive cells exhibited brown nuclear staining. The intensity of staining was evaluated on a scale of The strongest nuclear staining equal to or greater than that of normal breast epithelium was considered 3+, moderate brown staining 2+, and light nuclear staining 1+. Cells without any staining or those showing only minute trace reactions were read as negative. Three hundred cells were characterized in each slide. For semiquantitative analysis, the staining intensity was multiplied by the fraction of positive cells, and this numeric value, the ICA score," was determined for each lesion. The pattern, intensity of ER positivity, and nuclear pleomorphism were evaluated independently by two pathologists (S.M. and R.B.). The pattern was characterized as diffuse if more than 70% cells stained ER positively versus focal if less than 70% cells stained positively, and homogeneous if more than 70% cells stained with optically equal intensity versus heterogeneous if less than 70% cells stained with optically equal intensity. Nuclear pleomorphism was evaluated with hematoxylin and eosin- (H and E) stained slides on the basis of variation in size, chromatin pattern, and hyperchromatism. They were then graded as follows: O no pleomorphism; 1 mild; 2 moderate; and 3 severe nuclear pleomorphism. For semiquantitative analysis, nuclear pleomorphism was graded as 1-3 and multiplied by the fraction of pleomorphic cells to determine the degree of nuclear pleomorphism. Results Positive staining for estrogen receptor proteins was limited to the nucleus of the epithelial cells (normal, hyperplastic, and carcinoma). Seventy-five percent of CIS, 73% of CIS/CA, and 100% of atypical hyperplastic lesions displayed ER positivity. The ICA score was highest in AH, intermediate in CIS, and lowest in CIS/CA and comedocarcinoma in situ (Table 1). A diffuse/heterogeneous pattern was the most frequent finding in all lesions (CIS 34% [Fig. 1], CIS/CA 53%, and atypical hyperplasia 55%). All lesions showed a remarkable degree of ER heterogeneity within each lobule as well as between lobules or portions of tumor. The premalignant lesions displayed a focal pattern of ER (CIS 41%, AH 44%) nearly twice as often as lesions with invasion (CIS/ CA 23%) but with the same degree of heterogeneous staining. The homogeneous pattern was rarely encountered and, when identified, was nearly always the expression of a lesion with low aggressiveness. In most (90%) of the cases with both CIS and invasive CA, the percentage of ER positivity, pattern, and intensity of positive staining were equivalent in the CIS and invasive components (Fig. 2). Nuclear pleomorphism was evaluated as a separate variable and found to correlate inversely with ER positivity in CIS and CIS/CA. A similar inverse relationship has also been described in invasive carcinoma of the breast. 17 Comedocarcinoma in situ was specifically examined as a subset of CIS. ER negativity was found in 46% (6 of 13 cases). A diffuse heterogeneous pattern was most commonly seen (3%) in positive comedocarcinoma. ERpositive comedocarcinoma in situ had a higher degree of nuclear pleomorphism than positive CIS of other types (solid cribriform, etc.) and closely resembled negative cribriform and solid CIS in terms of nuclear pleomorphism. negative comedocarcinoma had the highest degree of nuclear pleomorphism (Table 2). Table 1. Summary of Estrogen Receptor Status and Staining Pattern in Atypical Hyperplasia and Carcinoma In Situ Atypical hyperplasia Carcinoma in situ CIS/CAt Comedo-CIS n %ER * Estrogen receptor immunocytochemical assay. t CIS/CA = carcinoma in situ with invasive cancer. % Diffuse % Focal ICA*

3 Vol. 94 No. 5 ER PATTERN IN BREAST CARCINOMA IN SITU 535 FIG. 1. Most lesions displayed a diffuse and heterogeneous staining pattern for ER. There was wide variation of both interlobular and intralobular staining. ICA, counterstain with methyl green (X200). FIG. 2. Most invasive carcinomas displayed the same ER staining pattern and level of intensity as the adjacent CIS component. ICA counterstain with methyl green (X100). Increasing age in patients with CIS was directly related to ER positivity and inversely related to nuclear pleomorphism (Table 3). This is consistent with studies of breast cancer that have shown nuclear pleomorphism inversely related to ER content and patient age.17 In the current study, the patient population was categorized by age rather than menopausal status, because studies have shown ER expression is a characteristic of the tumor in- dependent of circulating estrogen levels.13 In our study invasive cancer was found more frequently in patients with CIS who were younger than age 55 (14 of 23) than those 55 and older (16 of 39). Atypical hyperplasia exhibited ER positivity in all cases (100%, n = 36). The pattern was most often heterogeneous and diffuse, with wide variations between lobules and within lobules. The patterns in atypical hyperplasia closely

4 536 BARNES AND MASOOD A.J.C.P. November 1990 resembled those of CIS. The nuclei exhibited minimal pleomorphism. There were no significant differences in any of the observed values between younger and older age groups. It is interesting that apocrine metaplasia was consistently ER negative in 21 slides examined. A loss of ER positivity could be traced in areas where ductal epithelium was in the process of undergoing apocrine metaplastic change. Table 3. Summary of Estrogen Receptor (ER) Status and Nuclear Pleomorphism by Age Groups AGE <55 ClSf CIS/CAt >55CIS CIS/CA N %ER* ICA* Nuclear Pleomorphism Discussion The results of this study suggest the dynamics of ER expression previously described in breast carcinoma 17 may apply to breast CIS as well. The ER staining intensity has been shown to be a quantifiable feature that correlates significantly with the ER content by cytosol measurement. The percentages of positive CIS and CIS/CA were equivalent (75% and 73%, respectively). However, the staining intensity reflected by the ICA score was generally greatest in the least aggressive lesions (AH and CIS) and lowest in the more aggressive lesions (CIS/CA). As with breast cancer, 17 the ER expression in our study of breast CIS is generally increased with age and decreased with nuclear pleomorphism. In most cases studied, the estrogen receptor status of invasive carcinoma could be predicted by observing ER status expressed in areas of CIS. In 90% (27 of 30) of our cases of CIS with invasive cancer, the pattern and intensity of the invasive component was similar to that of the CIS component. The three exceptions were one case of ERnegative comedocarcinoma giving rise to an positive ductal carcinoma and two cases of weakly positive CIS developing into a moderately positive carcinoma. Only one case demonstrated discordance in terms of ER positivity and negativity (a 97% concordance rate). Table 2. Summary of Nuclear Pleomorphism Related to Estrogen Receptor (ER) Status in Atypical Hyperplasia (AH) and Carcinoma In Situ (CIS) AH CIS C1S/CA* Comedo-CIS n CIS/CA = carcinoma in situ with invasive cancer. Nuclear Pleomorphism * Estrogen receptor immunocytochemical assay. t Carcinoma in situ. % Carcinoma in situ with invasive cancer. The discrepancies may possibly be explained by regional differences in relative tissue hypoxia or lack of other nutrients. Studies have shown that positive breast tumor cells grown in nutrient-deficient media may lose their ER expression until the deficient nutrient is resupplied. Alternatively, these discrepancies may be associated with tumor heterogeneity. Heterogeneous expression of ER in normal breast epithelium and invasive tumors with the use of monoclonal antibodies has previously been described. 419 Simultaneous investigations of multiple, separate areas of the same tumor mass using biochemical receptor assays have demonstrated discordance rates (ER positive vs. ER negative) ranging from 12.5% to 32%. I9 Intratumor quantitative values in positive cancers show similar discordance. 19 Two previous studies have characterized ER expression in atypical hyperplasia and breast CIS using monoclonal antibodies. Fabris and colleagues 4 studied 150 cases each of benign and malignant breast lesions. CIS and atypical hyperplasia of breast expressed homogeneous ER patterns. Normal breast epithelium and invasive breast cancer stained heterogeneously. The differences in the patterns of ER staining in CIS may be related to the use of different antibodies. Bur and Chowdhary, 1 using a technique similar to that of the current study, reported ER expression predominately diffuse in CIS. ER expression was frequently negative in comedocarcinoma 1/36, especially when associated with nuclear pleomorphism, a finding supported by our study. In the current study ER expression in CIS with and without invasive carcinoma was predominately diffuse and heterogeneous. The degree of nuclear pleomorphism observed is generally inversely related to ER positivity. In comedocarcinoma this relationship is so strong, our study indicates that nuclear pleomorphism may serve as one potential indicator of ER expression. The ER expression of carcinoma could be predicted from the ER expression in the associated CIS lesion with such consistency that a discordant ER expression between a biopsy of CIS

5 Vol. 94. No. 5 and subsequent invasive carcinoma necessitates additional study to evaluate the heterogeneity of ER expression within the tumor and to establish true ER status. References ER PATTERN IN BREAST CARCINOMA IN SITU Bur M, Chowdhary S. Estrogen and progesterone receptor immunohistochemistry in carcinoma in situ of the breast with paraffin embedded sections. Abstract presented at the International Academy of Pathology Annual Meeting, San Francisco, March Cheny L, Binder S, Fu Y, Lewin K. Demonstration of estrogen receptors by monoclonal antibody in formalin fixed breast tumor. Lab Invest 19.5: Dupont WD, Page OL. Risk factors for breast cancer in women with proliferative breast disease. N Engl J Med 195;312: Fabris G, Marchetti E, Morgola A, Bogni A, Quezoli P, Nenci I. Pathophysiology of estrogen receptor in mammary tissue by monoclonal antibodies. J Steroid Biochem 197;27: Ghosh L, Ghosh BC, Dasgupta TK. Immunocytological localization of estrogen in human mammary carcinoma cells by horseradishanti-horseradish peroxidase complex. J Surg Oncol 197; 10: Greene GL, Jensen EB. Monoclonal antibodies as probes for estrogen receptor detection and characterization. J Steroid Biochem 192;16: Hayward JL, Rubens RD, Carbone PP, Heuson J-C, Kumaoka S, SegalaffA. Assessment of response to therapy in advanced breast cancer. A project of the Programme on Clinical Oncology of the International Union Against Cancer, Geneva, Switzerland. Br J Cancer 1977;35: Jakesy R, Smith CA, Aitken S, et al. Influence of cell proliferation and cell cycle phase on expression of estrogen receptor in MCF- 7 breast cancer cells. Cancer Res 194;44: King WJ, DeSombre ER, Jensen EV. et al. Comparison of immunocytochemical and steroid binding assays for estrogen receptors in human breast tumor. Cancer Res 195;45: Kute TE, Muss HB, Anderson D, Crumb K. et al. Relationship of steroid receptor, cell kinetics and clinical status in patients with breast cancer. Cancer Res 191;41: Masood S. Use of monoclonal antibody for assessment of estrogen receptor content in fine-needle aspiration biopsy specimens from patients with breast cancer. Arch Pathol Lab Med 199:113: Masood S, Johnson H. The value of imprint cytology in cytochemical detection of steroid hormone receptors in breast cancer. Am J Clin Pathol 197;7: Mass H, Engel B, Trems G. Steroid hormone receptors in human breast cancer and the clinical significance. J Steroid Biochem 1975;6: McCarthy KS Jr, Miller LS, Cox EB, et al. Estrogen receptor analysis. correlation of biochemical and immunohistochemical methods using monoclonal anti-receptor antibodies. Arch Pathol Lab Med 195;109: Meredith JT, McBride RC, Cerejo L. Estrogen receptors in breast cancer. J Fla Med Assoc 19;75: Meyer JS, Baier WC, Rao BR. Subpopulation of breast carcinoma defined by S-phase fraction, morphology and estrogen receptor content. Lab Invest 197;39: Meyer JS, Lee JY. Relationships of S-phase fraction of breast carcinomas in relapse to duration of remission estrogen receptor content, therapeutic responsiveness and duration of survival. Cancer Res 190;40: Meyer JS, Rao BR, Stevens SC, White WL. Low incidence of estrogen receptors in breast carcinomas with rapid rates of cellular replication. Cancer 197:40: Osborne CK. Heterogeneity in hormone receptor status in primary and metastatic breast cancer. Semin Oncol 195:12: Shintaku IP, Said JW. Detection of estrogen receptors with monoclonal antibodies and routinely processed formalin fixed paraffin sections of breast carcinoma. Am J Clin Pathol 196;7: