[CANCER RESEARCH 51. 239-244. January I, 1991) Immunocytochemical Determination of Estrogen Receptor, Progesterone Receptor, and 1,25-Dihydroxyvitamin D3 Receptor in Breast Cancer and Relationship to Prognosis1
Uta Berger, Richard A. McClelland, Patricia Wilson, Geoffrey L. Greene, Mark R. Haussler, J. VV.Pike, Kay Colston, Douglas Easton, and R. Charles Coombes2 Ludwig Institute for Cancer Research (London-St. George's Group) [L'. B., R. A. M., P. H'.. R. C. C.J and Department of Chemical Pathology [K. C.]t St. George's Hospital Medical School, Cranmer Terrace, London SH 17 ORE, L'nited Kingdom: University of Chicago, The Ben May Institute, Chicago, Illinois 60637 [G. L. G.j; Department of Biochemistry, University of Arizona, Tucson, Arizona 85721 ¡M.R. //./; Baylor College of Medicine, Houston, Texas 77030 ¡J.W. P.]; and Section of Epidemiology, Institute of Cancer Research, Sutton, Surrey SM2 5PX, L'nited Kingdom [D. E.]
ABSTRACT breast cancer specimens using biochemical methods is limited due to the amount of tissue required for such assays. In many We have determined the estrogen receptor, progesterone receptor cases sufficient material is available for measurement of only (PR), and 1,25-dihydroxyvitamin I), receptor content of 136 breast one receptor. The utilization of recently developed monoclonal carcinomas by an immunocytochemical method. The presence of the antibodies to both ER and PR has led to the development of three receptors was not related to clinical features of presentation such as T-stage or to age or menopausa! status. However, each of the three immunocytochemical techniques for the visualization of these receptors has a different relationship to the course of the disease in these two receptors in tissue sections. This method has been shown patients. The presence of PR was significantly associated with an im to correlate well with biochemical assays and can be applied proved overall survival (v = 4.61, /' = 0.032). Patients whose tumors both to frozen sections and fine needle aspirates of breast cancer contained immunocytochemically detectable 1,25-dihydroxyvitamin I). and to normal breast tissue and bone marrow aspirates (9-12). receptor had a longer disease-free interval than those patients with We have shown previously, using a recently developed im negative tumors (x2 = 4.01, P = 0.045). The presence of estrogen receptor munocytochemical technique (13), that approximately 80% of and PR were found to correlate with an increased survival between relapse and death (/' = 0.027 and /' = 0.09, respectively). The relationships breast tumors demonstrate the presence of l,25(OH)2DiR. Furthermore the active hormonal form of vitamin D., is known between estrogen receptor and PR and prognosis are more apparent when to inhibit cell proliferation and promote differentiation in the degree of cell staining is considered. Combined receptor analysis improves our ability to predict the course of the disease and may therefore certain cell lines (14, 15). The clinical significance of facilitate better management of the patients. 1,25(OH)2D.,R status in breast cancer is not yet known. We have studied a group of women with breast cancer and determined the ER, PR, and 1,25(OH)2D.,R using immunocy INTRODUCTION tochemical assays. We now report the analysis of correlation between receptor Steroid receptors are important markers for hormone de status of the primary tumor or metastasis and outcome of the pendence or responsiveness in breast cancer. The knowledge of the presence of ER-' and PR status of breast tumors is a useful disease. means of predicting response to endocrine therapy (1, 2), and patients with ER negative tumors are less likely to benefit from such treatment. However, it has been reported that only around MATERIALS AND METHODS 59% of patients with appreciable levels of ER show objective response to endocrine therapy and the presence of PR in breast Patients cancer is more indicative of the hormone responsiveness of the tumor (3). One hundred thirty-six patients diagnosed with primary breast cancer The presence of PR has been shown to give a more reliable were studied between 1979 and 1984. A tissue sample from each indication of predicting disease free survival time in the lymph patient's primary tumor was immediately snap frozen in liquid nitrogen node positive group of patients with breast cancer (4). Further and stored in the vapor phase of a liquid nitrogen bank. more there are reports that PR may be of more value in Of the studied group, the age of the patients ranged between 32 and predicting time of relapse (5) while the absence of PR is 85 years. Twenty-seven patients were premenopausal; 106 were post- associated with tumor aneuploidy (6). Less differentiated tu menopausal. In three cases data were not available. When these patients were grouped according to their tumor (T)-stage, in 26 patients no data mors show significantly lower levels of ER and PR (7). ER and were available, and of the remaining patients 20 had T, tumors, 57 had PR levels and the nuclear grade of tumor are independent T2 tumors, 23 had T3 tumors, and 10 had T4 tumors. markers in breast cancer, but a more accurate prediction may Fifty-six patients were lymph node negative tumors, 63 had lymph be made when these are combined (8). node involvement (35 «3 and 26 > 3) in 17 cases lymph nodes were At the present time the determination of steroid receptor in not investigated. A total of 52 relapses and 32 deaths have occurred in the study population after a median follow-up of 4 years. Received 2/3/89; accepted 10/22/90. The costs of publication of this article were defrayed in part by the payment Nineteen of the patients (14%) had received some adjuvant chemo of page charges. This article must therefore be hereby marked advertisement in therapy, but none had received any form of adjuvant hormonal therapy. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Concerning treatment at relapse, all patients with non-life threatening 1Supported in part by the Cancer Research Campaign. visceral disease had therapy using tamoxifen. After relapse, aminoglu- 2To whom requests for reprints should be addressed. ' The abbreviations used are: ER. estrogen receptor; PR. progesterone receptor: tethimide was given until second progression. Patients with progressive l,25(OH)2DjR, 1,25-dihydroxyvitamin Dj receptor: SII, staining intensity index; visceral disease or those who had relapsed after two forms of endocrine PBS, phosphate buffered saline. therapy were treated with combination chemotherapy. 239
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Methods RESULTS
The rat monoclonal antibody H222 recognizing the human ER was Receptor Analysis. Ninety-six of the 136 patients (71%) had used in the form of a kit (Abbott) to demonstrate the ER immunocy- ER-positive tumors, 71 of 134 (53%) had PR positive tumors, tochemically (16). H222 was raised in male Lewis rats, which were and 123 of 136 (90%) showed positive immunoreactivity for immunized with ER receptor complex derived from the human breast 1,25(OH)2D3R receptor (Table 1). cancer cell line MCF-7 (17). The combined receptor status of ER, PR, and 1,25(OH)2D.,R The monoclonal antibody KD68 (kindly supplied by G. Greene, is shown in Table 2. The distribution of the steroid hormone Chicago, IL) has been utilized for PR detection. KD68 is a rat IgG2a receptors according to the percentage of stained cells and ad monoclonal antibody which recognizes the occupied and unoccupied ditionally for PR and ER according to the staining intensity PR in the target cell. It was raised to PR partially purified from T47D index is displayed in Tables 3 and 4, including 2 patients where cells and detects both the A and B subunits of PR (18). insufficient material allowed only the ER and 1,25(OH)2D3R For visualizing the 1,25(OH)2D3R the rat monoclonal antibody 9A77 assay. None of the three receptors assessed was associated with was introduced (kindly provided by M. R. Haussler). This antibody, an IgG2b subclass, was raised against chick intestinal 1,25(OH)2D3R (19, age, menopausal status, T stage, and lymph node involvement 20). at presentation. Staining Procedure. The methods of staining for the antibody to ER Overall Survival. ER positivity is only marginally signifi and 1,25(OH)2D.,R have been described previously (20, 21). The stain cantly related to overall survival (Table 5; P = 0.063). If ER ing for PR follows the same method. The procedure includes the positive patients are divided into those with SII less than and modified PAP technique after Sternberger (22). greater than 2, there is a somewhat more significant association Briefly, 4 cryostat sections of each specimen (4-8 ^m) were prepared between ER and the overall survival (trend test x2 = 3.98; P = on poly-L-lysine coated glass slides. The sections were fixed for 10 min 0.046; Fig. 1). After adjustment for T stage and nodal involve in 3.6% formaldehyde in 0.1 M PBS (pH 7.2) immediately after sec ment, however, this effect was not significant. Simple measure tioning, then transferred to a PBS bath for 10 min and placed in cold methanol (-20°C) for 4 min followed by cold acetone (-20°C) for 1 ment of ER by percentage of cells stained does not help define prognosis. Immunocytochemically detectable 1,25(OH)2D3R is min, and then washed in PBS. Slides were incubated for 15 min with not related to overall survival (P > 0.1; Fig. 2). 2% normal goat serum in PBS. The slides were then incubated with The PR status showed the clearest evidence of influence to the appropriate first antibody: one with H222 antibody; one with KD68; and one with 9A7-y antibody. The fourth slide was used as a control the overall survival time. Patients with PR positive tumors have a significantly longer survival time than PR negative patients. and was incubated with normal rat immunoglobulin. H222 antibody is (x2 = 4.61, P = 0.032; Fig. 3). Furthermore the degree of PR undiluted as part of the kit. The PR antibody was used in a concentra tion of 5.11 Mg/ml and 9A7-y was diluted 1:750 in PBS/bovine serum immunoreactivity as assessed by increasing percentage of albumin. Slides were incubated for 0.5 and 1 h, respectively and then stained tumor cells (<50% or >50%) was positively related to washed in PBS, followed by the indirect peroxidase-antiperoxidase the survival time (trend x2 = 7.05; P = 0.008; Fig. 4). The procedure. The receptors were visualized by diaminobenzidine hydro- evaluation of the SII (>2 and <2) showed similar significant chloride:hydrogen peroxide chromogen substrate reaction. The slides correlation (P trend = 0.015). These differences remain mar were counterstained in 1% (v/v) Harris hematoxylin in distilled H2O ginally significant after adjusting for T stage and nodal involve for 5 min. Slides were dehydrated and mounted in a xylene soluble ment (e.g., P = 0.05 for trend in survival with increasing SII). mountant. Disease Free Survival. There is no evidence of an effect of This staining procedure showed a positive reaction as light to dark either ER status or PR status, assessed either by percentage of brown precipitate in the nuclei of the tumor cells. cells stained or SII, on the time between first tumor detection Evaluation. The appearance of any staining was counted as positive. and relapse. There is, however, some suggestion that disease The reaction was semiquantitatively assessed by estimation of the free survival is related to the 1,25(OH)2D.,R status of the tumor; number and the intensity of stained tumor cells per slide and a SII was the few patients with 1,25(OH)2D,R negative tumors relapsed calculated (10). In this way, 4 groups of reaction were observed: 0, no significantly earlier than the receptor positive group (x2 = 4.01, reaction; +, weak reaction; ++, moderate reaction; +++, strong reac P = 0.045; Fig. 5). This does not depend on the amount of tion. The reaction of the 1,25(OH)2D3R was only reported in the percent immunoreactivity for 1,25(OH)2D3R present in positive tu age of stained tumor cells, because the reacting cells were, in general, mors. There is no suggestion of a trend in disease free survival relatively homogeneous and often weakly stained. with increasing percentage of cells stained (divided into 0, <50%, and >50% (trend x2 = 0.52 P > 0.1). The difference Statistical Analysis. The principal analyses in this study were of the effect of each receptor assay on overall survival, disease free survival becomes nonsignificant after adjustment for T and nodal stage. and survival from first relapse. These were assessed using the Kaplan- Survival from Relapse to Death. The ER status was signifi Meier survival curves and the logrank test (23). For overall survival cantly related to the time between relapse and death (P = 0.027) and disease free survival, entry to the study is taken to be the date of (Table 5). This relationship is more evident if ER positive the primary surgery. AH significance levels are two sided. The effect of patients are divided into those with high SII (>2) and low SII each receptor on prognosis was assessed (a) by comparing patients with (<2) (P trend = 0.007). This was, however, not so clearly no staining (negative patients) and those with any degree of staining demonstrated if percentage (<50% or >50%) stained cells (P (positive patients) and (b) by comparing patients on the basis of per trend = 0.08) is taken into account. PR status is also signifi centage of cells stained (none, <50%, >50%). The 50% boundary was cantly related to the survival from relapse to death when as preferred to the 20% because relatively few patients had 0 to 20%. No sessed on the degree of SII as for ER (P trend = 0.028). There material differences in the results were obtained using a 20% boundary. ER and PR were also evaluated in terms of SII, in the categories of was also a significant relationship with survival from relapse if 0, <2, and >2. PR is analyzed on the basis of percentage of cell staining (0, The logrank analyses were also carried out stratified by T stage and <50%, >50%) (P trend = 0.023; Fig. 6). 1,25(OH)2D,R content nodal involvement to determine whether receptor status were prognos is not related to survival from first relapse. tic factors independent of these known factors. Although the presence of ER and that of PR are associated, 240
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Table 1 Distribution of baseline characteristics in patients positive and negative for each receptor Distribution of steroid hormone receptor content of breast cancers determined by immunocytochemical methods and their relation to clinical stage, age. menopausa! status, and nodal involvement. Positivi!} for each receptor is assumed when any number of tumor cells demonstrated immunoperoxidase staining of the nucleus. receptorTotalAge Estrogen 1Positive76612191722IX57133486332211Negative5833192211947623154221317%receptor receptorNegative13016152114330433'.'< Positive715687725485687370795270737158ProgesteronePositive57678050446767556860.1560606339Positive123914333829259516542110523225,25(OH);D,ofpositive90100938597859390809591100939189
(yr)Under 4040-4950-5960-6970+Menopausal
status*PrePostT
stage*T,T;T,T,Nodes
involved'None1-34+Positive9651328212917771445127412515Negative404211185829612113151011%
°Three cases mcnopausal status unrecorded. * Twenty-six patient tumor (T) stage unrecorded. c Seventeen N stage unrecorded.
Table 2 Distribution of steroid hormone receptors in 136 breast cancers Table 4 Staining intensity index for ER and PR Distribution of immunocytochemically determined steroid receptor content of The staining intensity index distribution of ER and PR as determined immu 136 breast cancers, where all three receptors were simultaneously assessed on nocytochemically. Numbers in parentheses, percentage of tumors assigned to each adjacent time sections. Receptor positivity is assumed when any number of tumor group. Staining intensity index calculations are described in the text but represent cell nuclei were observed to express a specific staining reaction as visualized by a quotient based on both staining intensity and percentage of cells stained. SII indirect immunopcroxidase cytochemistry. for 1.25(OH)2D,R has not been determined due to low intensities of staining for this receptor observed overall. 1.25(OH);Dj receptor indexReceptorER Staining intensity Receptors Positive Negative Total ER+ (%)34(25) (%)14(10) PR+ 68 73 (%)48 PR- 20 22 (30)58 (35) PR0(%)40 (43)<1 35 (26)1-2 35 (26)>2 6(5)Total136(100)134(100) Not done ER-PR+PR-Not DISCUSSION Our study of 136 patients indicates that analysis of tumor done3320123041133361136 steroid hormone receptor content measured by immunocyto chemical techniques can help in judging prognosis in patients with primary breast cancer. We have assessed ER, PR, and 1,25(OH)2D,R and confirm Table 3 Percentage of cells stained for ER, PR, and I,25(OH)2DSK earlier reports that no correlation exists between the presence The percentage of tumor cells stained for each of the three steroid receptors of 1,25(OH)2D,R and ER or PR (13, 24-26). was analyzed immunocytochemically. Numbers in parentheses, percentage of Each of the three steroid hormone receptors gave information tumors found in each category. The percentage of tumor cells is estimated from an assessment of the whole tumor section. regarding a different period of survival. Increased overall sur vival is correlated with the presence of PR and these findings % of cells stained are in accordance with Mason et al. (4) who reported that biochemically estimated PR positivity is predictive of the time ReceptorER to recurrence of disease and of increased overall survival time. (29) (56) PR 58 (43) 30 (22) 46 (34) 134(100) Clark and McGuire (5) have shown a increase in relapse free 1.25
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Table 5 Logrank analysis of overall survival, relapse free survival, and survival from relapse with respect to ER. PR ana l,25(OH)iD,R The logrank analysis illustrâtesthe prognostic significance of ER. PR. and 1,25(OH)¡D,R positivi!) in breast cancer. ERPositive
NegativeOverall Positive140.0631727NS23120.02717L.'•Positive19.8 P P29.2NS-2.847.80.0454.225.8NS4.2j'--3 survivalObserved 23.6Expected 19 270.03211.2
8.4Relapse 13 530.7
free survival Observed37.7Expected 37 44NS19.3
14.3Survival 15 816.5
from re lapse Observed22.3Expected 17 250.0912.5
7.7" 13 5100 NS,no100
- - •-•' "^) 90- 90 - ^_1 •-.~^^3 g 80- g 80- |70-Wo 170-l/lo 60-f 60-1 50-2 50-fOo 40 - 40-C SS 30-20-10 5? 30-20-10-o
-o -Csignificant.S - 4Years 2P (""•-.. 1 4Years 21.25(OH)2D,RNegative
takenFig. since sample takenFig. since sample 1. O ill sun ¡valaccording to the presence and stainings intensity index 3. Pi of overall sun ¡valaccording to the presence or absence of PR.,significantlv PR negative cases; . PR positive cases. The presence of PR is of ER. , ER negative cases, , ER positive cases with SII <2, , related4.61,^i to a longer overall survival. Trend test significant (x2 = ER positive cases with SII >2. A higher level of ER is associated with a better overallsum100val.0.046).i Trend test is significant (x2 = 3.987; P = P = 0.032).100
- -
90- ~^-- ..1 90-g g80-| * ...LPRNegative 80-1 70- 70-i/i w 0 60-I o 60-1 50-o 50-o 40-CäS 40-Û.a? 30-20-10 30-20-10-o
-o -t-r. i i i i i , , . i i . , i , , i . i i ii* -obabilityiiiiiiiiiiiiiiiiiiiiiiiiiiii iiiiiiiiiiiii 01234
Years since sample taken Years since sample taken Fig. 2. Overall survival according to the presence of l,25(OH)2DjR. . Fig. 4. The overall survival of PR related to the percentage of stained cells is l,25(OH):DjR negative cases, , 1,25(OH)¡D,R positive cases showing no significantly influenced by the percentage of positively reactive cells. Difference significant difference (x2 = 1.81: P> 0.1). is significant (x2 trend = 7.05; P = 0.008). . negative cases; , less than 50% stain cases; , cases with >50% stained cells. tients with tumors positive for both receptors. This is of impor tance as it has been found that in the group with both receptors PR and survival after relapse and overall survival appear to be ER and PR positive more than 80% of the metastatic tumors independent of tumor size and nodal involvement. There are responded to hormonal therapy (28). The relationships between too few data to assess whether ER measured in this way is also 242
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100 lated with late lymph node métastases.However, these patients apparently did not attend a single specialized clinic as in our study. Rather, information regarding disease outcome was ob g tained by questionnaire and it is unclear if treatment was standardized with regard to axillary clearance and irradiation. In our study, of the patients with 1,25(OH)2D3R positive tumors only 27% have relapsed or died. Our findings indicate that the S presence of this receptor may be of some clinical significance and assessment of the tumor 1,25(OH)2D.,R status in patients with breast cancer may be of value in predicting progress of disease. We would emphasize that care should be taken in the interpretation of these results since the number of patients with 1,25(OH)2D,R negative tumors is small and thus a chance finding cannot be excluded. A larger study will be needed to confirm or refute these preliminary findings, and continued Years since sample taken follow-up in this group of patients will be important. Fig. 5. l,25(OH)2DjR and disease free survival. The disease free survival is The functional response to steroid hormones of human breast related to the presence of 1,25(OH)2D3R. , negative cases; , positive cancer cell lines in vitro may be relevant to these clinical studies. cases. Trend test x! = 4.01, P = 0.045. The ER positive cell line MCF-7 shows induction of PR in response to 17/3-estradiol but the stimulation of cell prolifera tion by the hormones is only moderate. The ER positive MDA- MB 134 cell line does not demonstrate an increase in PR but proliferation is modulated by estrogen (32). 1,25(OH)2D.,R is present not only in the ER positive cell lines MCF-7 (33), T47D (15), and ZR-75-1 (34) but also in the ER negative cell line HS o i- 05787 (35). Furthermore 1,25(OH)2D, has been shown to mod 'S ulate replication in the T47D and MCF-7 cell lines (15, 29). Therefore the steroid hormone receptor status and the func tional activity of these receptors may reflect breast tumor activ ity and predict the course of the disease. Thus determination of ER and PR and additionally 1,25(OH)2D.,R taken together may offer valuable prognostic information for clinical management of patients with breast cancer. We conclude that multiple steroid hormone receptor deter mination utilizing immunocytochemistry is a rapid and conven Years since sample taken ient method of assessment which does not require large Fig. 6. Probability of survival after relapse according to progesterone receptor amounts of tissue. Assessment of receptor content may well content. The postrelapse survival time is significantly related to the level of PR. , PR negative cases; cases with less than 50% stained cells, , >50% lead to prediction of response and survival. stained cells, x2 trend = 5.16, P = 0.023.
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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1991 American Association for Cancer Research. Immunocytochemical Determination of Estrogen Receptor, Progesterone Receptor, and 1,25-Dihydroxyvitamin D 3 Receptor in Breast Cancer and Relationship to Prognosis
Uta Berger, Richard A. McClelland, Patricia Wilson, et al.
Cancer Res 1991;51:239-244.
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