Estrogen and progesterone receptor content of endometrial carcinomas: Comparison of total tissue versus cancer component analysis

GYNECOLOGIC

ONCOLOGY

36, 363-368 (1990)

Estrogen and Progesterone Receptor Content of Endometrial Carcinomas: Comparison of Total Tissue versus Cancer Component Analysis’ JOHN T. SOPER, M.D., EILEEN M. SEGRETI, M.D., DEBRA B. NOVOTNY, WILLIAM T. CREASMAN, M.D.,4 AND KENNETH S. MCCARTY, Departments

of Obstetrics

and Gynecology,

Internal Medicine and Pathology, Durham, North Carolina 27710

M.D.,3

M.D. ,* DAVID MUTCH, JR., M.D.,

Duke

University

PH.D. Medical

Center,

Received July 24, 1989

Estrogen(ER) and progesterone(PgR) receptor valuesin 105 endometrialcarcinomaswere comparedusingimmunocytochemical and standardbiochemicaltechniques.Peroxidase-antiperoxidase staining for location of anti-ER (H222) and anti-PgR (JZB39) primary antibodieswas usedto generatea semiquantitative (HSCORE) assessment of receptor content in tissuecomponentsand the total specimen.Both total HSCORE and cancer componentHSCORE correlatedwith logbiochemicalassayvalues for ER and PgR. Biochemicalassayvalues, total HSCORE, and cancer component HSCORES all demonstratedinternal correlations betweenER and PgR levels. Correlation was somewhat closer for cancer componentHSCORE values of ER and PgR than the valuesfor total tissueHSCORE. When receptorcontent was analyzed by histologicgrade, all three estimatesof receptor status demonstrateda decreasingproportion of ER and PgR positive lesionswith decreasinghistologic differentiation; however, the proportion of receptornegativelesionsin grade3 lesions wasmuchhigherwhen usingtotal HSCOREor cancercomponent HSCORE than whenusingbiochemicalassayvalues(P < 0.005). Immunocytochemicaltechniquesfor localization of ER and PgR in endometrialcarcinomaspecimensmay allow a more focused evaluationof the receptorcontent in the malignantelementsthan standard biochemicaltechniques. o 1990 Academic press, IX. INTRODUCTION

The development and characterization of estrogen receptor (ER) [l-3] and progesterone receptor (PgR) [461 monoclonal antibodies have allowed localization of sex steroid receptors in human tissues using immuno’ Supported by NIH I ROl CA 39635-02 and Abbott Laboratories. * Current address: University of North Carolina, Chapel Hill, NC. 3 Current address: Washington University, St. Louis, MO. 4 Current address: Medical University of South Carolina, Charleston, SC.

cytochemical analysis (ICA) techniques. Prior studies with the H222 anti-ER and JZB39 anti-PgR monoclonal antibodies established the ICA technique as producing information about ER content in endometrial carcinomas that complements information derived from biochemical assays of binding in whole tissue homogenates of these malignancies [7-g]. Biochemical and ICA techniques measure different properties of receptors: biochemical assays are dependent on high-affinity specific steroid binding within tissue homogenates, while localization of receptor by ICA technique is dependent on recognition of specific antigenic epitopes. Biochemical assay values could be elevated due to a variable contribution of receptor in benign tissue elements that are intermingled with specimens from individual endometrial carcinomas [7] or by inclusion of “nonreceptor” binding in the total measured value. Monoclonal antibodies used for ICA might detect similar epitopes on nonreceptor proteins or epitopes on nonfunctional receptor molecules, which could also lead to overestimation of receptor in content in tissues. Since PgR is thought to be induced by estrogen action mediated through ER in tissues having an intact sex steroid regulatory system [IO], this study was designed to investigate the relationships between levels of ER and PgR in endometrial carcinomas as defined by ICA techniques and biochemical assays. Specifically, the relationships of ER and PgR levels in the cancer component were compared to receptor levels estimated for the whole tissue specimen. MATERIALS

AND METHODS

Two hundred eighty-two consecutive specimens from women with endometrial adenocarcinomas accessioned 363 0090~8258190 $1 SO

Copyright 0 1990 by Academic Press, Inc.

All rights of reproduction

in any form reserved.

364

SOPER

for steroid receptor analysis between 1980 and 1986 formed the basis for this study. Requirements for eligibility included (1) histologic confirmation of malignancy in the specimen, (2) biochemical analysis of both ER and PgR, and (3) sufJicient tissue in the specimen for ICA determinations of both ER and PgR as described subsequently. Patients were derived from two cohorts: Those from cohort I were selected from 173 specimens consecutively accessioned between 1980 and 1983 which were previously analyzed by ICA technique for ER content [7]. Of 100 evaluable specimens in this cohort, 43 had biochemical analysis of both ER and PgR while 4 had no residual cancer in the remaining specimen for PgR-ICA, resulting in 39 cases from this group. Cohort II consisted of 107 consecutively submitted specimens from 1984 to 1986. Excluded were 12 cases with insufficient tissue for biochemical analysis of both ER and PgR, 6 with insufficient tissue remaining after biochemical assays for ICA, 6 with extensive tissue necrosis or desiccation, and 19 cases because no residual malignancy was evident on cryostat tissue sections submitted for ICA. The study population consisted of 105 women with primary or recurrent endometrial carcinomas. In all cases, tissue was obtained prior to initiation of radiation therapy, chemotherapy, or hormonal therapy. Histologic differentiation of lesions was assigned by one reviewer (K.M.) using FIG0 criteria [l l] on the H&E-stained cryostat sections of the tissue submitted for receptor analysis. Tissues were received fresh, rinsed in cold buffer (10 mM/liter Tris-HCl, 5 m/liter Hepes HCI, 1.5 mM/liter EDTA, 1 .O mM/liter thioglycerol, and 0.2% NaN3, pH 7.4) at 4°C snap-frozen in liquid nitrogen and maintained at -80°C. Biochemical assays of ER and PgR content consisted of dextran-coated charcoal (DCCA) and sucrose density gradient analysis (SDGA) as previously described [ 111. Biochemical receptor values were derived from the multiconcentration DCCA except for cases limited by tissue quantity. In these, the total receptor as measured by SDGA was used since these methods have comparable ability to detect threshold levels of receptor [12]. For the purposes of this study binding 210 fmole/mg cytosol protein of estradiol or Org 2058 was used as the threshold positive level for each receptor. Monoclonal antibody H222, developed against MCF7 human breast carcinoma ER [l-3], and monoclonal antibody JZB39 (B39), developed against T37D human breast carcinoma PgR [4-61, were selected for ICA analyses. Serial 4- to 6-pm cryostat sections were mounted on poly-L-lysine-coated slides. The first section was stained with H&E for purposes of assessing tissue content, assigning histologic grade and tumor type, and determining the relative proportions of various tissue com-

ET AL.

ponents (i.e., malignant and benign epithelium, stroma, myometrium, and necrosis) in the specimen. The remaining sections were fixed in 3.7% formaldehyde-phosphate-buffered saline (PBS) for 10 min and transferred to a PBS bath for 5 min. Slides were placed in cold absolute methanol at - 15 to -20°C Celsius for 1 min, followed by two PBS washes for 5 min each at ambient temperature. Slides were treated with 2% normal goat serum in PBS for 15 min in a humidified chamber to reduce nonspecific binding of bridging antibody. Sections were incubated with primary antibody (H222, minimum concentration 0.1 pg/ml) and B39 (minimum concentration 16 pg/ml), for 30 min at ambient temperature in a humidified chamber. Control slides from each tissue specimen were incubated with control antibody (nonimmunized rat immunoglobulin in PBS) in place of the primary antibody for 30 min. Positive control slides of receptor positive MCF-7 human breast cancer cells Were incubated with primary and control antibody. Slides were washed in PBS twice for 5 min each. Sections were incubated with the bridging antibody (goat anti-rat immunoglobulin in PBS) for 30 min followed by two 5-min washes. Peroxidase-antiperoxidase (PAP) complex in PBS was applied to the sections for 30 min followed by two 5-min PBS washes. Slides were flooded with DAB:H202 solution (8 mg diaminobenzidine/l6 ml Hz02 in PBS) for 6 min in the dark. The DAB:Hz02 solution had been prepared in minimal light conditions and was used within 30 min. Slides were rinsed in running water for 5 min, dehydrated in serial alcohols to xylene and coverslipped with Pro-Texx. In the presence of hydrogen peroxide, the tissue bound peroxidase converts the diaminobenzidine chromagen to an insoluble brown pigment that can be visualized with the light microscope. By the PAP technique, the minute quality of receptor monoclonal antibody complex is amplified. The ICA for each receptor was scored in a semiquantitative fashion incorporating both the intensity and distribution of specific staining. The intensity of specific staining was characterized as not present (0), weak but detectable above control (1 +), distinct (2 +), and intense (3 + ). A value was derived for each tissue component consisting of the sum of the percentage of positively stained cells multiplied by a weighted intensity of staining for each tissue component, HSCORE

= W,(I

+ l),

where HSCORE is the histologic score, Pi is the percentage of stained cells at each intensity level, and i is the intensity for i = 1,2,3 [7-91. A total tissue HSCORE was calculated as the sum of the component HSCOREs weighted by the fraction of each component observed. HSCOREs of 75 or more were considered positive. Each section was reviewed independently by two observers

ER AND

PgR IN ENDOMETRIAL

(E.S. and K.S.M.). Differences >lO% were jointly rereviewed and scored as a consensus value. Results of biochemical assays, ICA values, and patient information were maintained as separate files until all data was analyzed. Quantitative comparisons of biochemical and HSCORE values were carried out using correlation analysis. x2 tests were used where appropriate .

RESULTS Both ER and PgR-ICA techniques localized primary antibody in cellular nuclei. Receptors were identified in all tissue corn-ponents at variable staining intensities within individual specimens. Within individual specimens, the epithelium frequently demonstrated heterogeneity of staining while stroma and myometrium were more uniform in the intensity of staining. As previously noted [7], ER-ICA total HSCOREs had strong intra- and interobserver correlation. Similar correlation was demonstrated for PgR-ICA total HSCOREs (Y = 0.89 interobserver correlation). Semiquantitative HSCOREs for total tissue and cancer component correlated with the log biochemical ER and PgR values (Figs. IA-1D). HSCORE values 275 identified threshold biochemical binding values (2 10 fmole/mg protein) with a range of sensitivity and specificity depending on whether total tissue HSCORE or cancer component HSCORE was utilized (Table 1). As these figures and sensitivities indicate, biochemical assays tended to detect a higher proportion of “receptor positive” lesions, particularly when compared to the cancer HSCORE (Table 1). Quantitative levels of ER and PgR were compared using biochemical assays, total tissue HSCORE, and cancer component HSCORE (Figs. 2A-2C). Similar quantitative relationships were observed in that increasing ER levels correlated with increased PgR levels. The association between ER and PgR content of the cancer component was slightly stronger than the associations observed between the ER and PgR content of the whole specimen (r = 0.61 vs r = 0.59 and 0.54 for biochemical and total HSCORE values, respectively). Quantitative assignment of receptor status was compared to histologic grade, using the threshold 10 fmole/mg protein for biochemical assays or 75 for both total HSCORE and cancer component HSCORE (Table 2). Significantly more poorly differentiated lesions were identified as having both negative ER and negative PgR status by ICA techniques than with biochemical assays (P < 0.005, biochemical assay vs total HSCORE or cancer HSCORE). In general, well- and moderately differentiated lesions had a much higher proportion of lesions

365

CANCER

that were positive for both ER and PgR than poorly differentiated lesions. DISCUSSION The presence of steroid hormone receptors in some malignancies provides insight into the prognosis and likelihood of response to hormonal therapies. The utilization of ER and PgR analysis in breast carcinomas has been well documented and clinicopathologic correlations of endometrial carcinoma sex steroid receptor content have been previously studied using biochemical analyses [8,11,13-151. In this report, biochemical and ICA techniques for measuring ER and PgR were used to analyze specimens of endometrial carcinoma. Correlations of semiquantitative assessment of receptor content (HSCORE) with biochemical binding assays for both ER and PgR were observed. Of note, the ER content as determined by ICA techniques correlated with the PgR content; this association was also observed when receptor content of the cancer component was analyzed. Unlike breast carcinomas, which are surrounded by tissues which contain relatively low levels of receptor, endometrial carcinomas are frequently intermingled with benign tissue elements which may contain considerable amounts of receptor and contribute to the total binding measured by biochemical analyses of whole tissue homogenates [7,8]. Using immunohistochemical techniques, the proportional contribution of each tissue element can be assessed and measured in a semiquantitative fashion (HSCORE) [7-91. In this study ICA techniques were used to estimate receptor content in both the total tissue specimen (total HSCORE) and in the malignant epithelium (cancer component HSCORE). Similar quantitative and qualitative relationships were observed between these estimates and receptor content as determined by biochemical assays, correlation of ER and PgR content, and relationships to histologic grade. This reflects the relatively large proportion of the cancer component that constituted the majority of the specimens. The study group specimens were selected in part because they were large lesions with ample material for conventional histologic analysis, biochemical assays, and ICA techniques. Further studies using ICA techniqes to localize receptor are needed to determine the relevance of stromal epithelial interactions in edometrial carcinomas. However, the current study suggests that analysis of the receptor content in the cancer component using ICA techniques can yield an estimate of receptor content which demonstrates the known correlations of ER and PgR content, and of receptor content with histologic differentiation. Although semiquantitative correlations between biochemical and ICA techniques for both ER and PgR were

366

SOPER ET AL. 4-

Iron:

4

j!:~: 4

8*.= 0 o- 0 000 0I

B

8

.a0 Q

I 100

0

200I

I 300

400I

o-o

TOTAL TISSUE ER HSCORE

0

300 100 200 TOTAL TISSUE PqR HSCORE

400

0

1 0

ooo 0 0

0 0 100 MALIGNANT

loo MALIGNANT

Oo I 200 EPITHELIAL

200 EPITHELIAL

0 300 ER HSCOAE

300 PqR HSCORE

400

400

FIG. 1. Quantitative correlations between immunocytochemistry assays and biochemical assays for steroid receptor content of endometrial carcinomas. (A) Total ER HSCORE vs log biochemical ER content (Y = 0.47). (B) Cancer component ER HSCORE vs log biochemical ER content (r = 0.47). (C) Total PgR HSCORE vs log biochemical PgR content (r = 0.53). (D) Cancer component PgR HSCORE vs log biochemical PgR content (r = 0.53).

observed, several differences in their associations were documented in this study. There was a positive correlation between levels of ER and PgR as determined by both biochemical and ICA techniques; however, both quantitative correlation of HSCORE with biochemical assay and sensitivity of ICA for detecting threshold levels of receptor content determined by biocemical assay were not as strong as the correlations observed for ERICA with the H222 monoclonal antibody in our original cohort of endometrial cancer study patients [7]. This decrease in sensitivity for ICA techniques may be accounted for by several factors. The current study was biased toward endometrial carcinomas with relatively large lesions. This is reflected by the relatively large proportion of poorly differentiated lesions in the study and this subgroup has the greatest divergence in receptor status when comparing ICA and biochemical assays. Some of these lesions may contain receptor proteins that have altered antigenic epitopes. Alternatively, prolonged storage of the frozen sample might result in degradation of the receptor protein and loss of receptor antigenic

properties. Indeed, several specimens were rejected for this study because of tissue desiccation; however, previous studies using the H222 antibody for staining breast carcinoma specimens stored up to 8 years under identical conditions demonstrated no significant loss in sensitivity for identifying threshold ER content using ER-ICA when compared to biochemical ER assays [15]. Although a lower proportion of lesions were receptor positive using ICA techniques than with biochemical assays, inspection of Figs. 2A-2D demonstrates that lowering the threshold for HSCORE or raising the threshold for biochemical assays would decrease the specificity of ICA while increasing the proportion of positive lesions determined by ICA techniques. In general, ICA techniques identified a lower proportion of ER and PgR positive lesions, particularly for poorly differentiated lesions when compared to biochemical assays, and conversely identified a higher proportion of ER and PgR negative lesions in this histologic group. The relative importance of these observations and threshold values will need to be reproduced and validated in further studies comparing

ER AND PgR IN ENDOMETRIAL

367

CANCER

TABLE 1 Comparison of Threshold Receptor Content: Biochemical Assay versus Immunocytochemistry Assays of Total Tissue and Cancer Component Receptor Content Biochemical assays”

ER-ICA Total HSCORE 275 Total HSCORE <75 Cancer HSCORE 275 Cancer HSCORE <75

ER+

ER-

41 26 40 27

5 33 5 33

Sensitivity Specificity Sensitivity Specificity

= = = =

60% 87% 57% 87%

I 2 3 LOO BIOCHEMICAL ER CONTEfiT ( fmol/sp protein)

0

Biochemical assays”

PgR-ICA Total HSCORE z-75 Total HSCORE ~75 Cancer HSCORE a75 Cancer HSCORE ~75

PgR +

PgR -

37 24 35 26

5 39 5 39

400-

Sensitivity Specificity Sensitivity Specificity

= = = =

4

B

61% 89% 57% 89%

Note. Abbreviations used: ER, estrogen receptor: PgR, progesterone receptor: ICA, immunocytochemistry assay. a Positive biochemical assays 210 fmole/mg protein.

receptor content with biological behavior, such as prognosis and response to therapy. The semiquantitative HSCORE generated by the ICA techniques is dependent upon the skills of the observer. However, studies using these techniques to generate HSCOREs have documented that with experienced observers intra- and interobserver results correlate well [7,81.

Compared to biochemical assays, the ICA technique has a potential source of bias which is not present in biochemical binding assays. Although the tissues used for monoclonal antibody staining are not counterstained for histologic detail, tissue sections do contain features of architectural or nuclear differentiation which might influence interpretation of the HSCORE. We have attempted to minimize this bias by using two independent reviewers, generating a consensus HSCORE for each tissue, and rereviewing specimens by the primary observer. However, determination of receptor content by ICA techniques cannot be totally blinded to features of histologic differentiation. This potential source of bias must be critically examined as experience with the ICA techniques accumulates. The ICA techniques of ER and PgR analyses are new tools for investigating receptor content of endometrial carcinomas. They are able to take into account the heterogeneous tissue components of these lesions and discriminate localization of receptor. These assays correlate well with histologic differentiation and appear to cor-

00 0

4004

“00 (Do 0 I 100 200 300 TOTAL TISSUE EA HSCORE

c

I-

w 8

E = 300z

4OO

0 I

0 0

I 0

I 100 MALIGNANT

0

1 200 EPITHELIAL

300 ER HSCORE

400

FIG. 2. Quantitative correlations between ER and PgR content of endometrial carcinomas. (A) Log biochemical ER vs log biochemical PgR content (r = 0.59). (B) Total ER HSCORE vs total PgR HSCORE (r = 0.54). (C) Cancer component ER HSCORE vs cancer component PgR HSCORE (r = 0.61).

relate with known receptor function in that increasing levels of ER are associated with increasing levels of PgR, especially when only the level of receptor in the malignant component is analyzed. Thus, they appear to complement the information derived from standard biochemical binding assays for analyses of receptor content of

SOPER ET AL.

368

TABLE

2

Distribution of ReceptorStatus versusHistologicGrade Receptor status Er-/PgR+

ER + /PgR +

ER + /PgR -

30(71) 17(61) 18(W

l(4) 3(11) 3(11)

4(14)

21(48) 12(28) I3(30)

X16) 8(19)

2(5) 3(7)

6(14

2G)

321)

3(9) 3(9)

Well differentiated Biochemical assay Total HSCORE Cancer HSCORE Moderately differentiated Biochemical assay Total HSCORE Cancer HSCORE Poorly differentiated* Biochemical assay Total HSCORE Cancer HSCORE

1 l(32) l(3)

5(15) 3(9)

2(6)

6(21) 3(11)

z(6)

Er-/PgR3(11) 2(7) 4(14) 13(30) 20(47) 22(5 1) 13(38) 25(73) 27(79)

Note. Abbreviations used: ER, estrogen receptor; PgR, progesterone receptor. * P < 0.005, distribution ER-PgRBiochemical assay vs total HSCORE or cancer HSCORE.

endometrial carcinomas. Further investigations are necessary to determine the relative utility of the techniques for predicting biological behavior of endometrial carcinemas .

8.

ACKNOWLEDGMENTS The authors acknowledge the excellent assistance of Ms. Donna Silva in the typing and preparation of this manuscript. The authors thank Dr. Geoffrey Greene for the monoclonal antibody JZB39 and Abbott Laboratories for the monoclonal antibody H222.

9.

REFERENCES 10. 1. Greene, G. L., Fitch, F. W., and Jensen, E. V. Monoclonal antibodies to estrophilim: Probes for the study of estrogen receptors, Proc. Natl. Acad. Sci. USA 77, 157-161 (1980). 2. Greene, G. L., Nondair, C., Engler, J. P., and Jensen, E. V. Monoclonal antibodies to human estrogen receptor, Proc. Nat/. Acad. Sci. USA 77, 5115-5119 (1980). 3. Greene, G. L., and Jensen, E. V. Monoclonal antibodies as probes for estrogen receptor detection and characterization, J. Steroid Biochem. 16, 353-359 (1982). 4. Greene, G. L., Harris, K., Bova, R., Kinders, R., Moore, B., and Nolan, C. Purification of T47D human progesterone receptor and immunochemical localization with monoclonal antibodies, Mol. Endocrinol. 2, 714-726 (1988). 5. Press, M. F., and Greene, G. L. Localization of progesterone receptor with monoclonal antibodies to the human progesterone receptor, Endocrinology 122, 1165-l 175 (1988). 6. Press, M. F., Uilave, J. A., and Greene, G. L. Progesterone receptor distribution in the human endometrium. Analysis using monoclonal antibodies to the human progesterone receptor, Amer. J. Pathol. 131, 112-124 (1988). 7. Budwit-Novotny, D. A., McCarty, K. S., Jr., Cox, E. B., Soper, J. T., Mutch, D. G., Creasman, W. T., Flowers, J. L., and

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McCarty, K. S., Sr. Immunohistochemical analysis of estrogen receptor in endometrial cancer using a monoclonal antibody, Cancer Res. 46, 5419-5425 (1986). Mutch, D. G., Soper, J. T., Budwit-Novotny, D. A., Creasman, W. T., McCarty, K. S., Sr., and McCarty, K. S., Jr. Endometrial adenocarcinoma estrogen receptor content: Association of clinicopathologic features with immunohistochemical analysis compared with standard biochemical methods, Amer. J. Obstet. Gynecol. 157, 925-931 (1987). Segreti, E. M., Novotny, D. B., Soper, J. T., Mutch, D. G., Creasman, W. T., and McCarty, K. S. Endometrial cancer: Histologic correlates of immunohistochemical localization of progesterone receptor and estrogen receptor, Ubstet. Gynecol. 73, 780785 (1989). McCarty, K. S., Jr., Lubahn, D. B., and McCarty, K. S., Sr. Oestrogen and progesterone receptors: Physiological and pathologicai considerations, Clin. Endocrinol. Metab. 12, 133-154 (1983). Kottmeier, H. L. (Ed.) Annual report on the results of treatment in gynecology cancer-FIGO, Stockholm: International Federation of Gynecology and Obstetrics, Vol. 18 (1982). McCarty, K. S., Jr., Barton, T. K., Fetter, B. F., et al. Correlations of estrogen and progesterone receptors with histologic differentiation in endometrial adenocarcinoma, Amer. J. Pathol. 96, 171-184 (1979). Smith, R. G. Quality control in steroid hormone receptor assays, Cancer Philadelphia 46, 2946-2949 (1980). Creasman, W. T., Soper, J. T., McCarty, K. S., Jr., McCarty, K. S., Sr., Hinshaw, W., and Clarke-Pearson, D. L. Influence of cytoplasmic steroid receptor content on prognosis of early stage endometrial carcinoma, Amer. J. Obstet. Gynecol. 151, 922-932 (1985). McCarty, K. S., Jr., Szabo, E., Flowers, J. L., Cox, E. B., Leight, G. S., Miller, L., Konrath, J., Soper, J. T., Budwit-Novotny, D. A., Creasman, W. T., Seigler, H. F., and McCarty, K. S., Sr. Use of a monoclonal anti-estrogen receptor antibody in the immunohistochemical evaluation of human tumors, Cancer Res. Suppl. 46, 42,445-42,485 (1986).