ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 7, No. 6 Copyright (g) 1977, Institute for Clinical Science

Barr Body Distribution and Estrogen Receptor Protein in Mammary Carcinoma*

PAUL PETER ROSEN, M.D.,f ANGELA SAVINO, CT (ASCP),t CELIA MENENDEZ-BOTET, Ph .D.,* JEROME A. URBAN, M.D.,§ VALERIE MIKE, Ph .D.," MORTON K. SCHWARTZ, Ph .D.,* and MYRON R. MELAMED, M.D.f

Departments of Pathology (Cytology Service), t Biochemistry t and Surgery (Breast Service),§ Memorial Hospital, and Biostatistics Laboratory," Memorial Sloan-Kettering Cancer Center, New York, NY 10021

ABSTRACT

This study was undertaken to investigate the relationship between Barr body distribution and estrogen receptor protein content of mammary car­ cinoma. The proportion of cells with one or more Barr body was deter­ mined in 105 specimens of mammary carcinoma from Guard stained im­ prints. Receptor protein content of the specimen was measured by the dextran charcoal method and compared with histopathologic features of the carcinomas. Primary carcinomas with Barr bodies in more than 10 percent of tumor cells were more likely to have higher levels of receptor protein than those with a lower proportion of Barr body containing cells (P < 0.005). The results obtained for primary carcinoma may explain why pa­ tients with carcinomas that have a high proportion of Barr body positive cells are more likely to respond to hormonal therapy. Furthermore, these observations, when correlated with other available data about ERP suggest that an X-chromosome is involved in the synthesis of and/or carries the locus of action for estrogen receptor protein.

Introduction Complexes formed through interaction of receptor and hormone in the cytoplasm of Assays of the content of receptor pro­ potentially responsive cells are translo­ tein for estrogen12 in mammary car­ cated to the nucleus. By binding with des­ cinoma have proven to be clinically oxyribonucleic acid (DNA) acceptor sites, useful indicators of the likelihood of the complexes influence the formation of response to hormonal therapy in patients specific ribonucleic acid (RNA) and ulti­ with recurrent mammary carcinoma. mately protein synthesis.13,23 Non-histone protein components of chromatin are ap­ * Address reprint requests to Paul Peter Rosen, M.D., Department of Pathology, Memorial Hospital, parently important determinants of the 1275 York Avenue, New York, NY 10021. specificity of binding sites,22 but there is 4 9 1 492 ROSEN, ET AL.

as yet no evidence as to which chromo­ analysis were obtained from the Pathol­ some bears this locus of action or the ogy Laboratory. Barr bodies (figure 1) locus for the synthesis of receptor protein. were identified in Guard stained direct It has been noted that patients whose imprints made from the tissue selected tumors had a greater proportion of cells in for ERP analysis. The procedure for which a distinct Barr body could be iden­ staining and counting cells was identical tified had a more favorable prog­ with that described in a prior report.29 A nosis.7,24'29'34 Recurrent carcinomas with a minimum of 100 carcinoma cells from higher proportion of Barr body positive each tumor was studied. The number of cells were found to be more likely to re­ cells with a Barr body was expressed as a gress in response to therapy.5,17'24'31 percentage of the total number. Cells Women whose breast carcinoma con­ with more than one Barr body were in­ tained a high proportion of Barr body pos­ frequent. When present, they were re­ itive cells derived some benefit from corded as positive but were not listed prophylactic oophorectomy, whereas separately. Four cases in which the prep­ those with Barr body negative tumors did aration was unsatisfactory were excluded not benefit.33 from analysis. This study was undertaken to evaluate A detailed pathological review of the the relationship between the estrogen re­ tissues was also undertaken, using a pro­ ceptor content and the proportion of Barr tocol developed in an earlier study of body positive cells in mammary car­ morphological correlations with estrogen cinoma. receptor protein.26 Results of biochemi­ cal, cytological and pathological investi­ Materials and Methods gations performed independently were One hundred and five specimens of combined at the time of data analysis. primary or recurrent mammary car­ ERP was measured by the dextran char­ cinoma submitted for frozen section and coal method described in detail for estrogen receptor protein (ERP) elsewhere.20'26 Incubations were carried out at room temperature for 60 minutes. As in earlier studies of ERP from this hospital, specimens with less than 10 femtomoles of ERP per mg of cytosol pro­ tein and in which less than 60 percent of the binding was inhibited by 1000 fold excess untritiated estradiol, diethylstil- f h besterol or 20,000 fold excess CI628 were % designated as ERP negative. Those with 10 femtomoles or more of ERP with at least 60 percent inhibition were desig­ nated as positive. Specimens with inter­ mediate combinations were classified as positive if the specific ERP were greater 9 % than six. Those with a lesser specific ERP <# were termed negative. Specific ERP was determined as the product of femtomoles F ig u r e 1. Photograph of mammary carcinoma per mg cytosol protein and percentage cell in imprint preparation stained with Guard’s stain. Barr body is indicated by arrow (original inhibition. The constant of dissociation magnification x570). (Kd) was determined from a Scratchard BARR BODY AND ESTROGEN RECEPTOR PROTEIN 4 9 3 plot based on analysis of three estradiol T A B L E X concentrations (1,2 and 5nM). Among Relationship Between Barr Body Distribution specimens found to be ERP positive on and Estrogen Receptor Protein in All Specimens the basis of binding in femtomoles and Barr Body Distribution percent inhibition at InM estradiol, all Less than 10% 10 to 20% More than 20% but two primary tumors and two metas- % % tases had a KD greater than 5 x 10~9 M. ERP Of No. Total No. Total No. Total Specimen Spec. Spec. Spec. Spec. Spec- Spec. Statistical significance of the results was evaluated by the chi-square test and for Positive 6 18 25 51 11 58 small samples by the Fisher exact proba­ Negative _27 82 24_ 49 8 42 bility ratio test. Total 33 49 19 Buccal smears from 20 other women P < 0.01 between the ages of 18 and 74 were also Counts in the table are related to the EBP content examined as controls. Four were hospital determined by the dextran charcoal method. employees with no history of breast dis­ ease. The others were a consecutive others were locally recurrent carcinoma series of women with breast disease seen or distant metastases. Analysis of these in the office of one of the authors (JAU). groups separately (table II) revealed a Half had been treated for mammary car­ positive correlation between Barr body cinoma and half had clinically diagnosed count and ERP among the primary fibrocystic disease or mastitis. tumors (P < 0.005) but not among the smaller number of recurrent tumors examined. Results

The distribution of Barr body counts A g e and the relationship of these counts to the Thirty-four patients were less than 50 estrogen receptor protein content of the years old and 67 were older. As shown in specimen are shown in table I. The table III, a greater percentage of the specimens were categorized according to older women had Barr body counts over the percentage of tumor cells with Barr 10 percent than did younger women; this bodies to obtain data comparable to a difference, however, was not statistically prior study in which similar groupings significant. were used.29 The percentage distribution of specimens was not significantly differ­ T u m o r M a r g i n s ent in the two series. Carcinomas with Barr bodies in more than 10 percent of Fourteen primary carcinomas were cells were more likely to be ERP pos­ considered to have circumscribed or itive. The difference in the frequency of pushing margins and 51 had irregular or ERP positivity between all specimens of infiltrative borders. Virtually the same carcinoma with less than 10 percent and percentages of tumors with pushing and more than 10 percent Barr body positive infiltrating margins (35 percent and 31 cells was significant (P < 0.01). percent, respectively) had Barr body counts below 10 percent. P r i m a r y a n d M e t a s t a t ic C a r c i n o m a Barr body counts ranged from zero to L y m p h o i d R e a c t i o n 30 percent. The distribution was uni- Intensity of the lymphoid reaction in modal. Approximately two-thirds of the and around primary carcinomas was specimens were primary tumors. The characterized as minimal to moderate (1 4 9 4 ROSEN, ET AL.

TABLE II

Estrogen Receptor Protein and Barr Body Distribution: Comparison o f Primary and M etastatic Breast Carcinoma

Barr Body Distribution in Carcinoma PRIMARY CARCINOMA METASTATIC CARCINOMA Less % 10% % Less % 10% % ERP of than 10% Total or more Total than 10% Total or more Total Specimen No. Spec. Spec. No. Spec. Spec. No. Spec. Spec. No. Spec. Spec.

Positive 4 1 9 29 66 2 17 7 29 Negative 17 81 15 34 10 83 17 7 1

Total

P < 0.005 Counts in the table are related to the ERP content determined by the dextran charcoal method. to 2+) and intense to very intense (3 to over 10 percent and two of these (or 40 4 + ). The majority (73 percent) of car­ percent of the five infiltrating lobular cinomas in which there was a sparse carcinomas) were over 20 percent. Barr lymphoid infiltrate had Barr body counts body counts were below 10 percent in over 10 percent (table IV). On the other two of three medullary carcinomas and in hand, only 33 percent of tumors with a single cases of tubular and intraductal dense lymphoid infiltrate had high Barr carcinoma. body counts (P < 0.05). H i s t o l o g i c G r a d e o f P r i m a r y H i s t o l o g i c T y p e o f P r i m a r y C a r c i n o m a C a r c i n o m a Histologic grade was evaluated in Sixty-seven percent of the infiltrating primary infiltrating duct carcinomas on duct carcinomas had Barr body counts the basis of the extent of gland or tubule above 10 percent (table V). Ten of these formation. Special types such as lobular, cases had counts over 20 percent (repre­ colloid, tubular and non-infiltrating car­ senting 18 percent of all infiltrating duct cinomas were not graded. The lesions carcinomas). Four of the five (80 percent) infiltrating lobular carcinomas had counts T A B L E IV

Relationship of Lymphoid Infiltrate in T A B L E I I I Primary Carcinoma to Barr Body D istribution

Age of Patients and Barr Body Distribution* Intensity of Lymphoid Infiltrate in Primary Carcinoma 1-2 (+) Slight 3-4 (+) Intense Age of Patient (Years) Barr Body No. % No. % 51 More than 51 or less Distribution Spec. Total Spec. Total Barr Body N o . % No. % Distribution Spec. Spe c . Spec. Spec. Less than 10% 15 27 6 67 10% or more 41_ 73 3_ 33 Less than 10% 14 41 19 28 10% of more 20. 59 48 72 Total 56 9

Total 34 67 P < 0.05 Counts in the table are related to the intensity of ♦Includes primary and metastatic carcinoma. lymphoid infiltration determined by examination of Counts in the table are related to age of patient. histological sections of the same carcinoma. BARR BODY AND ESTROGEN RECEPTOR PROTEIN 4 9 5 were distributed on a scale of 1 (low- TABLE V grade, well differentiated) to 4 (high H istologic Type of Primary Carcinoma grade, poorly differentiated). Grades 1 and Barr Body D istribution and 2 were compared with grades 3 and Number of Cases with Indicated 4. Forty-seven percent of carcinomas Percentage of Cells with Barr Body with counts below 10 percent and 42 per­ % of Cases with Counts cent of those with higher Barr body Histologic Type Less 10% or of 10% counts were in the group of better differ­ of Carcinoma than 10% More or More entiated lesions. The differences be­ Infiltrating duct 16 38 67 tween low and high grade carcinomas Infiltrating lobular 1 4 80 were not significant. Medullary 2 1 33 Colloid 0 1 Tubular 1 0 Intraductal 1 0 S t a t u s o f A x i l l a r y L y m p h N o d e s There was no correlation between Barr body count in primary carcinomas and the presence or absence of axillary lymph that the nuclear chromatin body is one rather than both x-chromosomes. As a node métastasés. Thus, patients with tumors that had low Barr body counts rule, it has been observed that the were not more likely to have axillary x-chromosomes complement of a normal cell is one more than the number of Barr lymph node métastasés. bodies. Moore and Barr21 reviewed, in 1957, C o n t r o l s several studies of the distribution of sex Counts of Barr body positive cells in chromatin in normal tissues and neo­ buccal smear preparations from control plasms. Most benign and malignant tumors patients ranged from 7 to 19 percent. The were found to have a sex chromatin dis­ mean count of Barr body positive cells in tribution similar to that of the normal tis­ female buccal smears in this laboratory sues of the host. Carcinoma of the female has been about 12. The average propor­ breast was a notable exception.21,32 In a tion of Barr body positive cells in buccal substantial number of carcinomas, the smear preparations was 13 percent for pa­ proportion of tumor cells with a Barr tients with breast carcinoma and the body was lower than or higher than in same for women with benign breast dis­ normal tissues. In this and two prior re­ ease. This result is almost identical with ports from this institution29,34 and in other the average result obtained in this labora­ studies,14 the distribution of cases has tory for buccal smears in “normal” wo­ been essentially unimodal. men. There is surprisingly little information available about the relationship between the Barr body and the actual chromosome Discussion complement of neoplastic cells. In a The nuclear sex chromatin or Barr body study of cervical carcinoma, Atkin1 found has been the subject of intense study a normal nuclear chromatin pattern in 80 since it was described in 1949 by Barr percent of tumors which were “near dip­ and Bertram.2 Graham and Barr originally loid.” Chromatin bodies were present in proposed that this structure represented a nuclei of only 20 percent of “near tetra- condensation of the heterochromatic por­ ploid” carcinomas. Gropp and Wolf s9 tions of both x-chromosomes.8 However studies indicated that nuclei in car­ subsequent investigations have shown cinomas composed of small, uniform cells 4 9 6 ROSEN, ET AL. were more likely to contain a Barr body primary mammary carcinomas. How­ than nuclei in tumors composed of larger ever, it was a surprise to find that this cells. relationship was not true for specimens of Kallenberger et al15 correlated the metastatic carcinoma although there was DNA content of Feulgen stained imprints a slightly higher percentage of metastatic with the Barr body incidence in mam­ carcinomas with positive ERP and Barr mary carcinoma. An increased frequency body counts over 10 percent. No readily of polyploidy was found in tumors with apparent explanation is available for the low chromatin body counts. Other inves­ difference between the two groups of tigators have described the DNA content specimens. However, the frequency of and chromosome constitution of benign16 ERP positive specimens is lower, overall, and malignant15,16,18,19 breast lesions. among metastases than it is in primary Many of these reports were summarized tumors.26 by Ludwig et al18 in 1973 who concluded Several observations about ERP, Barr that most mammary carcinomas were body count and characteristics of the aneuploid. None of these studies provide tumors or patients appear to be interre­ any information about the relationship lated. Primary carcinomas in post meno­ between the Barr body and x-chromo- pausal patients are more likely to be estro­ some content of mammary carcinoma gen receptor positive than are carcinomas cells. in younger or premenopausal women. In Little attention has been given to cor­ the current study, it has been noted that relating the Barr body distribution in primary tumors in older patients were also breast carcinoma with histologic charac­ more likely to have higher Barr body teristics of the tumors. Kimel17 concluded counts. ERP26 and Barr body count both that Barr body count and tumor differ­ were negatively correlated with lymphoid entiation, as described by nuclear grade, infiltration in the primary lesion. The rela­ were inversely related. However, in a tionships previously reported between prior study29 from this institution and in histologic type of mammary carcinoma and the current investigation, there was no ERP26 were similar to those between Barr association between Barr body count and body count and histologic type. Medullary tumor grade as expressed in terms of the carcinomas tended to have low Barr body degree of glandular differentiation. Sev­ counts and to be ERP negative. A signifi­ eral authors7,17,29 have failed to find a cantly high proportion of infiltrating lobu­ significant correlation between the pres­ lar carcinomas were ERP positive and they ence or absence of axillary lymph node tended to have high Barr body counts. métastasés and Barr body count in the These differences in the ERP properties primary tumor. Evidently the positive of lobular and medullary carcinoma paral­ correlation between Barr body count in lel the finding noted by Gropp and WoIP the primary tumor and survival7,24,29,34 is that tumor cells with small, uniform nuclei not due to differences in the frequency of were likely to have higher Barr body métastasés between tumors with high counts. Lobular carcinoma is charac­ and low Barr body counts. terized by the relatively small size and The reported positive correlation be­ uniformity of its nuclei whereas medullary tween response to therapy and Barr body carcinoma features larger more anaplastic count might be explained by the finding nuclei. now being reported, that there was a In view of the foregoing, it seems un­ highly significant relationship between likely that the correlation between ERP the ERP content and Barr body count in and Barr body count in primary mammary BARR BODY AND ESTROGEN RECEPTOR PROTEIN 4 9 7 carcinomas is simply coincidental. The zyme are thought to arise from a single cytoplasmic complex of estrogen and ERP cell (clonal origin). When a mixture of have been shown to be translocated to the enzymes is present, it has been post­ nucleus where they are bound to “accep­ ulated that the tumor had a multicellular tor” sites in the chromatin. Many of the origin. biochemical steps have been studied in Limited data previously reported indi­ great detail,4,22 butthe chromosomal site of cated a mixed pattern of G-6-PD in this activity remains to be identified. Nor is mammary carcinoma. Recent studies of it known which chromosomes carry the the enzyme content of mammary car­ information for the synthesis of receptor cinoma revealed higher levels of G-6-PD protein. in comparison to non-carcinomatous The direct correlation between E RP and mammary tissue10 owing especially to an the Barr body content of these cells increased proportion of the faster migrat­ suggests that one or more x-chromosome is ing isoenzyme G-6-PD-I. Increased involved in the synthesis of ERP and/or G-6-PD-I activity has been observed after may carry locus for estrogen-ERP action. estrogen exposure25 in the rodent mam­ This possibility is supported by our obser­ mary tumor. While the mean level of vation that those morphological properties G-6-PD activity was lower in human of primary mammary carcinomas which mammary carcinomas characterized as were significantly associated with ERP estrogen receptor positive,30 this result bore a similar relationship to the Barr body did not necessarily reflect the levels of count. On the other hand, factors that did enzyme that would have been found in not relate significantly to receptor protein an ERP positive tumor after hormone also proved not to correlate with Barr body stimulation. No report has been found counts in the primary carcinoma. that compared G-6-PD activity in ERP Salih et al28 have suggested that the es­ positive and negative human mammary trogen sensitivity of breast cancer can be carcinomas after hormone stimulation. assayed by a histochemical method for Finally, our failure to find a significant measuring dehydrogenase activity of the correlation between ERP and Barr body pentose shunt pathway. They observed count in metastases is puzzling. It has that the dehydrogenase concentration been reported elsewhere by us that tended to increase when samples of a metastases of mammary carcinoma may breast carcinoma were incubated in vitro differ in their ERP properties from those with hormones that influence the growth of the primary tumor in nearly a third of of breast cancer.11 Increased activity of instances when the specimens compared glucose-6-phosphate dehydrogenase, were not obtained simultaneously.27 The (G-6-PD) an x-linked enzyme3,6 under the mean interval between the treatment of a influence of estrogen is additional evi­ primary tumor and the appearance of a dence that one or more loci involved in metastasis was 9.6 months in cases in estrogen-ERP activity may be located on which there was no discrepancy and 19.3 an x-chromosome. months when the ERP differed between As noted previously, in the normal the primary and metastasis. Discrepan­ female somatic cell only one of the two cies were observed in 26 percent of cases x-chromosomes is active. As a conse­ in which non-simultaneous metastases quence, in an individual heterozygous for were compared with one another and in the G-6-PD gene, either the fast or slow 15 percent when simultaneously occur­ varient would be expressed in a given ring but anatomically separate specimens cell. Tumors found to contain one isoen­ were compared.27 4 9 8 ROSEN, ET AL.

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