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Clinical Cancer Research Vol. 7, 1937-1945, July 2001
© 2001 American Association for Cancer Research

Regular Articles

Breast Tumor Immunophenotype of BRCA1-Mutation Carriers Is Influenced by Age at Diagnosis

Susan A. J. Vaziri, Lisa M. Krumroy, Paul Elson, G. Thomas Budd, Gerarda Darlington, Jonathan Myles, Raymond R. Tubbs and Graham Casey1

Departments of Cancer Biology [S. A. J. V., L. M. K., G. C.], Biostatistics [P. E.], Hematology and Oncology [G. T. B.], and Anatomic Pathology [J. M., R. R. T.], Cleveland Clinic Foundation, Cleveland, Ohio 44195; and Department of Mathematics and Statistics, University of Guelph, Guelph, Ontario, Canada N1G-2W1 [G. D.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Breast tumors of BRCA1 mutation carriers and those of early onset breast cancer cases share similar histological features, being generally high-grade, highly proliferative, aneuploid tumors that are predominantly estrogen- and progesterone-receptor negative. Because histological features of tumors of premenopausal women differ from those of tumors of older women, we sought to determine whether the immunophenotype of breast tumors of BRCA1 mutation carriers was influenced by age at diagnosis.

Experimental Design: We examined 31 breast tumors from BRCA1 mutation carriers and compared them with 81 tumors of age-matched (plus or minus 5 years) breast cancer patients unselected for family history. Tumors were further matched for histology, grade, and size. Paraffin-embedded tumor tissues were examined for protein expression of estrogen receptor (ER), PR, Ki-67, cyclin D1, TP53, HER2, ß-catenin, and cyclin E using immunohistochemical approaches.

Results: ER (P = 0.01), PR (P = 0.06), and cyclin D1 (P = 0.002) were less frequently expressed and Ki-67 (P = 0.01) and ß-catenin (P = 0.04) were more frequently expressed in tumors of BRCA1 mutation carriers than controls. After age stratification, we found a significant difference in the frequency of tumors of BRCA1 mutation carriers diagnosed before 50 years of age compared with age-matched controls that stained positive for ER (P = 0.01), PR (P = 0.03), Ki-67 (P = 0.008), cyclin D1 (P < 0.001), HER2 (P = 0.04), and ß-catenin (P = 0.05). However, no significant differences were observed in tumors of BRCA1 mutation carriers diagnosed at age 50 or older compared with age-matched controls.

Conclusions: These data suggest that age at diagnosis, possibly related to menopausal status, may be an important factor in the expression of specific proteins in breast tumors of BRCA1 mutation carriers.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Breast tumors of BRCA1 mutation carriers generally occur at an earlier age and have distinct pathological features when compared with nonfamilial breast cancers (1, 2, 3, 4, 5, 6) . Tumors are generally higher grade, highly proliferative, and aneuploid and are predominantly ER2 - and PR-negative compared with nonfamilial cases (2 , 3 , 7 , 8) . An increased frequency of medullary carcinomas in BRCA1 mutation carriers has also been reported (4 , 9) . Many of these features are similar to those seen in early-onset breast cancers. Indeed, there appear to be few molecular markers that distinguish tumors of BRCA1 mutation carriers from those of early-onset nonfamilial cases. Several studies (10, 11, 12) have reported a significantly lower frequency of HER2 expression in tumors of BRCA1 mutation carriers compared with nonfamilial cases, but no other consistent differences have been reported.

Tumors of premenopausal women differ from those of older women in that tumors from premenopausal women are generally higher grade, are ER- and PR-negative, and are clinically more aggressive compared with those of older cases (13 , 14) . This suggests that some of the reported features of tumors of BRCA1 mutation carriers may be age-related. To examine this possibility, we compared the immunophenotype of breast tumors of BRCA1 mutation carriers with those of breast cancer patients unselected for family history but matched for age at diagnosis, tumor histological type, grade, and size. Expression of protein markers ER, PR, Ki-67, cyclin D1, TP53, HER2, ß-catenin, and cyclin E was examined in tumors of 31 BRCA1 mutation carriers and those of 81 matched controls. ER, PR, Ki-67, TP53, and HER2 protein expression in tumors have been reported to differ between BRCA1 mutation carriers and noncarriers (10, 11, 12) . One study (12) has also reported that cyclin D1 is expressed less frequently in tumors of BRCA1 mutation carriers. We also studied ß-catenin and cyclin E expression because both have been implicated in breast cancer (15, 16, 17, 18) .

We report that tumors of BRCA1 mutation carriers diagnosed before the age of 50 years display less frequent ER, PR, and cyclin D1 staining and more frequent Ki-67 and ß-catenin staining than age-matched controls. However, no differences in marker staining were observed between tumors of BRCA1 mutation carriers and controls diagnosed at 50 years and older. This result suggests that the reported characteristic immunophenotype of breast tumors of BRCA1 mutation carriers may be influenced by age of diagnosis and may be reflective of menopausal status.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Case probands were recruited through the Familial Cancer Registry of the Cleveland Clinic Foundation between January 1992 and December 1998. Criteria for inclusion of cases were based on cancer family history that included at least three first-degree cases of cancer, two of which were breast cancer (under age 50) and/or ovarian cancer, and at least one case of breast, ovarian, colon, prostate, or pancreatic cancer. The affected cancer cases were present in at least two generations. All of the probands meeting these criteria for which we could obtain consent and blood samples were recruited into the study. In addition, breast cancer patients of any age that were self-identified as Ashkenazi Jewish by descent were recruited irrespective of family history. BRCA1 mutation carrier status was determined in affected probands with a cancer family history and confirmed in tumor tissue. All of the affected family members with confirmed BRCA1 mutations were eligible for entry into the study. Five family members were also recruited into the study, of which three attended the Cleveland Clinic Foundation for surgery. All of the five were diagnosed with breast cancer during the recruitment period. Written informed consent was obtained from all of the subjects. The study protocol was approved by the Institutional Review Board of the Cleveland Clinic Foundation.

Controls were breast cancer patients identified through the tumor registry of the Cleveland Clinic Foundation and were unselected for cancer family history. All of the controls were diagnosed with breast cancer during the same period as the cases. Controls were matched to each hereditary case based on age of diagnosis (plus or minus 5 years), histological type, tumor grade, and tumor size. For the majority of cases, three controls were matched/case. Case to control matching was as follows: 21 cases were matched to 3 controls; 8 cases were matched to 2 controls; and 2 cases were matched to 1 control each. The two BRCA1 mutation carriers that were matched only to one control both had early onset medullary cancer, and few candidate control matches could be identified in the tumor registry. Clinical features of BRCA1 mutation carriers and controls are shown in Table 1Citation .


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  		Table 1 Clinical features of BRCA1 mutation carriers (n = 31) and controls (n = 81)

 
Information on menopausal status was not collected. For our analyses, the age of 50 years was chosen as a surrogate for menopause.

Mutation Detection
The presence of germ-line BRCA1 mutations was determined in lymphocyte-derived DNA of cases with a family history using three techniques described below. Breast cancer patients of Ashkenazi descent without a reported family history were screened only for the presence of the BRCA1 185 delAG or BRCA1 5382 insC mutations. Mutations were confirmed in archived tumor tissue of BRCA1 mutation-positive cases and other affected family members. Controls were not examined for the presence of BRCA1 mutations.

DNA Isolation.
Genomic DNA was isolated from peripheral blood lymphocytes using a Qiamp Blood Kit (Qiagen, Valencia, CA). DNA from archived paraffin-embedded breast tumor tissue was isolated from one 5-µm tumor tissue section using a Qiamp Tissue Kit (Qiagen).

Allele-specific Amplification.
Allele-specific amplification was used to detect the BRCA1 185 delAG and BRCA1 5382 insC mutations. PCR amplification was performed using 100 ng of DNA derived from peripheral blood as described previously (19 , 20) with the following primers and PCR amplification conditions. To detect the 185 delAG mutation, a first-round PCR was performed using the forward (5'-GAAGTTGTCATTTTATAAACC-3') and the reverse (5'-TGTCTTTTCTTCCCTAGTATGT-3') primers at an annealing temperature of 53°C. This was followed by a nested PCR reaction using a nested forward primer harboring the 185 delAG mutation (5'-GCTATGCAGAAAATCTTAGTG-3') and the initial reverse primer at an annealing temperature of 63°C. To detect the 5382 ins C mutation, a first-round PCR was performed using the forward (5'-ATATGACGTGTCTGCTCCAC-3') and the reverse (5'-GGGAATCCAAATTACACAGC-3') primers at an annealing temperature of 58°C. A nested PCR was then performed using a nested forward primer harboring the 5382 insC mutation (5'-AAGCGAGCAAGAGAATCCCC-3') and the initial reverse primer at an annealing temperature of 67°C. The protocol was identical for paraffin-embedded tumor tissue-derived DNA except that the PCR mix included 1 to 10 µl of DNA.

CSGE.
CSGE (21) was used to screen for mutations in exons 2–24 of BRCA1, excluding exons 4 and 11. The BRCA1-coding region was amplified in 26 PCR fragments using primers outlined in Table 2Citation . The PCR reactions were performed with 100 ng of DNA obtained from peripheral blood. The PCR mix included, in a final volume of 15 µl, 0.45 µl of MgCl2 (50 mM stock; Life Technologies, Inc.), 0.75 µl of 10 x deoxynucleotide triphosphate (12.5 mM dATP, dCTP, dGTP, dCTP stock; Amersham/Pharmacia, Piscataway, NJ), 1.5 µl of 10 x PCR buffer [200 mM Tris-HCl (pH 8.4), 500 mM KCl stock], 0.15 µl each of forward and reverse primers (50 pmol/µl stock), and Taq polymerase (Life Technologies, Inc.). The PCR program included 5 min at 94°C, 35 cycles of 45 s at 94°C, 1 min at the specified annealing temperature (Table 2)Citation , and 1 min at 72°C, followed by a 7-min extension at 72°C.


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  		Table 2 PCR primers used for CSGE assaysa

 
The PCR products were run on a 20 x 50-cm semi-denaturing gel for 8.5 h at room temperature in 0.5 x TTE buffer [10 x stock (pH 9): 53.89 g of Tris, 17.83 g of taurine, and 372.2 mg of EDTA] after a 15-min preloading run of the gel. Before loading, the PCR products were heated for 5 min at 95°C followed by incubation at 68°C for 30 min to 1 h. PCR products were visualized using a StormImager (Molecular Dynamics, Sunnyvale, CA) set at blue luminescence mode after SybrGold (Molecular Probes, Eugene, OR) staining. Fragments displaying banding patterns that were different to the wild-type pattern were sequenced using an ABI 377 automated sequencer (Perkin-Elmer, Foster City, CA).

Protein Truncation Test.
A protein truncation test was used to screen for truncating mutations in exon 11 of BRCA1, using methods described previously (22) . Briefly, three overlapping segments of exon 11 were amplified by PCR, but the forward primer included a T7 polymerase promoter and Kozak consensus sequence. The PCR products were transcribed and translated using either a TnT coupled reticulocyte lysate system or wheat germ extract system (Promega, Madison, WI), incorporating either [35S]methionine (NEN, Boston, MA) or [35S]cysteine (Amersham/Pharmacia). Products were resolved on a 15% SDS-polyacrylamide gel and detected using a StormImager set at phosphorimager mode (Molecular Dynamics) after exposure to a phosphorimager screen. Fragments displaying banding patterns that were distinct from the wild-type pattern were sequenced using an ABI 377 automated sequencer (Perkin-Elmer).

Immunohistochemistry
Four-µm sections of archived paraffin-embedded tumor tissues of cases and controls were mounted on positively charged microscope slides and baked overnight at 60°C. Tumor grading was determined by a single pathologist (J. M.) based on the Scarff-Bloom-Richardson system (23) .

ER and PR status for the cases and controls was obtained using immunohistochemical approaches (Ventana, Tucson, AZ) and information obtained from pathology reports. The following dilutions of mouse monoclonal antibodies were used for detection of the remaining markers: Ki-67 (dilution, 1:100; Immunotech, Marseille France), cyclin D1 (dilution, 1:200; Cell Marque, Austin, TX), TP53 (dilution, 1:200; DAKO, Carpinteria, CA), HER2 (prediluted; Ventana), ß-catenin (dilution, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA), and cyclin E (dilution, 1:20; Novocastra, Burlingame, CA). Assays were performed using a Ventana 320 automated immunostainer. An antigen retrieval protocol was used for all of the protein markers. This involved microwaving the specimens on a high-power setting for either 15 min (KI-67, cyclin D1, and TP53) or 30 min (HER2, ß-catenin, and cyclin E) in 10 mM sodium citrate (pH 6.0). Tissue sections were incubated with each of the primary antibodies at 42°C for 32 min except for cyclin E. Sections incubated with the cyclin E antibody were incubated at room temperature for 60 min. For cyclin D1, slides were preincubated with trypsin (1:100 dilution; Zymed Laboratories, San Francisco, CA) for 4 min before application of the primary antibody. All of the tissue sections were incubated with a biotin-conjugated secondary antibody (Ventana) and processed for detection using the Ventana DAB detection kit and counter stained with hematoxylin. Both positive and negative controls were included in each assay. Positive controls included tissues that were previously determined to stain positive for the respective antibodies. For negative controls, the primary antibody was replaced with rabbit/mouse nonimmune IgG (Ventana).

All of the immunostaining was evaluated by a single pathologist (R. R. T.) blinded to the case and control status. For each antibody, only the cellular compartment that was expected to express the antigen of interest was scored. Positive (+) scores shown in Table 3Citation represent 5% or greater positive-staining cells, with the exception of HER2 and cyclin D1 (10% or greater) and Ki-67 (50% or greater).


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  		Table 3 BRCA1 mutation carriers; genotype, clinical features, and immunophenotypes

 
Statistical Methods
{chi}2 tests or Fisher’s exact tests for case-control studies involving variable numbers of controls were used to compare the cases and controls with respect to ER and PR status and the comparison of protein marker expression (24) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We screened affected probands of 104 breast cancer families and identified 18 cases with germ-line BRCA1 mutations (25) . Tumor blocks of sufficient quality for the proposed studies were available for 13 of these cases. An additional five family members were recruited into the immunotyping phase of the study after confirmation of mutation status. Fifty-three Ashkenazi Jewish breast cancer patients unselected for family history were also screened, and 13 patients with the BRCA1 185 delAG mutation, but none with the BRCA1 5382 insC mutation, were identified. Clinical features of BRCA1 mutation carriers and controls are shown in Table 1Citation . The overall frequency of high-grade tumors in BRCA1 mutation carriers is 52%, which is lower than in published reports (10 , 12) . However, all except three of the high-grade cases were under 50 years of age at diagnosis. When restricted only to cases under 50 years of age, the frequency of high-grade tumors is more consistent with published studies. A summary of the mutation and immunostaining data for cases is shown in Table 3Citation .

A comparison of the frequency of marker expression in tumors of all of the cases and controls is shown in Table 4Citation . Overall, ER (P = 0.01), PR (P = 0.06), and cyclin D1 (P = 0.002) expression was observed less frequently in tumors of the BRCA1 mutation cases than controls, whereas Ki-67 (P = 0.01) and ß-catenin (P = 0.04) expression was observed more frequently in tumors of the BRCA1 mutation cases than controls. No statistically significant differences were found in the frequency of HER2 or TP53 expression in tumors of BRCA1 mutation cases compared with controls. Cyclin E was detected in tumors of only two BRCA1 mutation cases and three controls and was further evaluated.


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  		Table 4 Comparison of immunohistochemical marker expression between cases and matched controls not stratified by age or grade

 
Most markers examined in the study were expressed in an age-dependent (under age 50 versus age 50 and over) manner in both cases and controls (Table 5)Citation . For BRCA1 mutation carrier cases, statistically significant age-related differences were observed in the frequency of tumors staining for ER (P = 0.01), PR (P = 0.01), and Ki-67 (P = 0.05). Fewer tumors stained positive for ER and PR, and more stained positive for Ki-67 in cases diagnosed before 50 years of age. A similar significant difference was found in tumors of controls for ER (P = 0.05) and PR (P = 0.03) but not Ki-67 (P = 1.0). In addition, highly significant differences were observed in the frequency of tumors staining for TP53 (P = 0.0002) and HER2 (P = 0.0005), indicating that TP53 and HER2 expression occurs infrequently in tumors of nonfamilial breast cancers diagnosed at 50 years of age or older.


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  		Table 5 Comparison of protein expression in tumors stratified by age within groups: BRCA1 mutation carriers and matched controls

 
Despite the similarities in the frequency of marker staining in tumors of cases and controls stratified by age, when BRCA1 mutation cases and controls were compared with each other, significant differences were found. ER, PR, and cyclin D1 were expressed less frequently (P = 0.01, P = 0.03, and P < 0.001, respectively) in tumors of BRCA1 mutation carriers than matched controls in breast cancer patients diagnosed before 50 years of age (Table 6)Citation . In contrast, no differences were seen in the frequency of ER, PR, or cyclin D1 staining in tumors of cases and controls diagnosed with breast cancer at age 50 or over (Table 6)Citation .


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  		Table 6 Comparison of protein expression in tumors of BRCA1 mutation carriers and matched controls stratified by age

 
Ki-67 and ß-catenin were expressed more frequently (P = 0.008 and P = 0.05, respectively) in tumors of BRCA1 mutation cases than matched controls diagnosed before 50 years of age. In contrast, no significant differences in expression frequencies between the case and control groups diagnosed at age 50 and older were observed for either Ki-67 or ß-catenin (Table 6)Citation . No differences were observed in the frequency of tumors expressing TP53 in tumors of BRCA1 mutation carriers and controls. TP53 overexpression was seen only in two BRCA1 mutation-associated tumors and one control tumor of patients diagnosed at 50 years of age or older. HER2 was more frequently (P = 0.04) expressed in tumors of controls than tumors of BRCA1 mutation carriers among patients diagnosed before 50 years of age. In tumors of patients diagnosed at age 50 years or older, the tumors of only one BRCA1 mutation carrier and no controls expressed HER2 (Table 6)Citation .

We also stratified by grade. ER and PR status were influenced by grade within both case and control populations. Among cases, 10 of 14 low-grade tumors were ER-positive compared with 2 of 14 high-grade tumors (P = 0.01). Among controls, 29 of 35 low-grade tumors were ER-positive compared with 16 of 31 high-grade tumors (P = 0.01). For PR, among cases, 9 of 14 low-grade tumors were PR-positive compared with 3 of 14 high-grade tumors (P = 0.05). Among controls, 32 of 35 low-grade tumors were positive for PR compared with 12 of 31 high-grade tumors (P = 0.001). We also observed a lower frequency of Ki-67 expression among low-grade tumors compared with high-grade tumors. Among cases, 9 of 14 high-grade tumors were Ki-67-positive compared with 9 of 14 low-grade tumors (P = 0.02). Among controls, none of 37 high-grade tumors were Ki-67-positive compared with 12 of 36 high-grade tumors (P = 0.001). No differences between cases and controls were observed with any of the other markers after stratification by grade (data not shown). We also statistically analyzed the 185delAG population alone, because all of the cases diagnosed over 50 years were 185delAG mutation carriers. No statistically significant differences were observed between 185delAG cases and matched controls alone (data not shown).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Several groups (4 , 5 , 12 , 26 , 27) have reported that breast tumors of BRCA1 mutation carriers display distinct pathological and molecular phenotypes. In this report, we demonstrate for the first time that the breast tumor phenotype of BRCA1 mutation carriers may be influenced by age at diagnosis. Although breast tumors of BRCA1 mutation carriers diagnosed with breast cancer before the age of 50 years show a distinct tumor phenotype compared with age-matched controls, tumors of carriers diagnosed at 50 years of age or older display a phenotype that is similar to that of age-matched breast cancer controls. We chose the age of 50 years at diagnosis as our cutoff because several studies (28, 29, 30) have indicated that this age represents a surrogate for menopausal status.

We compared the molecular phenotypes of tumors of 31 BRCA1 mutation carriers matched with tumors of 81 breast cancer controls on age at diagnosis (within ±5 years), histology, grade, and size of tumor. Overall, we found a lower frequency of tumors expressing ER, PR, and cyclin D1 and a higher frequency of tumors expressing Ki-67 and ß-catenin in BRCA1-mutation cases compared with controls. These data are consistent with published reports (10 , 12 , 27 , 31 , 32) . However, we also observed an even greater difference in the number of tumors expressing these proteins in BRCA1 mutation carriers compared with controls diagnosed before the age of 50 years but no differences between carriers and controls diagnosed at age 50 years or older.

This age-related difference in marker expression suggests that menopausal status may affect marker expression in tumors. How age may influence marker expression is unclear, but BRCA1 has been shown to inhibit ER-{alpha} transcription (33) , and a direct interaction has been reported recently (34) . This raises the possibility that BRCA1 (and possibly BRCA2) may inhibit estrogen-dependent pathways in mammary epithelial cells and that loss of this ability contributes to cancer development (33) . Indeed, studies (35 , 36) have reported that use of dated oral contraceptive formulations with elevated estrogen levels may confer increased breast cancer risk for women with a hereditary predisposition. This suggests that estrogen supplementation may exacerbate pathways affecting mammary cell proliferation that may proceed unchecked in the absence of functional BRCA1. By inference, a reduction in circulating estrogens after menopause may reduce the impact that loss of BRCA1 expression may have on estrogen-related pathways, leading to the appearance of similar tumor phenotypes in BRCA1 mutation carriers and nonfamilial cases who develop breast cancer after menopause.

Estrogens have been hypothesized to play a dual role in breast cancer risk (37) . Directly or indirectly, they may promote risk through stimulating growth of mammary cells and inducing DNA damage. Estrogens have also been proposed to play a role in reducing risk of developing breast cancer through the activation of tumor suppressor genes critical to the maintenance of genomic stability and repair of DNA damage, such as BRCA1 and TP53 (37) . If this were the case, estrogens would not have any protective effect and may only induce genetic instability in women who harbor a BRCA1 mutation and subsequently lose BRCA1 function.

Does this suggest that inactivation of TP53 is critical to BRCA1 mutation-associated tumor development? Several studies (38, 39, 40) have reported that a significantly higher frequency of tumors of BRCA1 mutation carriers overexpresses TP53 protein (and presumably contain mutant TP53) than controls and that loss of TP53 function may be critical in the development of BRCA1-related tumors. In the present study, no differences were observed between the frequency of tumors of carriers or controls overexpressing TP53 protein with or without age stratification. However, it should be noted that we used an immunohistochemical approach for TP53 mutation detection, and this approach does not identify all of the mutations (41) . Several other studies (10 , 12 , 31 , 42) have reported TP53 protein expression frequencies similar to our own. It is unclear whether or not other critical genes within the TP53 damage response pathway may be inactivated in apparently wild-type TP53 cells.

More lobular carcinomas were seen in cases diagnosed over 50 years of age than under 50 years of age (four versus one). Because lobular carcinomas are generally ER-positive, this raises the possibility that histology may also play some role in the differences observed in immunophenotype in this study. Unfortunately, in our study the number of lobular cases is too small to draw any conclusions.

The implication of these findings on survival in BRCA1 mutation-associated breast cancer cases is not known. In the present study, follow-up data were not available. Several studies (1 , 2 , 27 , 43, 44, 45) have evaluated survival of patients with BRCA1-associated cancers; however, findings have been inconsistent. It remains a possibility that this inconsistency may be attributable to the relative number of pre- and post-menopausal BRCA1 mutation carriers in these study populations. However, two published studies (3 , 10) did not find age to be a prognostic factor in BRCA1-mutation cases. Given the inconsistency in findings related to survival of BRCA1-mutation cases, future studies should take age of diagnosis into account when assessing prognosis in these patients. It is conceivable that the age-related difference in the expression of certain tumor markers could significantly impact disease course.

In summary, this study highlights differences in marker expression in tumors of BRCA1 mutation carriers that appear to be associated with age at diagnosis. These findings imply that breast cancer development in BRCA1 mutation carriers may involve molecular mechanisms that are influenced by estrogen, possibly related to menopausal status. However, the mechanism by which these changes occur and the effect that they have on prognosis remain unknown. It should be noted that we cannot discount the possibility that some of the breast tumors developing in BRCA1 mutation carriers at later ages were unrelated to BRCA1 status and were in fact sporadic phenocopies. Larger studies will be needed to confirm our findings, and those studies should include menopausal status in patient assessment.


   ACKNOWLEDGMENTS
 
We thank Linda Webster and Cindy Sebrasky for assistance in patient recruitment and Linda Vargo for technical assistance in immunostaining.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Department of Cancer Biology, ND50, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. Phone: (216) 445-9754; Fax: (216) 445-0610; E-mail: caseyg{at}ccf.org Back

2 The abbreviations used are: ER, estrogen receptor; PR, progesterone receptor; CSGE, conformation-sensitive gel electrophoresis; DAB, diaminobenzidine; PTT, protein truncation test. Back

Received 11/20/00; revised 4/16/01; accepted 4/17/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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