Isolevuglandins as a gauge of lipid peroxidation in human tumors.

The cellular production of free radicals or reactive oxygen species (ROS) can lead to protein, lipid or DNA modifications and tumor formation. The cellular lipids undergo structural changes through the actions of enzymes (e.g. cyclooxygenases) or free radicals to form a class of compounds called Isolevuglandins (IsoLGs). The recruitment and continued exposure of tissue to ROS and IsoLGs causes increased cell proliferation, mutagenesis, loss of normal cell function and angiogenesis. The elevated concentration of ROS in cancerous tissues suggests that these mediators play an important role in cancer development. We hypothesized that tumors with elevated ROS levels would similarly possess an increased concentration of IsoLGs when compared with normal tissue. Using D11, an ScFv recombinant antibody specific for IsoLGs, we utilized immunohistochemistry to visualize the presence of IsoLG in human tumors compared to normal adjacent tissue (NAT) to the same tumor. We found that IsoLG concentrations were elevated in human breast, colon, kidney, liver, lung, pancreatic and tongue tumor cells when compared to NAT and believe that IsoLGs can be used as a gauge indicative of lipid peroxidation in tumors.

Alterations in oestrogen metabolism: implications for higher penetrance of familial pulmonary arterial hypertension in females.

Mutations in bone morphogenetic protein receptor type 2 (BMPR2) cause familial pulmonary arterial hypertension (FPAH), but the penetrance is reduced and females are significantly overrepresented. In addition, gene expression data implicating the oestrogen-metabolising enzyme CYP1B1 suggests a detrimental role of oestrogens or oestrogen metabolites. We examined genetic and metabolic markers of altered oestrogen metabolism in subjects with a BMPR2 mutation. Genotypes for CYP1B1 Asn453Ser (N453S) were determined for 140 BMPR2 mutation carriers (86 females and 54 males). Nested from those subjects, a case-control study of urinary oestrogen metabolite levels (2-hydroxyoestrogen (2-OHE) and 16alpha-hydroxyoestrone (16alpha-OHE(1))) was conducted in females (five affected mutation carriers versus six unaffected mutation carriers). Among females, there was four-fold higher penetrance among subjects homozygous for the wild-type genotype (N/N) than those with N/S or S/S genotypes (p = 0.005). Consistent with this finding, the 2-OHE/16alpha-OHE(1) ratio was 2.3-fold lower in affected mutation carriers compared to unaffected mutation carriers (p = 0.006). Our findings suggest that variations in oestrogens and oestrogen metabolism modify FPAH risk. Further investigation of the role of oestrogens in this disease with profound sex bias may yield new insights and, perhaps, therapeutic interventions.

Metabolic gene polymorphism frequencies in control populations.

Using the International Project on Genetic Susceptibility to Environmental Carcinogens (GSEC) database containing information on over 15,000 control (noncancer) subjects, the allele and genotype frequencies for many of the more commonly studied metabolic genes (CYP1A1, CYP2E1, CYP2D6, GSTM1, GSTT1, NAT2, GSTP, and EPHX) in the human population were determined. Major and significant differences in these frequencies were observed between Caucasians (n = 12,525), Asians (n = 2,136), and Africans and African Americans (n = 996), and some, but much less, heterogeneity was observed within Caucasian populations from different countries. No differences in allele frequencies were seen by age, sex, or type of controls (hospital patients versus population controls). No examples of linkage disequilibrium between the different loci were detected based on comparison of observed and expected frequencies for combinations of specific alleles.

Catechol-O-methyltransferase (COMT)-mediated metabolism of catechol estrogens: comparison of wild-type and variant COMT isoforms.

The oxidative metabolism of 17beta-estradiol (E2) and estrone (E1) to catechol estrogens (2-OHE2, 4-OHE2, 2-OHE1, and 4-OHE1) and estrogen quinones has been postulated to be a factor in mammary carcinogenesis. Catechol-O-methyltransferase (COMT) catalyzes the methylation of catechol estrogens to methoxy estrogens, which simultaneously lowers the potential for DNA damage and increases the concentration of 2-methoxyestradiol (2-MeOE2), an antiproliferative metabolite. We expressed two recombinant forms of COMT, the wild-type (108Val) and a common variant (108Met), to determine whether their catalytic efficiencies differ with respect to catechol estrogen inactivation. The His-tagged proteins were purified by nickel-nitrilo-triacetic acid chromatography and analyzed by electrophoresis and Western immunoblot. COMT activity was assessed by determining the methylation of 2-OHE2, 4-OHE2, 2-OHE1, and 4-OHE1, using gas chromatography/mass spectrometry for quantitation of the respective methoxy products. In the case of 2-OHE2 and 2-OHE1, methylation occurred at 2-OH and 3-OH groups, resulting in the formation of 2-MeOE2 and 2-OH-3-MeOE2, and 2-MeOE1 and 2-OH-3-MeOE1, respectively. In contrast, in the case of 4-OHE2 and 4-OHE1, methylation occurred only at the 4-OH group, yielding 4-MeOE2 and 4-MeOE1, respectively. Individual and competition experiments revealed the following order of product formation: 4-MeOE2 > 4-MeOE1 >> 2-MeOE2 > 2-MeOE1 > 2-OH-3-MeOE1 > 2-OH-3-MeOE2. The variant isoform differed from wild-type COMT by being thermolabile, leading to 2-3-fold lower levels of product formation. MCF-7 breast cancer cells with the variant COMT 108Met/Met genotype also displayed 2-3-fold lower catalytic activity than ZR-75 breast cancer cells with the wild-type COMT 108Val/Val genotype. Thus, inherited alterations in COMT catalytic activity are associated with significant differences in catechol estrogen and methoxy estrogen levels and, thereby, may contribute to interindividual differences in breast cancer risk associated with estrogen-mediated carcinogenicity.

No elevation in long-term breast carcinoma risk for women with fibroadenomas that contain atypical hyperplasia.

BACKGROUND: The authors previously showed that women with a fibroadenoma have a relative risk of invasive breast carcinoma of approximately 2.0 compared with women of similar age from the general population. This relative risk approaches 1.0 when family history and proliferative changes in the adjacent parenchyma are removed and rises to > 3.0 if the fibroadenoma has complex histology. The risk for developing breast carcinoma in women with atypical lobular hyperplasia (ALH) and atypical ductal hyperplasia (ADH) or their minimal variants within a fibroadenoma is unknown. METHODS: The authors conducted a long-term, retrospective cohort study of 1834 women with adequate follow-up who presented with fibroadenoma at three local hospitals between 1950 and 1968. Histology was reviewed using established criteria, and the patients were categorized with ALH, ADH, minimal atypia, or no atypia. RESULTS: The overall prevalence of ALH or ADH within fibroadenomas was 0.81%. Minimal or true atypia within a fibroadenoma appeared to be correlated with proliferative disease in the adjacent parenchyma but could not predict for the presence there of well-established atypia. Only 7% of women with well-developed atypia developed invasive carcinoma on follow-up. Three women with minimal atypia developed invasive carcinoma. CONCLUSIONS: In this study of a large cohort of women with fibroadenoma, the authors found that atypia within a fibroadenoma cannot predict for the presence of atypia within adjacent breast parenchyma. They also found that atypia confined to a fibroadenoma does not incur a clinically meaningful risk of future breast carcinoma development greater than that of fibroadenoma alone.

Multifactor-dimensionality reduction reveals high-order interactions among estrogen-metabolism genes in sporadic breast cancer.

One of the greatest challenges facing human geneticists is the identification and characterization of susceptibility genes for common complex multifactorial human diseases. This challenge is partly due to the limitations of parametric-statistical methods for detection of gene effects that are dependent solely or partially on interactions with other genes and with environmental exposures. We introduce multifactor-dimensionality reduction (MDR) as a method for reducing the dimensionality of multilocus information, to improve the identification of polymorphism combinations associated with disease risk. The MDR method is nonparametric (i.e., no hypothesis about the value of a statistical parameter is made), is model-free (i.e., it assumes no particular inheritance model), and is directly applicable to case-control and discordant-sib-pair studies. Using simulated case-control data, we demonstrate that MDR has reasonable power to identify interactions among two or more loci in relatively small samples. When it was applied to a sporadic breast cancer case-control data set, in the absence of any statistically significant independent main effects, MDR identified a statistically significant high-order interaction among four polymorphisms from three different estrogen-metabolism genes. To our knowledge, this is the first report of a four-locus interaction associated with a common complex multifactorial disease.

Aberrant methylation of the estrogen receptor and E-cadherin 5' CpG islands increases with malignant progression in human breast cancer.

Loss of expression for both the estrogen receptor-alpha and E-cadherin genes has been linked to disease progression in human ductal breast carcinomas and has been associated with aberrant 5' CpG island methylation. To assess when, during malignant progression, such methylation begins and whether such methylation increases with advancing disease, we have surveyed 111 ductal carcinomas of the breast for aberrant methylation of the estrogen receptor-alpha and E-cadherin 5' CpG islands. Hypermethylation of either CpG island was evident prior to invasion in approximately 30% of ductal carcinoma in situ lesions and increased significantly to nearly 60% in metastatic lesions. Coincident methylation of both CpG islands also increased significantly from approximately 20% in ductal carcinoma in situ to nearly 50% in metastatic lesions. Furthermore, in all cases, the pattern of methylation displayed substantial heterogeneity, reflecting the well-established, heterogeneous loss of expression for these genes in ductal carcinomas of the breast.

Cytochrome P450 1B1 (CYP1B1) pharmacogenetics: association of polymorphisms with functional differences in estrogen hydroxylation activity.

Activation of 17beta-estradiol (E2) through the formation of catechol estrogen metabolites, 2-OH-E2 and 4-OH-E2, and the C-16alpha hydroxylation product, 16alpha-OH-E2, has been postulated to be a factor in mammary carcinogenesis. Cytochrome P450 1B1 (CYP1B1) exceeds other P450 enzymes in both estrogen hydroxylation activity and expression level in breast tissue. To determine whether inherited variants of CYP1B1 differ from wild-type CYP1B1 in estrogen hydroxylase activity, we expressed recombinant wild-type and five polymorphic variants of CYP1B1: variant 1 (codon 48Arg-->Gly), variant 2 (codon 119Ala-->Ser), variant 3 (codon 432Val-->Leu), variant 4 (codon453Asn-->Ser), variant 5 (48Gly, 119Ser, 432Leu, 453Ser). The His-tagged proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography and analyzed by electrophoresis and spectrophotometry. We performed assays of E2 hydroxylation activity and quantitated production of 2-OH-E2, 4-OH-E2, and 16alpha-OH-E2 by gas chromatography/mass spectrometry. Wild-type CYP1B1 formed 4-OH-E2 as main product (Km, 40+/-8 microM; k(cat) 4.4+/-0.4, min(-1); k(cat)/Km, 110 mM(-1) min(-1)), followed by 2-OH-E2 (Km, 34+/-4 microM; k(cat), 1.9+/-0.1 min(-1); k(cat)/Km, 55 mM(-1)min(-1)) and 16alpha-OH-E2 (Km, 39+/-5.7 microM; k(cat), 0.30+/-0.02 min(-1); k(cat)/Km, 7.6 mM(-1)min(-1)). The CYP1B1 variants also formed 4-OH-E2 as the main product but displayed 2.4- to 3.4-fold higher catalytic efficiencies k(cat)/Km than the wild-type enzyme, ranging from 270 mM(-1)min(-1) for variant 4, to 370 mM(-1)min(-1) for variant 2. The variant enzymes also exceeded wild-type CYP1B1 with respect to 2- and 16alpha-hydroxylation activity. Thus, inherited alterations in CYP1B1 estrogen hydroxylation activity may be associated with significant changes in estrogen metabolism and, thereby, may possibly explain interindividual differences in breast cancer risk associated with estrogen-mediated carcinogenicity.

Estrogen replacement therapy in women with a history of proliferative breast disease.

BACKGROUND: Little information is available regarding the invasive breast carcinoma risk associated with estrogen replacement therapy (ERT) in women with histories of histologically defined breast lesions. METHODS: A retrospective cohort study of a consecutive series of women who underwent breast biopsies that proved to be benign between 1952-1978 was conducted. Follow-up data were obtained for 9494 women (87.6% of women eligible for follow-up). To investigate the effect of ERT on invasive breast carcinoma risk, the analysis was restricted to women with premenopausal breast disease whose follow-up extended through menopause and who did not develop premenopausal breast carcinoma. Relative risks were calculated with respect to women who took ERT but whose benign breast biopsies had neither atypical hyperplasia (AH), complex fibroadenoma (CFA), nor proliferative disease without atypia (PDWA). RESULTS: During 190,845 woman-years of follow-up there were 444 confirmed cases of invasive breast carcinoma in the entire cohort. Women with a history of AH had relative risks of invasive breast carcinoma of 2.87 (95% confidence interval [95% CI], 1.3-6.3) and 2.53 (95% CI, 1.0-6.3) if they did or did not take ERT, respectively. For women with a history of CFA these risks were 1.57 (95% CI, 0.72-3.4) and 1.46 (95% CI, 0.53-4.0), respectively, whereas for women with a history of PDWA they were 1.37 (95% CI, 0.88-2.1) and 1.13 (95% CI, 0.69-1.9), respectively. CONCLUSIONS: ERT does not significantly elevate the risk of invasive breast carcinoma in women with previous histologically defined benign breast disease. Therefore, ERT is not contraindicated in these women.

Association of cytochrome P450 1B1 (CYP1B1) polymorphism with steroid receptor status in breast cancer.

A key enzyme involved in the production of potentially carcinogenic estrogen metabolites and the activation of environmental carcinogens is cytochrome P450 1B1 (CYP1B1), the predominant member of the CYP1 family expressed in normal breast tissue and breast cancer. Because of the preeminent role of CYP1B1 in mammary estrogen/carcinogen metabolism, we examined the CYP1B1 gene to determine whether genetic differences could account for interindividual differences in breast cancer risk. We focused on exon 3, because it encodes the catalytically important heme binding domain of the enzyme, and discovered three polymorphisms of which two are associated with amino acid substitutions in codons 432 (Val-->Leu) and 453 (Asn-->Ser), designated as m1 and m2, respectively. Approximately 40% of Caucasian women have the m1 Val allele compared with nearly 70% of African-American women (P < 0.0001). The allele frequency also differs significantly in m2, with the rare Ser allele being present in 17.4% of Caucasians but only in 3.4% of African Americans (P < 0.0003). To determine whether the polymorphic CYP1B1 alleles hold implications as potential breast cancer risk factors, we compared the CYP1B1 genotypes in 164 Caucasian and 59 African-American breast cancer cases with those in age-, race-, and frequency-matched controls. Odds ratio calculations failed to show a significant association between any of the genotypes and breast cancer. Because CYP1B1 is known to be involved in mammary estrogen metabolism, we investigated whether the estrogen receptor status is influenced by the CYP1B1 genotypes. Caucasian patients with the m1 Val/Val genotype have a significantly higher percentage of estrogen receptor-positive (P = 0.02) and progesterone receptor-positive breast cancers (P = 0.003). There was no correlation with the m2 genotypes. These data suggest that the CYP1B1 polymorphisms in exon 3 are not associated with increased breast cancer risk but that the m1 polymorphism may be functionally important for steroid receptor expression in breast cancer of Caucasian patients.

Mapping of ER gene CpG island methylation-specific polymerase chain reaction.

Southern analysis has shown that DNA from 25% of primary estrogen receptor (ER) alpha-negative breast tumors displays aberrant methylation at one site within the ER gene CpG island. To examine more sites and increase sensitivity, we developed a methylation-specific PCR assay to map methylation of the entire ER CpG island. The island was unmethylated in normal breast tissue and ER-positive breast cancer cell lines, but extensively methylated in all ER-negative cell lines and breast tumors examined. In addition, some of the ER-positive/progesterone receptor-negative and ER-positive/progesterone receptor-positive tumors (about 70% and 35%, respectively) displayed methylation of the ER CpG island, suggesting that this heterogeneity within tumor cell populations could potentially shed light on the etiology of ER-negative recurrent tumors arising from ER-positive tumors.

Breast cancer and CYPIA1, GSTM1, and GSTT1 polymorphisms: evidence of a lack of association in Caucasians and African Americans.

Genetically based differences in carcinogen metabolism have been related to polymorphisms of the cytochrome P450IA1 gene (CYPIA1) and the null genotypes of glutathione S-transferase classes mu and theta (GSTM1 and GSTT1). By PCR we examined the genotypes of CYPIA1, GSTM1, and GSTT1 in relation to breast cancer risk in Caucasian and African-American women. The study included 164 Caucasian and 59 African-American women with primary invasive breast cancer and age-matched female controls. Enzyme polymorphisms included in this study were the null deletions of GSTM1 and GSTT1 and the m1 (MspI), m2 (codon 462: isoleucine-->valine), m3 (MspI-AA), and m4 (codon 461: threonine-->asparagine) polymorphisms of CYPIA1. Contrary to previous reports by other investigators, none of the enzyme genotypes, individually or combined, appear to associate with an increased risk for breast cancer in Caucasian or African-American women. We also report that the recently described m4 allele occurs at a lower frequency in African-Americans than Caucasians and is not linked with breast cancer in either race. Thus, it is unlikely that polymorphisms of GSTM1, GSTT1, or CYPIA1 represent susceptibility factors for breast cancer in Caucasians or African-Americans.

Impact of managed care on the economics of laboratory operation in an academic medical center.

BACKGROUND: Throughout the 1980s, the number of laboratory tests performed in the United States grew at an annual rate of over 10%, and laboratory costs accounted for approximately 10% of overall health care expenditures. Recently, the influence of capitation, emphasis on cost-effectiveness, and changing roles among specialists and primary care physicians have begun to affect the growth of laboratory testing. We examined the impact of managed care on the economics of the clinical chemistry laboratory at Vanderbilt University Medical Center, Nashville, Tenn, to define the relative position of the clinical laboratory in the managed care environment of an academic medical center. METHODS: The following data were prospectively collected between fiscal years 1984/1985 and 1995/1996: number of inpatients and outpatients, average length of stay, number of laboratory tests, total laboratory revenue, direct costs (consisting of salary and consumable costs), and number of full-time-equivalent (FTE) personnel. Using these data, we derived the following parameters: revenue and direct cost per patient, and revenue and productivity per FTE. RESULTS: Between 1984/1985 and 1995/1996 the number of inpatients and outpatients increased 33% and 155%, respectively. Laboratory utilization, expressed as tests per patient, increased from 17 to 22 for inpatients between 1984/1985 and 1991/1992, and then sharply declined to 14.5 tests by 1995/1996, a 34% decrease compared with the 1991/1992 level. Laboratory utilization for outpatients increased from 0.23 in 1984/1985 to 0.45 tests in 1991/1992, decreased to 0.38 in 1993/1994, but then rose again to 0.43 in 1995/1996. Total revenue more than doubled between 1984/1985 and 1991/1992, mostly owing to increased inpatient revenue. Since 1992/1993, inpatient revenue has steadily declined, leading to a decrease in total revenue, which was partially offset by a continuous increase in outpatient revenue. In 1995/1996, outpatient revenue accounted for 32.1% of total revenue, compared with 7.7% in 1984/1985. Direct test cost per patient increased approximately 20% between 1984/1985 and 1991/1992, followed by a decline below the 1984/1985 level. The number of FTEs increased in parallel to the rising test volume through 1991/1992 and subsequently was reduced in response to the decrease in test volume and productivity. In 1995/1996, a 22.7% reduction in staff was imposed despite an upward trend in test volume, resulting in a sharp increase in revenue and productivity per FTE. The staff reduction did not decrease direct laboratory costs, which have remained constant since 1992/1993. CONCLUSIONS: After three decades of continued growth, managed care has caused a sharp reversal in the upward trend in the number of laboratory tests, the number of tests per inpatient, test costs per patient, laboratory revenue, and productivity. A recent staff reduction significantly increased revenue and productivity per FTE, but showed no effect on direct laboratory costs.

High-mobility group (HMG) protein HMG-1 and TATA-binding protein-associated factor TAF(II)30 affect estrogen receptor-mediated transcriptional activation.

The estrogen receptor (ER) belongs to a family of ligand-inducible nuclear receptors that exert their effects by binding to cis-acting DNA elements in the regulatory region of target genes. The detailed mechanisms by which ER interacts with the estrogen response element (ERE) and affects transcription still remain to be elucidated. To study the ER-ERE interaction and transcription initiation, we employed purified recombinant ER expressed in both the baculovirus-Sf9 and his-tagged bacterial systems. The effect of high-mobility group (HMG) protein HMG-1 and purified recombinant TATA-binding protein-associated factor TAF(II)30 on ER-ERE binding and transcription initiation were assessed by electrophoretic mobility shift assay and in vitro transcription from an ERE-containing template (pERE2LovTATA), respectively. We find that purified, recombinant ER fails to bind to ERE in spite of high ligand-binding activity and electrophoretic and immunological properties identical to ER in MCF-7 breast cancer cells. HMG-1 interacts with ER and promotes ER-ERE binding in a concentration- and time-dependent manner. The effectiveness of HMG-1 to stimulate ER-ERE binding in the electrophoretic mobility shift assay depends on the sequence flanking the ERE consensus as well as the position of the latter in the oligonucleotide. We find that TAF(II)30 has no effect on ER-ERE binding either alone or in combination with ER and HMG-1. Although HMG-1 promotes ER-ERE binding, it fails to stimulate transcription initiation either in the presence or absence of hormone. In contrast, TAF(II)30, while not affecting ER-ERE binding, stimulates transcription initiation 20-fold in the presence of HMG-1. These results indicate that HMG-1 and TAF(II)30 act in sequence, the former acting to promote ER-ERE binding followed by the latter to stimulate transcription initiation.

Methylation of estrogen and progesterone receptor gene 5' CpG islands correlates with lack of estrogen and progesterone receptor gene expression in breast tumors.

Hormonal factors have a profound influence on the development, treatment, and outcome of breast cancer. The absence of steroid hormone receptors is highly correlated with resistance to antihormonal treatments. Work in cultured human breast cancer cell lines has shown that the absence of estrogen receptor (ER) gene expression in ER- cells is associated with extensive methylation of the ER gene 5' CpG island, and treatment with agents that demethylate the ER gene CpG island results in the production of functional ER protein. The current study shows that CpG islands in the 5' region of the ER and progesterone receptor (PR) genes are methylated in a significant fraction of primary human breast cancer tissues. The ER CpG island is methylated at the methylation-sensitive NotI restriction site in 9 of 39 (25%) of primary ER- breast cancers but remains unmethylated in 53 ER+ breast cancers and 9 normal breast specimens. Three methylation-sensitive restriction sites in the PR gene CpG island are not methylated in normal breast specimens and PR+ human breast cancers but are hypermethylated in 40% of PR- human breast tumors. These data demonstrate that methylation of the ER and PR gene CpG islands is associated with the lack of ER and PR gene expression in a significant fraction of human breast cancers.

Estrogen receptor gene analysis in estrogen receptor-positive and receptor-negative primary breast cancer.

BACKGROUND: In breast cancer patients, about two thirds of the tumors are estrogen receptor (ER)-positive and one third are ER-negative. The molecular mechanisms leading to the ER-negative phenotype are poorly understood. Nearly all ER-negative and about 40% of ER-positive cancers are resistant to endocrine therapy. PURPOSE: In this study, we examined the entire coding region of the ER gene in ER-positive and ER-negative primary breast tumors to determine whether deletions/insertions or point mutations might account for the ER-negative phenotype. METHODS: We amplified exons 1 through 8 of the ER gene in 118 ER-positive and 70 ER-negative primary breast tumors and searched for mutations by single-strand conformation polymorphism analysis, denaturing gradient gel electrophoresis, and DNA sequencing. RESULTS: Both ER-negative and ER-positive tumors contained neutral polymorphisms in codons 10 [TCT-->TCC (Ser)], 87 [GCG-->GCC (Ala)], 243 [CGC-->CGT (Arg)], 325 [CCC-->CCG (Pro)], and 594 [ACA-->ACG (Thr)]. There was no correlation of any of the polymorphic alleles with the ER phenotype or other clinicopathologic parameters including tumor type, size, grade, or stage. However, the polymorphism in codon 325 showed a strong association with a family history of breast cancer (P = .0005). This association was observed both in premenopausal and postmenopausal patients. Despite extensive searching in exons 1 through 8, we found no deletions/insertions and only two missense mutations in codons 69 [AAC (Asn)-->AAG (Lys)] and 396 [ATG (Met)-->GTG (Val)] of the same ER-negative tumor. Thus, only 1% of the primary breast cancers had point mutations in the ER gene. CONCLUSIONS: In the majority of primary breast cancers, the ER-negative phenotype is not the result of mutations in the coding region of the ER gene, but is due to deficient ER expression at the transcriptional or post-transcriptional level. IMPLICATIONS: The correlation reported previously, as well as our current findings, suggest that further investigations are warranted to understand the possible linkage of the ER gene locus to hereditary breast cancer.

Long-term risk of breast cancer in women with fibroadenoma.

BACKGROUND: Fibroadenomas are benign breast tumors that are commonly diagnosed in young women and are associated with a slight increase in the risk of breast cancer. These lesions vary considerably in their histologic characteristics. We assessed the correlation between the histologic features of fibroadenomas and the risk of subsequent breast cancer. METHODS: We conducted a retrospective cohort study of a consecutive series of patients with fibroadenoma diagnosed between 1950 and 1968. Follow-up data were obtained for 1835 patients (90 percent of those eligible). Fibroadenomas with cysts, sclerosing adenosis, epithelial calcifications, or papillary apocrine changes were classified as complex. The rate of subsequent breast cancer among the patients was compared with the rates in two control groups, women listed in the Connecticut Tumor Registry and women chosen from among the patients' sisters-in-law. RESULTS: The risk of invasive breast cancer was 2.17 times higher among the patients with fibroadenoma than among the controls (95 percent confidence interval, 1.5 to 3.2). The relative risk increased to 3.10 among patients with complex fibroadenomas (95 percent confidence interval, 1.9 to 5.1) and remained elevated for decades after diagnosis. Patients with benign proliferative disease in the parenchyma adjacent to the fibroadenoma had a relative risk of 3.88 (95 percent confidence interval, 2.1 to 7.3). Patients with a family history of breast cancer in whom complex fibroadenoma was diagnosed had a relative risk of 3.72, as compared with controls with a family history (95 percent confidence interval, 1.4 to 10). Two thirds of the patients had noncomplex fibroadenomas and no family history of breast cancer and did not have an increased risk. CONCLUSIONS: Fibroadenoma is a long-term risk factor for breast cancer. The risk is increased in women with complex fibroadenomas, proliferative disease, or a family history of breast cancer.

Methylation of the estrogen receptor gene CpG island marks loss of estrogen receptor expression in human breast cancer cells.

Breast cancer is the most common malignancy in women and hormone resistance is a challenging problem in its treatment. Loss of estrogen receptor expression is an important means of hormone resistance, but the mechanisms involved are poorly understood. We now demonstrate a potential role for abnormal DNA methylation in transcriptional inactivation of the estrogen receptor gene. Estrogen receptor-negative human breast cancer cells growing in culture lack estrogen receptor mRNA, have a higher capacity to methylate DNA, and display extensive methylation of the CpG island in the 5' promoter region of the estrogen receptor gene, which would correlate with silencing of expression. These results suggest that abnormal methylation could account for transcriptional inactivation of the estrogen receptor gene and subsequent hormone resistance in some human breast cancers.

Microsatellite instability and loss of heterozygosity in breast cancer.

Microsatellite instability (MSI) has been described in colorectal and other cancers. The purpose of this study was to determine the presence of MSI in breast cancer and to correlate its occurrence with clinicopathological parameters. For microsatellite markers we examined mono-, di-, tri-, and tetranucleotide repeats that, due to their polymorphic nature, may also be used to investigate loss of heterozygosity. In 20 paired breast cancer-peripheral blood DNA samples we identified four tumors (20%) with somatic MSI. All four tumors were stage I or II, grade 1 or 2, and estrogen receptor positive. To study MSI in relation to tumor progression we also examined paired DNA samples from two ipsilateral and three contralateral breast cancers, as well as two matched tumor-metastatic lymph node specimens. None of these seven cases showed MSI, but two of the contralateral tumors revealed allelic loss of polymorphic repeats. These data suggest that MSI is an early event in mammary tumorigenesis while loss of heterozygosity may occur at a later stage.