Breast Hyperplasias, Risk Signature, and Breast Cancer.

We address the dilemma faced by oncologists in administering preventative measures to "at risk" patients diagnosed with atypical and nonatypical hyperplasias due to lack of any molecular means of risk stratification and identifying high-risk subjects. Our study purpose is to investigate a four marker risk signature, MMP-1, CEACAM6, HYAL1, and HEC1, using 440 hyperplastic tissues for identifying high-risk subjects who will benefit from preventative therapies. We assayed the markers by IHC and combined their expression levels to obtain a composite value from 0-10, which we called a "Cancer Risk Score." We demonstrate that the four marker-based risk scores predict subsequent cancer development with an accuracy of 91% and 86% for atypical and nonatypical subjects, respectively. We have established a correlation between risk scores and cancer rates by stratifying the samples into low risk (score ≤ 0.5); intermediate risk (score ≤ 5.4), and high risk (score >5.4) groups using Kaplan-Meier survival analysis. We have evaluated cancer rates at 5, 10, and 15 years. Our results show that the average cancer rates in the first 5 years among low- and intermediate-risk groups were 2% and 15%, respectively. Among high-risk group, the average cancer rates at 5 years were 73% and 34% for atypical and nonatypical subjects, respectively. The molecular risk stratification described here assesses a patient's tumor biology-based risk level as low, intermediate, or high and for making informed treatment decisions. The outcomes of our study in conjunction with the available prophylactic measures could prevent approximately 20%-25% of sporadic breast cancers.

Molecular constitution of breast but not other reproductive tissues is rich in growth promoting molecules: a possible link to highest incidence of tumor growths.

In the current study we tested if highest incidence of benign as well as cancer growths in breast tissue is due to constitutive molecular composition of this tissue. To delineate the molecular basis, we compared the expression of nine functional gene modules (total 578 genes) that regulate major positive growth and negative inhibitory signals in normal breast with two other reproductive tissues, ovary and uterus. We present data to demonstrate that breast tissues constitutively have very highly elevated levels of several growth promoting molecules and diminished levels of inhibitory molecules which may, in part, contribute for highest incidence of tumor growths in this tissue.

Molecular risk assessment for breast cancer development in patients with ductal hyperplasias.

PURPOSE: It has been reported that approximately a million women are diagnosed with benign breast lesions that include ductal hyperplasias per year in the United States. Recent studies that followed women with benign lesions have established that about 8% to 9% of them will subsequently develop invasive breast cancer (IBC). However, currently, there are no means of identifying a subclass of "true precancerous tissues" in women with ductal hyperplasias who will subsequently develop cancer. The purpose of this study is to investigate whether expression of hyaluronoglucosaminidase 1 (HYAL1), a known tumor promoter, in hyperplastic tissues identifies a "true precancerous stage" and predicts subsequent IBC development. EXPERIMENTAL DESIGN: A retrospective study was conducted with archival benign tissues of various histologic types and clinical information on development/nondevelopment of IBC. The control group was hyperplastic tissues from women who had no prior history of IBC and did not develop cancer in 5 to 7 years after diagnosis (n = 81). The test group was hyperplastic tissues from patients who developed cancer (n = 82). HYAL1 expression was studied by immunohistochemistry, and the results were statistically analyzed for significant association to develop cancer (P value), specificity, sensitivity, positive predictive value, and negative predictive value. RESULTS: Statistical analysis of HYAL1 expression data showed very highly significant association between its expression and subsequent cancer development (P = 0) and very high sensitivity (0.83), specificity (0.84), positive predictive value (0.84), and negative predictive value (0.83). CONCLUSIONS: The expression of HYAL1 in ductal hyperplastic tissues is a strong predictor of subsequent development of IBC; therefore, it can be applied as a diagnostic marker either singly or in combination with other marker(s) to screen benign tissues to predict subsequent development of IBC. Detection at the precancerous stage and treatment could drastically cut down breast cancer incidence and deaths from it.

The Cell Surface Estrogen Receptor, G Protein- Coupled Receptor 30 (GPR30), is Markedly Down Regulated During Breast Tumorigenesis.

BACKGROUND: GPR30 is a cell surface estrogen receptor that has been shown to mediate a number of non-genomic rapid effects of estrogen and appear to balance the signaling of estrogen and growth factors. In addition, progestins appear to use GPR30 for their actions. Therefore, GPR30 could play a critical role in hormonal regulation of breast epithelial cell integrity. Deregulation of the events mediated by GPR30 could contribute to tumorigenesis. METHODS: To understand the role of GPR30 in the deregulation of estrogen signaling processes during breast carcinogenesis, we have undertaken this study to investigate its expression at mRNA levels in tumor tissues and their matched normal tissues. We compared its expression at mRNA levels by RT quantitative real-time PCR relative to GAPDH in ERα"-positive (n = 54) and ERα"-negative (n = 45) breast cancer tissues to their matched normal tissues. RESULTS: We report here, for the first time, that GPR30 mRNA levels were significantly down-regulated in cancer tissues in comparison with their matched normal tissues (p < 0.0001 by two sided paired t-test). The GPR30 expression levels were significantly lower in tumor tissues from patients (n = 29) who had lymph node metastasis in comparison with tumors from patients (n = 53) who were negative for lymph node metastasis (two sample t-test, p < 0.02), but no association was found with ERα, PR and other tumor characteristics. CONCLUSIONS: Down-regulation of GPR30 could contribute to breast tumorigenesis and lymph node metastasis.

Estrogen receptor alpha (ER alpha) mRNA copy numbers in immunohistochemically ER alpha-positive-, and negative breast cancer tissues.

BACKGROUND: The presence of ERalpha is the basis for treating breast cancer patients with targeted molecular therapies that block estrogen stimulation of breast cancer cell division. To select patients for the above therapies, currently, the ERalpha presence in breast cancer tissues is determined in clinical laboratories by microscopically scoring the slides subjected to immunohistochemistry (IHC). This method is not quantitative, highly subjective and requires large amount of tumor tissue, therefore, cannot be applied to sterotactic and ultrasound guided biopsy samples. To circumvent these problems, we previously developed quantitative real-time PCR based molecular assay that can be applied to determine mRNA copies of ERalpha in picogram amounts of total RNA from tumor samples. However, it is not known how the mRNA copy numbers correlate to IHC positive and negative status. METHODS: In the current study we determined the copy numbers of ERalpha mRNA by Q RTPCR in breast cancer tissues that were graded as ERalpha-positive and negative by 1) IHC and 2) functional estrogen binding assay and statistically analyzed the data. RESULTS: We demonstrate here that ERalpha mRNA copy numbers are not significantly different in tissues that are graded as positive by IHC and ligand binding assays. We establish here a cut of value of 5 x 106 copies per 1010 mRNA copies of GAPDH with an Odds Radio of 39.4, Sensitivity of 0.81 and Specificity of 0.90 in breast cancer tissues that are negative for ERalpha protein by IHC and estrogen binding assays. ROC analysis of the data gave an area of 0.8967 under the curve. CONCLUSION: We expect that the cut off values determined here will be highly significant for applying molecular assay in the place of IHC in clinical laboratories for evaluating the presence of ERalpha for prognostic and therapeutic purposes.

Expression of carcinoembryonic antigen cell adhesion molecule 6 oncoprotein in atypical ductal hyperplastic tissues is associated with the development of invasive breast cancer.

BACKGROUND: Epidemiologic studies have established that women with prior atypical ductal hyperplastic (ADH) lesions have a 5-fold increased risk of developing invasive breast cancer (IBC). However, there is currently no means of identifying a subclass of ADH from women who will most likely develop cancer. The purpose of this study is to investigate whether elevated expression of carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) in ADH tissues is associated with the development of IBC. METHODS: A retrospective study was conducted with archival ADH tissues and clinical information on the development/nondevelopment of IBC. The control group was ADH from patients who had no prior history of IBC and did not develop cancer within 5 years after the diagnosis of ADH (n = 44). The test group was ADH from patients who either developed cancer concurrently or subsequently after diagnosis (ADHC; n = 44). The expression of CEACAM6 was studied by immunohistochemistry and the results were statistically analyzed for significant association to develop cancer (P value), specificity, sensitivity, positive predictive value, and negative predictive value. RESULTS: Of the 44 control ADH tissues from patients with no history of cancer, 9 were positive for CEACAM6. Among the ADHC tissues, 40 of 44 samples were positive. Statistical analysis of CEACAM6 expression data showed a significant association between its expression and cancer development, high sensitivity, specificity, positive predictive value, and negative predictive value. CONCLUSIONS: The expression of CEACAM6 in ADH lesions is strongly associated with the development of IBC, therefore, it can be applied as a diagnostic marker either singly or in combination with other marker(s) to predict IBC development in women with ADH lesions. It could also be a potential molecular therapeutic target for preventing IBC.

Estrogen receptors beta4 and beta5 are full length functionally distinct ERbeta isoforms: cloning from human ovary and functional characterization.

We describe here the cloning and functional characterization of two unique ER isoforms, ERbeta4 and ERbeta5. The full length ERbeta4 and ERbeta5 were identified by asymmetric PCR using human ovary cDNA, cloning, and sequence analyses. Both receptors share identical sequences with ERbeta1 from exon 1 to exon 7. In the place of exon 8, ERbeta4 has unique sequences arising from a region downstream of the ERbeta gene and upstream of the SYNE2 gene. ERbeta5 has sequences arising from retention of the 5' end of the intron between exon 7 and 8. Both receptors bind promoter sequences on DNA but do not bind estrogen. They translocate to the nucleus and exhibit three to four times higher estrogen-independent transcriptional activity than ERbeta1. When co-transfected with ERalpha, they predominantly form heterodimers and negatively regulate its transcriptional activity. Estrogen-independent transcriptional activity of ERbeta5, but not ERbeta4, was inhibited by ERalpha, demonstrating for the first time that ERalpha regulates ERbeta. Tissue-specific expression of ERbeta4 and ERbeta5, together with their ligand-independent transcriptional properties and ERalpha modulating activities, could have a number of implications in seemingly unlinked biological processes regulated by estrogen.

Estrogen receptor alpha-negative breast cancer tissues express significant levels of estrogen-independent transcription factors, ERbeta1 and ERbeta5: potential molecular targets for chemoprevention.

We have investigated the expression of two estrogen receptor beta (ERbeta) isoforms, ERbeta1 and ERbeta5, which activate gene transcription independent of estrogen or growth factors, in ERalpha-negative breast cancer tissues. We report here, for the first time, that ERalpha-negative tissues express significant levels of ERbeta1 and ERbeta5, and their expression levels are not different from levels in ERalpha positive tumors. However, significant differences exist between the two racial groups, African American and Caucasian, in that the patients from the former group express higher levels of ERbeta1 and ERbeta5 but not ERalpha. These two transcription factors could be potential molecular targets for designing chemopreventive drugs to treat ERalpha-negative breast cancers.

Identification of MMP-1 as a putative breast cancer predictive marker by global gene expression analysis.

Breast cancer is the second leading cause of cancer death for women in the United States. In 2005, about 215,000 cases of invasive breast cancer (IBC) and 50,000 cases of ductal carcinoma in situ will be diagnosed and 40,000 women will die of IBC in the US. Yet there is presently no molecular marker that can be used to detect a precancerous state or identify which premalignant lesions will develop into invasive breast cancer. Here we report the gene expression analysis of atypical ductal hyperplastic tissues from patients with and without a history of breast cancer. We identify MMP-1 as a candidate marker that may be useful for identification of breast lesions that can develop into cancer.

Molecular assays to profile 10 estrogen receptor beta isoform mRNA copy numbers in ovary, breast, uterus, and bone tissues.

Estrogens regulate various biological processes in a diverse range of reproductive and nonreproductive tissues through two genetically distinct but structurally related high affinity nuclear receptors, the estrogen receptor alpha and beta (ERalpha and ERbeta). The physiological significance of the presence of two ERs that have redundant functions is not known. Several unique properties of ERbeta together with its distinct expression patterns are considered to be, in part, the basis for diverse functional actions of estrogens and opposing actions of selective estrogen receptor modulators (SERMs) in different tissues. To understand how relative expression levels of two ERs correlate to seemingly dissimilar actions of estrogens and SERMs, quantitative methods are required that can precisely measure the levels of every isoform. Previously, methods to quantify eight ERalpha isoforms have been described [Poola I. (2003) Anal. Biochem. 314, 217-226]. In this article, real-time PCRbased molecular assays are described that can distinguish and quantify as low as 100 copies of 10 ERbeta isoform mRNAs, the ERbeta1, ERbeta2, ERbeta4, ERbeta5, and ERbeta exon 2Delta, exon 3Delta, exon 4Delta, exon 5Delta, exon 6Delta, and exons 5-6Delta. Each isoform mRNA is quantified using a specific primer pair and a 5'FAM (carboxy-fluorescein)- and 3'TAMARA (6-carboxy tetraethyl-rhodamine)-labeled probe and in comparison with a standard curve constructed with known copy numbers of its respective reverse transcribed cRNA. The devised assays were applied to profile 10 ERbeta isoforms in four estrogen-sensitive tissues-ovary, breast, uterus, and bone. The sensitivity of detection of each isoform in these tissues varied from picograms to nanograms of reverse-transcribed total RNA depending on the isoform and the tissue. The results presented also show that each tissue has a distinct profile of 10 isoform mRNAs. Interestingly, ERalpha- negative breast cancer cell lines and tumors expressed significant amounts of ERbeta isoforms suggesting that mitogenic stimulation by estrogen exists in these tissues. Bone tissues expressed several isoforms, although wild type was not present. In addition to the assay development, evidence is presented to demonstrate for the first time that ERbeta4 and ERbeta5 are full length receptors, contrary to previous reports that they are short receptors of exon 7-8.

Correlation of expression of BP1, a homeobox gene, with estrogen receptor status in breast cancer.

BACKGROUND: BP1 is a novel homeobox gene cloned in our laboratory. Our previous studies in leukemia demonstrated that BP1 has oncogenic properties, including as a modulator of cell survival. Here BP1 expression was examined in breast cancer, and the relationship between BP1 expression and clinicopathological data was determined. METHODS: Total RNA was isolated from cell lines, tumors, and matched normal adjacent tissue or tissue from autopsy. Reverse transcription polymerase chain reaction was performed to evaluate BP1 expression. Statistical analysis was accomplished with SAS. RESULTS: Analysis of 46 invasive ductal breast tumors demonstrated BP1 expression in 80% of them, compared with a lack of expression in six normal breast tissues and low-level expression in one normal breast tissue. Remarkably, 100% of tumors that were negative for the estrogen receptor (ER) were BP1-positive, whereas 73% of ER-positive tumors expressed BP1 (P = 0.03). BP1 expression was also associated with race: 89% of the tumors of African American women were BP1-positive, whereas 57% of those from Caucasian women expressed BP1 (P = 0.04). However, there was no significant difference in BP1 expression between grades I, II, and III tumors. Interestingly, BP1 mRNA expression was correlated with the ability of malignant cell lines to cause breast cancer in mice. CONCLUSION: Because BP1 is expressed abnormally in breast tumors, it could provide a useful target for therapy, particularly in patients with ER-negative tumors. The frequent expression of BP1 in all tumor grades suggests that activation of BP1 is an early event.

Molecular assay to generate expression profile of eight estrogen receptor alpha isoform mRNA copy numbers in picogram amounts of total RNA from breast cancer tissues.

The presence of estrogen receptor alpha (ERalpha) is the basis for treating breast cancer patients with targeted molecular therapies that block estrogen stimulation of breast cancer cell division. Currently, the ERalpha presence is determined by microscopically scoring the slides subjected to immunohistochemistry. This method is not quantitative, cannot distinguish between all the known ERalpha isoforms, and requires large amounts of tumor tissue. We describe here a real-time PCR-based molecular approach that can be applied to determine mRNA copies of eight ERalpha isoforms in picogram amounts of total RNA from clinical samples. Each isoform mRNA is quantified using a specific primer pair and a 5'FAM- and 3'TAMARA-labeled probe in comparison with a standard curve constructed with known copy numbers of its respective reverse-transcribed cRNA. Seven alternatively spliced isoforms were quantified using splice-targeted primers. The cRNAs for eight isoforms were generated by in vitro transcription of their respective coding sequences. The sensitivity of detection with reverse-transcribed cRNAs is as low as 100 copies. The devised assays can detect ERalpha cDNAs reverse transcribed from as low as 50-100 pg of total RNA from breast cancer tissues. The applicability of the devised assays for profiling eight ERalpha isoform mRNAs is demonstrated using 6 breast cancer cell lines and 10 breast cancer tissues. It is expected that these assays could be applied to profile ERalpha isoforms in any estrogen-responsive tissues. In addition, these methods could highly facilitate the design of tissue-specific selective estrogen receptor modulators to treat breast cancers and other estrogen-related abnormalities.