Whole-genome analysis informs breast cancer response to aromatase inhibition.

To correlate the variable clinical features of oestrogen-receptor-positive breast cancer with somatic alterations, we studied pretreatment tumour biopsies accrued from patients in two studies of neoadjuvant aromatase inhibitor therapy by massively parallel sequencing and analysis. Eighteen significantly mutated genes were identified, including five genes (RUNX1, CBFB, MYH9, MLL3 and SF3B1) previously linked to haematopoietic disorders. Mutant MAP3K1 was associated with luminal A status, low-grade histology and low proliferation rates, whereas mutant TP53 was associated with the opposite pattern. Moreover, mutant GATA3 correlated with suppression of proliferation upon aromatase inhibitor treatment. Pathway analysis demonstrated that mutations in MAP2K4, a MAP3K1 substrate, produced similar perturbations as MAP3K1 loss. Distinct phenotypes in oestrogen-receptor-positive breast cancer are associated with specific patterns of somatic mutations that map into cellular pathways linked to tumour biology, but most recurrent mutations are relatively infrequent. Prospective clinical trials based on these findings will require comprehensive genome sequencing.

HER2 intratumoral heterogeneity analyses by concurrent HER2 gene and protein assessment for the prognosis of HER2 negative invasive breast cancer patients.

HER2 gene-protein assay (GPA) is a new method for the simultaneous evaluation of HER2 immunohistochemistry (IHC) and HER2 dual in situ hybridization (DISH) on single tissue sections of breast cancer. We investigated the presence of HER2 gene and protein discrepancy and HER2-heterogeneity using HER2-GPA. HER2 status was analyzed for the correlation between the presence of HER2-heterogeneity and patient prognosis. Consecutive 280 invasive breast cancer were examined. Statuses of HER2 protein and gene were evaluated in whole tumor sections of HER2 GPA slides. HER2 protein and gene combination patterns were classified to six phenotypic and genotypic types for each case, as well as at individual cell levels: (A) IHC and DISH positive; (B) IHC positive and DISH negative; (C) IHC equivocal and DISH positive; (D) IHC equivocal and DISH negative; (E) IHC negative and DISH positive; and (F) IHC and DISH negative. The presence of HER2-heterogeneity was determined by the existence of at least two of six types within one tumor. HER2-IHC positive patients had significantly worse survival than IHC negative patients and HER2-DISH positive patients had significantly worse survival than DISH negative patients. HER2 IHC negative and DISH positive patients had significantly worse recurrence-free survival than IHC and DISH negative patients. In the HER2 IHC and DISH negative group, the HER2 heterogeneous group had significantly worse survival than the nonheterogeneous group. Notably, among triple negative breast cancer (TNBC), the HER2 heterogeneous group had significantly worse survival than the nonheterogeneous group. Our study suggests that the presence of HER2-heterogeneity might be a prognostic factor in HER2 negative breast cancer patients, especially in TNBC.

Intra-mammary ductal transplantation: a tool to study premalignant progression.

Ductal carcinoma in situ (DCIS) is a non-obligate precursor to invasive breast cancer. Although there is extensive information on the cellular and molecular changes in DCIS, there is limited ability to functionally test. The critical changes in premalignant progression. This review summarizes our experience with a recently developed method which provides. The opportunity to functionally test the molecular events occuring to functionally test the molecular events occurring in the initial changes in premaligant progression; i.e., the step from non-invasive to invasive behavior.

Estrogen Receptor Status by Immunohistochemistry Is Superior to the Ligand-Binding Assay for Predicting Response to Adjuvant Endocrine Therapy in Breast Cancer.

PURPOSE: Immunohistochemistry (IHC) is a newer technique for assessing the estrogen receptor (ER) status of breast cancers, with the potential to overcome many of the shortcomings associated with the traditional ligand-binding assay (LBA). The purpose of this study was to evaluate the ability of ER status determination by IHC, compared with LBA, to predict clinical outcome-especially response to adjuvant endocrine therapy-in a large number of patients with long-term clinical follow-up. PATIENTS AND METHODS: ER status was evaluated in 1,982 primary breast cancers by IHC on formalin-fixed paraffin-embedded tissue sections, using antibody 6F11 and standard methodology. Slides were scored on a scale representing the estimated proportion and intensity of positive-staining tumor cells (range, 0 to 8). Results were compared with ER values obtained by the LBA in the same tumors and to clinical outcome. RESULTS: An IHC score of greater than 2 (corresponding to as few as 1% to 10% weakly positive cells) was used to define ER positivity on the basis of a univariate cut-point analysis of all possible scores and disease-free survival (DFS) in patients receiving any adjuvant endocrine therapy. Using this definition, 71% of all tumors were determined to be ER-positive by IHC, and the level of agreement with the LBA was 86%. In multivariate analyses of patients receiving adjuvant endocrine therapy alone, ER status determined by IHC was better than that determined by the LBA at predicting improved DFS (hazard ratios/P = 0.474/.0008 and 0.707/.3214, respectively) and equivalent at predicting overall survival (0.379/.0001 and 0.381/.0003, respectively). CONCLUSION: IHC is superior to the LBA for assessing ER status in primary breast cancer because it is easier, safer, and less expensive, and has an equivalent or better ability to predict response to adjuvant endocrine therapy.

FGFR1 amplification and the progression of non-invasive to invasive breast cancer.

The incidence of invasive breast cancer (IBC) can be dramatically reduced by improving our abilities to detect and treat ductal carcinoma in situ (DCIS). Progress will be based on a detailed understanding of molecular mechanisms responsible for tumor progression. An interesting study by Jang and colleagues evaluated and compared the frequency of amplification of four oncogenes (HER2, c-MYC, CCND1 and FGFR1) in large cohorts of pure DCIS, in the DCIS component of IBC, and in corresponding IBC. Of particular interest, they found a twofold increase in FGFR1 amplification in IBC versus pure DCIS, and significantly reduced disease-free survival in amplified versus unamplified IBC - leading the authors to conclude that FGFR1 plays an important role in the development and progression of IBC. These observations indeed provide hints that FGFR1 is important in this setting, although the issue is very complex and far from resolved.

Pathologic outcomes of nonmalignant papillary breast lesions diagnosed at imaging-guided core needle biopsy.

PURPOSE: To determine the upstage rate from nonmalignant papillary breast lesions obtained at imaging-guided core needle biopsy (CNB) and if there are any clinical, imaging, or pathologic features that can be used to predict eventual upstaging to malignancy. MATERIALS AND METHODS: This retrospective case review was institutional review board approved and HIPAA compliant, with a waiver of informed consent. A database search (from January 2001 to March 2010) was performed to find patients with a nonmalignant papillary breast lesion diagnosed at CNB. Of the resulting 128 patients, 86 (67%) underwent surgical excision; 42 (33%) patients were observed with imaging, for a median observation time of 4.1 years (range, 1.0-8.6 years). Chart review was performed to determine pertinent features of each case. RESULTS: Fourteen of 128 patients were subsequently found to have malignancy at excision, for an upstage rate of 11%. Nine (7%) of the 128 patients were subsequently found to have atypia at excision. Comparisons between patients with upstaged lesions and patients whose lesions were not upstaged demonstrated patients with upstaged lesions to be slightly older (65 vs 56 years, P=.01), more likely to have a mass than calcifications at imaging (P=.03), and to have had less tissue obtained at biopsy (three vs five cores obtained, P=.02; 14- vs 9-gauge needle used, P<.01; no vacuum assistance used, P<.01). Most strongly predictive of eventual malignancy, however, was whether the interpreting pathologist qualified the benign diagnosis at CNB with additional commentary (P<.01). CONCLUSION: Given the substantial upstage rate (11%) of papillary lesions diagnosed at imaging-guided CNB, surgical excision is an appropriate management decision; however, careful evaluation in concert with an expert breast pathologist may allow for observation in appropriately selected patients.

Metastatic breast cancer, version 1.2012: featured updates to the NCCN guidelines.

These NCCN Guidelines Insights highlight the important updates/changes specific to the management of metastatic breast cancer in the 2012 version of the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Breast Cancer. These changes/updates include the issue of retesting of biomarkers (estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2) on recurrent disease, new information regarding first-line combination endocrine therapy for metastatic disease, a new section on monitoring of patients with metastatic disease, and new information on endocrine therapy combined with an mTOR inhibitor as a subsequent therapeutic option.

Human primary ductal carcinoma in situ (DCIS) subtype-specific pathology is preserved in a mouse intraductal (MIND) xenograft model.

Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer. The current recognition that DCIS lesions exhibit inter- and intra-lesion diversity suggests that the process of evolution to invasive breast cancer is more complex than previously recognized. Here we demonstrate the reproducible growth of primary DCIS cells derived from patient's surgical and biopsy samples by the mouse intraductal (MIND) model. MIND involves injection of cells into the NOD-SCID IL2Rgamma$^{{\rm{null}}}$ (NSG) mouse mammary ducts. Twelve (eight unique and four repeats) DCIS and two atypical hyperplasia specimens, heterogeneous with respect to biomarker expression and histology, were injected into 48 mouse mammary glands and analysed for successful xenotransplantation. Overall, 14/34 and 11/14 MIND xenotransplanted glands contained human DCIS and atypical hyperplastic cells, respectively, after 8 weeks, which formed single and multi-layered epithelium inside the ducts, and were heterogeneous with respect to expression of human cytokeratins, oestrogen receptor α (ER), and HER2. ER protein expression was recapitulated in MIND xenografts at ratios similar to the corresponding patient biopsies. In both patient biopsies and corresponding MIND xenografts, HER2 protein expression and nuclear HER2 gene overexpression were restricted to the DCIS lesions and were not found in the surrounding stroma or normal ducts. The xenografted DCIS lesions recapitulate the pathology and heterogeneity of human disease, thus providing a powerful tool for the characterization of the distinct cellular and molecular basis of inter- and intra-tumoural heterogeneity and the processes of DCIS to early invasive breast cancer progression.

Prognostic and predictive value of the 21-gene recurrence score assay in postmenopausal women with node-positive, oestrogen-receptor-positive breast cancer on chemotherapy: a retrospective analysis of a randomised trial.

BACKGROUND: The 21-gene recurrence score assay is prognostic for women with node-negative, oestrogen-receptor-positive breast cancer treated with tamoxifen. A low recurrence score predicts little benefit of chemotherapy. For node-positive breast cancer, we investigated whether the recurrence score was prognostic in women treated with tamoxifen alone and whether it identified those who might not benefit from anthracycline-based chemotherapy, despite higher risks of recurrence. METHODS: The phase 3 trial SWOG-8814 for postmenopausal women with node-positive, oestrogen-receptor-positive breast cancer showed that chemotherapy with cyclophosphamide, doxorubicin, and fluorouracil (CAF) before tamoxifen (CAF-T) added survival benefit to treatment with tamoxifen alone. Optional tumour banking yielded specimens for determination of recurrence score by RT-PCR. In this retrospective analysis, we assessed the effect of recurrence score on disease-free survival by treatment group (tamoxifen vs CAF-T) using Cox regression, adjusting for number of positive nodes. FINDINGS: There were 367 specimens (40% of the 927 patients in the tamoxifen and CAF-T groups) with sufficient RNA for analysis (tamoxifen, n=148; CAF-T, n=219). The recurrence score was prognostic in the tamoxifen-alone group (p=0.006; hazard ratio [HR] 2.64, 95% CI 1.33-5.27, for a 50-point difference in recurrence score). There was no benefit of CAF in patients with a low recurrence score (score <18; log-rank p=0.97; HR 1.02, 0.54-1.93), but an improvement in disease-free survival for those with a high recurrence score (score > or =31; log-rank p=0.033; HR 0.59, 0.35-1.01), after adjustment for number of positive nodes. The recurrence score by treatment interaction was significant in the first 5 years (p=0.029), with no additional prediction beyond 5 years (p=0.58), although the cumulative benefit remained at 10 years. Results were similar for overall survival and breast-cancer-specific survival. INTERPRETATION: The recurrence score is prognostic for tamoxifen-treated patients with positive nodes and predicts significant benefit of CAF in tumours with a high recurrence score. A low recurrence score identifies women who might not benefit from anthracycline-based chemotherapy, despite positive nodes. FUNDING: National Cancer Institute and Genomic Health.

Nuclear IRS-1 predicts tamoxifen response in patients with early breast cancer.

Insulin receptor substrate-1 (IRS-1) is a cytoplasmic scaffolding protein that is phosphorylated by insulin-like growth factor-I receptor and recruits downstream effectors. Recent evidence suggests that IRS-1 has a nuclear localization and function. Here we investigated whether nuclear and cytoplasmic IRS-1 levels are associated with clinico-pathological characteristics and clinical outcome in breast cancer patients. Tissue microarrays from 1,097 patients with stage I-II breast cancer were stained by immunohistochemistry for IRS-1. Nuclear and cytoplasmic IRS-1 were scored separately according to the Allred score. Nuclear IRS-1 showed a positive association with estrogen receptor (ER) (r = 0.09, P = 0.003) and progesterone receptor (PR) (r = 0.08, P = 0.008) status and a negative correlation with lymph node involvement (r = -0.10, P = 0.001). Cytoplasmic IRS-1 did not correlate with ER or PR but showed a positive correlation with tumor size (r = 0.10, P = 0.001) and S-phase fraction (r = 0.16, P < 0.001). In univariate analysis, tamoxifen-treated patients with tumors showing positive nuclear IRS-1 had a better recurrence-free survival (RFS) (P = 0.009) and overall survival (OS) (P = 0.0007), while no association was shown between cytoplasmic IRS-1 and RFS or OS in the same group of patients. In multivariate analysis of patients receiving tamoxifen, negative nuclear IRS-1 showed a significantly reduced RFS (P = 0.046) and OS (P = 0.018). Combining both PR and nuclear IRS-1, tamoxifen-treated patients with PR+/IRS-1+ tumors had a better RFS (P = 0.0003) and OS (P < 0.0001) when compared with patients with PR-/IRS-1- tumors. In conclusion, nuclear IRS-1 may be a useful marker to predict tamoxifen response in patients with early breast cancer, particularly when assessed in combination with PR.

Issues and updates: evaluating estrogen receptor-alpha, progesterone receptor, and HER2 in breast cancer.

There are currently three prognostic/predictive biomarkers used in routine clinical management of patients with breast cancer, and their assessment is mandatory. They include estrogen receptor-alpha (ERalpha), progesterone receptor (PgR), and the HER2 oncogene/oncoprotein. This paper briefly reviews the assessment of ERalpha, PgR, and HER2 in breast cancer, emphasizing recent progress and persistent controversies.

An intraductal human-in-mouse transplantation model mimics the subtypes of ductal carcinoma in situ.

INTRODUCTION: Human models of noninvasive breast tumors are limited, and the existing in vivo models do not mimic inter- and intratumoral heterogeneity. Ductal carcinoma in situ (DCIS) is the most common type (80%) of noninvasive breast lesions. The aim of this study was to develop an in vivo model whereby the natural progression of human DCIS might be reproduced and studied. To accomplish this goal, the intraductal human-in-mouse (HIM) transplantation model was developed. The resulting models, which mimicked some of the diversity of human noninvasive breast cancers in vivo, were used to show whether subtypes of human DCIS might contain distinct subpopulations of tumor-initiating cells. METHODS: The intraductal models were established by injection of human DCIS cell lines (MCF10DCIS.COM and SUM-225), as well as cells derived from a primary human DCIS (FSK-H7), directly into the primary mouse mammary ducts via cleaved nipple. Six to eight weeks after injections, whole-mount, hematoxylin and eosin, and immunofluorescence staining were performed to evaluate the type and extent of growth of the DCIS-like lesions. To identify tumor-initiating cells, putative human breast stem/progenitor subpopulations were sorted from MCF10DCIS.COM and SUM-225 with flow cytometry, and their in vivo growth fractions were compared with the Fisher's Exact test. RESULTS: Human DCIS cells initially grew within the mammary ducts, followed by progression to invasion in some cases into the stroma. The lesions were histologically almost identical to those of clinical human DCIS. This method was successful for growing DCIS cell lines (MCF10DCIS.COM and SUM-225) as well as a primary human DCIS (FSK-H7). MCF10DCIS.COM represented a basal-like DCIS model, whereas SUM-225 and FSK-H7 cells were models for HER-2+ DCIS. With this approach, we showed that various subtypes of human DCIS appeared to contain distinct subpopulations of tumor-initiating cells. CONCLUSIONS: The intraductal HIM transplantation model provides an invaluable tool that mimics human breast heterogeneity at the noninvasive stages and allows the study of the distinct molecular and cellular mechanisms of breast cancer progression.

Mammary tumorigenesis and metastasis caused by overexpression of insulin receptor substrate 1 (IRS-1) or IRS-2.

Insulin receptor substrates (IRSs) are signaling adaptors that play a major role in the metabolic and mitogenic actions of insulin and insulin-like growth factors. Reports have recently noted increased levels, or activity, of IRSs in many human cancers, and some have linked this to poor patient prognosis. We found that overexpressed IRS-1 was constitutively phosphorylated in vitro and in vivo and that transgenic mice overexpressing IRS-1 or IRS-2 in the mammary gland showed progressive mammary hyperplasia, tumorigenesis, and metastasis. Tumors showed extensive squamous differentiation, a phenotype commonly seen with activation of the canonical beta-catenin signaling pathway. Consistent with this, IRSs were found to bind beta-catenin in vitro and in vivo. IRS-induced tumorigenesis is unique, given that the IRSs are signaling adaptors with no intrinsic kinase activity, and this supports a growing literature indicating a role for IRSs in cancer. This study defines IRSs as oncogene proteins in vivo and provides new models to develop inhibitors against IRSs for anticancer therapy.

Ductal carcinoma in situ and the emergence of diversity during breast cancer evolution.

PURPOSE: Human invasive breast cancers (IBC) show enormous histologic and biological diversity. This study comprehensively evaluated diversity in ductal carcinoma in situ (DCIS), the immediate precursors of IBCs. EXPERIMENTAL DESIGN: The extent of diversity for conventional histologic grade and standard prognostic biomarkers assessed by immunohistochemistry was evaluated in a series of pure DCIS (n = 200) compared with a contemporaneous series of IBCs (n = 200). A subset of the DCIS (n = 25) was evaluated by DNA microarrays for the presence of luminal, basal, and erbB2 intrinsic subtypes. The extent of diversity within individual cases of DCIS (n = 120) was determined by assessing multiple regions independently for histologic (nuclear) grade and several biomarkers by immunohistochemistry, which approximate microarrays in determining intrinsic subtypes. RESULTS: DCIS showed a broad distribution of conventional histologic grades and standard biomarkers ranging from well to poorly differentiated, nearly identical to IBCs. Microarrays showed the same intrinsic subtypes in DCIS as in IBCs. However, higher resolution analysis showed that multiple histologic grades, biomarker phenotypes, and intrinsic subtypes often coexist within the same DCIS, and these diverse regions probably compete for dominance. Diversity within cases of DCIS was highly correlated with mutated p53 (P = 0.0007). CONCLUSIONS: These results support the hypothesis that poorly differentiated DCIS gradually evolve from well-differentiated DCIS by randomly acquiring genetic defects resulting in increasingly abnormal cellular features. This diversity is amplified by defects resulting in genetic instability (e.g., p53 mutation), and the alterations are propagated to IBC in a manner independent of progression to invasion.

The relevance of mouse models to understanding the development and progression of human breast cancer.

Mouse modeling of human breast cancer has developed tremendously over the past ten years. Human breast cancer is characterized by enormous biological diversity and, collectively, the new models have come much closer to encompassing this diversity. They have provided a deeper understanding of the fundamental events that mediate the initiation, development, and progression of breast cancer, and they offer new opportunities to develop and test strategies to treat and, perhaps, even prevent the disease. This chapter reviews the historical development of mouse models of breast cancer and highlights some of their major strengths, weaknesses, and contributions.

Perturbed myoepithelial cell differentiation in BRCA mutation carriers and in ductal carcinoma in situ.

Myoepithelial cells play key roles in normal mammary gland development and in limiting pre-invasive to invasive breast tumor progression, yet their differentiation and perturbation in ductal carcinoma in situ (DCIS) are poorly understood. Here, we investigated myoepithelial cells in normal breast tissues of BRCA1 and BRCA2 germline mutation carriers and in non-carrier controls, and in sporadic DCIS. We found that in the normal breast of non-carriers, myoepithelial cells frequently co-express the p63 and TCF7 transcription factors and that p63 and TCF7 show overlapping chromatin peaks associated with differentiated myoepithelium-specific genes. In contrast, in normal breast tissues of BRCA1 mutation carriers the frequency of p63+TCF7+ myoepithelial cells is significantly decreased and p63 and TCF7 chromatin peaks do not overlap. These myoepithelial perturbations in normal breast tissues of BRCA1 germline mutation carriers may play a role in their higher risk of breast cancer. The fraction of p63+TCF7+ myoepithelial cells is also significantly decreased in DCIS, which may be associated with invasive progression.

Response and resistance to the endocrine prevention of breast cancer.

The data from observational studies and clinical trials indicates that it is possible to prevent BC for prolonged periods using various endocrine manipulations. Ovarian suppression is thought to give lifelong protection and recent data indicate that the effectiveness of Tam continues after cessation of treatment at 5-8 years. It is clear from three randomised trials that SERMs prevent ERalpha+ tumors only in women at increased risk and at population risk of BC entered into these trials. The data from the Ral trials also suggests that this agent appears less effective than Tam in preventing DCIS. This is surprising since a large proportion of DCIS is ERalpha+. Equally surprising is the effectiveness of oophorectomy and Tam in mutation carriers, particularly BRCA1, which is associated with ERalpha+ tumors. The fact that ERT can be given without apparently abrogating the effect of oophorectomy and also to naturally postmenopausal women without increasing BC risk suggests that cyclical estrogen or estrogen + progestin are important for BC initiation and/or progression. The question arises whether the information we have concerning the responsiveness of ERalpha+ cells in TDLU, premalignant lesions, and invasive cancers give an indication of the targets for endocrine prevention. Data summarised in Table 1 indicate that TDLU are responsive to estrogen, ED, and SERMs/SERDs in premenopausal women and there may be the targets for the preventative effect of early oophorectomy particularly in BRCA1 carriers where we have demonstrated endocrine responsiveness of TDLU, which at this heterozygote stage are ERalpha+. The decline in numbers of atypical lobules in breasts without invasive cancer suggests that these are targets for the 'preventive' effect of the menopause, as suggested by Wellings. The data also suggest that ERalpha+ DCIS is responsive to estrogen and ED supporting premalignant lesions is a target as does the data from the NSABP P1 trial indicating a marked preventive effect of Tam in women previously diagnosed with atypical ductal hyperplasia and a preventative effect on CIS.

Estrogen receptor positive breast cancer metastasis: altered hormonal sensitivity and tumor aggressiveness in lymphatic vessels and lymph nodes.

Breast cancers commonly spread to lymph nodes (LNs). If the primary tumors are estrogen receptor (ER) and/or progesterone receptor (PR) positive, then the likelihood that LN metastases express receptors exceeds 80%. However, due to lack of ER+ models, little is known about the role of hormones in breast cancer spread or the effects of the LN microenvironment on hormone responsiveness. We have developed metastasis models using ZsGreen labeled MCF-7 and T47D human breast cancer cells. Tumors are tracked in living mice by whole-body imaging, and macrometastases or micrometastases are detected by intravital imaging or fluorescence microscopy. Tumor growth is estrogen dependent and required for intratumoral lymphangiogenesis. Seventy-five percent of all tumors and >95% of larger tumors generate LN metastases. Occasionally more distant metastases are also observed. "Triads" of primary tumors, tumor-filled draining lymphatic vessels, and tumor-filled LNs from the same mouse show that (a) proliferation, as measured by 5-bromo-2'-deoxyuridine uptake, is higher in the LN than in the primary tumor. (b) High ER levels are extensively down-regulated by estradiol in primary tumors. However, there is partial failure of ER down-regulation in LNs associated with (c) reduced PR expression. This suggests that ER are dysfunctional in the LN microenvironment and perhaps hormone resistant. (d) CD44 is sparsely expressed in primary tumor cells but homogeneously overexpressed in cells transiting the lymphatics and populating LNs. We hypothesize that CD44 expression targets tumor cells for transport to, and uptake in, LNs. If so, the CD44 pathway could be targeted therapeutically to slow or prevent LN metastases.

Progesterone-action in the murine uterus and mammary gland requires steroid receptor coactivator 2: relevance to the human.

The importance of the progesterone receptor (PR) in female reproductive and mammary gland biology is well recognized; however, the coregulators selectively enlisted by PR have yet to be comprehensively defined in vivo. To evaluate the involvement of steroid receptor coactivator (SRC)/p160 family members in these physiological systems, a mouse model (PRCre/+SRC-2flox/flox) was generated in which SRC-2 function was ablated specifically in cell-types that express the PR. Although PRCre/+SRC-2flox/flox ovarian activity was normal, uterine function was severely compromised. Absence of SRC-2 in PR positive uterine cells led to an early block in embryo implantation, a defect not ascribed to SRC-1 or -3 knockouts. While the PRCre/+SRC-2flox/flox uterus can display a partial decidual response, removal of SRC-1 in the PRCre/+SRC-2flox/flox uterus results in a block in decidualization, confirming that uterine SRC-2 and -1 are both necessary for PR-mediated transcriptional responses which lead to complete decidualization. The absence of significant branching and alveolar morphogenesis in the hormone-treated PRCre/+SRC-2flox/flox mammary gland establishes an important role for mammary SRC-2 in cellular proliferative programs that require PR. Finally, the observation that SRC-2 is also expressed in many of the same cell-types in the human, underscores the importance of further study of this coregulator's role in both peri-implantation biology and mammary development.

Development of standard estrogen and progesterone receptor immunohistochemical assays for selection of patients for antihormonal therapy.

Estrogen receptor (ER) and progesterone receptor (PR) status in breast carcinomas are considered validated predictive factors for selecting patients for antihormonal therapy. Published surveys have shown a significant rate of disagreement and lack of reproducibility of immunohistochemistry (IHC) results from laboratories around the world. To address these limitations IHC assays for ER and PR were developed using characterized reagents, after careful calibration of the sensitivity and specificity to match established assays previously validated in large clinical studies. The ER assay uses a cocktail of 2 mouse monoclonal antibodies (1D5 and ER-2-123) and the PR assay uses 1 mouse monoclonal antibody (PgR 1294); both are followed by a polymer-peroxidase-based detection system. All antibodies were tested for specificity by epitope mapping. The sensitivity of the new assays was calibrated to be equivalent to previously validated IHC assays followed by a comparison with the validated assays in a concordance study involving over 200 specimens. All slides were scored with the "Allred Score," also used for scoring of the original validated assays. The overall concordance between the new and the established IHC assays was nearly perfect (99%). The concordance study demonstrated greater than 98% positive agreement and 100% negative agreement of the new IHC assays with the previously validated IHC assays. This equivalence establishes the clinical validation of the assays and, as they are based on newer generation reagents and are produced and tested under stringent quality control conditions to ensure their consistency, they add additional advantages to the user and patients.