Sofia D. Merajver

BRCA1 regulates human mammary stem/progenitor cell fate

Although it is well established that women with germ-line mutations in the BRCA1 gene have a greatly increased lifetime incidence of breast and ovarian cancer, the molecular mechanisms responsible for this tissue-specific carcinogenesis remain undefined. The majority of these breast cancers are of the basal-like phenotype characterized by lack of expression of ER, PR, and ERBB2. Because this phenotype has been proposed to resemble that of normal breast stem cells, we examined the role of BRCA1 in human mammary stem cell fate. Using both in vitro systems and a humanized NOD/SCID mouse model, we demonstrate that BRCA1 expression is required for the differentiation of ER-negative stem/progenitor cells to ER-positive luminal cells. Knockdown of BRCA1 in primary breast epithelial cells leads to an increase in cells displaying the stem/progenitor cell marker ALDH1 and a decrease in cells expressing luminal epithelial markers and estrogen receptor. In breast tissues from women with germ-line BRCA1 mutations, but not normal controls, we detect entire lobules that, although histologically normal, are positive for ALDH1 expression but are negative for the expression of ER. Loss of heterozygosity for BRCA1 was documented in these ALDH1-positive lobules but not in adjacent ALDH1-negative lobules. Taken together, these studies demonstrate that BRCA1 plays a critical role in the differentiation of ER-negative stem/progenitor cells to ER-positive luminal cells. Because BRCA1 also plays a role in DNA repair, our work suggests that loss of BRCA1 may result in the accumulation of genetically unstable breast stem cells, providing prime targets for further carcinogenic events.

Ten-Year Multi-Institutional Results of Breast-Conserving Surgery and Radiotherapy in BRCA1/2-Associated Stage I/II Breast Cancer

PURPOSE We compared the outcome of breast-conserving surgery and radiotherapy in BRCA1/2 mutation carriers with breast cancer versus that of matched sporadic controls. METHODS A total of 160 BRCA1/2 mutation carriers with breast cancer were matched with 445 controls with sporadic breast cancer. Primary end points were rates of in-breast tumor recurrence (IBTR) and contralateral breast cancers (CBCs). Median follow-up was 7.9 years for mutation carriers and 6.7 years for controls. RESULTS There was no significant difference in IBTR overall between carriers and controls; 10- and 15-year estimates were 12% and 24% for carriers and 9% and 17% for controls, respectively (hazard ratio [HR], 1.37; P = .19). Multivariate analyses for IBTR found BRCA1/2 mutation status to be an independent predictor of IBTR when carriers who had undergone oophorectomy were removed from analysis (HR, 1.99; P = .04); the incidence of IBTR in carriers who had undergone oophorectomy was not significantly different from that in sporadic controls (P = .37). CBCs were significantly greater in carriers versus controls, with 10- and 15-year estimates of 26% and 39% for carriers and 3% and 7% for controls, respectively (HR, 10.43; P < .0001). Tamoxifen use significantly reduced risk of CBCs in mutation carriers (HR, 0.31; P = .05). CONCLUSION IBTR risk at 10 years is similar in BRCA1/2 carriers treated with breast conservation surgery who undergo oophorectomy versus sporadic controls. As expected, CBCs are significantly increased in carriers. Although the incidence of CBCs was significantly reduced in mutation carriers who received tamoxifen, this rate remained significantly greater than in controls. Additional strategies are needed to reduce contralateral cancers in these high-risk women.

International expert panel on inflammatory breast cancer: consensus statement for standardized diagnosis and treatment

BACKGROUND Inflammatory breast cancer (IBC) represents the most aggressive presentation of breast cancer. Women diagnosed with IBC typically have a poorer prognosis compared with those diagnosed with non-IBC tumors. Recommendations and guidelines published to date on the diagnosis, management, and follow-up of women with breast cancer have focused primarily on non-IBC tumors. Establishing a minimum standard for clinical diagnosis and treatment of IBC is needed. METHODS Recognizing IBC to be a distinct entity, a group of international experts met in December 2008 at the First International Conference on Inflammatory Breast Cancer to develop guidelines for the management of IBC. RESULTS The panel of leading IBC experts formed a consensus on the minimum requirements to accurately diagnose IBC, supported by pathological confirmation. In addition, the panel emphasized a multimodality approach of systemic chemotherapy, surgery, and radiation therapy. CONCLUSIONS The goal of these guidelines, based on an expert consensus after careful review of published data, is to help the clinical diagnosis of this rare disease and to standardize management of IBC among treating physicians in both the academic and community settings.

Characterization of RhoC Expression in Benign and Malignant Breast Disease: A Potential New Marker for Small Breast Carcinomas with Metastatic Ability

The most important factor in predicting outcome in patients with early breast cancer is the stage of the disease. There is no robust marker capable of identifying invasive carcinomas that despite their small size have a high metastatic potential, and that would benefit from more aggressive treatment. RhoC-GTPase is a member of the Ras-superfamily and is involved in cell polarity and motility. We hypothesized that RhoC expression would be a good marker to identify breast cancer patients with high risk of developing metastases, and that it would be a prognostic marker useful in the clinic. We developed a specific anti-RhoC antibody and studied archival breast tissues that comprise a broad spectrum of breast disease. One hundred eighty-two specimens from 164 patients were used. Immunohistochemistry was performed on formalin-fixed tissues. Staining intensity was graded 0 to 3+ (0 to 1+ was considered negative and 2 to 3+ was considered positive). RhoC was not expressed in any of the normal, fibrocystic changes, atypical hyperplasia, or ductal carcinoma in situ, but was expressed in 36 of 118 invasive carcinomas and strongly correlated with tumor stage (P = 0.01). RhoC had high specificity (88%) in detecting invasive carcinomas with metastatic potential. Of the invasive carcinomas smaller than 1 cm, RhoC was highly specific in detecting tumors that developed metastases. RhoC expression was associated with negative progesterone receptor and HER-2/neu overexpression. We characterized RhoC expression in human breast tissues. RhoC is specifically expressed in invasive breast carcinomas capable of metastasizing, and it may be clinically useful in patients with tumors smaller than 1 cm to guide treatment.

Molecular biology of breast cancer metastasis Inflammatory breast cancer: clinical syndrome and molecular determinants

Inflammatory breast cancer (IBC) is an aggressive form of locally advanced breast cancer (LABC) that effects approximately 5% of women with breast cancer annually in the USA. It is a clinically and pathologically distinct form of LABC that is particularly fast growing, invasive, and angiogenic. Nearly all women have lymph node involvement at the time of diagnosis, and approximately 36% have gross distant metastases. Despite recent advances in multimodality treatments, the prognosis of patients with IBC is poor, with a median disease-free survival of less than 2.5 years. Recent work on the genetic determinants that underlie the IBC phenotype has led to the identification of genes that are involved in the development and progression of this disease. This work has been aided by the establishment of primary human cell lines and animal models. These advances suggest novel targets for future interventions in the diagnosis and treatment of IBC.

BRCA1 Mutation Analysis of 41 Human Breast Cancer Cell Lines Reveals Three New Deleterious Mutants

Germ line mutations of the BRCA1 gene confer a high risk of breast cancer and ovarian cancer to female mutation carriers. The BRCA1 protein is involved in the regulation of DNA repair. How specific tumor-associated mutations affect the molecular function of BRCA1, however, awaits further elucidation. Cell lines that harbor BRCA1 gene mutations are invaluable tools for such functional studies. Up to now, the HCC1937 cell line was the only human breast cancer cell line with an identified BRCA1 mutation. In this study, we identified three other BRCA1 mutants from among 41 human breast cancer cell lines by sequencing of the complete coding sequence of BRCA1. Cell line MDA-MB-436 had the 5396 + 1G>A mutation in the splice donor site of exon 20. Cell line SUM149PT carried the 2288delT mutation and SUM1315MO2 carried the 185delAG mutation. All three mutations were accompanied by loss of the other BRCA1 allele. The 185delAG and 5396 + 1G>A mutations are both classified as pathogenic mutations. In contrast with wild-type cell lines, none of the BRCA1 mutants expressed nuclear BRCA1 proteins as detected with Ab-1 and Ab-2 anti-BRCA1 monoclonal antibodies. These three new human BRCA1 mutant cell lines thus seem to be representative breast cancer models that could aid in further unraveling of the function of BRCA1.

Persistent E-Cadherin Expression in Inflammatory Breast Cancer

E-cadherin is a transmembrane glycoprotein that mediates epithelial cell-to-cell adhesion. Because loss of E-cadherin expression results in disruption of cellular clusters, it has been postulated that E-cadherin functions as a tumor suppressor protein. The role of E-cadherin in inflammatory breast cancer (IBC), a distinct and highly aggressive form of breast cancer, is largely unknown. The aim of our study was to elucidate whether E-cadherin expression contributes to the development and progression of the IBC phenotype and to investigate any differences in E-cadherin expression between IBC and stage-matched non-IBC. Forty-two breast cancer cases (20 IBC and 22 non-IBC) were identified. Strict and well-accepted criteria were used for the diagnosis of IBC. Clinical and pathologic features were studied, and formalin-fixed, paraffin-embedded tissue sections were immunostained for E-cadherin, estrogen and progesterone receptors (ER and PR, respectively), and HER2/neu. Statistical analysis was performed using Fishers exact test. All IBC uniformly expressed E-cadherin, whereas 15 of the 22 (68%) of the non-IBC expressed the protein (P = .006). Intralymphatic tumor emboli in the IBC cases were also all E-cadherin positive. Two IBC tumors demonstrated invasive lobular histology, and both cases were positive for E-cadherin. Of the non-IBC cases, three were invasive lobular carcinomas, and all were positive for E-cadherin. No association was found between E-cadherin expression and ER, PR status, or HER2/neu overexpression. Our study demonstrates that there is a strong association between E-cadherin expression and IBC and suggests that E-cadherin may be involved in the pathogenesis of this form of advanced breast cancer. In our study, we demonstrate that circulating IBC tumor cells strongly express E-cadherin, thereby providing an important exception to the positive association between E-cadherin loss and poor prognosis in breast cancer.

Downregulation of EZH2 decreases growth of estrogen receptor-negative invasive breast carcinoma and requires BRCA1

Increased levels of enhancer of zeste homolog 2 (EZH2), a critical regulator of cellular memory, are associated with negative estrogen receptor (ER) expression and disease progression in breast cancer. High levels of EZH2 signal the presence of metastasis and poor outcome in breast cancer patients. To test the hypothesis that deregulation of EZH2 contributes to ER-negative breast cancer progression, EZH2 expression was inhibited in ER-negative breast cancer cells MDA-MB-231 and CAL51 using a lentivirus system. EZH2 knockdown decreased proliferation and delayed the G2/M cell-cycle transition, although not affecting apoptosis. In vivo, EZH2 downregulation significantly decreased breast xenograft growth and improved survival. EZH2 knockdown upregulated BRCA1 protein. Of note, BRCA1 knockdown was sufficient to rescue the effects of EZH2 downregulation on proliferation, G2/M arrest, and on the levels of hyperphosphorylated mitotic Cdc25C and Cyclin B1 proteins, crucial for entry into mitosis. Invasive ER-negative breast carcinomas show significant overexpression of EZH2 and downregulation of BRCA1 proteins. Taken together, we show that EZH2 is important in ER-negative breast cancer growth in vivo and in vitro, and that BRCA1 is required for the proliferative effects of EZH2. Blockade of EZH2 may provide a prime target to prevent and/or halt ER-negative breast cancer progression.

Nanoroughened Surfaces for Efficient Capture of Circulating Tumor Cells without Using Capture Antibodies

Circulating tumor cells (CTCs) detached from both primary and metastatic lesions represent a potential alternative to invasive biopsies as a source of tumor tissue for the detection, characterization and monitoring of cancers. Here we report a simple yet effective strategy for capturing CTCs without using capture antibodies. Our method uniquely utilized the differential adhesion preference of cancer cells to nanorough surfaces when compared to normal blood cells and thus did not depend on their physical size or surface protein expression, a significant advantage as compared to other existing CTC capture techniques.

Prognostic factors in oral cavity and oropharyngeal squamous cell carcinoma: The impact of tumor-associated macrophages

The survival of patients with head and neck squamous cell carcinoma (HNSCC) remains unaffected despite recent therapeutic advances. To reverse this trend, reliable and clinically applicable markers of tumor aggressiveness must be identified. One such marker may be the tumor‐associated macrophage content. The authors hypothesized that tumor‐associated macrophages contribute to HNSCC aggressiveness, and the objective of the current study was to prove this hypothesis using mRNA expression analysis and a large cohort of clinical specimens.