Leslie K. Diaz

αB-Crystallin is a novel oncoprotein that predicts poor clinical outcome in breast cancer

Recent gene profiling studies have identified a new breast cancer subtype, the basal-like group, which expresses genes characteristic of basal epithelial cells and is associated with poor clinical outcomes. However, the genes responsible for the aggressive behavior observed in this group are largely unknown. Here we report that the small heat shock protein alpha-basic-crystallin (alphaB-crystallin) was commonly expressed in basal-like tumors and predicted poor survival in breast cancer patients independently of other prognostic markers. We also demonstrate that overexpression of alphaB-crystallin transformed immortalized human mammary epithelial cells (MECs). In 3D basement membrane culture, alphaB-crystallin overexpression induced luminal filling and other neoplastic-like changes in mammary acini, while silencing alphaB-crystallin by RNA interference inhibited these abnormalities. alphaB-Crystallin overexpression also induced EGF- and anchorage-independent growth, increased cell migration and invasion, and constitutively activated the MAPK kinase/ERK (MEK/ERK) pathway. Moreover, the transformed phenotype conferred by alphaB-crystallin was suppressed by MEK inhibitors. In addition, immortalized human MECs overexpressing alphaB-crystallin formed invasive mammary carcinomas in nude mice that recapitulated aspects of human basal-like breast tumors. Collectively, our results indicate that alphaB-crystallin is a novel oncoprotein expressed in basal-like breast carcinomas that independently predicts shorter survival. Our data also implicate the MEK/ERK pathway as a potential therapeutic target for these tumors.

Estrogen receptor analysis for breast cancer: current issues and keys to increasing testing accuracy.

The estrogen receptor (ER) is a regulator of cellular growth, proliferation, and differentiation. In addition to having prognostic value, ER is the most important biologic marker of therapeutic response in breast cancer. Some level of measurable ER protein is expressed in 70-80% of human breast. Immunohistochemistry is the current method of choice for ER assessment, and its predictive value has been shown to be superior to that of biochemically based assays. Although accurate ER protein assessment is critical for optimal treatment of patients with breast cancer, studies have demonstrated inter-laboratory variability in ER detection. False-negative results for tumors with low ER protein levels have been a subject of recent concern. Lack of standardization for immunohistochemistry between laboratories is thought to be the major reason for testing errors, although variability in scoring methods and reporting practices, which can affect results, also plays a role. In this article, we review studies addressing interlaboratory variability and recommend optimal testing techniques and reporting procedures for ER testing, with the goal of increasing interlaboratory standardization for ER analysis by immunohistochemistry. We additionally highlight recent biologic, molecular, and gene expression profiling data related to ER in breast cancer.

Triple negative breast carcinoma and the basal phenotype: From expression profiling to clinical practice

Triple negative breast carcinomas (TNBCs) are a group of primary breast tumors with aggressive clinical behavior. Most TNBCs possess a basal phenotype (BP) and show varying degrees of basal cytokeratin and myoepithelial marker expression. The importance of recognizing these tumors came to light largely as the result of gene expression profiling studies that categorized breast cancer into 3 major groups. Two of these groups are defined by their respective expression of estrogen receptor and HER2. TNBCs represent a third group and are defined by negativity for hormone receptors and HER2. TNBCs currently lack effective targeted therapies and are frequently resistant to standard chemotherapeutic regimens. These tumors tend to occur in premenopausal women and members of specific ethnic groups and a subset are associated with heritable BRCA1 mutations. For patients with sporadic TNBCs and BP tumors, BRCA1 dysfunction seems to play a major role in the development and progression of disease. The pathologists role in the diagnosis and characterization of TNBCs and BP tumors is currently being defined as we are acquiring knowledge of the biologic and genetic underpinnings that drive this heterogeneous group of diseases. This review will provide a historical prospective on TNBCs and tumors that express basal cytokeratins and myoepithelial makers. Additionally, we will discuss the molecular biologic, genetic and pathologic aspects of these tumors. Guidelines will be provided on how to best approach the diagnosis of these cases and on what input pathologists should provide clinicians to help develop optimal therapeutic and preventative strategies against this aggressive group of breast cancers.

Benign metastasizing leiomyoma: clonality, telomere length and clinicopathologic analysis.

Benign metastasizing leiomyoma is a rare condition affecting women with a history of uterine leiomyomata and is characterized by multiple histologically benign pulmonary smooth muscle tumors. Speculations on its pathogenesis include a benign uterine leiomyoma colonizing the lung, a metastatic low-grade uterine leiomyosarcoma, and primary pulmonary leiomyomatosis. To elucidate its pathogenesis, we analyzed the clinical, pathological and immunohistochemical features, clonality, and telomere length of multiple lung and uterine tumors in three patients with benign metastasizing leiomyoma. In all cases, pulmonary tumors had benign histology and immunohistochemical profiles (estrogen receptor positive, progesterone receptor positive, and very low proliferative index) identical to uterine leiomyoma. In eight tumors from three patients, clonality was assessed by analyzing the variable length of the polymorphic CAG repeat sequence within the human androgen receptor gene. In the two informative patients pulmonary and uterine tumors showed identical patterns of androgen receptor allelic inactivation, indicating that they were clonal. The telomere length measured by fluorescence in situ hybridization in pulmonary leiomyomas of all three patients were either long or very long and were identical to the uterine counterparts, indicating significant telomere shortening is not a crucial step for developing metastases. Our evidence supports the notion that benign metastasizing leiomyoma is clonally derived from benign-appearing uterine leiomyomas.

Clinical, histopathologic, and immunohistochemical features of microglandular adenosis and transition into in situ and invasive carcinoma.

Microglandular adenosis (MGA) of the breast is widely known as a benign lesion that can mimic invasive carcinoma. In situ and invasive carcinomas have been described as arising in MGA, but which cases of MGA will progress to carcinoma is unclear. Criteria for distinguishing uncomplicated MGA, MGA with atypia (AMGA), and carcinoma arising in MGA (MGACA) are not standardized. The primary objective of this study was to illustrate the clinical, histopathologic, and immunophenotypical characteristics of MGA, AMGA, and MGACA in an effort to provide criteria for distinguishing the 3 types. We retrospectively identified 108 cases seen at M.D. Anderson Cancer Center between 1983 and 2007 that had a diagnosis of MGA. Of the 108 cases, 65 cases had available material for review. Inclusion criteria were glands of MGA expressing S-100 protein and lacking myoepithelial layer (smooth muscle actin negative). Eleven out of 65 cases qualified to have an MGA component; myoepithelial layer was detected in the remaining 54 cases and were classified as adenosis. Out of the 11 MGA patients, there were 3 patients with uncomplicated MGA, 2 had AMGA, and 6 had MGACA. Staining indices for the cell cycle markers p53 and Ki-67 were used to compare the 3 tumor categories. Additional staining for other tumor markers [estrogen and progesterone receptors, HER2, epidermal growth factor receptor (EGFR), c-kit, CK5/6, and CK18] were performed. Patient demographics, tumor radiologic features, and clinical follow-up data were collected for all cases. Multiple invasive histologic components were identified in each of the MGACA cases. All invasive MGACAs had a duct-forming component. In addition, basal-like component was present in 2 cases, aciniclike in 2, matrix producing in 4, sarcomatoid in 1, and adenoid cystic in 1. All tumors had strong and diffuse CK8/18 and EGFR expression but no estrogen receptor, progesterone receptor, HER2 (ie, triple negative), or CK5/6 expression. C-kit was focally expressed in 2 of the MGACAs. Ki-67 and p53 labeling indices was <3% in all MGAs, 5% to 10% in the AMGAs, and >30% in MGACAs. In a follow-up ranging from 14 days to 8 years, none of the MGA cases recurred. One of the AMGA cases recurred as invasive carcinoma in a background of AMGA after 8 years following incomplete excision of the lesion. Three out of 6 MGACA cases (50%) required multiple consecutive resections ending up with mastectomy due to involved margins by invasive or in situ carcinoma. Two out of 6 MGACA cases (34%) developed metastasis and died of disease. Our data showed that Ki-67 and p53 expression, in conjunction with the morphologic features, could be a reliable marker to distinguish MGA from AMGA and MGACA. Although 11 tumors were only included in our study, 64% of the tumors were carcinomas arising in MGA. This high incidence of MGACA may not represent the actual frequency of MGAs progressing into carcinoma and is likely due to referral bias in our institution. Nonetheless, the high association of carcinoma with MGA necessitates complete excision of MGA to rule out invasion. Although all the MGACA cases were triple negative and express EGFR (basal-like features), all the cases in our study showed a luminal type of differentiation by CK8/18 expression, indicating that MGACA may not fit well into the current proposed molecular classification of breast cancer.

αB-crystallin: A novel marker of invasive basal-like and metaplastic breast carcinomas

Basal-like tumors are a newly recognized estrogen receptor (ER) negative and HER2 negative breast cancer subtype that express basal epithelial genes and are associated with poor survival. Metaplastic carcinomas are thought to belong within the basal-like group. We have recently demonstrated that the small heat shock protein alphaB-crystallin is commonly expressed in basal-like tumors and contributes to their aggressive phenotype. The current study examined the rates and patterns of alphaB-crystallin expression in whole tissue sections of human breast, including normal tissue, proliferative lesions, in situ and invasive carcinomas (ER positive, HER2 positive, basal-like, and metaplastic cancers). In normal breast tissue, proliferative lesions and in situ carcinomas, alphaB-crystallin expression was restricted to the myoepithelial cell compartment of ductal and lobular units. Most basal-like and metaplastic carcinomas demonstrated cytoplasmic expression of alphaB-crystallin (81% and 86%, respectively). Conversely, no staining for alphaB-crystallin was observed in nonbasal-like (ie, ER positive or HER2 positive) breast carcinomas. Taken together, our results indicate that alphaB-crystallin is a sensitive (81%) and specific (100%) marker for basal-like breast carcinomas. Moreover, the high rates of expression of alphaB-crystallin in metaplastic breast carcinomas (86%) suggest that these tumors may represent a histologically distinctive subset of basal-like breast tumors with a similar underlying molecular etiology.

A comparison of ductoscopy-guided and conventional surgical excision in women with spontaneous nipple discharge.

Introduction:Radiologic imaging is routinely used to evaluate women with spontaneous nipple discharge (SND), but definitive diagnosis is usually only achieved by surgical terminal duct excision (TDE). Ductoscopy has been reported to result in improved localization of intraductal lesions and may avoid surgery in women with endoscopically normal ducts. Materials and Methods:We conducted a retrospective review of the records of 117 consecutive women who underwent ductoscopy to guide ductal excision (scope-DE) or received conventional TDE without ductoscopy. Two women had atypical ductal lavage cytology and the remainder presented with SND from 1 or more duct. Preoperative evaluation included radiologic imaging as clinically indicated. Results:Fifty-nine women underwent scope-DE, and 58 underwent conventional TDE. There were no significant differences in age, race, discharge characteristics, or radiologic findings. The proportion of women with intraductal neoplasia was slightly greater in the group undergoing scope-DE (88% vs. 81%, P = 0.2). In the conventional TDE group, 8.5% were found to have atypical hyperplasia or duct carcinoma in situ compared with 18.6% in the scope-DE group. In the ductoscopy group, 22 of 59 (37.3%) had lesions >5 cm from the nipple, compared with 1 of 17 women for whom distance of the lesion from the nipple was known in the conventional group (P = 0.02). Of the ductoscopy-detected cancers, 5 of 6 had no symptoms other than SND, whereas 1 of the 4 malignancies in the conventional group presented as SND alone. Discussions:Ductoscopy identifies intraductal lesions in a high proportion of women with SND, and it may contribute to more accurate resection of these. A prospective study is required to obtain an unbiased assessment of these possible advantages.

The Use of TMA for Interlaboratory Validation of FISH Testing for Detection of HER2 Gene Amplification in Breast Cancer

HER2 fluorescence in situ hybridization (FISH) testing for breast cancer is largely limited to academic centers and commercial laboratories. As testing demands increase, methods for rapid and cost-effective technical validation and quality assessment will be required. Tissue microarray (TMA), a technique for high-throughput biomarker evaluation, could help facilitate these needs. Our objective was to assess the usefulness of TMA technology for validation of HER2 FISH testing. Two TMA blocks containing paired cores from 41 breast cancers were constructed. HER2 FISH was performed in parallel at two institutions and the results compared. One institution, with considerable HER2 FISH experience, served as the reference laboratory. HER2 chromogenic in situ hybridization (CISH) and immunohistochemistry (IHC) were compared to the FISH results. For positive and negative results, the concordance rate between laboratories was 100%. Using kappa statistical analysis to determine interobserver agreement, HER2 to chromosome 17 gene copy ratios showed strong agreement between laboratories with kappa = 0.85 (perfect agreement = 1.0). Four cases displaying low-level amplification by CISH contained chromosome 17 polysomy and gene copy ratios of <2.0 by FISH. Good concordance was observed between HER2 IHC and in situ hybridization testing. TMA is a robust and effective method for the technical validation of HER2 FISH testing and should be considered for use by quality assessment programs.

Coexpression of α6β4 Integrin and Guanine Nucleotide Exchange Factor Net1 Identifies Node-Positive Breast Cancer Patients at High Risk for Distant Metastasis

Preclinical data indicate that α6β4 integrin signaling through Ras homolog gene family, member A, plays an important role in tumor cell motility. The objective of this study was to determine whether the combined expression of α6β4 integrin and neuroepithelioma transforming gene 1 (Net1), a guanine nucleotide exchange factor specific for Ras homolog gene family member A, is associated with adverse clinical outcome in breast cancer patients. Immunohistochemical expression of each protein was evaluated in a tumor tissue microarray prepared from the primary tumors of 94 node-positive patients with invasive breast carcinoma treated with total mastectomy and doxorubicin-based chemotherapy without radiation with a median follow-up of 12.5 years. Associations between staining results and multiple clinicopathologic variables were investigated. Although there was no significant association between α6β4 integrin or Net1 expression and clinical outcome when each marker was considered individually, coexpression of α6β4 and Net1 was associated with decreased distant metastasis–free survival (P = 0.030). In the subset of patients with hormone receptor–positive tumors, coexpression of α6β4 and Net1 was associated with a decrease in distant metastasis–free and overall survival (P < 0.001 and P = 0.006, respectively). Although an association between human epidermal growth factor receptor 2 expression and coexpression of α6β4 and Net1 (P = 0.008) was observed, coexpression of α6β4 and Net1 (hazard ratio, 1.63; P = 0.02) and lymphovascular invasion (hazard ratio, 2.35; P = 0.02) were the only factors independently associated with the development of distant metastasis in multivariate analysis. These findings suggest that coexpression of α6β4 integrin and Net1 could be a useful biomarker for aggressive disease in node-positive breast cancer patients. (Cancer Epidemiol Biomarkers Prev 2009;18(1):80–6)

COX-2 and PPARγ expression are potential markers of recurrence risk in mammary duct carcinoma in-situ

BackgroundIn women with duct carcinoma in-situ (DCIS) receiving breast conservation therapy (BCT), in-breast recurrences are seen in approximately 10%, but cannot be accurately predicted using clinical and histological criteria. We performed a case-control study to identify protein markers of local recurrence risk in DCIS.MethodsWomen treated for DCIS with BCT, who later developed in-breast recurrence (cases) were matched by age and year of treatment to women who remained free of recurrence (controls).ResultsA total of 69 women were included in the study, 31 cases and 38 controls. Immunohistochemical evaluation of DCIS tissue arrays was performed for estrogen receptor, progesterone receptor, HER-2/neu, cyclin D1, p53, p21, cycloxygenase-2 (COX-2) and peroxisome proliferator activated receptor γ (PPARγ). Two markers were significantly different between cases and controls on univariate analysis: strong COX-2 expression was associated with increased risk of recurrence, with 67% vs. 24% positivity in cases and controls p = 0.006; and nuclear expression of PPARγ was associated with protection from recurrence with 4% vs. 27% positivity in cases and controls, p = 0.024. In a multivariate model which included size, grade, COX-2 and PPARγ positivity, we found COX-2 positivity to be a strong independent risk factor for recurrence (OR 7.90, 95% CI 1.72–36.23)., whereas size and grade were of borderline significance. PPARγ expression continued to demonstrate a protective trend, (OR 0.14, 95% CI 0.06–1.84).ConclusionOur findings suggest that COX-2 and PPARγ should be investigated further as biologic markers to predict DCIS recurrence, particularly since they are also potential therapeutic targets.