Shannon Tarr

Breast cancers with brain metastases are more likely to be estrogen receptor negative, express the basal cytokeratin CK5/6, and overexpress HER2 or EGFR

Brain metastases (BM) from breast cancer are associated with significant morbidity and mortality. In the current study, we have examined a cohort of breast cancer patients who went on to develop BM for clinical-pathologic features and predictive markers that identify this high-risk subgroup of patients at the time of diagnosis. The primary tumors from 55 patients who developed BM were used to construct a tissue microarray. The clinical and pathologic features were recorded and the tissue microarray was stained for estrogen receptor, human epidermal growth factor receptor 2, cytokeratin 5/6, and epidermal growth factor receptor by immunohistochemistry . This cohort of patients was compared against a group of 254 patients who remain free of metastases (67 mo mean follow-up), and another cohort of 40 patients who developed mixed visceral and bone metastatic disease without brain recurrence over a similar period of time. Breast cancer patients who went on to develop BM were more likely to be <50 years old (P<0.001), and the primary tumors were more likely to be estrogen receptor negative (P<0.001) and high grade (P=0.002). The primary tumors were also more likely to express cytokeratin 5/6 (P<0.001) and epidermal growth factor receptor (P=0.001), and to overexpress human epidermal growth factor receptor 2 (P=0.001). The data presented above suggest a profile for breast cancer patients at increased risk for developing BM. Predictive factors to help identify patients with metastatic breast cancer who are at an increased risk for developing central nervous system recurrence might allow for screening of this population for early detection and treatment or for the development of targeted strategies for prevention.

Novel Prognostic Immunohistochemical Biomarker Panel for Estrogen Receptor–Positive Breast Cancer

PURPOSE Patients with breast cancer experience progression and respond to treatment in diverse ways, but prognostic and predictive tools for the oncologist are limited. We have used gene expression data to guide the production of hundreds of novel antibody reagents to discover novel diagnostic tools for stratifying carcinoma patients. PATIENTS AND METHODS One hundred forty novel and 23 commercial antisera, selected on their ability to differentially stain tumor samples, were used to stain paraffin blocks from a retrospective breast cancer cohort. Cox proportional hazards and regression tree analysis identified minimal panels of reagents able to predict risk of recurrence. We tested the prognostic association of these prospectively defined algorithms in two independent cohorts. RESULTS In both validation cohorts, the Kaplan-Meier estimates of recurrence confirmed that both the Cox model using five reagents (p53, NDRG1, CEACAM5, SLC7A5, and HTF9C) and the regression tree model using six reagents (p53, PR, Ki67, NAT1, SLC7A5, and HTF9C) distinguished estrogen receptor (ER)-positive patients with poor outcomes. The Cox model was superior and distinguished patients with poor outcomes from patients with good or moderate outcomes with a hazard ratio of 2.21 (P = .0008) in validation cohort 1 and 1.88 (P = .004) in cohort 2. In multivariable analysis, the calculated risk of recurrence was independent of stage, grade, and lymph node status. A model proposed for ER-negative patients failed validation in the independent cohorts. CONCLUSION A panel of five antibodies can significantly improve on traditional prognosticators in predicting outcome for ER-positive breast cancer patients.

Loss of Breast Cancer Metastasis Suppressor 1 Protein Expression Predicts Reduced Disease-Free Survival in Subsets of Breast Cancer Patients

Purpose: This study aims to determine the effect of loss of breast cancer metastasis suppressor 1 (BRMS1) protein expression on disease-free survival in breast cancer patients stratified by estrogen receptor (ER), progesterone receptor (PR), or HER2 status, and to determine whether loss of BRMS1 protein expression correlated with genomic copy number changes. Experimental Design: A tissue microarray immunohistochemical analysis was done on tumors of 238 newly diagnosed breast cancer patients who underwent surgery at the Cleveland Clinic between January 1, 1995 and December 31, 1996, and a comparison was made with 5-year clinical follow-up data. Genomic copy number changes were determined by array-based comparative genomic hybridization in 47 breast cancer cases from this population and compared with BRMS1 staining. Results: BRMS1 protein expression was lost in nearly 25% of cases. Patients with tumors that were PR negative (P = 0.006) or HER2 positive (P = 0.039) and <50 years old at diagnosis (P = 0.02) were more likely to be BRMS1 negative. No overall correlation between BRMS1 staining and disease-free survival was observed. A significant correlation, however, was seen between loss of BRMS1 protein expression and reduced disease-free survival when stratified by either loss of ER (P = 0.008) or PR (P = 0.029) or HER2 overexpression (P = 0.026). Overall, there was poor correlation between BRMS1 protein staining and copy number status. Conclusions: These data suggest a mechanistic relationship between BRMS1 expression, hormone receptor status, and HER2 growth factor. BRMS1 staining could potentially be used in patient stratification in conjunction with other prognostic markers. Further, mechanisms other than genomic deletion account for loss of BRMS1 gene expression in breast tumors.

A new rabbit monoclonal antibody (4B5) for the immunohistochemical (IHC) determination of the HER2 status in breast cancer: comparison with CB11, fluorescence in situ hybridization (FISH), and interlaboratory reproducibility.

The 2 methodologies in current clinical use to assess HER2 status in breast cancer are: fluorescence in situ hybridization (FISH) (gene amplification) and immunohistochemistry (protein over-expression). A consistent finding has been that 3% to 15% of breast cancers over-express HER2 protein without evidence for gene amplification. Accurate determination of the HER2 status has implications for selecting patients most likely to respond to trastuzumab. We report here our preliminary experience with a new anti-HER2 rabbit monoclonal antibody, 4B5. The evaluation of HER2 status in 2 different cohorts of breast cancer cases (Single Institution (SI) and Multinational (MN)) with a total of 322 breast cancer cases was performed on an automated staining system (Ventana Medical Systems, Inc, Tucson, AZ) and scored by 3 pathologists (0-3+), for comparison with CB11 staining results (PATHWAY) and FISH (PathVysion). Interlaboratory reproducibility of automated staining results and interpretation was determined on a subset of the SI cohort at 3 separate laboratories. Rabbit monoclonal 4B5 demonstrated sharper membrane staining with less cytoplasmic and stromal background staining than CB11. In the SI cohort, the staining results for 4B5 were highly comparable with those obtained for CB11 with an overall concordance of 93.3%. In the multinational cohort, the overall concordance with CB11 was 84.7%. This lower level of concordance was associated with a much higher overall agreement of 4B5 with FISH (89.5%), compared with agreement of CB11 with FISH (81.2%). The difference in the performance of CB11 in the MN cohort versus the SI cohort may be due to differences in tissue fixation and processing in a centralized, high volume laboratory in an academic medical center versus multiple sites in the international community with potentially nonstandardized techniques. The staining results with 4B5 indicate that it has a more robust performance than CB11 because the correlation of 4B5 with FISH was nearly equivalent (88.2% MN; 89.3% SI) in both cohorts. Interlaboratory reproducibility was also excellent (κ 1.0). RMoAb 4B5 provides excellent sensitivity, specificity, and interlaboratory reproducibility for the detection of HER2 status in breast cancer.

The Expression of the Cytoskeletal Focal Adhesion Protein Paxillin in Breast Cancer Correlates with HER2 Overexpression and May Help Predict Response to Chemotherapy: A Retrospective Immunohistochemical Study

Abstract:  Paxillin, a cytoskeletal focal adhesion adaptor protein, has been shown to be transcriptionally up‐regulated and phosphorylated by human epidermal growth factor receptor‐2 (HER2) signaling in vitro. Paxillin expression may also correlate with HER2 amplification in breast cancer patients. In the current study, we sought to explore the relationship further between paxillin expression and clinicopathologic features and clinical outcome in breast cancer. A total of 314 primary invasive breast carcinomas were assessed for paxillin expression via immunohistochemistry. Paxillin immunoreactivity was compared with estrogen receptor/progesterone receptor status, HER2 status by silver in situ hybridization, age, tumor size, stage, Bloom–Richardson grade, nodal status, disease‐free survival (DFS), and overall survival (OS). Paxillin expression was identified in 27.7% of breast carcinomas as diffuse cytoplasmic staining and the expression correlated with HER2 overexpression (p < 0.001). The influence of paxillin on clinical outcome, in particular the response to chemotherapy, appeared to differ depending on the HER2 status of the tumor. For the subset of HER2 nonamplified cases treated with chemotherapy, patients whose tumor showed a loss of paxillin expression demonstrated a significantly lengthened DFS and OS. In contrast, loss of paxillin expression in the HER2 amplified subset of patients who received chemotherapy correlated with a significantly worse outcome. These data suggest that paxillin up‐regulation may be a part of the HER2 pathway in some breast cancers and, furthermore, paxillin expression may also influence the clinical response to chemotherapy, depending upon the HER2 status of a given patient’s tumor. Further study of a role for paxillin expression in predicting response to cytotoxic regimens or targeted treatments is warranted.

In situ hybridization in the pathology laboratory: General principles, automation, and emerging research applications for tissue-based studies of gene expression

Diagnostic anatomic pathologists play an important role in the care of patients through their careful evaluation of morphological features in routinely prepared histological sections stained with Hematoxylin and Eosin. Morphological assessment of tissue sections, backed by over one hundred years of experience is powerful and allows for the accurate classification and diagnosis of the majority of disease states within pathologically altered tissues. However, the appearance of cells and their architectural arrangement within a morphologically complex tissue represents only a fraction of the information, which is contained within a histological section. These tissues also contain all of the cellular proteins and expressed genes, which help to ultimately determine the biological behavior of cells, as well as provide clues to the origins and pathogenesis of disease states. Technical and theoretical advances in our understanding of cellular biology have provided pathologists with powerful tools to probe beyond pure morphology into the abnormalities in both protein and gene expression that underlie human disease. These tools, which include immunohistochemistry and in situ hybridization, are playing an increasingly important role in diagnostic pathology, as well as in translational research. This review will focus on the emerging role of in situ hybridization within clinical and research laboratories, and will highlight a number of technical advances that have expanded the application of this technology.

Comparison of obstructive sleep apnea patients with and without leg edema.

BACKGROUND To determine the proportion of patients with obstructive sleep apnea (OSA) who have leg edema, and to identify differences between edematous and non-edematous OSA patients. METHODS Retrospective, cross-sectional study of 378 patients with OSA (apnea/hypopnea index [AHI] >or=15) who had neither heart failure nor chronic lung disease. RESULTS Thirty-five percent (133/378) of the subjects with OSA had bilateral leg edema. Eighty-one percent (108/133) of the edematous subjects had mild pitting that was 1+. Compared to the non-edematous OSA subjects, the edematous subjects were older (age=51+/-13 versus 45+/-13 years, p=0.001), more obese (body mass index=39+/-9 versus 33+/-8kg/m(2), p=0.001), had more severe OSA (AHI=46+/-71 versus 27+/-29, p=0.004), spent a greater proportion of sleep time with an oxygen saturation <90% (20+/-26 versus 11+/-18%, p=0.001), and were more likely to have diabetes mellitus (11% versus 3%, p=0.001) and hypertension (32% versus 10%, p=0.001). Age, obesity, hypertension and diabetes mellitus correlated significantly with edema status. After adjusting for these confounding variables, the AHI means remained different between the edema and non-edema groups (41+/-5 versus 28+/-3, p=0.04). CONCLUSIONS Approximately one-third of OSA patients have edema. Edematous OSA patients are older, more obese, more likely to have diabetes mellitus and hypertension, and have more severe OSA than OSA patients who lack edema.

Using novel protein antibodies on tissue microarrays (TMAs) for breast cancer prognostication

9629 Background: Estrogen receptor (ER) and progesterone receptor (PR) are important prognostic and predictive factors for breast cancer patients (pts). Murine monoclonal antibodies (MuAb) are currently used in immunohistochemistry (IHC) assays to determine ER/PR status. Rabbit monoclonal antibodies (RAb) may have greater affinity for human antigens than MuAb. We compared the concordance of MuAb- and RAb-based IHC assays of ER and PR and the prognostic value of these and other factors in pts with Stage I-III breast cancer. METHODS TMAs were produced from formalin-fixed paraffin-embedded tissue sections of primary breast cancer pts. IHC assays were performed for the following: ER/PR status using RAb SP1CLONE (Labvision™) and using MuAb (Ventana™), fascin (a tumor cell motility protein) using clone 55K-2 (DAKO/M3567™), cytokeratin 5/6 (CK5/6) using clone D5/164B (Roche Biomed/1273396™), and HER2 using CB11. These were correlated with stage, progression-free (PFS) and overall survival (OS). ER/PR positivity was defined as ≥5% estimated proportion of positive-staining tumor cells. RESULTS 199 pts were evaluable for ER/PR. The concordance rate for RAb- and MuAb- assessed ER status was 90% (180/199). The level of agreement for RAb- and MuAb- assessed PR status was 81% (162/199). ER/PR positivity by RAb correlated with each other, HER2 negativity and earlier stage. In 159 pts with evaluable follow up, pts with ER positivity by RAb had better 5-year PFS (p<.001) and OS (p=.03), similar to ER positivity by MuAb. Pts with PR positivity by RAb had superior 5-year PFS (p=.001) and OS (p=.04) and correlated better with survival than PR positivity by MuAb. Fascin was expressed in 34 pts. Using univariate analysis, fascin expression correlated with higher stage, negative ER/PR, shorter PFS and OS. CK5/6 positivity did not correlate with any variable, except ER/PR positivity by RAb. After multivariate analysis, significant factors for PFS were stage, HER2, and PR by RAb, and for OS were stage, HER2, and fascin positivity. CONCLUSION 1) The RAb for PR may be superior to the MuAb in predicting prognosis. 2) Fascin deserves further investigation as a prognostic factor. No significant financial relationships to disclose.