Breast Neoplasms: Hicks DG

Vance GH, Barry TS, Bloom KJ, Fitzgibbons PL, Hicks DG, Jenkins RB, Persons DL, Tubbs RR, Hammond ME, Anonymous00034. · Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202, USA. · Arch Pathol Lab Med. · Pubmed #19391661 No free full text.

Abstract: CONTEXT: Intratumoral heterogeneity of HER2 gene amplification has been well documented and represents subclonal diversity within the tumor. The reported incidence of intratumor HER2 amplification genetic heterogeneity ranges in the literature from approximately 5% to 30%. The presence of HER2 genetic heterogeneity may increase subjectivity in HER2 interpretation by the pathologist. OBJECTIVES: To define HER2 genetic heterogeneity and to provide practice guidelines for examining and reporting breast tumors with genetic heterogeneity for improvement of HER2 testing in breast cancer. DESIGN: We convened an expert panel to discuss HER2 gene amplification testing by fluorescence in situ hybridization. Components addressed included a definition of HER2 amplification heterogeneity, practice guidelines for examination of the tissue, and reporting criteria for this analysis. RESULTS: Genetic heterogeneity for amplification of HER2 gene status in invasive breast cancer is defined and guidelines established for assessing and reporting HER2 results in these cases. These guidelines are additive to and expand those published in 2007 by the American Society of Clinical Oncology and the College of American Pathologists. CONCLUSION: Standardized methods for analysis will improve the accuracy and consistency of interpretation of HER2 gene amplification status in breast cancer.

Yaziji H, Taylor CR, Goldstein NS, Dabbs DJ, Hammond EH, Hewlett B, Floyd AD, Barry TS, Martin AW, Badve S, Baehner F, Cartun RW, Eisen RN, Swanson PE, Hewitt SM, Vyberg M, Hicks DG, Anonymous00020. · Vitro Molecular Laboratories, Miami, FL daggerKeck School of Medicine, University of Southern California, Los Angeles, USA. · Appl Immunohistochem Mol Morphol. · Pubmed #18931614 No free full text.

Abstract: Estrogen receptor (ER) status in breast cancer is currently the most important predictive biomarker that determines breast cancer prognosis after treatment with endocrine therapy. Although immunohistochemistry has been widely viewed as the gold standard methodology for ER testing in breast cancer, lack of standardized procedures, and lack of regulatory adherence to testing guidelines has resulted in high rates of "false-negative" results worldwide. Standardized testing is only possible after all aspects of ER testing--preanalytical, analytical, and postanalytical, have been closely controlled. A meeting of the "ad-hoc committee" of expert pathologists, technologists, and scientists, representing academic centers, reference laboratories, and various agencies, issued standardization testing recommendations, aimed at optimization of clinical ER testing environment, as a step toward improved standardized testing.

Hicks DG, Kulkarni S, Hammond ME. · No affiliation provided · Arch Pathol Lab Med. · Pubmed #18684020 No free full text.

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Hicks DG, Dawson A, Mattingly S, Crowe JP. · No affiliation provided · Arch Pathol Lab Med. · Pubmed #16253013 No free full text.

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Hicks DG, Kulkarni S. · Department of Pathology and Laboratory Medicine, University of Rochester, Rochester, NY 14642, USA. · Arch Pathol Lab Med. · Pubmed #18517261 No free full text.

Abstract: CONTEXT: Overexpression of human epidermal growth factor receptor 2 (HER2) is an adverse prognostic parameter for patients with breast cancer. Patients with HER2-positive tumors are, however, likely to gain significant benefit from targeted therapy with trastuzumab. Four large-scale trials have assessed the efficacy and safety of adjuvant trastuzumab. In all 4 trials, disease-free survival and overall survival were significantly improved with adjuvant trastuzumab (36%-52% and 33%-41%, respectively), and improvements were observed in all patient subgroups. OBJECTIVE: To describe, in detail, the clinical benefits of using trastuzumab in the adjuvant setting, and to emphasize the importance this places on the pathologist to correctly identify all patients with HER2-positive tumors with a high degree of accuracy. DATA SOURCES: Published literature on both HER2 testing and the adjuvant use of trastuzumab. CONCLUSIONS: Immunohistochemistry and/or fluorescence in situ hybridization are routinely used to determine HER2 status. Maintaining quality assurance throughout a standardized testing process is essential to achieve accurate and reproducible assay results. Adherence to the new American Society of Clinical Oncology/College of American Pathologists HER2 testing guidelines will help to ensure correct identification of all patients who may benefit from adjuvant trastuzumab and has significant implications for patient outcomes.

Meguerditchian AN, Malik DA, Hicks DG, Kulkarni S. · Department of Surgical Oncology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York 14263, USA. · Breast J. · Pubmed #18384484 No free full text.

Abstract: Benign stromal tumors of the breast are rare mesenchymal neoplasms that have significant clinical and morphologic overlap. We report the case of a spindle cell tumor occurring in the mammary gland with mixed features of solitary fibrous tumor and mammary myofibroblastoma. The significance of this finding as well as the pathologic and radiologic diagnostic strategies used to differentiate these two lesions are presented based on a review of pertinent literature.

Hicks DG, Kulkarni S. · Department of Pathology and Laboratory Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263, USA. · Am J Clin Pathol. · Pubmed #18208807 links to  free full text

Abstract: Clinical laboratory testing for human epidermal growth factor receptor 2 (HER2) status in newly diagnosed breast cancer is critically important for therapeutic decision making. Unlike most pathologic testing, which serves as an adjunct to establishing a diagnosis, the results of HER2 testing stand alone in determining which patients are likely to respond to trastuzumab, a monoclonal antibody against HER2. Given the significant clinical impact of the testing results on subsequent patient management, the accuracy, precision, and reproducibility of HER2 testing are critical. At present, several preanalytic factors, including the time from tissue removal to tissue fixation, are underappreciated as important variables that have the potential to negatively impact the consistency and reliability of HER2 testing. Rigorous quality control and standardization of the testing process, from the handling of tissue samples to interpretation and reporting of results, are essential for achieving accurate and reproducible assay results.

Hicks DG, Tubbs RR. · Department of Anatomic Pathology, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA. · Hum Pathol. · Pubmed #15791569 No free full text.

Abstract: Diagnostic assays for HER2 in breast cancer provide important prognostic information and independently help guide management by identifying patients who are the most likely to benefit from Herceptin-targeted therapy. The biological events underlying HER2 -driven breast cancer that can be assessed in routine clinical specimens include the evaluation of gene amplification by fluorescence in situ hybridization (FISH), enhanced messenger RNA expression by real-time polymerase chain reaction, and the assessment of protein overexpression at the tumor cell membrane by immunohistochemistry (IHC). Immunohistochemistry and FISH methodologies have the advantage of being morphologically driven, allowing for correlations between HER2 expression and morphologic features. However, each has important advantages and disadvantages, which are discussed in detail. Although immunohistochemistry is familiar and readily accommodated in most surgical pathology laboratories, increasing demands for FISH testing in the clinical setting will require greater familiarity with the technical aspects of FISH assays and their interpretation by the greater laboratory community. In this review, we provide an overview of FISH testing for HER2 in breast cancer, with an emphasis on technical considerations, interpretive guidelines, scoring criteria, and quality control. The development of automated platforms for hybridization, image analysis for signal enumeration, and experience with FISH interpretation should broaden the availability of this technology for clinical diagnostic testing.

Kulkarni S, Hicks DG. · Department of Surgical Oncology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA. · Ann Surg Oncol. · Pubmed #18398661 No free full text.

Abstract: Although the treatment of patients with early-stage breast cancer is provided by a multidisciplinary team, surgeons must ensure they are well informed about all aspects of patient care. For example, understanding the importance of human epidermal growth factor receptor 2 (HER2) gene amplification and/or protein overexpression and the effect on patient prognosis can guide therapeutic decision making. In addition, surgeons should also be knowledgeable about the wide variety of available postsurgical treatments, from traditional chemotherapy and radiotherapy to novel agents such as the HER2-targeted monoclonal antibody trastuzumab. Trastuzumab was recently approved for adjuvant treatment of invasive, HER2-positive, node-positive breast cancer. Its approval was based on the combined results of two large-scale trials, which demonstrated that adding trastuzumab to chemotherapy significantly improves disease-free and overall survival. Knowledge about the indications, schedules, and safety profiles of therapies such as trastuzumab will enable surgeons to optimize the timing of surgery in relation to these therapies, and to make informed decisions about the team member to whom a particular patient should ideally be referred for postsurgical care. In the future, results from large-scale trials evaluating the clinical utility of neoadjuvant trastuzumab will become available. Early results from ongoing phase III trials of the addition of trastuzumab to presurgical chemotherapy suggest that high response rates, including pathologic complete responses, are achievable. If trastuzumab is approved for use in neoadjuvant regimens, the need for surgeons to be well informed about the appropriate use of this particular agent will become even more important.

Tubbs RR, Hicks DG, Cook J, Downs-Kelly E, Pettay J, Hartke MB, Hood L, Neelon R, Myles J, Budd GT, Moore HC, Andresen S, Crowe JP. · Anatomic and Clinical Pathology, Cleveland Clinic Foundation and the Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, USA. · Diagn Mol Pathol. · Pubmed #18043283 No free full text.

Abstract: The demand for both reflexed and primary fluorescence in-situ hybridization (FISH) testing in the clinical setting is increasing. Relevant literature has reported the incidence of HER2 overexpression in 20% to 30% of cases, but some reports suggest that HER2 gene amplification rates are substantially lower. Published data, however, on primary FISH assessment from a single institution is limited, especially information about the frequency of the anomalous genotypes defined by FISH. We report our experience with primary FISH testing in 742 consecutive cases of breast cancer, in the calendar year 2006. Eighty percent (595/742) of the breast cancer cases were not amplified for HER2 (HER2/CEP17=0.8-1.9), whereas 19% (142/742) of cases were HER2 amplified (HER2/CEP17>or=2.0). Among the HER2-amplified cases, 3% (19/742) were low-level amplified (HER2/CEP17 ratio=2.0-2.5). Genotypic heterogeneity, defined as >5% but <50% of the tumor cells demonstrating HER2 gene amplification, was observed in 5% (40/7242) of the cases. HER2 monoallelic deletion (HER2/CEP17<or=0.7) was demonstrated in 2% (12/742) of the cases and CEP17 monosomy (1 CEP17 signal in >80% of tumor cells) was observed in 2% (13/742). Polysomy, if defined as CEP17 spot count 3.0 or more in at least 80% of tumor cells, was observed in 3% (20/742) of the cases. These data may be helpful as benchmarks for other institutions initiating primary FISH analysis for HER2 genotyping.

Powell WC, Hicks DG, Prescott N, Tarr SM, Laniauskas S, Williams T, Short S, Pettay J, Nagle RB, Dabbs DJ, Scott KM, Brown RW, Grogan T, Roche PC, Tubbs RR. · Ventana Medical Systems Inc,; Tucson, AZ, USA. · Appl Immunohistochem Mol Morphol. · Pubmed #17536315 No free full text.

Abstract: 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 (kappa 1.0). RMoAb 4B5 provides excellent sensitivity, specificity, and interlaboratory reproducibility for the detection of HER2 status in breast cancer.

Sossey-Alaoui K, Safina A, Li X, Vaughan MM, Hicks DG, Bakin AV, Cowell JK. · Roswell Park Cancer Institute, Department of Cancer Genetics, Elm and Carlton Streets, Buffalo, NY 14263, USA. · Am J Pathol. · Pubmed #17525277 links to  free full text

Abstract: The expression of WAVE3, an actin-cytoskeleton and reorganization protein, is elevated in malignant human breast cancer, yet the role of WAVE3 in promoting tumor progression remains undefined. We have recently shown that knockdown of WAVE3 expression in human breast adenocarcinoma MDA-MB-231 cells using small interfering RNA resulted in a significant reduction of cell motility, migration, and invasion, which correlated with a reduction in the levels of active p38 mitogen-activated protein kinase. Here, we investigated the effect of stable suppression of WAVE3 by short hairpin RNA on tumor growth and metastasis in xenograft models. Breast cancer MDA-MB-231 cells expressing short hairpin RNA to WAVE3 (shWAVE3) showed a significant reduction in Matrigel invasion and formation of lung colonies after tail-vein injection in SCID mice. In the orthotopic model, we observed a reduction in growth rate of the primary tumors, as well as in the metastases to the lungs. We also show that suppression of p38 mitogen-activated protein kinase activity by dominant-negative p38 results in comparable phenotypes to the knockdown of WAVE3. These studies provide direct evidence that the WAVE3-p38 pathway contributes to breast cancer progression and metastasis.

Short SM, Yoder BJ, Tarr SM, Prescott NL, Laniauskas S, Coleman KA, Downs-Kelly E, Pettay JD, Choueiri TK, Crowe JP, Tubbs RR, Budd TG, Hicks DG. · Department of Anatomic and Clinical Pathology L25, Cleveland, Ohio, USA. · Breast J. · Pubmed #17319853 No free full text.

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.

Crowe JP, Patrick RJ, Rybicki LA, Budd GT, Escobar PF, Tubbs RR, Hicks DG. · Departments of General Surgery-Breast Center, The Cleveland Clinic, Cleveland, Ohio 44195, USA. · Int J Fertil Womens Med. · Pubmed #17269588 No free full text.

Abstract: BACKGROUND: The purpose of this study was to identify correlates of HER2 status for patients with bilateral breast cancer. METHODS: Data were collected prospectively in our institutional review board approved patient registry for all patients with asynchronous (ABBC) and synchronous (SBBC) bilateral infiltrating breast cancer whose HER2 assays were performed at our laboratory using FISH. Data were analyzed using the Wilcoxon rank sum and Chi-square test. RESULTS: Data were available for 98 tumors in 49 patients. Patients diagnosed with SBBC were more likely to be white (P = 0.023); to have bilateral HER2 non-amplified tumors (P = 0.022) and to be older at diagnosis (P = 0.025) compared to those with ABBC. Patients with one HER2 amplified tumor were likely to have at least one tumor be hormone receptor negative and to have ABBC. CONCLUSIONS: Only 16% of patients in this study had one tumor that was HER2 amplified; no patient had bilateral HER2 amplified tumors.

Tubbs RR, Swain E, Pettay JD, Hicks DG. · Departments of Anatomical and Clinical Pathology, The Cleveland Clinic Foundation, The Lerner College of Medicine, Cleveland, OH 44195, USA. · J Mol Histol. · Pubmed #17211577 No free full text.

Abstract: Tissue microarrays (TMA) are valuable tools for validating results of array-based comparative genomic hybridization (ACGH) and other translational research applications requiring independent verification of genomic gains and losses by fluorescence in situ hybridization (FISH). However, spatial orientation and accurate manual tracking of the TMA cores is challenging and prone to error. Image analysis combined with core tracking software, implemented via an automated FISH scanning workstation, represents a new approach to FISH and TMA-based validation of novel genomic changes discovered by ACGH in breast and other cancers. Automated large-scale tissue microarray validation FISH studies of genomic gains and losses identified by ACGH for breast cancer are feasible using an automated imaging scanner and tracking/classifying software. Furthermore, by leveraging the bifunctional fluorescent and chromogenic properties of the alkaline phosphatase chromogen fast red K and combining the technology with FISH, correlative and simultaneous phenotype/genotype studies may be enabled.

Tubbs RR, Pettay JD, Swain E, Roche PC, Powell W, Hicks DG, Grogan T. · Department of Anatomic and Clinical Pathology, The Cleveland Clinic Foundation and the Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA. · Appl Immunohistochem Mol Morphol. · Pubmed #17122642 No free full text.

Abstract: Determination of HER2 status by fluorescence in situ hybridization (FISH) in breast carcinoma correlates well with response to targeted therapy and prognosis. However, manual time consuming methods and quantification aspects of the procedure may be challenging for some laboratories. We examined the feasibility of automating these components of the FISH assay using a tissue microarray (TMA-118 clinically annotated cases) and a series of 41 whole sections. An in situ hybridization automated staining workstation was used to automate a programmed overnight start, on line baking, deparaffinization, cell conditioning, protease digestion, and prehybridization buffer washing. Dual label probe/target codenaturation/hybridization and stringency washing were done off line. The HER2 and CEP17 spot counts were quantified, and the HER2/CEP17 ratio calculated, via an imaging workstation. Results were benchmarked against manual counts for whole sections, and bright field in situ hybridization [silver in situ hybridization (SISH)] for the TMA. Automated FISH results using whole sections correlated well with manual results: HER2/CEP17 ratio correlation coefficient r = 0.9154, r = 0.8380, P < 0.0001. Correlation between automated and manual TMA FISH results was also excellent, and disease-free survival was significantly shorter (P < 0.001) for the HER2 amplified cases. Automation of the laborious manual prehybridization and image quantification components of FISH using directly labeled probes is feasible. Operational gains and enhanced consistency are inherent in this automated approach to HER2 clinical FISH testing.

Hicks DG, Yoder BJ, Short S, Tarr S, Prescott N, Crowe JP, Dawson AE, Budd GT, Sizemore S, Cicek M, Choueiri TK, Tubbs RR, Gaile D, Nowak N, Accavitti-Loper MA, Frost AR, Welch DR, Casey G. · Department of Clinical and Anatomic Pathology, General Surgery-Breast Center, Lerner Research Institute, The Cleveland Clinic, Cleveland, Ohio 44195, USA. · Clin Cancer Res. · Pubmed #17121889 links to  free full text

Abstract: 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.

Hicks DG, Short SM, Prescott NL, Tarr SM, Coleman KA, Yoder BJ, Crowe JP, Choueiri TK, Dawson AE, Budd GT, Tubbs RR, Casey G, Weil RJ. · Clinical and Anatomic Pathology, Cleveland Clinic Foundation, OH 44195, USA. · Am J Surg Pathol. · Pubmed #16931954 No free full text.

Abstract: 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.

Ghadersohi A, Pan D, Fayazi Z, Hicks DG, Winston JS, Li F. · Department of Pharmacology & Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Street, Buffalo, NY 14263, USA. · Breast Cancer Res Treat. · Pubmed #16897429 No free full text.

Abstract: Previous studies using immunohistochemistry suggest that loss of the expression of the prostate-derived Ets transcription factor (PDEF) is a strong indicator for cancer cell malignancy. However, the underlying mechanism for this has not been well elucidated. We determined the role of PDEF in breast cancer cell growth and tumor formation using a series of experiments including Western blotting, promoter-luciferase reporter assay, RNA interference technology and a mouse xenograft model. We also determined the relationship between PDEF expression in human breast tumor specimen and cancer patient survivability. These studies revealed that PDEF expression is inversely associated with survivin expression and breast cancer cell xenograft tumor formation. PDEF-specific shRNA-mediated silencing of PDEF expression resulted in the upregulation of survivin expression in MCF-7 cells, which was associated with increased cell growth and resistance to drug-induced DNA fragmentation (apoptosis). In contrast, survivin-specific siRNA-mediated silencing of survivin expression decreased MCF-7 cell growth. Ectopic expression of PDEF inhibited both survivin promoter activity and endogenous survivin expression. Importantly, shRNA-mediated silencing of PDEF expression in MCF-7 breast cancer cells enhanced survivin expression and xenograft tumor formation in vivo. Furthermore, loss of PDEF expression in breast cancer tissues tends to be associated with unfavorable prognosis. These studies provide new information for the role of PDEF and survivin in breast cancer cell growth and tumor formation.

Ring BZ, Seitz RS, Beck R, Shasteen WJ, Tarr SM, Cheang MC, Yoder BJ, Budd GT, Nielsen TO, Hicks DG, Estopinal NC, Ross DT. · Applied Genomics Inc, 863 Mitten Rd #103, Burlingame, CA, USA. · J Clin Oncol. · Pubmed #16809728 No free full text.

Abstract: 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.

Crowe JP, Patrick RJ, Rybicki LA, Escobar PF, Weng D, Thomas Budd G, Tubbs RR, Procop GW, Hicks DG. · Department of General Surgery-Breast Center, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Desk A-80, Cleveland, OH 44195, USA. · Breast. · Pubmed #16716590 No free full text.

Abstract: Recently published clinical trial data have produced compelling evidence for increased survival when Herceptin is administered to patients whose tumors are HER2 amplified. Therefore, the accuracy of HER2 status is essential to determine which patients should or should not receive Herceptin. Although HER2 results obtained by FISH and IHC are often in agreement, there is a persistent group of cases in which results are discordant, particularly among tumors with intermediate results. A multivariable analysis was undertaken to determine relative significance of various clinical and pathologic findings for patients diagnosed with infiltrating ductal carcinoma, and a data model was produced that predicts which patients are most likely to have HER2 amplified tumors. Correlates of HER2 amplification were higher Scarff-Bloom-Richardson grade, younger age at diagnosis, and a comedo ductal carcinoma in situ component.

Tubbs R, Pettay J, Powell R, Hicks DG, Roche P, Powell W, Grogan T, Hainfeld JF. · Department of Anatomic and Clinical Pathology, the Cleveland Clinic Foundation, OH 44195, USA. · Appl Immunohistochem Mol Morphol. · Pubmed #16280669 No free full text.

Abstract: Ultrasensitive bright field in situ hybridization assays using enzyme metallography (EnzMet) have been developed and validated, but little is known regarding the applicability of EnzMet for immunophenotypic detection of protein via IHC. Superior resolution via discrete metallographic deposits offers the potential for enhancing high-resolution immunophenotyping. Using high-complexity tissue microarrays (TMAs), 88 common solid tumors were evaluated by automated EnzMet (Nanoprobes and Ventana). Targets were chosen to assess the ability of EnzMet to specifically localize encoded antigens in the nucleus (estrogen receptor), cytoplasm (cytokeratins), and cytoplasmic membrane (HER2) in TMAs. Results were compared with conventional IHC diaminobenzidine (DAB) immunostaining. There was full concordance between the EnzMet and conventional IHC results. Furthermore, the EnzMet reaction products did not appreciably diffuse, were dense and sharply defined, and provided excellent high-resolution differentiation of cellular compartments in paraffin sections for the nuclear, cytoplasmic, and cell membrane-localized antigens evaluated. The higher density of elemental silver deposited during enzyme metallography permitted evaluation of core immunophenotypes at a relatively low magnification, allowing more tissue to be screened in an efficient manner. This preliminary study shows the utility of using enzyme metallography for high-resolution immunophenotyping in TMAs.

Downs-Kelly E, Yoder BJ, Stoler M, Tubbs RR, Skacel M, Grogan T, Roche P, Hicks DG. · Department of Anatomical and Clinical Pathology, Cleveland Clinic Foundation, Cleveland, OH 44195, USA. · Am J Surg Pathol. · Pubmed #16096413 No free full text.

Abstract: Breast carcinomas with amplification of HER2 on chromosome 17 are associated with HER2 protein overexpression, adversely affecting prognosis and predicting response to Herceptin therapy. Chromosome 17 polysomy is encountered in assessing HER2 gene status, and its impact on HER2 gene and protein expression remains unclear. This impact was investigated in breast carcinomas identified by fluorescence in situ hybridization (FISH) to have a gain of chromosome 17 (CEP17+; n = 56), using a dual probe assay, which detects HER2 gene copy number and enumerates chromosome 17 (HER2/CEP17; Vysis). Cases were immunostained for HER2 protein (CB-11, Ventana), and scored blinded to FISH. A subgroup was evaluated by isotopic in situ hybridization for HER2 mRNA expression. Controls included ten HER2 amplified and ten nonamplified tumors, eusomic for chromosome 17. Immunohistochemistry (IHC) for HER2 protein was negative (0 or 1+) in 69% (39 of 56), 2+ in 27% (15 of 56), and 3+ in 3% (2 of 56) of CEP17+ cases. The mean CEP17 copy number among the three groups was similar (3.1, 3.0, and 3.1 for IHC 0/1+, 2+, and 3+, respectively). Isotopic in situ hybridization for HER2 mRNA performed on 26 CEP17+ cases (16 IHC 0-1+, 10 IHC 2+ or 3+) showed no increased HER2 mRNA expression (normalized to beta-actin mRNA). The mRNA expression and the IHC staining of the HER2-amplified and nonamplified controls was concordant with their FISH status. These results suggest that chromosome 17 polysomy in the absence of HER2 amplification does not have a significant biologic influence on HER2 gene expression in breast carcinoma.

Hicks DG, Yoder BJ, Pettay J, Swain E, Tarr S, Hartke M, Skacel M, Crowe JP, Budd GT, Tubbs RR. · Department of Anatomic Pathology, Cleveland Clinic Foundation, Cleveland, OH 44195, USA. · Hum Pathol. · Pubmed #15891995 No free full text.

Abstract: Clinical and in vitro evidence supports the concept that human epidermal growth factor receptor-2 ( HER2 ) gene amplification prediction of response to anthracycline-based chemotherapy in breast cancer is not a direct effect of HER2 overexpression, but the result of coamplification of topoisomerase II-alpha ( TOP2A ). We investigated the relationship of TOP2A to HER2 genomic alterations by fluorescence in situ hybridization (FISH) and the correlations with polysomic states for chromosome 17 (CEP17). One hundred thirty-eight cases of breast cancer HER2 gene amplified by 2-color FISH ( HER2 /CEP17) were reevaluated with a 3-color probe set ( HER2 /CEP17/ TOP2A ) to investigate the frequency of coamplification and deletion of TOP2A . TOP2A was never amplified in the absence of HER2 amplification and was coamplified with HER2 in 68 (50%) of 137 cases; HER2 gene copy number was higher than the TOP2A copy number ( P < .01). Of the 137 cases with HER2 amplification, 23 (16%) showed a monoallelic deletion of TOP2A . Of the 43 cases not amplified for HER2 , 27 (63%) were CEP17 eusomic, 13 (30%) polysomic, and 3 (7%) monosomic. Of the HER2 nonamplified cases, 2 (5%) showed monoallelic deletion of both the HER2 and TOP2A . The current study demonstrates the complex interrelationship between the HER2 and TOP2A genes in breast cancer. The clinical implications of TOP2A amplification and deletion in breast cancer need to be further defined. If TOP2A gene dosage can be confirmed to correlate with tumor responsiveness to anthracycline-based therapy in the clinical setting, FISH testing for TOP2A status may be warranted to aid in the selection of the most appropriate therapy.

Yoder BJ, Tso E, Skacel M, Pettay J, Tarr S, Budd T, Tubbs RR, Adams JC, Hicks DG. · Department of Anatomic Pathology, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA. · Clin Cancer Res. · Pubmed #15671545 links to  free full text

Abstract: The invasion and metastasis of tumor cells is a major cause of mortality in cancer patients. In the current study, we investigated the expression of fascin, an actin-bundling motility-associated protein, in 210 invasive breast carcinomas with corresponding 5-year clinical follow-up. Fascin expression was compared with hormone receptor (ER/PR) status, HER2 status, cancer grade, cancer stage, metastasis pattern, disease-free survival, and overall survival. Fascin expression was seen in 16% (33/210) of the cases and correlated with ER negativity (22/33, P < 0.001), PR negativity (21/33, P < 0.001), Bloom-Richardson grade 3 (19/29, P < 0.001), and advanced stage (stage 3 or 4, P = 0.04).There was no correlation between fascin expression and HER2 status or pattern of metastases. Patients whose tumors were positive for fascin showed both a decreased mean disease-free survival (74.44 versus 100.52 months, P = 0.002) and mean overall survival (77.58 versus 98.98 months, P = 0.002), independent of tumor stage and HER2 status, but not independent of ER/PR status or cancer grade. Given fascin's role in altering cell motility, overexpression may contribute to a more aggressive clinical course in ER/PR-negative breast cancers. If so, then fascin may represent a new molecular target for therapeutic intervention in patients with ER-negative breast cancer.