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Ephrin B2 Receptor and Microsatellite Status in Lymph Node-Positive

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Ephrin B2 Receptor and Microsatellite Status in Lymph Node-Positive Translational Oncology Volume 6 Number 5 October 2013 pp. 520–527 520 www.transonc.com Ephrin B2 Receptor and Arik Drucker*, Thomas Arnason†,‡, Sen Rong Yan§, Mohammed Aljawad¶, Kara Thompson# Microsatellite Status in and Weei-Yuarn Huang† Lymph Node–Positive *Capital Health and Department of Medicine, Division Colon Cancer Survival1 of Medical Oncology, Dalhousie University, Halifax, Nova Scotia, Canada; †Capital Health and Department of Pathology and Laboratory Medicine, Dalhousie University, Halifax, Nova Scotia, Canada; ‡Pathology Service, Massachusetts General Hospital and Harvard Medical School, Boston, MA; §Dr. Everett Chalmers Hospital, Fredericton, New Brunswick, Canada; ¶London Health Sciences Centre and Division of Gastroenterology, University of Western Ontario, London, Ontario, Canada; #Capital Health and Research Methods Unit and Department of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada Abstract BACKGROUND: Ephrin B2 receptor (EphB2) is a target of the canonical wnt pathway implicated in colorectal carci- nogenesis, and its down-regulation may be associated with adverse prognosis. We evaluated its prognostic value in resected colon cancer stratified by microsatellite status and other clinicopathologic characteristics. METHODS: We identified all cases of resected stage III colon cancer from 1995 to 2009 managed in the Capital Health district of Nova Scotia. Tissue microarrays were constructed and immunohistochemistry (IHC) for tumor EphB2 staining assigned into quartiles. Microsatellite status was evaluated by IHC for MutL homolog 1 (MLH1) and MutS homolog 2 (MSH2). Microsatellite stable tumors were defined as both MLH1/MSH2 (+/+); tumors staining otherwise were classified with microsatellite instability (MSI-H). Primary and secondary outcomes were disease-free survival (DFS) and overall survival (OS), respectively. RESULTS: We identified 159 cases with sufficient tissue for microarray analysis having a median follow-up of 3.47 years (range, 0.14-14). Median age was 61, 52% were male, 40% had an event, and 29% died. MSI-H was present in 18 (13%). Univariate analysis of EphB2 expression on DFS and OS showed a hazard ratio (HR) of 2.00 (P = .01) and 2.14 (P = .03), respectively. Multivariate analysis of EphB2 expression on DFS and OS showed an HR of 2.24 and 2.23, respectively, with tumor IHC ≤ 50%. CONCLUSIONS: In this cohort, decreased EphB2 expression was an independent prognostic factor for recurrence and death and may have prognostic relevance in tumors with MSI-H. However, this would require prospective validation in a larger study. Translational Oncology (2013) 6, 520–527 Address all correspondence to: Weei-Yuarn Huang, MD, PhD, Capital Health and Introduction Department of Pathology and Laboratory Medicine, Dalhousie University, 5788 University Treatment advances for colorectal cancer have changed significantly Avenue, Halifax, Nova Scotia B3H 1V8, Canada. E-mail: [email protected] over the last decade in both early-stage and metastatic settings. How- 1This study was funded by the Capital Health Category 3 Competitive Research grant ever, colorectal cancer remains one of the leading causes of mortality, and the Department of Pathology and Laboratory Medicine, Queen Elizabeth II Health Sciences Centre, both in Halifax, Nova Scotia. None of the authors have with rectal cancer having a worse stage-specific prognosis than colon any financial or other conflicts of interest to disclose. cancer. Despite the development and adoption of targeted systemic Received 1 May 2013; Revised 28 June 2013; Accepted 8 July 2013 therapies for metastatic disease, the median survival remains less than Copyright © 2013 Neoplasia Press, Inc. All rights reserved 1944-7124/13/$25.00 2 years, consumes significant healthcare resources, and is associated DOI 10.1593/tlo.13385 Translational Oncology Vol. 6, No. 5, 2013 Ephrin B2 Receptor and Microsatellite Status Drucker et al. 521 with rising treatment costs [1–5]. A patient selection strategy that is lymph nodes, or those with mesenteric or small omental tumor de- currently lacking is one that may further guide systemic therapy by posits without lymph node involvement (i.e., nodal stage N1c), were stratifying patients based on their tumor biology as well as stage-specific excluded. No patients received neoadjuvant chemotherapy. Pathology prognosis. This may allow foregoing adjuvant chemotherapy in some specimens were reviewed centrally at the Queen Elizabeth II Health patients with a sufficiently good prognosis or, conversely, treating those Sciences Centre, and two pathology reviewers (T.A. and W.Y.H.) were with higher stage-specific risk. blinded to outcomes. On the basis of these criteria, 159 cases had Significant progress has been made in solid tumor oncology with sufficient tissue for microarray analysis and constitute our target cohort. the identification of biologically distinct cancer-specific subsets. The This study was approved by the CDHA Research Ethics Board (File application of this knowledge to treatment decision-making has led No. CDHA-RS/2011-048). to clinically improved patient outcomes in metastatic colorectal can- cer; testing specific k-RAS mutations for epidermal growth factor Tissue Microarray and IHC Analysis receptor inhibitor therapy is one example [6,7]. Furthermore, colon Original hematoxylin and eosin slides from all cases were reviewed cancer has two dominant models of carcinogenesis, the microsatellite to confirm the diagnosis of colon adenocarcinoma. Tissue micro- instability pathway and chromosomal instability, almost exclusive of arrays including triplicate 0.5-mm or duplicate 2-mm punches were each other [8,9]. Testing tumors for expression of the mismatch re- constructed from formalin-fixed tissue in archived paraffin wax pair proteins MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), blocks. The 0.5-mm punches were collected using the Manual Tissue MutS homolog 6 (MSH6), and postmeiotic segregation increased 2 Arrayer MTA-1 (Beecher Instruments, Sun Prairie, WI), and the 2-mm (PMS2) by immunohistochemistry (IHC) is a practical way to assess punches were collected using the Tissue-Tek Quick-Ray Microarray microsatellite instability. In lymph node–positive colon cancer, obser- System (Sakura Finetek, Torrance, CA). IHC stains for MLH1 vational studies and retrospective analyses of randomized clinical trials (G168-15; BD Pharmingen, Franklin Lakes, NJ), MSH2 (FE11; demonstrate an improved prognosis for tumors with high levels of BD Pharmingen), MSH6 BC/44 (Biocare Medical, Concord, CA), microsatellite instability (MSI-H) and reduced benefit from adjuvant and PMS2 EPR3947 (Cell Marque, Rocklin, CA) were applied to chemotherapy [10,11]. 5-μm sections according to our previously published protocol [24]. Ephrins were initially identified when screening for a tyrosine IHC staining for EphB2 was performed manually as follows. After kinase domain of the viral oncogene v-fps [12]. Their roles are diverse dewaxing and rehydration, sections were pretreated with peroxidase but are largely related to maintaining homeostasis of the cellular blocking buffer (120 mM Na2HPO4, 43 mM citric acid, 30 mM environment, particularly in cellular orientation, motility, and micro- NaN3, 0.2% H2O2; pH 5.8) for 15 minutes. Antigen retrieval was vasculature [13–17]. The diverse roles of ephrin signaling in the performed by autoclaving sections for 20 minutes at 121°C and regulation of cell migration and tissue assembly have led to their study 1 atm in 20 mM sodium citrate buffer (pH 6.0). Slides were washed in multiple fields, including the pathogenesis of solid tumors. The three times in phosphate-buffered saline (PBS) before blocking for ephrin B2 receptor (EphB2) is a subtype of the ephrin receptor family, 20 minutes with 0.05% BSA Fraction V (Roche, Indianapolis, IN). the largest receptor tyrosine kinase family of transmembrane proteins. Slides were washed three times in PBS and incubated with goat EphB2 is a multifunctional tyrosine kinase receptor that has showed anti-EphB2 (AF467; R&D Systems, Minneapolis, MN) at 1:200 dilu- prognostic significance in various tumor types, including colorectal tion in 0.05% BSA at 4°C. Sections were washed three times in PBS cancer [15,18–20]. The extracellular domain is capable of recognizing and incubated with rabbit anti-goat IgG (Jackson ImmunoResearch, signals from the cells’ environment and influencing cell-cell interaction West Grove, PA) for 1 hour at room temperature at 1:5000 dilution and cell migration. Preclinical evidence and observational studies in 0.05% BSA, followed by incubation with Powervision HRP anti- have shown that progressive loss of functional EphB2 may be asso- rabbit IgG (Novocastra/Leica Microsystems, Wetzlar, Germany) ciated with a worse prognosis in multiple stages of colorectal cancer for 45 minutes at room temperature. Slides were washed in PBS, [18,19,21]. EphB2 is also believed to be a tumorigenic marker in developed with DAB, and counterstained with hematoxylin. colorectal cancer [22]. However, the largest clinicopathologic correla- IHC stains were interpreted by consensus of two study authors tive study on EphB2 as a prognostic factor for colorectal cancer had (T.A. and W.Y.H.). The stains were interpreted independently by evaluated patient cohorts with both colon and rectal cases analyzed each reviewer, and in cases where there was disagreement on indepen- together, limited information on systemic therapy, and mixed disease dent review, a consensus was reached by a secondary
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