Diffuse Myogenin Expression by Immunohistochemistry is an Independent Marker of Poor Survival in Pediatric Rhabdomyosarcoma A Tissue Microarray Study of 71 Primary Tumors Including Correlation With Molecular Phenotype Amy Heerema-McKenney, MD,*w z Liliane C. D. Wijnaendts, MD, PhD,y Joseph F. Pulliam, MD,w Dolores Lopez-Terrada, MD, PhD,w Jesse K. McKenney, MD,*z Shirley Zhu, MD,* Kelli Montgomery, BA,* Janet Mitchell, HT,* Robert J. Marinelli, PhD,J Augustinus A. M. Hart, MSc,z Matt van de Rijn, MD, PhD,* and Sabine C. Linn, MD, PhD*z Abstract: The pathologic classification of rhabdomyosarcoma (RMS) into embryonal or alveolar subtype is an important prognostic factor guiding the therapeutic protocol chosen for an individual patient. Unfortunately, this classification is not always straightforward, and the diagnostic criteria are con- troversial in a subset of cases. Ancillary studies are used to aid in the classification, but their potential use as independent prognostic factors is rarely studied. The aim of this study is to identify immunohistochemical markers of potential prognostic significance in pediatric RMS and to correlate their expression with PAX-3/FKHR and PAX-7/FKHR fusion status. A single tissue microarray containing 71 paraffin-embedded pediatric RMSs was immunostained with antibodies against p53, bcl-2, Ki-67, CD44, myogenin, and MyoD1. The tissue microarray and whole paraffin blocks were studied for PAX-3/FKHR and PAX-7/FKHR gene fusions by fluorescence in situ hybridization and reverse transcription-polymerase chain reaction. Clinical follow-up data were available for each patient. Immunohisto- chemical staining results and translocation status were corre- lated with recurrence-free interval (RFI) and overall survival (OS) using the Kaplan-Meier method, the log-rank test, and Cox proportional hazard regression. The minimum clinical follow-up interval was 24 months (median follow-up = 57 mo). On univariable analysis, immunohistochemical expression of myogenin, bcl-2, and identification of a gene fusion were associated with decreased 5-year RFI and 10-year OS (myogenin RFI P = 0.0028, OS P = 0.0021; bcl-2 RFI P = 0.037, OS P = 0.032; gene fusion RFI P = 0.0001, OS P = 0.0058). After adjustment for Intergroup Rhabdomyosarcoma Study-TNM stage, tumor site, age, tumor histology, and translocation status by multivariable analysis, only myogenin retained an indepen- dent association with RFI (P = 0.034) and OS (P = 0.0069). In this retrospective analysis, diffuse immunohistochemical reac- tivity for myogenin in RMS correlates with decreased RFI and OS, independent of histologic subtype, translocation status, tumor site, or stage. Key Words: rhabdomyosarcoma, myogenin, PAX gene fusion, immunohistochemistry (Am J Surg Pathol 2008;32:1513–1522) R habdomyosarcoma (RMS) is a malignant childhood tumor comprised of neoplastic cells with evidence of skeletal muscle differentiation. The annual incidence of RMS is 4 to 7 per million children 15 years of age or younger and the overall 5-year survival rate is 70%. 2 Prognostic factors are critical for determining the patient’s risk of recurrent or progressive disease and for tailoring the appropriate therapy. Overall risk stratifica- tion for current therapy is based upon tumor site, resectability, stage, and histologic subtyping as alveolar rhabdomyosarcoma (ARMS) or embryonal rhabdomyo- sarcoma (ERMS). ARMS is considered more clinically aggressive than ERMS, and may require more intensive therapy, depending upon the other clinical factors. 2,27 The histologic classification of RMS may be difficult in some cases, 1 which has led to the search for other surrogate markers of ARMS. Some studies have reported the prognostic importance of immunohistochemical expression of various markers such as p53 and MIB-1, but most do not adjust for other adverse prognostic Copyright r 2008 by Lippincott Williams & Wilkins From the Departments of *Pathology; JBiochemistry, Stanford University School of Medicine, Stanford, CA; wDepartment of Pathology, Texas Children’s Hospital, Baylor College of Medicine, Houston, TX; zDepartment of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR; yDepartment of Pathology, Free University Medical Center; and zDepartment of Medical Oncology, Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands. Sabine C. Linn was a recipient of a Dutch Cancer Society fellowship. This work has been presented in part at the United States and Canadian Academy of Pathology Meeting, 2002, Atlanta, GA and the Society for Pediatric Pathology Meeting 2006, Atlanta, GA. Correspondence: Amy Heerema-McKenney, MD, Department of Pathology, Stanford University School of Medicine, Stanford, CA, 94305 (e-mail: Amy.McKenney@stanford.edu). ORIGINAL ARTICLE Am J Surg Pathol  Volume 32, Number 10, October 2008 1513