[CANCER RESEARCH 62, 4704 – 4710, August 15, 2002] Genetic Heterogeneity in the Alveolar Rhabdomyosarcoma Subset without Typical Gene Fusions 1 Frederic G. Barr, 2 Stephen J. Qualman, Michele H. Macris, Natalya Melnyk, Elizabeth R. Lawlor, Donna M. Strzelecki, Timothy J. Triche, Julia A. Bridge, and Poul H. B. Sorensen Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 [F. G. B., M. H. M., D. M. S.]; Department of Laboratory Medicine, Children’s Hospital, Columbus, Ohio 43205 [S. J. Q.]; Department of Pathology, Children’s and Women’s Hospital of British Columbia, Vancouver, British Columbia, V5Z 4H4 Canada [N. M., E. R. L., P. H. B. S.]; Department of Pathology and Laboratory Medicine, Children’s Hospital, Los Angeles, California 90027 [T. J. T.]; and Department of Pathology/Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 69198 [J. A. B.] ABSTRACT Previous studies of the PAX3-FKHR and PAX7-FKHR gene fusions in alveolar rhabdomyosarcoma (ARMS) indicated that the corresponding fusion transcripts are not detectable in 20% of ARMS cases. To investi- gate the genetic features of this ARMS subset, we identified 23 ARMS cases in which PAX3-FKHR and PAX7-FKHR transcripts were not de- tected by a standard sensitivity reverse transcription-PCR (RT-PCR) assay. Subsequent analysis with a high sensitivity RT-PCR assay identified low-level expression of PAX3-FKHR or PAX7-FKHR in three cases. Analysis with a Southern blot assay for PAX3 and PAX7 rearrangements and a fluorescence in situ hybridization assay for FKHR rearrangements identified three cases with variant fusions in which PAX3 or PAX7 is postulated to be joined to novel genomic loci. In one such case, RT-PCR analysis of candidate partners identified a fusion of PAX3 to AFX, which is highly similar in structure and function to FKHR. Additional fluores- cence in situ hybridization analysis identified two cases in which a PAX3- FKHR or PAX7-FKHR genomic fusion is present but is not associated with a fusion transcript detectable by RT-PCR. Finally, our analyses of the PAX3, PAX7, and FKHR loci did not identify rearrangements in >50% of cases, consistent with the possibility that there is a true fusion-negative subset. In summary, our analysis of ARMS cases without characteristic PAX3-FKHR or PAX7-FKHR transcripts identified several genetically distinct subsets including low expression or atypical presentation of stand- ard fusions, variant fusions with other genes, and possibly true fusion- negative cases. INTRODUCTION RMS 3 is a family of soft tissue tumors related to the skeletal muscle lineage that occur in children and young adults (1). On the basis of histopathologic appearance, RMS has been divided into two major sub- types, ARMS and ERMS. Cytogenetic analyses have revealed recurrent t(2;13)(q35;q14) and t(1;13)(p36;q14) chromosomal translocations in ARMS but not in ERMS cases. Molecular genetic studies demon- strated that these translocations disrupt chromosomes 2 and 1 within the PAX3 and PAX7 loci, respectively, that encode related members of the paired box family of transcription factors (2, 3). These genes are juxtaposed with portions of the FKHR gene on chromosome 13, which encodes a member of the fork head family of transcription factors (3, 4). The end result is the formation of chimeric genes that are ex- pressed as chimeric transcripts, which can be readily detected with RT-PCR methodology (5, 6). These novel transcripts are in turn translated into chimeric proteins in which the DNA binding domains of PAX3 or PAX7 are joined to the transcriptional activation domain of FKHR to generate novel transcription factors with oncogenic activity (7–10). In a clinical correlative study of ARMS cases from IRS-IV, differ- ences were detected between cases with PAX3-FKHR and PAX7- FKHR fusion transcripts (11). In particular, PAX7-FKHR-expressing tumors occurred in younger children, were locally less invasive, and yet showed a comparable frequency of metastasis compared with PAX3-FKHR-expressing tumors. Although there was no significant survival difference between the two fusions in patients with locore- gional tumors, PAX7-FKHR was associated with a significantly better outcome than PAX3-FKHR in patients with metastatic disease. This difference in outcome may be related to the higher propensity of PAX3-FKHR-expressing tumors to metastasize to bone marrow. In the above-described IRS-IV study (11) as well as in previous molecular diagnostic studies of ARMS (5, 12–16), PAX3-FKHR and PAX7-FKHR transcripts were detected in 80% of ARMS cases, and thus there is a fusion-negative subset comprising 20% of ARMS cases. In the IRS-IV study, these fusion-negative cases had clinical characteristics that were generally intermediate between the PAX3- FKHR and PAX7-FKHR categories without statistically significant differences with either category (11). On the basis of the lack of a distinctive clinical phenotype, we hypothesized that this fusion- negative category may be genetically heterogeneous. To address this hypothesis, we investigated whether this category may contain cases with alterations of the PAX3, PAX7, or FKHR loci that were not detectable with the standard RT-PCR assays. Using RT-PCR, FISH, and Southern blot methodologies, we identified several distinct ge- netic categories within this “fusion-negative” subset. MATERIALS AND METHODS Tumor Specimens. Frozen tumor samples were retrieved from the Inter- group Rhabdomyosarcoma Study Group/Pediatric Cooperative Human Tissue Network tumor bank in Columbus, Ohio, as well as institutional tumor banks of the participating investigators. All of the cases were reviewed at the Intergroup Rhabdomyosarcoma Study Group Biopathology Center (Columbus, OH), and histopathological diagnoses were based on the International Classi- fication of Rhabdomyosarcoma criteria (17). All of the ARMS cases showed either the classic cystic or solid alveolar growth patterns. Dependent on the degree of intervening stroma, cells grew in nests or cords/trabeculae with either nascent (micro-alveolar) or frank central cystic change. Characteristic nuclear features of ARMS were present with monomorphic cells showing nuclei with coarse chromatin and prominent nucleoli or evenly distributed nuclear chro- matin. Tumor giant cells were also present in a subset of these cases. Finally, in all of the cases, the majority of tumor cells were viable, as assessed by histological analysis. RNA Extraction and RT-PCR Analysis. Primers and probes are listed in Table 1 and illustrated in Fig. 1. Total RNA was extracted from frozen primary tumors using the acid-guanidinium-phenol-chloroform method as described previously (6). In the standard sensitivity consensus RT-PCR assay for the PAX3-FKHR and PAX7-FKHR fusion transcripts, RNA was pretreated with DNase I, reverse transcribed from random hexamers, and the cDNA was amplified with PAX3/PAX7-1 and FKHR-R primers, as described previously Received 3/19/02; accepted 6/20/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by funds from NIH Grants CA24507, CA64202, CA71838, CA81659, and CA89461. 2 To whom requests for reprints should be addressed, at Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 36 th Street and Hamilton Walk, Philadelphia, PA 19104-6082. E-mail: barrfg@mail.med.upenn.edu. 3 The abbreviations used are: RMS, rhabdomyosarcoma; ARMS, alveolar rhabdo- myosarcoma; RT-PCR, reverse transcription-PCR; FISH, fluorescence in situ hybridiza- tion; ERMS, embryonal rhabdomyosarcoma; IRS-IV, Intergroup Rhabdomyosarcoma Study IV. 4704 Research. on November 27, 2021. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from