Modern Pathology (2011) 24, 1327–1335 & 2011 USCAP, Inc. All rights reserved 0893-3952/11 $32.00 1327 FOXO1–FGFR1 fusion and amplification in a solid variant of alveolar rhabdomyosarcoma Jinglan Liu1,2, Miguel A Guzman1,2, Donna Pezanowski1, Dilipkumar Patel1, John Hauptman1, Matthew Keisling3, Steve J Hou2,3, Peter R Papenhausen4, Judy M Pascasio1,2, Hope H Punnett1,5, Gregory E Halligan2,6 and Jean-Pierre de Chadare´vian1,2 1Department of Pathology and Laboratory Medicine, St Christopher’s Hospital for Children, Philadelphia, PA, USA; 2Drexel University College of Medicine, Philadelphia, PA, USA; 3Department of Pathology and Laboratory Medicine, Hahnneman University Hospital, Philadelphia, PA, USA; 4Laboratory Corporation of America, The Research Triangle Park, NC, USA; 5Department of Pathology and Laboratory Medicine, Temple University School of Medicine, Philadelphia, PA, USA and 6Department of Pediatrics, Section of Oncology, St Christopher’s Hospital for Children, Philadelphia, PA, USA Rhabdomyosarcoma is the most common pediatric soft tissue malignancy. Two major subtypes, alveolar rhabdomyosarcoma and embryonal rhabdomyosarcoma, constitute 20 and 60% of all cases, respectively. Approximately 80% of alveolar rhabdomyosarcoma carry two signature chromosomal translocations, t(2;13)(q35;q14) resulting in PAX3–FOXO1 fusion, and t(1;13)(p36;q14) resulting in PAX7–FOXO1 fusion. Whether the remaining cases are truly negative for gene fusion has been questioned. We are reporting the case of a 9-month-old girl with a metastatic neck mass diagnosed histologically as solid variant alveolar rhabdomyosarcoma. Chromosome analysis showed a t(8;13;9)(p11.2;q14;9q32) three-way translocation as the sole clonal aberration. Fluorescent in situ hybridization (FISH) demonstrated a rearrangement at the FOXO1 locus and an amplification of its centromeric region. Single-nucleotide polymorphism-based microarray analysis illustrated a co-amplification of the FOXO1 gene at 13q14 and the FGFR1 gene at 8p12p11.2, suggesting formation and amplification of a chimerical FOXO1–FGFR1 gene. This is the first report to identify a novel fusion partner FGFR1 for the known anchor gene FOXO1 in alveolar rhabdomyosarcoma. Modern Pathology (2011) 24, 1327–1335; doi:10.1038/modpathol.2011.98; published online 10 June 2011 Keywords: alveolar rhabdomyosarcoma; FOXO1–FGFR1 fusion; gene amplification; genetics; microarray; solid variant Rhabdomyosarcoma accounts for 4–10% of pedia- poor outcome, while embryonal rhabdomyosarcoma tric malignancies. Four pathological subtypes have primarily occurs in children younger than 4 years of been traditionally defined: alveolar rhabdomyosar- age and is usually associated with a better prog- coma, embryonal rhabdomyosarcoma, botyroid nosis.2 A rare solid variant of alveolar rhabdomyo- rhabdomyosarcoma, and pleiomorphic rhabdomyo- sarcoma has been described that is often sarcoma.1,2 Alveolar rhabdomyosarcoma and embry- morphologically indistinguishable from poorly dif- onal rhabdomyosarcoma have distinct clinical ferentiated embryonal rhabdomyosarcoma and manifestations, biological behavior, genetic altera- may be confused with other ‘small, round, blue tions, and account for B20 and B60% of all cases, cell tumors’.3 respectively.1 Alveolar rhabdomyosarcoma is most A study of a large pediatric cohort of 171 patients often seen in older children and is associated with a with rhabdomyosarcoma by the children’s oncology group (COG) has demonstrated the role of genetics in the development of rhabdomyosarcoma.4 In the Correspondence: Dr J-P de Chadare´vian, MD, Department of subtype of alveolar rhabdomyosarcoma, approxi- Pathology and Laboratory Medicine, St Christopher’s Hospital for mately 80% of cases harbored two signature Children, Drexel University College of Medicine, Philadelphia, chromosomal translocations, t(2;13)(q35;q14) in PA 19134, USA. 60%, and t(1;13) (p36;q14) in 20% of cases. These E-mail: [email protected] Received 31 December 2010; revised 22 February 2011; accepted translocations resulted in the formation and over- 22 February 2011; published online 10 June 2011 expression of chimerical genes PAX3–FOXO1 www.modernpathology.org FGFR1–FOXO1 fusion in alveolar rhabdomyosarcoma 1328 J Liu et al (FOXO1, also known as FKHR) and PAX7–FOXO1, muscle actin (clone 1A4), myosin (clone SMMS-1), respectively.4 Chromosome rearrangements not in- CD99 (clone HO36-1.1), CD1a (clone MTB1), alfa volving the FOXO1 locus have also been described fetoprotein (polyclonal), CD45 (clones 2B11 & PD7/ in both alveolar rhabdomyosarcoma and embryonal 26), and epithelial membrane antigen (EMA) (clone rhabdomyosarcoma.5 In embryonal rhabdomyosar- E29) at the dilution suggested by the manufacturer. coma, gains of all or portions of chromosomes 2, 7, All antibodies were obtained from Cell Marque (Hot 8, 11, 12, 13, 17, 19, and 20, with a particularly high- Springs, AR, USA). level gain of chromosome 8 material are consistently 6–8 observed, and loss of heterozygosity (LOH) on the Conventional Cytogenetic Analysis short arm of chromosome 11 (11p15.5) is frequently detected, suggesting that imprinting might be A piece of the resected lymph node was minced, involved.9 In both alveolar rhabdomyosarcoma and cultured in conditioned RPMI 1640 medium sup- embryonal rhabdomyosarcoma, amplification of plemented with 25% fetal bovine serum for 72 h. genes such as MYCN, MDM2, CDK4 and IFG-R1 are Metaphase chromosomes for Giemsa banding pat- often observed.10–14 Despite the usually sporadic tern by trypsin digestion with Wright stain (GTW occurrence of rhabdomyosarcoma, it has also been banding) were prepared according to standard reported in various congenital disorders, including procedures. In all, 20 metaphases were karyotyped cancer predisposition syndromes and genetic with CytoVison system (Applied Imaging, Santa disorders with multi-organ system defects such Clara, CA, USA), and karyograms were described as Li-Fraumeni syndrome, Beckwith–Wiedemann according to the International System for Human syndrome, neurofibromatosis type 1, Costello Cytogenetic Nomenclature 2009.22 Coordinates of syndrome, Nijmegen breakage syndrome, Rubin- cells with chromosome aberrations were documen- stein–Taybi syndrome, Dubowitz syndrome, multi- ted, and the slides were subsequently prepared for ple endocrine neoplasia type 2A, Roberts syndrome, metaphase FISH analysis. and Duchenne muscular dystrophy.15–17 Subtype classification and gene fusion status closely corre- Interphase and Metaphase FISH Assays late with the prognosis.18 However, a question remains, whether alveolar rhabdomyosarcoma cases Touch preparation slides were made from a frozen without PAX3/7–FOXO1 fusion are truly negative portion of the lymph node. Interphase FISH was for any gene fusion.18–21 In this study, we describe undertaken using LSI FOXO1 (FKHR) dual color the clinicopathological assessment, cytogenetic test break-apart probe at 13q14 (Vysis, Abbott Park, IL, results, and single nucleotide polymorphism (SNP)- USA) and FGFR1 dual color break-apart probe at based whole-genome microarray evaluation of a 8p12p11.2 (LabCorp, Research Triangle Park, NC, solid variant embryonal rhabdomyosarcoma with USA). Metaphase FISH was performed on the de- a novel FGFR1–FOXO1 fusion and amplification. stained GTW slides. Standard FISH protocol was followed. FISH images were captured and analyzed Materials and methods with a CytoVison system (Applied Imaging). Case Report SNP-Based Microarray Analysis A 9-month-old female was admitted with a chief Microarray assay was performed by LabCorp, using complaint of left neck mass present for a month, and Affymetrix (Santa Clara, CA, USA) whole-genome- a left shoulder mass present for a week. Chest CT human SNP array 6.0 platform containing 1.8 showed a 3 Â 1.9 Â 2-cm soft tissue mass in the million SNP and non-SNP markers. Genomic DNA region of the supraspinal muscle, and multiple extraction, probe labeling and hybridization were enlarged cervical lymph nodes. The remainder carried out following the manufacturer’s instruction of the physical examination was within the normal (Affymetrix). The publicly available HapMap set of range. The patient underwent excisional biopsy of 270 control individuals and an internal cohort of one of the cervical lymph nodes. controls were implemented as control groups. Genotyping Console (GTC) 4.0 software (Affymetrix) Pathology was applied to assess copy number alterations and long contiguous stretches of homozygosity (LCSH) Formalin-fixed, paraffin-embedded tissue sections using the software default settings. The cut-off for a were stained with hematoxylin–phloxine–saffron genomic imbalance was set up as 200 Kb for the for routine histological assessment. Immunohisto- deletion and 500 Kb for the duplication. NCBI Build chemical studies were conducted using an 36.1 (hg18) was used as reference sequence. automated immunostainer (Thermo Scientific, Freemont, CA, USA). The following primary Gene Ontology and Data Mining antibodies were used: desmin (clone D33), muscle- specific actin (clone HHF35), myogenin (clone F5D), Gene ontology analysis was conducted using cytokeratin cocktail (clone AE1/AE3), smooth UCSC genome browser (http://genome.ucsc.edu). Modern Pathology (2011) 24, 1327–1335 FGFR1–FOXO1 fusion in alveolar rhabdomyosarcoma J Liu et al 1329 Functional annotation, pathway hunting, and net- (Figure 1a). In some areas, especially toward the work construction were performed with Ingenuity periphery, the tumor cells stood