1. S.Shetty 1, N. Uddin 1,2,K.B. Geiersbach 1, S.T. South 1,2. 1)University of Utah, School of Medicine, Department of Pathology, Salt lake City, UT and Cytogenetics/Molecular Cytogenetics, ARUP Laboratories, Salt Lake City, UT 2)University of Utah, School of Medicine, Department of Pediatrics, Salt lake City, UT and Cytogenetics/Molecular Cytogenetics, ARUP Laboratories, Salt Lake City, UT Objectives of Study Cytogenetics in diagnosis of blue round cell tumors Molecular based approach to identify unknown translocation partners Abstract Ewing sarcoma is a common malignant round cell tumor of childhood and adolescence. Cytogenetic analysis to identify a common t(11;22)(q23;q12) or less frequently a t(21;22)(q22;q12) or t(7;22)(p22;q12) plays an important role in confirmation of the clinical diagnosis. The genetic mechanism, in majority of the cases, involves fusion of EWSR1 gene on 22q12 with a member of the ETS family of transcription factors. We report a case of a 20 year old male who had a soft tissue tumor on the left scapula. Conventional cytogenetic analysis on unstimulated suspension culture revealed a complex karyotype with multiple numerical and structural abnormalities; all cells from the monolayer culture were karyotypically normal. No chromosomal rearrangement involving the EWSR1 region was recognized. However, the biopsy results were consistent with the diagnosis of Ewing sarcoma. Immunohistochemistry was positive for CD99 and vimentin and negative for AE1/3, CAM 5.2, CD45 and Fli1. To identify a cryptic rearrangement, fluorescence in situ hybridization (FISH) on interphase cells was performed utilizing the LSI EWSR1, dual-color break-apart rearrangement probe. FISH findings revealed two normal fusion signals and an extra signal for the 5’ EWSR1 region suggestive of a rearrangement and deletion of the 3’ EWSR1. FISH on G banded metaphases from unstimulated suspension culture identified that the extra 5’ EWSR1 signal was on chromosome 2. Based on G-banding and FISH findings, the derivative chromosome 2 was shown to consist of a translocation between 2q and 5q with an insertion of the long arm of the 22q12 (approximately between bands 2q12~13 and 5q13. These findings suggest that an insertion event involving the EWSR1 locus on chromosome 22 is another mechanism that could lead to EWSR1-unknown partner gene fusion. To our knowledge, this is the first case report of an insertion of a segment of the EWSR1 region with a possible novel translocation partner on either chromosome 2q12~13 or 5q13 in Ewing sarcoma. This case also highlights the importance of chromosome studies in addition to FISH to understand the genetic mechanism and aid in identifying new partners for the EWSR1 gene on unstimulated suspension cultures in round cell tumors. Based on FISH findings alone, rearrangement of EWSR1 in this case would have been just a postulate and the rearrangements would have been missed if only monolayer culture was analyzed. Discussion and Conclusion Cytogenetic studies have been instrumental in mapping cancer-related genes located at genomic sites that are visibly involved in neoplasia-associated chromosomal rearrangements. The importance of cytogenetic characterization of solid tumors is twofold. First, recurrent aberrations provide insight into the pathogenetic mechanisms. They point to the areas in the human genome that might carry genes or regulatory sequences whose function is disrupted in neoplastic cells. Secondly, even before a fundamental understanding of the mechanism is achieved, the cytogenetic aberrations have direct clinical importance. The finding of an acquired clonal chromosomal abnormality identifies the presence of a neoplastic process, and the specific type of aberration may reveal the true nature of the tumor and thus improve the diagnostic precision. Our experience with culturing round cell tumors have shown that the suspension cultures (tube culture) work better than monolayer cultures (flask culture) and invariably clonal abnormalities are detected in the suspension culture. Our hypothesis is that the round cells tumors may be derived from the primitive and pluripotent cells that do not necessarily grow as a monolayer on a support (e.g., flask or coverslip), unlike other neoplastic cells (fibroblasts, endothelial, adipocytes etc.) that grow well in monolayer cultures. In our patient, all cells from the monolayer culture showed a normal chromosome complement; these normal cells likely represent non-neoplastic stromal cells. All cells from the suspension culture showed complex clonal abnormalities. This patient had diagnosis of Ewing sarcoma based on morphology and immunohistochemistry. Despite the complex chromosomal abnormalities, there was no cytogenetic evidence of a EWSR1 rearrangement. FISH analysis using the EWSR1 break-apart probe showed FFR signal pattern consistent with two normal chromosomes 22 (two fusion EWSR1 signals) and an additional red signal for the 5’ end of EWSR1 probe. FISH was also performed on a G-banded slide in order to locate the abnormal metaphases and understand the mechanism of the abnormal signal pattern. Metaphase FISH revealed insertion of the 5’ EWSR1 probe into the derivative chromosome 2 due to an unbalanced translocation between chromosomes 2 and 5. This has prompted us to look for a novel translocation partner for EWSR1 on chromosomes 2 and 5 using molecular based approaches (study ongoing). In conclusion, this case emphasizes on the role cytogenetics plays in mapping genes and identifying new translocation partners that may be prognostically significant. In addition, this case highlights the importance of an understanding of the biological nature of the tumor cells in culture in order to successfully grow them in culture. A novel unknown translocation partner on chromosome 2 or chromosome 5 for the EWSR1 gene in Ewing Sarcoma Methods Abnormality detected by Immunohistochemistry, Chromosomes, and FISH 1) Immunohistochemistry: CD99, Vimentin, AE1/3, CAM5.2,CD45 and FLI1 2) Culture type: Monolayer and Suspension 3) Molecular cytogenetics: Commercially available EWSR1 FISH Probe 4) Molecular approach to identify unknown partner (Study in progress) Introduction Ewing sarcoma/peripheral primitive neuroectodermal tumor (PNET) is a round-cell sarcoma that may show varying degrees of neuroectodermal differentiation. These tumors are identified by a characteristic round-cell morphology and immunohistochemical profile, as well as by specific translocations involving the EWS gene on chromosome 22 and the 3' portion of the E26 transformation-specific (ETS) family of transcription factors. These translocations result in fusion proteins that act as aberrant transcription factors. The majority of Ewing sarcoma cases are characterized by a balanced t(11;22)(q24;q12) translocation resulting in a EWSR1-FLI1 fusion. Specific chromosomal abnormalities often correlate with distinct morphologic or phenotypic subtypes of tumors and play an important role in prognosis 1,2. Here we describe the molecular cytogenetic investigation of a case of Ewing sarcoma in the left scapula of a 22-year-old male using fluorescent in situ hybridization and G-banding. This case shows the importance of combining cytogenetics and molecular cytogenetics to identify uncommon rearrangements in sarcomas and emphasizes the developing understanding of the biological nature of different tumor types. References 1)Pfeifer JD: Molecular Genetic Testing in Surgical Pathology Edited by Pfeifer JD Philadelphia, Lippincott Williams and Wilkins, 2006, pp.186-231 2)Delattre et al. The Ewing family of tumors--a subgroup of small-round-cell tumors defined by specific chimeric transcripts. N Engl J Med. 1994: 331:294-299. Acknowledgement: We would like to thank Joyce Stratton for her technical expertise with round cell tumor culturing and analysis. References 1)Pfeifer JD: Molecular Genetic Testing in Surgical Pathology Edited by Pfeifer JD Philadelphia, Lippincott Williams and Wilkins, 2006, pp.186-231 2)Delattre et al. The Ewing family of tumors--a subgroup of small-round-cell tumors defined by specific chimeric transcripts. N Engl J Med. 1994: 331:294-299. Acknowledgement: We would like to thank Joyce Stratton for her technical expertise with round cell tumor culturing and analysis. Fig 1B Fig 3 Fig 2 Results Figure 1A: Photomicrograph of the tumor (Hematoxylin-Eosin, X40): It is showing a confluent sheet of small, uniform looking cells with round to oval nuclei, fine chromatin, moderate amount of lightly eosinophilic cytoplasm & indistinct cytoplasmic membranes. Figure 1B: Photomicrograph of the CD99/O13 Immunostain (immunoperoxidase, X40): Majority of the tumor cells are showing a characteristic membranous staining by O-13 antibody against CD99 antigen. Figure 2: Representative abnormal G- banded Karyotype: One of the abnormal karyotyped cells from the suspension culture is captured, showing the der(2) chromosome with its long arm composed of a translocation between the long arms of chromosomes 2 & 5. In addition, also shown are the aberrations of -5, +?del(12)(q24.1), all marked by arrows. The -11 & -21 present in this cell were not found to be a clonal aberration after complete analysis of 20 cells. Figure 3: Interphase FISH with Vysis® LSI EWSR1 (22q12) dual color, break apart rearrangement probe: Showing two yellow (fusion) signals for two intact EWSR1 loci & one red signal (for 5’ EWSR1) suggestive of EWSR rearrangement with deletion of the 3’ EWSR1 (FISH probe fluorophore designation: 5’ EWSR1 – Red, 3’ EWSR1 – Green) Figure 4 A & B: Metaphase FISH with Vysis® LSI EWSR1 (22q12) dual color, break apart rearrangement probe: The metaphase FISH on abnormal metaphase (4A) shows two yellow (fusion) signals for intact EWSR1 at two normal chromosome 22 & one red signal (for 5’ EWSR1) on the der(2) consistent with EWSR rearrangement with deletion of the 3’ EWSR1. On the right (4B) the same abnormal metaphase is shown by inverted dapi staining method to identify the specific chromosomes showing hyberdization signals. (FISH probe fluorophore designation: 5’ EWSR1 – Red, 3’ EWSR1 – Green) Fig 4AFig 4B Fig 2Fig 3Fig 1A Fig 1B