Letter to the editor

Cytogenetic characterization of Ewing tumors: further update on 20 cases

The advent of the human genome map and advances in powerful, high throughput technologies, such as array-based profiling and next generation sequencing, have enabled researchers to better elucidate the molecular genetic changes underlying the pathogenesis of cartilaginous and bone tumor development and progression. Prior to this, preclinical development of treatment strategies was impeded by the large number of tumor subtypes, their genetic and clinical heterogeneity, as well as their paucity. Confounding these issues were inconsistent study results stemming from different study designs and treatment protocols, complicated variables associated with retrospective analyses and the use of diverse molecular methods. The unprecedented view of the tumor genomes that is afforded by these new technologies have enabled a better understanding of the biology of some of these cancers, and provided an important adjunct to standard morphological and immunohistochemical diagnoses. This increased understanding of the biology of cartilaginous and bone tumors provide not only the promise of short-term potential for improving tailored therapies, but also in the longer term, where it will enable the development of new drugs for patients who otherwise had limited treatment options.

Ewing sarcoma (ES) is an aggressive malignant small round cell tumor that arises in bone or soft tissue of adolescents and young adults. A characteristic molecular finding in ES is EWSR1 gene fusion with ETS (erythroblast transformation-specific) family genes including FLI1 (~90%) and ERG (>5%). Here we report our experience using integrated clinicopathologic, cytogenetic, fluorescence in situ hybridization (FISH), and reverse transcriptase polymerase chain reaction (RT-PCR) analyses of 32 pediatric patients with ES diagnosed in a single institution between 2005 and 2011. Diagnostic EWSR1 rearrangements were detected in 30 (93.8%) of 32 patients. Cytogenetics detected t(11;22) (n = 14) and t(21;22) (n = 1) in 15 (46.9%) patients. FISH detected EWSR1 rearrangements in 27 (96.4%) of 28 patients tested. RT-PCR was positive in 27 (84.4%) of 32 patients, including 24 EWSR1-FLI1 and 3 EWSR1-ERG. RT-PCR defined breakpoints and fusion partners in 7 cases with EWSR1 rearrangements detected by FISH. Sanger sequencing further delineated breakpoints in 21 (77.8%) of 27 RT-PCR positive cases. In summary, conventional cytogenetic analysis provided a global view but had a lower detection rate and longer turnaround time than other methods. FISH is a rapid method and theoretically can detect all EWSR1 rearrangements, but it cannot identify all partners and is not completely specific for ES. RT-PCR and sequencing are more sensitive and useful in identifying fusion partners and refining breakpoints; however, these methods can be compromised by poor RNA preservation and primer design. In conclusion, an integrated approach that uses all methods capable of detecting EWSR1 rearrangements has value in the workup of suspected cases of ES.

Cytogenetic analysis of a lumbar soft tissue Ewing sarcoma (ES) in a 7-month-old female child showed a t(17;22)(q21;q12), a rare translocation leading to an EWSR1-ETV4 chimeric transcript. These findings were confirmed by reverse transcription–polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) techniques. The breakpoints were characterized by direct sequencing of the chimeric fusion gene. Tumor genotyping using the Affymetrix Genome-Wide Human single nucleotide polymorphism (SNP) array 6.0 Genechip identified deletions of both chromosomal regions involved in the translocation, resulting in partial deletion of ETV4, but an uninvolved EWSR1 gene. The creation of a fusion between EWSR1 and an ETS family gene consecutive to a chromosomal translocation is characteristic of the Ewing family of tumors (EFT). This is the first report of a deletion involving the two breakpoints in an EWS-ETS translocation. To date, only two cases of t(17;22)(q21;q12) in Ewing sarcoma have been reported, with no associated deletion. Interestingly, both cases had also occurred in soft tissue tumors, which are less common than their bone-involving counterparts.

Over the past few years, the molecular genetic changes underlying cartilaginous and bone tumor development and progression have been increasingly elucidated. However, the development of optimal treatment strategies for some of these entities has been greatly complicated by the large number of subtypes, the heterogeneity in their genetics and biological behavior, and the rarity of these tumors. In addition, inconsistencies have emerged from different studies examining prognostic factors, probably as a result of different study designs and treatment protocols, confounding variables associated with retrospective analyses and the use of diverse molecular methods. In order to resolve these issues, prognostic factors should be validated using uniform and multiple methods in both retrospective investigations and prospective multi-center studies. The advent of the human genome map and molecular techniques, such as microarray-based copy number and gene expression profiling, provide a new approach to classifying tumors and promise to improve our ability to predict both the probability of metastasis and overall clinical course. In addition, an understanding of the molecular biology of these tumors is leading to the identification of targets for novel therapeutic approaches.

The formation of a high-ploidy is rare in Ewing tumor (ET) and is in contrast to some other childhood tumors such as neuroblastoma. In a series of 37 Ewing tumors analyzed by conventional cytogenetics, 4 of the 34 tumors with an abnormal clone (11.8%) demonstrated the presence of a high-ploidy clone, with a chromosome number that ranged from hypotriploid to pentaploid. All 4 contained a t(11;22)(q24;q12) and the karyotypes had further aberrations of the type that would be generally expected in ET. Numerical aberrations represented the majority of the karyotypic events identified and gain of chromosome 8 and loss of chromosomes 3, 10, 16, 19, and 22 occurred in at least 3 tumors. However, no single mechanism could be implicated to explain the karyotypic picture of the 4 cases. It is proposed that high-ploidy subgroups exist in ET and it would be potentially erroneous to group these and other cases together when determining their clinical implications.