We report our experience using conventional
We report our experience using conventional cytogenetic analysis, FISH using EWSR1 break-apart probes, RT-PCR, and Sanger sequencing to detect characteristic translocations in a total of 32 patients with ES diagnosed at Texas Children's Hospital over a 5-year interval.
Materials and methods
Discussion ES histopathology and immunophenotypic features often overlap with other SRBCTs in pediatric patients , and therefore, cytogenetic testing  and molecular testing  are necessary for a definitive diagnosis. Conventional cytogenetic analysis for the detection of t(11;22) or t(21;22) translocations has been used traditionally in ES , , . Chromosome analysis can provide a global view of tumor genome, detect variant t(11;22) translocations ,  or novel translocations , discover new fusion partners for EWSR1, , and detect other aberrations such as trisomy for ak7 australia 8 and 12 associated with relapse  or additional translocations, that is, der(16)t(1;16)(q12;q11.2) frequently reported in ES , . In the current study, only 7 of the 23 cases with abnormal cytogenetic findings had the sole t(11;22) abnormality (Table 1), with the majority (n = 16) showing additional chromosomal aberrations, including 12 cases with complex chromosomal aberrations that could predict an unfavorable prognosis in ES. Although our study showed an 87.5% success rate, we only detected the diagnostic t(11;22) or t(11;22) in 46.9% of the patients. In addition, chromosome analysis is limited by cell culture failure and normal fibroblast contamination. FISH is a very rapid and sensitive method to detect EWSR1 rearrangements. Our study showed a 100% success rate with an overall 93.5% detection rate and rapid turnaround time. In this study, only 1 primary tumor (case 30) and 1 recurrent tumor (case 12) showed normal EWSR1 by FISH. Case 30 was a true-negative case correlating with RT-PCR and karyotype results. FISH can also be reliably applied to the FFPE samples , , needle biopsies , or tumors with extensive necrosis . However, it is universally recognized that FISH is unable to detect fusion partner genes, sometimes even when combined with chromosome analysis, as observed in case 27. RT-PCR and/or sequencing plays an important role in the diagnosis of ES by targeting the specific fusion gene transcripts, significantly improving clinical sensitivity and specificity , and for the purpose of definitive diagnoses in soft tissue sarcomas . In this study, our method success rate was 93.8% (30/32) and a detection rate of 84.4%. RT-PCR was not informative in 2 cases owing to compromised RNA quantity and quality. In addition, only FLI1 and ERG were targeted in the current study, and the false-negative (case 29) or noninformative results could be explained by the presence of other fusion partners or variant breakpoints. It is important to note that RT-PCR was extremely sensitive in detecting cryptic fusions, as observed in 7 cases in this study (such as cases 2 and 27). The translocation of t(7;22) analyses in case 27 was initially indicative of a potential new fusion partner at 7p13 region for EWSR1. After RT-PCR showing the typical ESWR1-ERG fusion, additional metaphase FISH using the EWSR1-ERG probe was able to reveal the complex rearrangements showing the cryptic ins(22;21). In case 2 with a hyperdiploidy, EWSR1 metaphase FISH revealed a cryptic ins(20;22), suggesting a possible EWSR1-NFATc2 fusion ; nevertheless, RT-PCR demonstrated the typical EWSR1-FLI1. Certainly, RT-PCR is more specific in accurately providing diagnostic information and detecting cytogenetically undetectable cryptic fusions; however, it could not demonstrate other chromosomal aberrations including complex genomic rearrangements that may be prognostic indicators . RT-PCR cannot detect clonal evolution. Sanger sequencing was more specific in further delineating the breakpoints in EWSR1-FLI1 and EWSR1-ERG positive cases. Owing to the small number of cases studied, patients with type 1 and type 2 EWSR1-FLI1 fusion types showed no significant difference in overall and event-free survival (P > .5; data not shown), and further investigation is needed, although similar results have been reported . Comparison studies between FISH and RT-PCR have been conducted extensively by different research groups and clearly showed that both techniques are complementary, although FISH has higher sensitivity on FFPE samples .