Molecular Diagnosis in Ewing Family Tumors

Abstract

The Ewing's family of tumors (EFTs) are characterized by chimeric transcripts generated by specific chromosomal rearrangements. The most common fusions are between the EWSR1 gene on chromosome 22 and the ETS family of transcription factors; rarely, FUS (on chromosome 16) substitutes for EWSR1. The detection of specific translocations using molecular analysis is now a routine part of the pathological examination of EFT. Here, we report our experience with molecular diagnosis of EFT during the 4 years (2006 -2009) at the Rizzoli Institute. We analyzed 222 consecutive tumors with a presumptive diagnosis of EFT using molecular techniques and IHC. We found five distinct types of EWSR1-FLI1 fusion transcripts resulting from translocation t(11;22), three types of EWSR1-ERG transcripts resulting from t(21;22), and one type of t(2;22) resulting in EWSR1-FEV fusion. Molecular investigation validated 92% of cases ultimately diagnosed as EFT; IHC validated 76% of the cases. Thus, despite the difficulties and limitations associated with both molecular and IHC analysis on fresh and formalin-fixed, paraffin-embedded tissue, a combination of these techniques is the best approach to enhancing the accuracy of EFT diagnosis. We also present our method for choosing which molecular techniques to apply. Finally, we collected the most prevalent breakpoints reported in the literature, indicating which exons are involved, the sequence breakpoints, and the NCBI reference sequences. Ewing's sarcoma (ES) is a highly malignant small round cell tumor (SRCT) that can arise in both bone and soft tissues of children and young adults. Rarely, it occurs also in adults.1,2 It is the second most common pediatric bone tumor after osteosarcoma, accounting for ϳ30% of all primary bone tumors in this age group. 1,3,4 Based on their shared immunophenotypes and molecular parameters, several diagnostic entities that were previously considered distinct are now assembled as a single entity, the Ewing's sarcoma family of tumors (EFTs). These include bone and extraskeletal Ewing's sarcoma, primitive neuroectodermal tumor (PNET), and Askin tumor of the thoracopulmonary region. 1,5-7The histological evaluation of EFT is based on morphological, immunophenotypic, and molecular features. The morphology of ES is variable. These tumors are frequently composed of uniform small round cells with round nuclei containing fine chromatin, scanty clear or eosinophilic cytoplasm, and indistinct cytoplasmic membranes. In less differentiated cases, tumor cells are larger, with prominent nucleoli and irregular contours. The cytoplasm frequently contains PAS-positive diastase-resistant glycogen. PNET is characterized by HomerWright rosettes, which are typical of neuroectodermic differentiation. 6,8,9 Immunohistochemical analysis (IHC), the first ancillary technique used in pathology to confirm the diagnosis of EFT, can distinguish among different variants. The immunohistochemical features of ES are positivity for CD99, FLI1, and caveolin 1 antigens, whereas PNET also shows immun...

Detection of circulating tumor cells in liquid biopsy from Ewing sarcoma patients

“…In this study, primary tumor tissues from all patients were available to confirm the presence of specific chromosomal translocation and type of breakpoint as previously described. 18 Total RNA from enriched CTCs CD99+ was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and RNeasy Mini Kit spin columns (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s manual. Total RNA (from 1–2.5 µg) was then reverse transcribed to cDNA using Super-Script VILO IV Master Mix (Applied Biosystems, Foster City, CA, USA).…”

Biomarkers of Osteosarcoma, Chondrosarcoma, and Ewing Sarcoma

“…The authors study the genome of a group of patients with Ewing’s sarcoma. The authors find different types of genetic abnormalities as a result of translocation of chromosomes: five forms of EWSR1-FLI1, three forms of EWSR1-ERG, and one EWSR1-FEV (Gamberi et al, 2011). …”

Frequency of Translocation t(11;22)(q24;q12) Using Fluorescence In Situ Hybridization (FISH) in Histologically and Immunohistochemically Diagnosed Cases of Ewing’s Sarcoma

“…More than 90% ES cases have the t(11;22) chromosomal translocation. The identification of the highly specific balanced chromosomal rearrangement t(11;22)(q24;q12) in most ESs provides a valuable tool for diagnosing this tumor at the molecular level [ 4 , 5 ].…”