[2010] Characterization of EWSR1 Break Apart FISH in Non-EWSR1 Related Samples: Avoiding the False Positive Pitfall

Navid Sadri, Paul J Zhang. University of Pennsylvania, Philadelphia, PA

Background: Hybridizing two colored DNA probes flanking break points of a specific gene can visualize a signal separation (SS) in tumor cells containing that translocation. This break-apart FISH assay is widely used on routine paraffin section for diagnosis. Currently there lacks standardization in interpreting the presence and incidence of SS. Although false positive SS are recognized to exist their characterization and incidence has not been sufficiently characterized.
Design: We performed EWSR1 break-apart probes (Vysis) on sarcomas with a non-EWSR1 translocations (synovisal sarcomas [SYS; n=3] and alveolar rhabdomyosarcomas [AR, n=4]), 3 breast carcinomas (BRCa) and 2 normal tonsils. Tumors known to have EWSR1 translocation (EWSR1+; 1 DSRCT, 3 EWS/PNET, 1 extraskeletal myxoid chondrosarcoma) were evaluated as a control. Each tumor cell was categorized to have: at least two intact alleles (IA); at least one IA with one SS; or unpaired signal(s) (UPS). Cells with no signal, only one unpaired signal, or only one intact allele were not counted. The average EWSR1 alleles per cell were determined. The signal width (SW) was calculated for SS.
Results: See table below.

DiagnosisIAUPSSS1 SW>2 SW>3 SW>4 SW
STS/AR (n =7)49±1824±1421±512±39±44±30.7±0.6
EWSR1+ (n=5)3.0±2.730±1067±129±458±1447±1029±12
BRCa (n=3)49±1023±829±1212±129±910±73±2
Tonsil (n=2)55±430±118±38±111±37±42±1
All values provided % of cells reported as mean +/- std.

Conclusions: A range of SS can be seen in all non-EWSR1 related samples. Given its similar frequency in all groups analyzed, 1 SW SS is likely a nonspecific finding. In non-EWSR1 related samples when using 2, 3 or 4 SW as cut off criteria for SS, abnormal signals could be found in up to 40%, 15% or 5% of the total countable cells respectively. However, in this group 50% of countable cells contained IA as compared to 3% in EWSR1+ tumors. Aneuploid EWSR1 signal was common in SS and AR, but not in EWSR1+ tumors, the significance of which is unknown. UPS are likely due to artifact owing to nuclear truncation as it remains constant across all groups. The observed SS in non-EWSR1 related tissue is likely an artifact related to assay procedure and tissue processing, and less likely due to tumor specific nuclear changes, as they also occur in non-tumor tissue. In addition to focusing on the width of SS and % of cells with SS, evaluating tumor cells with IA might help to recognize a false SS result.
Category: Quality Assurance

Monday, March 4, 2013 9:30 AM

Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 301, Monday Morning


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