Ewing's sarcoma of bone: the detection of specific transcripts in a large, consecutive series of formalin-fixed, decalcified, paraffin-embedded tissue samples using the reverse transcriptase-polymerase chain reaction.
OBJECTIVE
(i) To report on the routine use of the reverse transcriptase-polymerase chain reaction (RT-PCR) technique on decalcified or non-decalcified, formalin-fixed, paraffin-embedded tissue (FFPET) for translocation detection, with particular emphasis on improved RNA extraction methodology and the use of PCR primers designed to generate small amplicons. (ii) To report on the relative incidences of translocation types and transcript variants in a large, single institution series of Ewing's sarcoma of bone.


RESULTS
Using RT-PCR to detect specific transcript variants, we analysed FFPET from 54 consecutive cases of Ewing's sarcoma of bone. We used 'gold standard' detection methods on corresponding fresh and fresh frozen tissue to validate the technique. We have demonstrated the effective use of RT-PCR on decalcified and non-decalcified FFPET samples for sarcoma-specific translocation detection (96% sensitivity, 100% specificity). Tissue decalcification did not affect the detection rate. The relative incidence of Ewing's sarcoma-specific translocation types and transcript variants was entirely consistent with previously published data.


CONCLUSIONS
With equal effectiveness, RT-PCR can be applied to both acid decalcified and non-decalcified FFPET for (Ewing's sarcoma) translocation detection and the technique can be introduced into routine practice in histopathology departments.
Department of Musculoskeletal Pathology, Royal Orthopaedic Hospital, The Medical School, University of Birmingham, Birmingham, UK. chas.mangham@rjah.nhs.uk
D C
DC
Mangham
A
A
Williams
D J
DJ
McMullan
J
J
McClure
V P
VP
Sumathi
R J
RJ
Grimer
A M
AM
Davies
D000293:Adolescent; D000328:Adult; D001859:Bone Neoplasms / Q000235:genetics / Q000473:pathology*; D002648:Child; D003648:Decalcification Technique; D003937:Diagnosis, Differential; D005260:Female; D005557:Formaldehyde; D006801:Humans; D008297:Male; D008875:Middle Aged; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D051789:Proto-Oncogene Protein c-fli-1 / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D034802:RNA-Binding Protein EWS; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012512:Sarcoma, Ewing / Q000235:genetics / Q000473:pathology*; D016610:Tissue Embedding / Q000379:methods; D016707:Tissue Fixation / Q000379:methods; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic
C450460:EWS-ERG fusion protein, human; C106031:EWS-FLI fusion protein; D015514:Oncogene Proteins, Fusion; D051789:Proto-Oncogene Protein c-fli-1; D012334:RNA, Neoplasm; D034802:RNA-Binding Protein EWS; D014157:Transcription Factors; D005557:Formaldehyde