Rearrangement analysis in archival thyroid tissues: punching microdissection and artificial RET/PTC 1-12 transcripts.
BACKGROUND
In few papillary thyroid carcinomas (PTC) and oxyphilic thyroid carcinoma, the clinical impact of the 15 known RET hybrid oncogene variants (RET/PTC 1 to 12, 1L, 3r2, 3r3) is subject to controversial discussions. Large patient cohorts and exploitation of pathological thyroid tissue archives are essential to study the prognostic significance of RET/PTC chimeras.


METHODS
Formalin-fixed and paraffin-embedded thyroid neoplasms were subjected to manual punching macrodissection and subsequent extraction of total RNA. Following reverse transcriptase polymerase chain reaction (RT-PCR)-based screening for RET rearrangements, hybrid-specific expression analyses were carried out for samples indicative of chimeric transcripts. Due to lack of tissue specimen harboring the rare RET chimeras, artificially constructed hybrid sequences of all known RET/PTC variants served as PCR controls.


RESULTS
Manual punching dissection successfully diminished RET wild-type contamination originating from C-cells dispersed throughout normal thyroid tissues. The average amount of 27.4 mug RNA extracted allowed for repeated molecular analyses (>60 PCRs). Hybrid-specific expression analysis identified 10 of 15 RET rearrangements (8x RET/PTC 1, 2x RET/PTC 3, 5x RET/PTC x) to be found in 54 oxyphilic thyroid tumors examined. Successful amplification of each artificial hybrid sequence ensured the absence of rare chimeric transcripts. Therefore, RET/PTC x represent either common chimeras not amplifiable due to archival RNA degradation or truly novel hybrid oncoproducts.


CONCLUSIONS
The fast and simple techniques described here were used to examine oxyphilic carcinomas and adenomas. These microdissection and RT-PCR procedures can easily be put into practice in any molecular biology research laboratory to enable screening of large numbers of archival thyroid tumors for known as well as yet unknown RET rearrangements.
Imkamp
Florian
F
Department of Urology, Hannover University Medical School, Hannover, Germany.
von Wasielewski
Reinhard
R
Musholt
Thomas J
TJ
Musholt
Petra B
PB
D018249:Adenoma, Oxyphilic / Q000235:genetics / Q000473:pathology; D000293:Adolescent; D000328:Adult; D000368:Aged; D001483:Base Sequence; D018070:Biological Specimen Banks; D002291:Carcinoma, Papillary / Q000235:genetics* / Q000473:pathology; D002454:Cell Differentiation; D047852:Chernobyl Nuclear Accident; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D015321:Gene Rearrangement; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D008297:Male; D042282:Microdissection / Q000379:methods*; D008875:Middle Aged; D008969:Molecular Sequence Data; D009381:Neoplasms, Radiation-Induced / Q000235:genetics / Q000473:pathology; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D013964:Thyroid Neoplasms / Q000235:genetics* / Q000473:pathology; D014158:Transcription, Genetic
D012333:RNA, Messenger; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human