title: Detection of PAX8/PPARG and RET/PTC rearrangements is feasible in routine air-dried fine needle aspiration smears.
abstract: BACKGROUND
  The diagnostic limitations of fine needle aspiration (FNA), like the indeterminate category, can be partially overcome by molecular analysis. As PAX8/PPARG and RET/PTC rearrangements have been detected in follicular thyroid carcinomas (FTCs) and papillary thyroid carcinomas (PTCs), their detection in FNA smears could improve the FNA diagnosis. To date, these rearrangements have never been analyzed in routine air-dried FNA smears, but only in frozen tissue, formalin-fixed paraffin-embedded (FFPE) tissue, and in fresh FNA material. Fixed routine air-dried FNA samples have hitherto been judged as generally not suitable for testing these rearrangements in a clinical setting. Therefore, the objective of the present study was to investigate the feasibility of extracting RNA from routine air-dried FNA smears for the detection of these rearrangements with real-time polymerase chain reaction (RT-PCR).
  
  
  METHODS
  A new method for RNA extraction from routine air-dried FNA smears was established, which allowed analysis for the presence of four variants of PAX8/PPARG and RET/PTC 1 and RET/PTC 3, which were analyzed in 106 routine FNA smears and the corresponding surgically obtained FFPE tissues using real-time quantitative PCR (RT-qPCR). To assess RNA quality, an intron-spanning PAX8 cDNA was amplified.
  
  
  RESULTS
  Acceptable RNA quality was obtained from 95% of the FNA samples and 92% of the FFPE samples. PAX8/PPARG was detected in 4 of 96 FFPEs and in 6 of 96 FNAs. PAX8/PPARG was present in 4 of 10 FTCs and in 3 of 42 follicular adenomas (FAs). Similarly, RET/PTC was found in 3 of 96 FFPEs and in 4 of 96 FNAs. Two of 21 PTC samples and 3 of 42 FA samples carried this rearrangement.
  
  
  CONCLUSIONS
  These data are the first to show the feasibility of extracting RNA from routine air-dried FNA smears for the detection of PAX8/PPARG and RET/PTC rearrangements with RT-qPCR. These promising methodological advances, if confirmed in larger series of FNA and FFPE samples, may lead to the introduction of molecular analysis of routine air-dried FNA smears in everyday practice.
authors.affiliation: Division of Endocrinology and Nephrology, University of Leipzig, Leipzig, Germany.
authors.forename: Carolina
authors.initials: C
authors.lastname: Ferraz
authors.forename: Christian
authors.initials: C
authors.lastname: Rehfeld
authors.forename: Annelise
authors.initials: A
authors.lastname: Krogdahl
authors.forename: Eva Magrethe
authors.initials: EM
authors.lastname: Precht Jensen
authors.forename: Eileen
authors.initials: E
authors.lastname: Bösenberg
authors.forename: Frank
authors.initials: F
authors.lastname: Narz
authors.forename: Laszlo
authors.initials: L
authors.lastname: Hegedüs
authors.forename: Ralf
authors.initials: R
authors.lastname: Paschke
authors.forename: Markus
authors.initials: M
authors.lastname: Eszlinger
mesh_terms: D018263:Adenocarcinoma, Follicular / Q000235:genetics* / Q000473:pathology; D044963:Biopsy, Fine-Needle / Q000379:methods; D002277:Carcinoma / Q000235:genetics* / Q000473:pathology; D002291:Carcinoma, Papillary; D015321:Gene Rearrangement; D006801:Humans; D015514:Oncogene Proteins, Fusion / Q000032:analysis*; D000072636:PAX8 Transcription Factor; D047495:PPAR gamma / Q000032:analysis / Q000235:genetics*; D051761:Paired Box Transcription Factors / Q000235:genetics*; D011505:Protein-Tyrosine Kinases / Q000032:analysis*; D051096:Proto-Oncogene Proteins c-ret / Q000032:analysis / Q000235:genetics*; D012313:RNA / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction; D012189:Retrospective Studies; D000077273:Thyroid Cancer, Papillary; D013964:Thyroid Neoplasms / Q000235:genetics* / Q000473:pathology
chemical_list: D015514:Oncogene Proteins, Fusion; D000072636:PAX8 Transcription Factor; C083325:PAX8 protein, human; C551995:PAX8-PPARgamma fusion protein, human; D047495:PPAR gamma; D051761:Paired Box Transcription Factors; D012313:RNA; D011505:Protein-Tyrosine Kinases; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; C093573:ret-PTC fusion oncoproteins, human