A Multiplex Quantitative Reverse Transcription Polymerase Chain Reaction Assay for the Detection of KIAA1549-BRAF Fusion Transcripts in Formalin-Fixed Paraffin-Embedded Pilocytic Astrocytomas.
Journal
Molecular diagnosis & therapy
ISSN: 1179-2000
Titre abrégé: Mol Diagn Ther
Pays: New Zealand
ID NLM: 101264260
Informations de publication
Date de publication:
08 2019
08 2019
Historique:
pubmed:
16
5
2019
medline:
6
5
2020
entrez:
16
5
2019
Statut:
ppublish
Résumé
Genomic duplications and fusion involving BRAF and KIAA1549 that create fusion proteins with constitutive B-RAF kinase activity are a hallmark of pilocytic astrocytomas (PAs). The detection of KIAA1549-BRAF fusion transcripts is of paramount importance to classify these tumors and to identify patients who could benefit from BRAF inhibitors. In a clinical setting, the available material for molecular analysis from these pediatric tumors is often limited to formalin-fixed paraffin-embedded (FFPE) tissue. The aim of the present study was to develop a new method to detect the three most frequent KIAA1549-BRAF fusion transcripts, 15-9, 16-11, and 16-9, where numbers refer to the exons fused together, using a FFPE-compatible multiplex quantitative reverse transcription polymerase chain reaction (qRT-PCR). We compared performance of the assay to a reference singleplex method on a collection of 46 FFPE PAs. The results showed that both methods are comparable. The multiplex method had an overall 97% sensitivity and 100% specificity compared to the singleplex method, and agreement between the two techniques was almost perfect (Cohen's kappa: 0.97). There was no evidence of a significant difference between the qRT-PCR efficiencies of the multiplex technique and of the singleplex assay for all fusion transcripts and for GAPDH, the latter used as a reference gene. The multiplex method consumed four times less complementary DNA (cDNA), cost less, and required half the hands-on technical time. The results show that it could be beneficial to implement the multiplex method in a clinical setting, where samples presenting low quantity of degraded RNA are not unusual.
Sections du résumé
BACKGROUND AND OBJECTIVE
Genomic duplications and fusion involving BRAF and KIAA1549 that create fusion proteins with constitutive B-RAF kinase activity are a hallmark of pilocytic astrocytomas (PAs). The detection of KIAA1549-BRAF fusion transcripts is of paramount importance to classify these tumors and to identify patients who could benefit from BRAF inhibitors. In a clinical setting, the available material for molecular analysis from these pediatric tumors is often limited to formalin-fixed paraffin-embedded (FFPE) tissue. The aim of the present study was to develop a new method to detect the three most frequent KIAA1549-BRAF fusion transcripts, 15-9, 16-11, and 16-9, where numbers refer to the exons fused together, using a FFPE-compatible multiplex quantitative reverse transcription polymerase chain reaction (qRT-PCR).
METHODS
We compared performance of the assay to a reference singleplex method on a collection of 46 FFPE PAs.
RESULTS
The results showed that both methods are comparable. The multiplex method had an overall 97% sensitivity and 100% specificity compared to the singleplex method, and agreement between the two techniques was almost perfect (Cohen's kappa: 0.97). There was no evidence of a significant difference between the qRT-PCR efficiencies of the multiplex technique and of the singleplex assay for all fusion transcripts and for GAPDH, the latter used as a reference gene. The multiplex method consumed four times less complementary DNA (cDNA), cost less, and required half the hands-on technical time.
CONCLUSION
The results show that it could be beneficial to implement the multiplex method in a clinical setting, where samples presenting low quantity of degraded RNA are not unusual.
Identifiants
pubmed: 31087282
doi: 10.1007/s40291-019-00403-3
pii: 10.1007/s40291-019-00403-3
doi:
Substances chimiques
BRAF-KIAA1549 fusion protein, human
0
Oncogene Proteins, Fusion
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
537-545Références
Biotechniques. 2005 Feb;38(2):287-93
pubmed: 15727135
Biomol Detect Quantif. 2015 Mar 11;3:9-16
pubmed: 27077029
Mod Pathol. 2018 Oct;31(10):1490-1501
pubmed: 29802359
Nat Genet. 2013 Aug;45(8):927-32
pubmed: 23817572
J Neurooncol. 2013 Dec;115(3):477-86
pubmed: 24057326
Case Rep Med. 2012;2012:418672
pubmed: 22548077
Acta Neuropathol. 2015 Jun;129(6):775-88
pubmed: 25792358
Int J Cancer. 2016 Feb 15;138(4):881-90
pubmed: 26314551
BMC Bioinformatics. 2012 Jun 18;13:134
pubmed: 22708584
Neuro Oncol. 2012 Sep;14 Suppl 4:iv90-9
pubmed: 23095836
J Neuropathol Exp Neurol. 2012 Jul;71(7):631-9
pubmed: 22710963
Nucleic Acids Res. 2003 Oct 15;31(20):e122
pubmed: 14530455
J Mol Diagn. 2018 Nov;20(6):765-776
pubmed: 30138724
Clin Transl Med. 2015 Mar 03;4:10
pubmed: 25883769
Cancer Res. 2008 Nov 1;68(21):8673-7
pubmed: 18974108
J Pathol Transl Med. 2017 May;51(3):205-223
pubmed: 28535583
Ann Pathol. 2014 Feb;34(1):74-86
pubmed: 24630640
J Neuropathol Exp Neurol. 2015 Jul;74(7):743-54
pubmed: 26083571
J Neuropathol Exp Neurol. 2013 Jan;72(1):2-7
pubmed: 23242278
J Mol Diagn. 2011 Nov;13(6):669-77
pubmed: 21884820
Methods. 2001 Dec;25(4):402-8
pubmed: 11846609
J Neuropathol Exp Neurol. 2017 Jul 1;76(7):562-570
pubmed: 28863456
J Neuropathol Exp Neurol. 2012 Jan;71(1):66-72
pubmed: 22157620
Adv Anat Pathol. 2015 Mar;22(2):94-101
pubmed: 25664944
Neuro Oncol. 2015 Oct;17 Suppl 4:iv1-iv62
pubmed: 26511214
Acta Neuropathol. 2016 Jun;131(6):803-20
pubmed: 27157931
Biometrics. 1977 Mar;33(1):159-74
pubmed: 843571
Neuro Oncol. 2017 Aug 1;19(8):1127-1134
pubmed: 28201752
J Mol Biol. 1990 Oct 5;215(3):403-10
pubmed: 2231712