A Multiplex SNaPshot Assay is a Rapid and Cost-Effective Method for Detecting POLE Exonuclease Domain Mutations in Endometrial Carcinoma.
Journal
International journal of gynecological pathology : official journal of the International Society of Gynecological Pathologists
ISSN: 1538-7151
Titre abrégé: Int J Gynecol Pathol
Pays: United States
ID NLM: 8214845
Informations de publication
Date de publication:
01 Nov 2022
01 Nov 2022
Historique:
pubmed:
16
12
2021
medline:
1
11
2022
entrez:
15
12
2021
Statut:
ppublish
Résumé
Determining the replicative DNA polymerase epsilon ( POLE) mutation status in endometrial carcinomas (ECs) has important clinical implications given that the majority of "ultramutated" tumors harboring pathogenic exonuclease domain mutations in POLE ( POLE mut) have a favorable prognosis, even among high-grade histotypes. Currently, there are no specific morphologic or immunophenotypic features that allow accurate detection of POLE mut tumors without molecular testing. Consequently, identifying POLE mut tumors has been challenging without employing costly and/or time-consuming DNA sequencing approaches. Here we developed a novel SNaPshot assay to facilitate routine and efficient POLE mutation testing in EC. The SNaPshot assay interrogates 15 nucleotide sites within exons 9, 11, 13, and 14 encoding the POLE exonuclease domain. The variant sites were selected based on recurrence, evidence of functional impact, association with high tumor mutation burden and/or detection in EC clinical outcome studies. Based on the pathogenic somatic variants reported in the literature, the assay is predicted to have a clinical sensitivity of 90% to 95% for ECs. Validation studies showed 100% specificity and sensitivity for the variants covered, with expected genotypic results for both the positive (n=11) and negative (n=20) patient controls on multiple repeat tests and dilution series. Analytic sensitivity was conservatively approximated at a 10% variant allele fraction (VAF), with documented detection as low as 5% VAF. As expected, the SNaPshot assay demonstrated greater sensitivity than Sanger sequencing for VAFs below 20%, an important characteristic for somatic mutation detection. Here we have developed and validated the first SNaPshot assay to detect hotspot POLE mutations. While next-generation sequencing and Sanger sequencing-based approaches have also been used to detect POLE mutations, a SNaPshot approach provides useful balance of analytical sensitivity, cost-effectiveness, and efficiency in a high-volume case load setting.
Identifiants
pubmed: 34907997
doi: 10.1097/PGP.0000000000000841
pii: 00004347-202211000-00001
doi:
Substances chimiques
Exonucleases
EC 3.1.-
Poly-ADP-Ribose Binding Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
541-551Informations de copyright
Copyright © 2021 by the International Society of Gynecological Pathologists.
Déclaration de conflit d'intérêts
The authors declare no conflict of interest.
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