Fast and reliable Sanger POLE sequencing protocol in FFPE tissues of endometrial cancer.
Endometrial cancer
POLE
Sanger sequencing
Journal
Pathology, research and practice
ISSN: 1618-0631
Titre abrégé: Pathol Res Pract
Pays: Germany
ID NLM: 7806109
Informations de publication
Date de publication:
Feb 2023
Feb 2023
Historique:
received:
12
11
2022
revised:
14
01
2023
accepted:
16
01
2023
pubmed:
5
2
2023
medline:
15
2
2023
entrez:
4
2
2023
Statut:
ppublish
Résumé
The new classification of endometrial carcinoma (EC) requires molecular interpretation of somatic polymerase epsilon (POLE) exonuclease domain mutations. The identification of pathogenic mutations within the POLE gene defines the important subtype of ultramutated tumours ("POLE-ultramutated") with specified prognostic and predictive utility. POLE somatic mutations are present in 7-12% of ECs, usually high-grade tumours with aggressive appearance. Molecular analysis of the POLE gene can be performed using a qPCR test, the Sanger sequencing method, a next generation sequencing (NGS) panel test and also in situ hybridisation (IHC) assay. We describe our current approach of identification of POLE mutations using Sanger sequencing technology, which is still the most robust, accurate and fast technique to sequence DNA. We present a reliable protocol for Sanger sequencing of the entire sequence coding exonuclease domain of POLE - exons 9, 10, 11, 12, 13 and 14 (codons 268-491) with 5-10 nucleotides in exon/intron boundaries (reference sequences: NM_006231.4, NP_006222.2). The protocol has been optimized for formalin-fixed, paraffin-embedded (FFPE) EC tissues. The method developed in our laboratory allows better diagnosis of patients with EC according to current standards.
Sections du résumé
BACKGROUND
BACKGROUND
The new classification of endometrial carcinoma (EC) requires molecular interpretation of somatic polymerase epsilon (POLE) exonuclease domain mutations. The identification of pathogenic mutations within the POLE gene defines the important subtype of ultramutated tumours ("POLE-ultramutated") with specified prognostic and predictive utility. POLE somatic mutations are present in 7-12% of ECs, usually high-grade tumours with aggressive appearance. Molecular analysis of the POLE gene can be performed using a qPCR test, the Sanger sequencing method, a next generation sequencing (NGS) panel test and also in situ hybridisation (IHC) assay. We describe our current approach of identification of POLE mutations using Sanger sequencing technology, which is still the most robust, accurate and fast technique to sequence DNA.
MATERIALS AND METHODS
METHODS
We present a reliable protocol for Sanger sequencing of the entire sequence coding exonuclease domain of POLE - exons 9, 10, 11, 12, 13 and 14 (codons 268-491) with 5-10 nucleotides in exon/intron boundaries (reference sequences: NM_006231.4, NP_006222.2).
RESULT
RESULTS
The protocol has been optimized for formalin-fixed, paraffin-embedded (FFPE) EC tissues.
CONCLUSION
CONCLUSIONS
The method developed in our laboratory allows better diagnosis of patients with EC according to current standards.
Identifiants
pubmed: 36738508
pii: S0344-0338(23)00015-8
doi: 10.1016/j.prp.2023.154315
pii:
doi:
Substances chimiques
Poly-ADP-Ribose Binding Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
154315Informations de copyright
Copyright © 2023 Elsevier GmbH. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of Competing Interest None.