快速可靠的 Sanger POLE 测序协议在子宫内膜癌的 FFPE 组织中。

Fast and reliable Sanger POLE sequencing protocol in FFPE tissues of endometrial cancer.

机构信息

Lower Silesian Oncology, Pulmonology and Hematology Center, Hirszfelda 12, Wroclaw 53-413, Poland; Department of Genetics, Wroclaw Medical University, Marcinkowskiego 1, Wroclaw 50-368, Poland.

Lower Silesian Oncology, Pulmonology and Hematology Center, Hirszfelda 12, Wroclaw 53-413, Poland.

出版信息

Pathol Res Pract. 2023 Feb;242:154315. doi: 10.1016/j.prp.2023.154315. Epub 2023 Jan 18.

DOI:10.1016/j.prp.2023.154315

PMID:36738508

Abstract

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

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

The protocol has been optimized for formalin-fixed, paraffin-embedded (FFPE) EC tissues.

CONCLUSION

The method developed in our laboratory allows better diagnosis of patients with EC according to current standards.

摘要

背景

新的子宫内膜癌（EC）分类要求对体细胞聚合酶 epsilon（POLE）外切酶结构域突变进行分子解读。POLE 基因中致病性突变的鉴定定义了具有特定预后和预测效用的超突变肿瘤的重要亚型（“POLE-超突变”）。POLE 体细胞突变存在于 7-12%的 EC 中，通常是外观具有侵袭性的高级别肿瘤。POLE 基因的分子分析可使用 qPCR 测试、Sanger 测序法、下一代测序（NGS）面板测试和原位杂交（IHC）检测进行。我们描述了当前使用 Sanger 测序技术鉴定 POLE 突变的方法，该技术仍然是最稳健、准确和快速的 DNA 测序技术。