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

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.

Detection of BRCA1/2 pathogenic variants in patients with breast and/or ovarian cancer and their families. Analysis of 3,458 cases from Lower Silesia (Poland) according to the diagnostic algorithm of the National Cancer Control Programme.

Breast and ovarian cancers are among the most common malignancies in the female population, with approximately 5-10% of cases being hereditary. BRCA1 and BRCA2 with other homologous recombination genes are the most tested genes in hereditary breast and ovarian cancer (HBOC) patients. As next-generation sequencing (NGS) has become a standard and popular technique, such as for HBOC, it has greatly simplified and accelerated molecular diagnosis of cancer. The study group included 3,458 HBOC patients or their relatives from Lower Silesia (Poland) (a voivodeship located in south-west Poland inhabited by 2.9 million people). All patients were tested according to the recommendations from the National Cancer Control Programme of the Ministry of Health for the years 2018-21. We tested 3,400 patients for recurrent pathogenic variants for the Polish population: five BRCA1 founder variants (c.5266dup, c.181T>G, c.4035del, c.3700_3704del, and c.68_69del), two PALB2 variants (c.509_510del, c.172_175del) and three CHEK2 variants [c.1100del, c.444+1G>A, g.27417113-27422508del (del5395)]. Next 260 patients from the study group were chosen for the BRCA1/2 NGS panel, and additionally selected marker pathogenic variants were tested using Sanger sequencing and MLPA methods in 45 and 13 individuals, respectively. The analysis of BRCA1/2 in the 3,458 patients with HBOC or their relatives revealed 144 carriers of 37 different pathogenic variants (22 in BRCA1 and 15 in BRCA2). Among all detected variants, 71.53% constituted founder pathogenic BRCA1 variants. Our study has revealed that for the Lower Silesian population, the first-line BRCA1/2 molecular test may be limited to only three variants in BRCA1-c.5266dup, c.181T>G, and c.4035del-but the aim should be to provide a full screening test of HBOC critical genes. The key and still growing role of molecular diagnostics of neoplasms, which includes HBOC, is undeniable. Therefore, it is necessary to provide complete and optimal therapeutic and prophylactic algorithms in line with current medical knowledge.