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.

Aberrant Expression of PIWIL1 and PIWIL2 and Their Clinical Significance in Ductal Breast Carcinoma.

BACKGROUND/AIM: P-Element-induced wimpy testis (PIWI) proteins in complex with PIWI-interacting RNA (piRNA) are involved in epigenetic regulation of gene expression in germline cells. Aberrant expression of piRNA and PIWI proteins have been identified in various types of tumour cells. The aim of this study was to evaluate the expression profiles of PIWI-like protein-1, -2 (PIWIL1 and PIWIL2), their immunohistochemical (IHC) characteristics in ductal breast cancer, and determine their correlation with clinicopathological parameters of this type of cancer. MATERIALS AND METHODS: Material for IHC studies comprised of 101 invasive ductal carcinoma (IDC) cases and 31 mastopathy tissues. Frozen fragments of paired tissue specimens (tumour and adjacent non-malignant breast tissue) taken from 55 patients with IDC and 18 samples of mastopathy were used for molecular studies using real-time polymerase chain reaction (RT-PCR). RESULTS: A statistically significantly higher level of PIWIL1 and PIWIL2 was found in IDC compared to mastopathy samples (p≤0.0001). Increased expression of PIWIL1 was correlated with increased PIWIL2 expression in breast cancer tissue. Surprisingly, PIWIL1 mRNA was detected only in cancer and mastopathy, but was not found in most normal breast tissues, although it is noteworthy that the PIWIL2 mRNA level was statistically significantly lower in mastopathy and IDC samples compared to normal breast tissues. CONCLUSION: Our results affirm the hypothesis that reactivation of PIWI expression in various caner types is crucial for cancer development.

Presence of Periodontopathic Bacteria DNA in Atheromatous Plaques from Coronary and Carotid Arteries.

OBJECTIVES: Interest in periodontitis as a potential risk factor for atherosclerosis and its complications resulted from the fact that the global prevalence of periodontal diseases is significant and periodontitis may induce a chronic inflammatory response. Many studies have analyzed the potential impact of the Porphyromonas gingivalis, major pathogen of periodontitis, on general health. The purpose of this study was to find the presence of the Porphyromonas gingivalis DNA in the atherosclerotic plaques of coronary and carotid arteries and in the periodontal pockets in patients with chronic periodontitis, who underwent surgery because of vascular diseases. METHODS AND RESULTS: The study population consisted of 91 patients with coronary artery disease or scheduled for carotid endarterectomy. The presence of Porphyromonas gingivalis DNA in atheromatous plaques and in subgingival samples was determined by PCR. Bacterial DNA was found in 21 of 91 (23%) samples taken from vessels and in 47 of 63 (74.6%) samples from periodontal pockets. CONCLUSIONS: Porphyromonas gingivalis DNA is frequently found in atheromatous plaques of patients with periodontitis. That is why more research should be conducted to prove if this periopathogen may have an impact on endothelium of patients at risk of atherosclerosis.

[The role of microRNA in ischemic diseases--impact on the regulation of inflammatory, apoptosis and angiogenesis processes]. / Rola mikroRNA w chorobach niedokrwiennych--wplyw na regulacje procesów zapalnych, apoptozy i angiogenezy.

Ischemic diseases, including coronary artery disease and critical limb ischemia, are one of the leading causes of mortality worldwide. The result of tissue hypoxia is activation of processes such as inflammatory, angiogenesis and cell death by apoptosis, autophagy or necrosis. Recenty special attention has been paid to the investigation of the microRNA's role in these processes. MicroRNAs (miRNAs) belong to a group of noncoding small RNAs with a length of 20-24 ribonucleotides, which play an important role in post-transcriptional regulation of gene expression. Most of them specifically recognizes the 3'-untranslated regions (UTR) of their target mRNAs, thereby blocking the process of protein translation or causing mRNA degradation. The purpose of this study was to describe the role of miRNA in processes of apoptosis, angiogenesis and inflammation during tissue ischemia. Particular attention was paid to the regulation of these molecules in cardiac cells, vascular smooth muscle and heart, and endothelium. In summary diagnostic and therapeutic use of microRNAs in ischemic disease was discussed.