Global and gene specific DNA methylation in breast cancer cells was not affected during epithelial-to-mesenchymal transition in vitro.

Epithelial-to-mesenchymal transition (EMT) significantly affects the risk of metastasising in breast cancer. Plasticity and reversibility of EMT suggest that epigenetic mechanisms could be the key drivers of these processes, but little is known about the dynamics of EMT-related epigenetic alterations. We hypothesised that EMT, mediated by autocrine and paracrine signals, will be accompanied by changes in DNA methylation profiles. Therefore, conditioned medium from adipose tissue-derived mesenchymal stromal cells was used for induction of EMT in human breast cancer SK-BR-3 cell line. EMT-related morphological alterations and changes in gene expression of EMT-associated markers were assessed. To reverse EMT, 20 nm size gold nanoparticles (AuNPs) synthesized by the citrate reduction method were applied. Finally, DNA methylation of LINE-1 sequences and promoter methylation of TIMP3, ADAM23 and BRMS1 genes were quantitatively evaluated by pyrosequencing. Despite the presence of EMT-associated morphological and gene expression changes in tumour cells, EMT induced by adipose tissue-derived mesenchymal stromal cells had almost no effect on LINE-1 and gene-specific DNA methylation patterns of TIMP3, ADAM23 and BRMS1 genes. Although treatment for 24, 48 or 72 hours with 20 nm AuNPs at a concentration of 3 µg/ml slightly decreased gene expression of EMT-associated markers in SK-BR-3 cells, it did not alter global or gene-specific DNA methylation. Our results suggest that changes in DNA methylation are not detectable in vitro in early phases of EMT. Previously published positive findings could represent rather the sustained presence of potent EMT-inducing signals or the synergistic effect of various epigenetic mechanisms. Treatment with AuNPs slightly attenuated EMT, and their therapeutic potential needs to be further investigated.

Evaluation of protein expression and DNA methylation profiles detected by pyrosequencing in invasive breast cancer.

Breast carcinoma is the most common cancer with high mortality caused by metastatic disease. New molecular biomarkers predicting the tumour's metastatic potential would therefore improve metastasis prevention and personalised care. The aim of the study was to investigate the relationship between DNA methylation levels in invasivity and metastasising associated genes with aberrant protein expression and also to evaluate whether a similar DNA methylation level is present in the tumour and circulating cell-free DNA for utilising plasma DNA methylation as prognostic biomarker. By using pyrosequencing, we analysed DNA methylation levels of 11 genes, namely APC, ADAM23, CXCL12, ESR1, PGR B, CDH1, RASSF1A, SYK, TIMP3, BRMS1 and SOCS1 in tumour, plasma and peripheral blood cells from 34 patients with primary breast cancer, as well as plasma and peripheral blood cells from 50 healthy controls. Simultaneously, the expression of related proteins in paraffin-embedded tumour samples was evaluated by immunohistochemistry. Statistical analysis was performed by SPSS statistics 15.0 software. Tumour DNA hypermethylation was found in most commonly methylated RASSF1A (71.9%), APC (55.9%), ADAM23 (38%) and CXCL12 (34.4%) genes with methylation levels up to 86, 86, 53 and 64 %, respectively. In tumours, significantly higher methylation levels were found in nine genes, compared with the patients´ peripheral blood cell DNA. Furthermore, in patients methylation levels in peripheral blood cell DNA were significantly higher than in controls in CXCL12, ESR1 and TIMP3 genes, but the values did not exceed 15%. On the other hand, no correlations were observed in patients between DNA methylation in tumours and cell-free plasma DNA. Moreover, in patients and controls nearly identical values of cumulative DNA methylation (43.6 % ± 20.1 vs. 43.7 % ± 15.0) were observed in plasma samples. A variable spectrum from high to none expressions presented in tumour tissues in all of the proteins evaluated, however in APC and CXCL12 genes a visible decreasing trend of mean DNA methylation level with increasing expression of the corresponding protein was observed. The DNA methylation profiles manifested in our group of breast carcinomas are cancer specific, but they are not the only cause that affects the silencing of evaluated genes and the decrease of relevant protein products. The clinical utility of DNA methylation testing in peripheral blood cell DNA for cancer diagnosis and therapy need to be further investigated.

Genetic analysis of KRAS mutation status in metastatic colorectal cancer patients.

Colorectal carcinoma (CRC) represents a serious problem worldwide: in the Slovak republic are diagnosed about 2600 new CRC cases annually and its incidence is increasing. Colorectal cancer patients may succumb to the disease because of local recurrence or local formation of metastasis. Therefore, it is necessary to modulate therapeutic algorithm with new methods, leading to early diagnostic of CRC or changing the existing therapeutic procedures. Recent progresses have been made in understanding of EGFR pathway involved in CRC carcinogenesis, especially the role of Ras protein. Mutations in KRAS oncogene are frequently found in human cancers, particularly colorectal, pancreatic, billiary tract and lung tumors. The presence of the KRAS mutations in metastatic colorectal cancer patients correlates with lack of response to the certain epidemal growth factor receptor (EGFR) inhibitor therapies, such as Panitumumab and Cetuximab. Consequently, screening for KRAS mutations status may be used as a prognostic marker, because the CRC patients with KRAS positive tumors have a worse prognosis. The aim of our study was to establish the methods for rapid and sensitive detection of KRAS mutation status in formalin fixed paraffin embedded (FFPE) tissues DNA. We applied Real Time PCR analysis (TheraScreen KRAS Mutation Test Kit) and sequencing analysis (optimised for the analysis of FFPE tissues) to detect somatic mutations in codon 12 and 13 of KRAS gene. Both methods were used concurrently in the panel of DNA isolated from 25 colorectal FFPE tissues tumor. The positive or negative results from all 25 samples were identified by both methods independently. The KRAS mutations were presented in 8 of 25 patients (32%). Our results demonstrate that the Real Time PCR analysis can be used for detection of somatic KRAS mutations in FFPE clinical samples. However, we also recognize that the sequencing analysis of approximately 200bp amplicons may be used for mutations status screening, but with care of method sensitivity.

Frequency of the three most common polymorphisms in the MDR1 gene in Slovak population.

UNLABELLED: The human multidrug resistance gene (MDR1) is encoding the transmembrane transporter P-glycoprotein (P-gp) which plays an important role in the efflux of various drugs and thus is potentially influencing the drug-treatment outcome. It has been indicated that the level of P-gp activity may be affected by the presence of single nucleotide polymorphisms (SNP) in the gene which led to the studies estimating MDR1-SNP frequencies in various populations. Here, we have investigated the occurrence of seven SNP in the MDR1 gene for the first time in Slovak population using multiplex SNaPshot genotyping method. The allelic frequencies of the most common gene variants, i.e. 1236C>T, 2677G>T, 2677G>A and 3435C>T were estimated to be 42.5%, 43.5%, 2%, and 44.5%, respectively. We found that the most prevalent haplotype in Slovak population is 1236C-2677G-3435C occurring in 42.2% of individuals. Our preliminary data show that it is reasonable and feasible to utilize MDR1 genotypes and haplotypes in Slovak patients, e.g. those with acute myeloid leukemia, in order to adjust the individual effective drug dosage and predict the patient's response to the treatment as well as the treatment outcome. KEYWORDS: MDR1 gene, P-glycoprotein, polymorphisms, MDR1 haplotypes, Slovak population.

Novel approaches in evaluation of pathogenicity of single-base exonic germline changes involving the mismatch repair genes MLH1 and MSH2 in diagnostics of Lynch syndrome.

Germline defects in the DNA mismatch repair genes MLH1 and MSH2 are the major cause of hereditary nonpolyposis colon cancer (HNPCC), also called Lynch syndrome. Detection of inherited pathogenic change in their DNA sequence in HNPCC families allows for identification of asymptomatic individuals who require appropriate medical surveillance. However, evaluation of clinical significance of identified DNA alteration is not always straight-forward and some changes maybe classified incorrectly depending on the method used. The aim of this review is to summarize rationale, practice and pitfalls in the characterization of substitutions localized in the exons and outline new experimental and in silico approaches used to determine mutation consequence. Our survey of variants identified in MLH1 and MSH2 genes which were confirmed to cause splicing defect but often appear characterized as missense, nonsense or silent mutations in various databases and publications as well as a list of true missense mutations may serve as a valuable aid for laboratories providing HNPCC diagnosis.

The role of clinical criteria, genetic and epigenetic alterations in Lynch-syndrome diagnosis.

Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC) represents 1-3% of all diagnosed colorectal cancers (CRCs). This study aimed to evaluate the benefit of clinical criteria and several molecular assays for diagnosis of this syndrome. We examined tumors of 104 unrelated clinically characterized colorectal cancer patients for causal mismatch repair (MMR) deficiency by several methods: microsatellite instability (MSI) and loss of heterozygosity (LOH) presence, MMR protein absence, hypermethylation of MLH1 promoter and germline mutation presence. Twenty-five (24%) patients developed CRCs with a high level of MSI (MSI-H). Almost all (96%) had at least one affected relative, while this simple criterion was satisfied in only 22% (17/79) of individuals with low level MSI or stable cancers (MSI-L, MSS). Using strict Amsterdam criteria, the relative proportion of complying individuals in both sets of patients (MSI-H vs. MSI-L and MSS) decreased to 68% and 9%, respectively. The right-sided tumors were located in 54% of MSI-H persons when compared to 14% of cancers found in MSI-L or MSS patients. In 16 MSI positive patients with identified germline mutation by DNA sequencing, the gene localization of mutation could be indicated beforehand by LOH and/or immunohistochemistry (IHC) in four (25%) and 14 cases (88%), respectively. The IHC findings in MSI-H cancers with methylation in distal or both regions of MLH1 promoter have not confirmed the epigenetic silencing of the MLH1 gene. None of the patients with MSIL or MSS tumors was a carrier of the MLH1 del616 mutation, despite seven of them meeting Amsterdam criteria. The effective screening algorithm of Lynch-syndrome-suspected patients consists of evaluation of Bethesda or Revised Bethesda Guidelines fulfilling simultaneous MSI, LOH and IHC analyses before DNA sequencing. Variable methylation background in MLH1 promoter does not affect gene silencing and its role in Lynch-syndrome tumorigenesis is insignificant.

STK11/LKB1 germline mutations in the first Peutz-Jeghers syndrome patients identified in Slovakia.

Peutz-Jeghers syndrome (PJS) is characterized by number of hamartomatous polyps in the gastrointestinal tract and by mucocutaneous hypermelanocytic lesions at different sites. Older patients have an increased risk of the cancers of small intestine, stomach, pancreas, colon, esophagus, ovary, testis, uterus, breast and lung. In majority of PJS cases, the germline mutations in serine/threonine kinase STK11/LKB1 gene were found to be associated with disease. Here we report the results of a first mutational screen of STK11/LKB1 in PJS patients characterized in Slovak population. The first patient with unusual carcinoma of duodenum was a sporadic case and carried c.842delC change residing in a mutational C6 repeat hotspot. Neither the polyp nor the tumor of the patient displayed the loss of heterozygosity at the site of mutation suggesting different mechanism involved in the formation of polyp and tumor in this case. The second patient belonged to a three-generation family with typical PJS features but not cancers. Interestingly, the patient displayed concomitant occurrence of adenomatous and hamartomatous polyps. Molecular analysis revealed an IVS2+1A>G mutation that alters the second intron 5' splice site and was shown to lead to aberrant splicing mediated by the U12-dependent spliceosome. The same mutation was present in the 9 affected members of the family but in none of their normal relatives. We also observed novel c. IVS2+61G>A unclassified variant, and recurrent IVS2+24G>T and 3UTR+129C>T polymorphisms. Based on the achieved results, we could offer predictive genetic testing and counseling to other members of the patient's families.

Novel and recurrent germline alterations in the MLH1 and MSH2 genes identified in hereditary nonpolyposis colorectal cancer patients in Slovakia.

Hereditary non-polyposis colorectal cancer (HNPCC) is associated with germline mutations in DNA mismatch repair genes, predominantly MSH2 and MLH1. Mutation carriers develop cancers in the colorectum, endometrium, ovary, stomach, small intestine and the upper urinary tract. We describe here the results of a mutational analysis of 11 unrelated HNPCC patients by direct genomic sequencing of MLH1 and MSH2. The alterations found include 7 novel changes and 4 different pathogenic mutations described previously in Poland, Moldavia, Finland, Germany, France and USA. Four novel pathogenic mutations in the MLH1 gene include two frameshift mutations (c.1150delG and c.1210_1211delCT), one missense mutation (c.793C>A) and one intron-exon border mutation (c.546- 2A>C). The last change resulted in the skipping of exon 7, as shown by sequencing of RT-PCR products. The only novel MSH2 pathogenic change was a nonsense mutation c.1129C>T. The novel intronic change c.381-41A>G in MLH1 was found in a patient carrying a previously-described mutation in the MSH2 gene. Interestingly, two unrelated patients carried also a novel change in the promoter region of MLH1 in one of the CpG islands (c.-269C>G). However, this alteration does not abrogate transcription, as shown by RT-PCR analysis. In summary, most (approximately 80%) pathogenic germline mutations detected in the studied group of patients by direct genomic sequencing of MLH1 and MSH2 were located in the MLH1 gene. These and previous data indicate that the majority of germline point mutations and small deletions/insertions in HNPCC families in Slovakia affect the MLH1 locus.