Tomas Krivulcik

RASSF1A and CDH1 hypermethylation as potential epimarkers in breast cancer

Breast cancer is the most common cancer in women worldwide, representing 28.2% of all female malignancies. In addition to genetic changes, epigenetic events, as aberrant DNA methylation and histone modification, are responsible for cancer development. Many tumour suppressor genes are inactivated by DNA hypermethylation, which could be utilized for identification of new epigenetic biomarkers. To investigate the relation between DNA methylation level and breast cancer progression, we analysed DNA methylation in RASSF1A and CDH1 promoters using quantitative multiplex methylation-specific PCR in paraffin-embedded tumour tissues and blood samples from 92 breast cancer patients and 50 controls, respectively. The associations between RASSF1A and CDH1 methylation levels and clinico-pathological parameters were tested by Kruskal-Wallis and van der Waerden ANOVA tests. Out of 92 breast cancer patients, 76 (82.6%) manifested various levels of RASSF1A (range from 1.20 to 92.63%) and 20 (21.7%) of CDH1 (range from 1.20 to 79.62%) methylation. However, no methylation was found in 50 controls. Increasing trends in RASSF1A methylation were observed in tumour size, lymph node status and TNM stage, but only CDH1 methylation levels showed statistically significant differences between the patient subgroups in lymph node status and IHC subtype. Overall, stable relatively high RASSF1A methylation could be utilised as universal tumour marker and the less frequent but highly methylated CDH1 promoter can serve for identification of potentially metastasising tumours.

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 tumours 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.

CXCL12 and ADAM23 hypermethylation are associated with advanced breast cancers

More than 25% of the patients with breast cancer (BC) develop metastatic disease. In the present study, we investigated the relationship between DNA methylation levels in genes regulating cell growth, invasiveness, and metastasis and advanced BCs and evaluated the clinical utility of methylation profiles for detecting metastatic potential. Pyrosequencing was used to quantify methylation levels in 11 cancer-associated genes in primary tumors (PTs), lymph node metastases (LNMs), plasma (PL), and blood cells from 206 patients with invasive BC. Protein expression was evaluated using immunohistochemistry. PTs showed hypermethylation of A isoform of the RAS-association domain family 1 (RASSF1A), adenomatous polyposis coli (APC), chemokine C-X-C motif ligand 12 (CXCL12), and disintegrin and metalloprotease domain 23 (ADAM23) (means 38.98%, 24.84%, 12.04%, and 10.01%, respectively). Positive correlations were identified between methylations in PTs and LNMs, but not between PL and PTs. The cumulative methylation of PTs and LNMs manifested similar spectrums of methylated genes that indicate the maintaining of aberrant methylation during breast tumorigenesis. Significantly increased methylation levels in RASSF1A, APC, CXCL12, and ADAM23 were found in estrogen receptor (ER) positive BCs in comparison with ER negative cases. Regarding these results, the evaluation of DNA methylation could be more informative in testing of patients with ER positive BC. The risk for LNMs development and higher proliferation of cancer cells measured through Ki-67 expression was increased by hypermethylation of CXCL12 and ADAM23, respectively. Therefore, the quantification of CXCL12 and ADAM23 methylation could be useful for the prediction of advanced stage of BC.

Multiplex SNaPshot Genotyping for Detecting Loss of Heterozygosity in the Mismatch-Repair Genes MLH1 and MSH2 in Microsatellite-Unstable Tumors

BACKGROUND In the workup of patients with suspected hereditary nonpolyposis colorectal cancer (HNPCC), detection of loss of heterozygosity (LOH) could help pinpoint the mismatch-repair (MMR) gene carrying the germline mutation, but analysis of microsatellite markers has proved unreliable for this purpose. We developed a simple, low-cost method based on single-nucleotide polymorphism (SNP) genotyping and capillary electrophoresis for the assessment of LOH at 2 MMR loci simultaneously. METHODS We used the Applied Biosystems SNaPshot Multiplex Kit with meticulously selected primers to assess 14 common SNPs in MLH1 [mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli)] and MSH2 [mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli)] and optimized the protocol for DNA isolated from peripheral blood and fresh/frozen or archival microsatellite-unstable tumors from patients with confirmed (n = 42) or suspected (n = 25) HNPCC. The 42 tumors from patients with confirmed MLH1 or MSH2 germline mutations were used to validate the methods diagnostic accuracy against results obtained with DNA sequencing or multiplex ligation-dependent probe amplification. RESULTS The SNaPshot assay provided better detection of certain SNPs than DNA sequencing. The MLH1 and MSH2 SNP marker sets were informative in 82% and 76% of the 67 cases analyzed, respectively. The new assay displayed 100% specificity for detecting LOH and predicted the location of the germline mutation in 40% of the cases (54% of those involving MLH1, 22% in MSH2). CONCLUSIONS Our SNP-based method for detecting LOH in MLH1 and MSH2 is simple to perform with instruments available in most clinical genetics laboratories. It can be a valuable addition to protocols now used to guide mutational screening of patients with suspected HNPCC.

Expression of SOCS1 and CXCL12 Proteins in Primary Breast Cancer Are Associated with Presence of Circulating Tumor Cells in Peripheral Blood.

Circulating tumor cells (CTCs) are independent prognostic factors in the primary and metastatic breast cancer patients and play crucial role in hematogenous tumor dissemination. The aim of this study was to correlate the presence of CTCs in peripheral blood with the expression of proteins in tumor tissue that have a putative role in regulation of cell growth and metastatic potential. This prospective study included 203 primary breast cancer patients treated by definitive surgery. CTCs were detected by quantitative real-time PCR for the expression of epithelial (CK19) or epithelial-to-mesenchymal transition–inducing transcription factor genes (TWIST1, SNAIL1, SLUG, and ZEB1). Expression of APC, ADAM23, CXCL12, E-cadherin, RASSF1, SYK, TIMP3, BRMS1, and SOCS1 proteins in primary breast tumor tissue was evaluated by immunohistochemistry. CTCs with epithelial markers were found in 17 (9.2%) patients. Their occurrence was associated with inhibition of SOCS1 expression (odds ratio [OR] = 0.07; 95% confidence interval [CI], 0.03-0.13; P < .001). CTCs with positive epithelial-to-mesenchymal transition markers were detected in 30 (15.8%) patients; however, no association with analyzed protein expressions was found. Overall, CTCs were detected in 44 (22.9%) patients. Presence of any CTC marker was significantly associated with positive CXCL12 expression (OR = 3.08; 95% CI, 1.15-8.26; P = .025) and lack of SOCS1 expression (OR = 0.10; 95% CI, 0.04-0.25; P < .001) in patient’s tumor tissues. As both CXCL12 and SOCS1 proteins are involved in cytokine signaling, our results provide support for the hypothesis that aberrant signaling cross talk between cytokine and chemokine responses could have an important role in hematogenous dissemination of tumor cells in breast cancer.

Down-regulation of traditional oncomiRs in plasma of breast cancer patients

Deregulated expression of microRNAs has the oncogenic or tumor suppressor function in cancer. Since miRNAs in plasma are highly stable, their quantification could contribute to more precise cancer diagnosis, prognosis and therapy prediction. We have quantified expression of seven oncomiRs, namely miR-17/92 cluster (miR-17, miR-18a, miR-19a and miR-20a), miR-21, miR-27a and miR-155, in plasma of 137 breast cancer (BC) patients. We detected down-regulation of six miRNAs in patients with invasive BC compared to controls; however, only miR-20a and miR-27a down-regulations were statistically significant. Comparing miRNA expression between early and advanced stages of BC, we observed statistically significant decrease of miR-17 and miR-19a. We identified down-regulation of miR-17 and miR-20a in patients with clinical parameters of advanced BC (lymph node metastasis, tumor grade 3, circulating tumor cells, higher Ki-67-related proliferation, hormone receptor negativity and HER2 amplification), when compared to controls. Moreover, decreased level of miR-17 was found from low to high grade. Therefore, miR-17 could represent an indicator of advanced BC. Down-regulated miR-27a expression levels were observed in all clinical categories regardless of tumor progression. Hence, miR-27a could be used as a potential diagnostic marker for BC. Our data indicates that any changes in miRNA expression levels in BC patients in comparison to controls could be highly useful for cancer-associated pathology discrimination. Moreover, dynamics of miRNA expression changes could be used for BC progression monitoring.

Partial loss of heterozygosity events at the mutated gene in tumors from MLH1/MSH2 large genomic rearrangement carriers

BackgroundDepending on the population studied, large genomic rearrangements (LGRs) of the mismatch repair (MMR) genes constitute various proportions of the germline mutations that predispose to hereditary non-polyposis colorectal cancer (HNPCC). It has been reported that loss of heterozygosity (LOH) at the LGR region occurs through a gene conversion mechanism in tumors from MLH1/MSH2 deletion carriers; however, the converted tracts were delineated only by extragenic microsatellite markers. We sought to determine the frequency of LGRs in Slovak HNPCC patients and to study LOH in tumors from LGR carriers at the LGR region, as well as at other heterozygous markers within the gene to more precisely define conversion tracts.MethodsThe main MMR genes responsible for HNPCC, MLH1, MSH2, MSH6, and PMS2, were analyzed by MLPA (multiplex ligation-dependent probe amplification) in a total of 37 unrelated HNPCC-suspected patients whose MLH1/MSH2 genes gave negative results in previous sequencing experiments. An LOH study was performed on six tumors from LGR carriers by combining MLPA to assess LOH at LGR regions and sequencing to examine LOH at 28 SNP markers from the MLH1 and MSH2 genes.ResultsWe found six rearrangements in the MSH2 gene (five deletions and dup5-6), and one aberration in the MLH1 gene (del5-6). The MSH2 deletions were of three types (del1, del1-3, del1-7). We detected LOH at the LGR region in the single MLH1 case, which was determined in a previous study to be LOH-negative in the intragenic D3S1611 marker. Three tumors displayed LOH of at least one SNP marker, including two cases that were LOH-negative at the LGR region.ConclusionLGRs accounted for 25% of germline MMR mutations identified in 28 Slovakian HNPCC families. A high frequency of LGRs among the MSH2 mutations provides a rationale for a MLPA screening of the Slovakian HNPCC families prior scanning by DNA sequencing. LOH at part of the informative loci confined to the MLH1 or MSH2 gene (heterozygous LGR region, SNP, or microsatellite) is a novel finding and can be regarded as a partial LOH. The conversion begins within the gene, and the details of conversion tracts are discussed for each case.