Clinical Significance of microRNAs in Hematologic Malignancies and Hematopoietic Stem Cell Transplantation.

Hematologic malignancies are a group of neoplastic conditions that can develop from any stage of the hematopoiesis cascade. Small non-coding microRNAs (miRNAs) play a crucial role in the post-transcriptional regulation of gene expression. Mounting evidence highlights the role of miRNAs in malignant hematopoiesis via the regulation of oncogenes and tumor suppressors involved in proliferation, differentiation, and cell death. In this review, we provide current knowledge about dysregulated miRNA expression in the pathogenesis of hematological malignancies. We summarize data about the clinical utility of aberrant miRNA expression profiles in hematologic cancer patients and their associations with diagnosis, prognosis, and the monitoring of treatment response. Moreover, we will discuss the emerging role of miRNAs in hematopoietic stem cell transplantation (HSCT), and severe post-HSCT complications, such as graft-versus-host disease (GvHD). The therapeutical potential of the miRNA-based approach in hemato-oncology will be outlined, including studies with specific antagomiRs, mimetics, and circular RNAs (circRNAs). Since hematologic malignancies represent a full spectrum of disorders with different treatment paradigms and prognoses, the potential use of miRNAs as novel diagnostic and prognostic biomarkers might lead to improvements, resulting in a more accurate diagnosis and better patient outcomes.

Clinical Relevancy of Circulating Tumor Cells in Breast Cancer: Epithelial or Mesenchymal Characteristics, Single Cells or Clusters?

Metastatic breast cancer (MBC) is typically an incurable disease with high mortality rates; thus, early identification of metastatic features and disease recurrence through precise biomarkers is crucial. Circulating tumor cells (CTCs) consisting of heterogeneous subpopulations with different morphology and genetic, epigenetic, and gene expression profiles represent promising candidate biomarkers for metastatic potential. The experimentally verified role of epithelial-to-mesenchymal transition in cancer dissemination has not been clearly described in BC patients, but the stemness features of CTCs strongly contributes to metastatic potency. Single CTCs have been shown to be protected in the bloodstream against recognition by the immune system through impaired interactions with T lymphocytes and NK cells, while associations of heterotypic CTC clusters with platelets, leucocytes, neutrophils, tumor-associated macrophages, and fibroblasts improve their tumorigenic behavior. In addition to single CTC and CTC cluster characteristics, we reviewed CTC evaluation methods and clinical studies in early and metastatic BCs. The variable CTC tests were developed based on specific principles and strategies. However, CTC count and the presence of CTC clusters were shown to be most clinically relevant in existing clinical trials. Despite the known progress in CTC research and sampling of BC patients, implementation of CTCs and CTC clusters in routine diagnostic and treatment strategies still requires improvement in detection sensitivity and precise molecular characterizations, focused predominantly on the role of CTC clusters for their higher metastatic potency.

Targeting DNA Methylation in Leukemia, Myelodysplastic Syndrome, and Lymphoma: A Potential Diagnostic, Prognostic, and Therapeutic Tool.

DNA methylation represents a crucial mechanism of epigenetic regulation in hematologic malignancies. The methylation process is controlled by specific DNA methyl transferases and other regulators, which are often affected by genetic alterations. Global hypomethylation and hypermethylation of tumor suppressor genes are associated with hematologic cancer development and progression. Several epi-drugs have been successfully implicated in the treatment of hematologic malignancies, including the hypomethylating agents (HMAs) decitabine and azacytidine. However, combinations with other treatment modalities and the discovery of new molecules are still the subject of research to increase sensitivity to anti-cancer therapies and improve patient outcomes. In this review, we summarized the main functions of DNA methylation regulators and genetic events leading to changes in methylation landscapes. We provide current knowledge about target genes with aberrant methylation levels in leukemias, myelodysplastic syndromes, and malignant lymphomas. Moreover, we provide an overview of the clinical trials, focused mainly on the combined therapy of HMAs with other treatments and its impact on adverse events, treatment efficacy, and survival rates among hematologic cancer patients. In the era of precision medicine, a transition from genes to their regulation opens up the possibility of an epigenetic-based approach as a diagnostic, prognostic, and therapeutic tool.

miR-205-5p Downregulation and ZEB1 Upregulation Characterize the Disseminated Tumor Cells in Patients with Invasive Ductal Breast Cancer.

BACKGROUND: Dissemination of breast cancer (BC) cells through the hematogenous or lymphogenous vessels leads to metastatic disease in one-third of BC patients. Therefore, we investigated the new prognostic features for invasion and metastasis. METHODS: We evaluated the expression of miRNAs and epithelial-to-mesenchymal transition (EMT) genes in relation to CDH1/E-cadherin changes in samples from 31 patients with invasive ductal BC including tumor centrum (TU-C), tumor invasive front (TU-IF), lymph node metastasis (LNM), and CD45-depleted blood (CD45-DB). Expression of miRNA and mRNA was quantified by RT-PCR arrays and associations with clinico-pathological characteristics were statistically evaluated by univariate and multivariate analysis. RESULTS: We did not verify CDH1 regulating associations previously described in cell lines. However, we did detect extremely high ZEB1 expression in LNMs from patients with distant metastasis, but without regulation by miR-205-5p. Considering the ZEB1 functions, this overexpression indicates enhancement of metastatic potential of lymphogenously disseminated BC cells. In CD45-DB samples, downregulated miR-205-5p was found in those expressing epithelial and/or mesenchymal markers (CTC+) that could contribute to insusceptibility and survival of hematogenously disseminated BC cells mediated by increased expression of several targets including ZEB1. CONCLUSIONS: miR-205-5p and potentially ZEB1 gene are promising candidates for markers of metastatic potential in ductal BC.

miR-497-5p Decreased Expression Associated with High-Risk Endometrial Cancer.

The current guidelines for diagnosis, prognosis, and treatment of endometrial cancer (EC), based on clinicopathological factors, are insufficient for numerous reasons; therefore, we investigated the relevance of miRNA expression profiles for the discrimination of different EC subtypes. Among the miRNAs previously predicted to allow distinguishing of endometrioid ECs (EECs) according to different grades (G) and from serous subtypes (SECs), we verified the utility of miR-497-5p. In ECs, we observed downregulated miR-497-5p levels that were significantly decreased in SECs, clear cell carcinomas (CCCs), and carcinosarcomas (CaSas) compared to EECs, thereby distinguishing EEC from SEC and rare EC subtypes. Significantly reduced miR-497-5p expression was found in high-grade ECs (EEC G3, SEC, CaSa, and CCC) compared to low-grade carcinomas (EEC G1 and mucinous carcinoma) and ECs classified as being in advanced FIGO (International Federation of Gynecology and Obstetrics) stages, that is, with loco-regional and distant spread compared to cancers located only in the uterus. Based on immunohistochemical features, lower miR-497-5p levels were observed in hormone-receptor-negative, p53-positive, and highly Ki-67-expressing ECs. Using a machine learning method, we showed that consideration of miR-497-5p expression, in addition to the traditional clinical and histopathologic parameters, slightly improves the prediction accuracy of EC diagnosis. Our results demonstrate that changes in miR-497-5p expression influence endometrial tumorigenesis and its evaluation may contribute to more precise diagnoses.

Discriminating miRNA Profiles between Endometrioid Well- and Poorly-Differentiated Tumours and Endometrioid and Serous Subtypes of Endometrial Cancers.

The discrimination of different subtypes of endometrial carcinoma (EC) is frequently problematic when using the current histomorphological classification; therefore, new markers for this differentiation are needed. Here, we examined differences in miRNA expression between well- and poorly-differentiated (grades 1 and 3) endometrioid endometrial carcinoma (EEC) and between EEC and serous endometrial carcinoma (SEC). The expression of 84 tumour-suppressor miRNAs was analysed by real-time polymerase chain reactions in 62 EC and 20 non-neoplastic endometrial specimens. The potential functions of the differentially expressed miRNAs were determined by bioinformatics analyses. The expression of let-7c-5p, miR-125b-5p, miR-23b-3p, and miR-99a-5p in grade 3 EEC was decreased compared to grade 1 EEC. To discriminate between EEC and SEC, let-7g-5p, miR-195-5p, miR-34a-5p, and miR-497-5p expression was significantly downregulated in SEC. In bioinformatic analyses, miRNAs that could discriminate grade 1 from grade 3 mainly targeted genes involved in PI3K-AKT signaling, whereas miRNAs that could discriminate EEC from SEC targeted genes involved in several signaling pathways, but mainly MAPK signaling. Taken collectively, our results indicate that the activation of certain signaling pathways can be useful in the molecular characterization of EEC and SEC.

MicroRNAs Contribute to Breast Cancer Invasiveness.

Cancer statistics in 2018 highlight an 8.6 million incidence in female cancers, and 4.2 million cancer deaths globally. Moreover, breast cancer is the most frequent malignancy in females and twenty percent of these develop metastasis. This provides only a small chance for successful therapy, and identification of new molecular markers for the diagnosis and prognostic prediction of metastatic disease and development of innovative therapeutic molecules are therefore urgently required. Differentially expressed microRNAs (miRNAs) in cancers cause multiple changes in the expression of the tumorigenesis-promoting genes which have mostly been investigated in breast cancers. Herein, we summarize recent data on breast cancer-specific miRNA expression profiles and their participation in regulating invasive processes, in association with changes in cytoskeletal structure, cell-cell adhesion junctions, cancer cell-extracellular matrix interactions, tumor microenvironments, epithelial-to-mesenchymal transitions and cancer cell stem abilities. We then focused on the epigenetic regulation of individual miRNAs and their modified interactions with other regulatory genes, and reviewed the function of miRNA isoforms and exosome-mediated miRNA transfer in cancer invasiveness. Although research into miRNA's function in cancer is still ongoing, results herein contribute to improved metastatic cancer management.

A disintegrin and metalloprotease 23 hypermethylation predicts decreased disease-free survival in low-risk breast cancer patients.

A Disintegrin And Metalloprotease 23 (ADAM23), a member of the ADAM family, is involved in neuronal differentiation and cancer. ADAM23 is considered a possible tumor suppressor gene and is frequently downregulated in various types of malignancies. Its epigenetic silencing through promoter hypermethylation was observed in breast cancer (BC). In the present study, we evaluated the prognostic significance of ADAM23 promoter methylation for hematogenous spread and disease-free survival (DFS). Pyrosequencing was used to quantify ADAM23 methylation in tumors of 203 BC patients. Presence of circulating tumor cells (CTC) in their peripheral blood was detected by quantitative RT-PCR. Expression of epithelial (KRT19) or mesenchymal (epithelial-mesenchymal transition [EMT]-inducing transcription factors TWIST1, SNAI1, SLUG and ZEB1) mRNA transcripts was examined in CD45-depleted peripheral blood mononuclear cells. ADAM23 methylation was significantly lower in tumors of patients with the mesenchymal CTC (P = .006). It positively correlated with Ki-67 proliferation, especially in mesenchymal CTC-negative patients (P = .001). In low-risk patients, characterized by low Ki-67 and mesenchymal CTC absence, ADAM23 hypermethylation was an independent predictor of DFS (P = .006). Our results indicate that ADAM23 is likely involved in BC progression and dissemination of mesenchymal CTC. ADAM23 methylation has the potential to function as a novel prognostic marker and therapeutic target.

Decreased methylation in the SNAI2 and ADAM23 genes associated with de-differentiation and haematogenous dissemination in breast cancers.

BACKGROUND: In breast cancer (BC), deregulation of DNA methylation leads to aberrant expressions and functions of key regulatory genes. In our study, we investigated the relationship between the methylation profiles of genes associated with cancer invasivity and clinico-pathological parameters. In detail, we studied differences in the methylation levels between BC patients with haematogenous and lymphogenous cancer dissemination. METHODS: We analysed samples of primary tumours (PTs), lymph node metastases (LNMs) and peripheral blood cells (PBCs) from 59 patients with sporadic disseminated BC. Evaluation of the DNA methylation levels of six genes related to invasivity, ADAM23, uPA, CXCL12, TWIST1, SNAI1 and SNAI2, was performed by pyrosequencing. RESULTS: Among the cancer-specific methylated genes, we found lower methylation levels of the SNAI2 gene in histologic grade 3 tumours (OR = 0.61; 95% CI, 0.39-0.97; P = 0.038) than in fully or moderately differentiated cancers. We also evaluated the methylation profiles in patients with different cancer cell dissemination statuses (positivity for circulating tumour cells (CTCs) and/or LNMs). We detected the significant association between reduced DNA methylation of ADAM23 in PTs and presence of CTCs in the peripheral blood of patients (OR = 0.45; 95% CI, 0.23-0.90; P = 0.023). CONCLUSION: The relationships between the decreased methylation levels of the SNAI2 and ADAM23 genes and cancer de-differentiation and haematogenous dissemination, respectively, indicate novel functions of those genes in the invasive processes. After experimental validation of the association between the lower values of SNAI2 and ADAM23 methylation and clinical features of aggressive BCs, these methylation profiles could improve the management of metastatic disease.

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.

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.

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.

Relationship between circulating tumor cells, blood coagulation, and urokinase-plasminogen-activator system in early breast cancer patients.

Cancer is a risk factor for venous thromboembolism (VTE) and plasma d-dimer (DD) and tissue factor (TF) are established VTE associated markers. Circulating tumor cells (CTCs) are associated with the risk of VTE in metastatic breast cancer. This study aimed to correlate CTCs, blood coagulation and the urokinase plasminogen activator (uPA) system in primary breast cancer (PBC) patients. This prospective study included 116 PBC patients treated by primary surgery. CTCs were detected by quantitative RT-PCR assay for expression of epithelial (CK19) or epithelial-mesenchymal transition (EMT) genes (TWIST1, SNAIL1, SLUG, ZEB1, FOXC2). Plasma DD, TF, uPA system proteins were detected by enzyme-linked immunosorbent assays, while expressions of uPA system in surgical specimens were evaluated by immunohistochemistry. CTCs were detected in 27.6% patients. Patients with CTCs had a significantly higher mean plasma DD (ng/mL) than those of patients without CTCs (632.4 versus 365.4, p = 0.000004). There was no association between plasma TF and CTCs. Epithelial CTCs exhibit higher expression of uPA system genes compared to EMT_CTCs. Patients with CTCs had higher plasma uPA proteins than those of patients without CTCs; there was no correlation between tissue expression of uPA system, CTCs, DD or TF levels. In multivariate analysis CTCs and patients age were independent factors associated with plasma DD. We found association between plasma DD and CTCs indicating a potential role for activation of the coagulation cascade in the early metastatic process. CTCs could be directly involved in coagulation activation or increased CTCs could be marker of aggressive disease and increased VTE risk.

RASSF1A Promoter Methylation Levels Positively Correlate with Estrogen Receptor Expression in Breast Cancer Patients.

The aim of this study was to investigate the relationship between the promoter methylation in five cancer-associated genes and clinicopathologic features for identification of molecular markers of tumor metastatic potential and hormone therapy response efficiency in breast cancer. The methylation levels in paraffin-embedded tumor tissues, plasma, and blood cells from 151 sporadic breast cancer patients and blood samples of 50 controls were evaluated by quantitative multiplex methylation-specific polymerase chain reaction. DNA methylation of RAS-association domain family member 1 (RASSF1A), estrogen receptor 1 (ESR1), cadherin 1, type 1, E-cadherin (CDH1), TIMP metallopeptidase inhibitor 3 (TIMP3) and spleen tyrosine kinase (SYK) genes was detected in the tumors of 124, 19, 15, 15, and 6 patients with mean levels of 48.45%, 3.81%, 2.36%, 27.55%, and 10.81%, respectively. Plasma samples exhibited methylation in the same genes in 25, 10, 15, 17, and 3 patients with levels of 22.54%, 17.20%, 22.87%, 31.93%, and 27.42%, respectively. Cumulative methylation results confirmed different spectra in tumor and plasma samples. Simultaneous methylation in tumors and plasma were shown in less than 17% of patients. RASSF1A methylation levels in tumor samples statistically differ according to tumor size (P = .029), estrogen receptor (ER) and progesterone receptor (PR) status (P = .000 and P = .004), and immunohistochemical subtype (P = .000). Moreover, the positive correlation was found between RASSF1A methylation levels and percentage of cancer cells expressing ER and PR. The direct relationship between RASSF1A promoter methylation and expression of ER could aid the prognosis of hormonal therapy response.

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.

Epigenetic tools in potential anticancer therapy.

Human cancer represents a heterogeneous group of diseases that are driven by progressive genetic and epigenetic abnormalities. The latter alterations involve hypermethylation and hypomethylation of DNA, and changed patterns of histone modification, with resultant remodeling of the chromatin structure that cause deregulation of the transcription activity of many genes. Unlike the remarkable progress in understanding the processes by which DNA methyltransferases can regulate gene expression and histone deacetylases can induce alteration of chromatin structure, the roles of epigenetic events in tumors remain insufficiently explained. In contrast to genetic changes, the epigenetic alterations in cancer cells can be reversed by the inhibition of DNA methylation and histone deacetylation. Therefore, many inhibition agents for re-expression, predominantly of tumor-suppressor genes, have been identified and tested in laboratory models and numerous clinical trials. Despite in-vitro evidence that a single drug can lead to reactivation of methylated genes, inhibitors of DNA methyltransferases and histone deacetylases have been investigated in combination, or together with cytotoxic chemotherapy, radiotherapy, immunotherapy, or hormonal therapy to improve the therapeutic effect. Ongoing trials are recognizing that the identification of a target group of patients who are more likely to respond to the epigenetic therapy, defining of an optimal dose and schedule of treatment, and the development of more specific inhibitors with minimal unwanted side effects are necessary. Thus, new combinations of anticancer agents, including epigenetic modulators, may lead to a more effective control of cancer.

Redundant DNA methylation in colorectal cancers of Lynch-syndrome patients.

Lynch syndrome is an inherited disease resulting predominantly in colorectal cancer (CRC). The crucial cause is DNA mismatch repair (MMR) malfunction that is associated mostly with MLH1 or MSH2 germline mutations. A significant hallmark of repair defects is a high level of instability in microsatellites (MSI-H). In many sporadic unstable CRCs, the MLH1 gene is inactivated by promoter hypermethylation in addition to extensive promoter methylation in many tumor-suppressor genes known as CpG island methylation phenotype (CIMP). To investigate the possible role of epigenetic alterations in causing MMR deficiency and thereby Lynch syndrome, we evaluated the MLH1 specific and global hypermethylation in hereditary CRCs. Of 22 Lynch-syndrome-related CRCs, 18 (81.8%) demonstrated various levels of DNA methylation; of these, 14 (63.6%) and 4 (18.2%) were methylated in distal and both distal and proximal regions of the MLH1 promoter, respectively. However, only 7/18 (38.9%) of results were confirmed by bisulfite sequencing. Similar methylation patterns in tumors and frequently in matched normal DNA were found in twelve and four patients with MLH1 and MSH2 alterations documented by the absence of protein or presence of germline mutation, respectively. Moreover, the same results were observed in five stable CRCs. None of 22 Lynch-syndrome-related tumors presented CIMP in contrast to 3/10 (30%) stable carcinomas. The rather randomly distributed weak methylation patterns in hereditary CRCs indicate that epigenetic events are redundant in Lynch-syndrome etiology, in contrast to the widespread DNA methylation in sporadic unstable CRCs. These methylation-profile differences can lead to more effective molecular diagnosis of Lynch syndrome.

The MRE11/RAD50/NBS1 complex destabilization in Lynch-syndrome patients.

Lynch syndrome is an inherited disease leading to the development predominantly of colorectal cancer (CRC). The crucial cause is malfunction of DNA mismatch repair that is characterized by high level of microsatellite instability; however, new knowledge of two MSI modes (types A and B) suggests a more complex molecular basis of this syndrome. To investigate, whether the extensive alterations in individual MSI markers (type B) can indicate the potential deficiency of DNA double-strand break (DSB) repair in Lynch-syndrome-related tumours, we evaluated the MSI type and alterations in the MRE11 and RAD50 repeats that are associated with the reduced protein expression and functional impairment of the MRE11-RAD50-NBS1 (MRN) complex. Of 27 CRCs, 21 samples manifested type B in at least one MSI+ marker. From type B tumours, the genetic alterations were identified in 16 (76%) samples; seven, one and eight cases manifested mutations in MRE11, RAD50 and both genes, respectively. However, predominantly biallelic MRE11 alterations with simultaneously developed RAD50 mutations impaired the protein expressions with different intensity and location in tumour. Of six tumours presenting changes

New aspects in molecular diagnosis of Lynch syndrome (HNPCC).

Colorectal carcinoma is one of the most common cancers that occurs in the human population, resulting in a mortality rate of more than 50%. Mismatch repair (MMR) defects are mostly manifested as high levels of microsatellite instability (MSI-H); this occurs in approximately 19% of all colorectal cancers including a smaller, but high-risk subgroup that is represented by hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome). The aim of molecular diagnosis is to distinguish these familial cases from of the majority of sporadic unstable cancers. Here, we review the widespread MSI-H phenotype in various tumours, assessing the genetic, epigenetic and morphological differences to distinguish between sporadic and familial unstable colorectal cancers. The most important morphological feature is the presence of tumour-infiltrating lymphocytes (TILs), the presence of which is typical in MSI cancers. The main discriminators for HNPCC patients are family history, age at onset up to 60 years and MMR defects caused by germline mutations. This is in contrast to the discriminators for sporadic colorectal cancers, which include onset at any age, BRAF mutation presence and epigenetically MMR inactivation by extensive CpG-island methylation. This article presents a modified strategy for molecular diagnosis of HNPCC by selective inclusion of recently recognized characteristics of tumours. The clear identification of affected families can improve the strategy of early detection, therapy and prevention of colorectal cancers.

Novel MLH1 and MSH2 germline mutations in the first HNPCC families identified in Slovakia.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a dominantly-inherited cancer predisposition syndrome, in which the susceptibility to cancer of the colon, endometrium and ovary is linked to germline mutations in DNA mismatch repair (MMR) genes. We have recently initiated a cancer prevention program in suspected HNPCC families in the Slovak Republic. The first ten families fulfilling Amsterdam criteria or Bethesda guidelines were screened for germline mutations in MLH1 and MSH2, two MMR genes most frequently mutated in HNPCC families. Six mutations were identified, five of which have not been reported previously. Two of the three new mutations in MLH1 (c.380+2T>A; c.307-2A>C) were absent from 100 chromosomes of healthy controls and probably cause a splicing defect, while the third was a 1 bp deletion (c.1261delA). In the MSH2 gene, one new nonsense (c.1030C>T [p.Q344X]) and one missense (c.524T>C [p.L175P]) mutation were identified. This latter variant was not found in 104 alleles of healthy control individuals. Moreover, a previously-reported pathogenic mutation (c.677G>T [p.R226L]) was found in one kindred. The clinical data and the genotypic and phenotypic evaluation of the tumors indicate that all the new alterations are pathogenic HNPCC mutations.