Search

Variations in the hepatitis C virus (HCV) core gene are related to the progression of liver fibrosis and therapy response. However, the influence of individual amino acid (aa) substitutions at different positions of HCV Core protein on the response to combined pegylated interferon/ribavirin (PEG-IFN/RBV) therapy and disease progression is not yet fully understood. The HCV Core protein can inactivate various genes in the host genome by affecting the methylation of their promoters, leading to liver damage and carcinogenesis. Two genes whose methylation status is affected by the HCV Core protein are the tumor suppressor genes RASSF1A and p16. We have previously shown that the methylation status of these two genes, together with the single nucleotide polymorphism (SNP) rs12979860 near the interleukin-28 beta subunit (IL-28B) gene, influences the response to combined therapy. Herein, we investigated a possible association between detected aa substitutions in HCV Core protein and response to combined therapy, liver disease progression, IL28B genotype, and the methylation status of the RASSF1A and p16 genes. In 29 examined patients we found no association between individual aa substitutions and therapy response. However, we observed that patients with HCV Core aa substitutions at position 75 and CT/TT IL28B genotypes were non-responders (NR) (p=0.023), which was associated with the presence of unmethylated RASSF1A gene. In contrast, even 75% of patients with aa substitutions at position 91 and CC IL28B genotype achieved a sustained virologic response (SVR) (p=0.030), and 70% of them had methylated RASSF1A gene. There was no significant association between the methylation status of the p16 gene and aa variations in the HCV core region. Our results suggest that combined analysis of aa substitutions in HCV Core protein, IL28B genotype, and methylation status of the RASSF1A gene may help predict response to PEG-IFN/RBV therapy in patients with chronic hepatitis C genotype 1b.x

Variations in the hepatitis C virus (HCV) core sequence have been related to disease progression and response to antiviral therapy. Previously we showed that the methylation status of RASSF1A and p16 genes, and IL28B genotypes affects the response to pegylated interferon/ribavirin (PEG-IFN/RBV) therapy. Herein we investigated whether amino acid (aa) substitutions in the HCV core region alone or in combination with IL28B genotypes and RASSF1A/p16 methylation affect the response to PEG-IFN/RBV therapy and liver disease progression. Among 29 examined patients, we found no association between single aa substitutions and response to therapy. However, we observed that patients with the HCV core aa substitution at position 75 and CT/TT IL28B genotypes were non-responders (NR), (P=0.023). Moreover, these patients had unmethylated RASSF1A. In contrast, most patients (75%) with aa substitutions at position 91 and CC IL28B genotype achieved sustained virologic response (SVR), (P=0.030), and 70% of them had methylated RASSF1A gene. Our results suggest that combined analysis of aa substitutions in the core protein, the IL28B rs12979860 polymorphism, and the methylation status of the RASSF1A gene may help in predicting treatment response to PEG-IFN/RBV in genotype 1b chronic hepatitis C patients.

Hepatocellular carcinoma (HCC) is one of the most frequent cancers in humans, characterised by a high resistance to conventional chemotherapy, late diagnosis, and a high mortality rate. It is necessary to elucidate the molecular mechanisms involved in hepatocarcinogenesis to improve diagnosis and treatment outcomes. The Runt-related (RUNX) family of transcription factors (RUNX1, RUNX2, and RUNX3) participates in cardinal biological processes and plays paramount roles in the pathogenesis of numerous human malignancies. Their role is often controversial as they can act as oncogenes or tumour suppressors and depends on cellular context. Evidence shows that deregulated RUNX genes may be involved in hepatocarcinogenesis from the earliest to the latest stages. In this review, we summarise the topical evidence on the roles of RUNX gene family members in HCC. We discuss their possible application as non-invasive molecular markers for early diagnosis, prognosis, and development of novel treatment strategies in HCC patients.

Prevention of chronic hepatitis C (CHC) and its complications is based on antiviral therapy and early detection of reliable molecular markers in persons under risk. We investigated whether the methylation status of RASSF1A and p16 genes, alone or in combination with host and viral factors, affects the response to therapy with pegylated interferon/ribavirin (PEG-IFN/RBV). Methylation-specific polymerase chain reaction (MSP) was used to determine the methylation status of the target promoter sequences of RASSF1A and p16 in circulating-free DNA from the peripheral blood of 49 patients with CHC genotype 1b. The methylation status of the examined genes did not affect the response to therapy. However, the simultaneous presence of either RASSF1A or p16 methylation and the CC genotype of IL28B was significantly related to a sustained virologic response (P=0.009 and P=0.032, respectively). After Bonferroni correction, only the result concerning the RASSF1A gene remained significant (P<0.0125). Methylation of RASSF1A was associated with the CC genotype of the IL28B gene (P=0.024) and a higher viral load (≥400 000 IU/mL, P=0.009). Our results suggest that combined analysis of RASSF1A gene methylation and IL28B rs12979860 polymorphism could potentially help in the prediction of therapy response in CHC genotype 1b patients.

Paper description:Patient responses to standard treatment of advanced stages of rectal carcinoma are variable, which emphasizes the need to define reliable predictive and prognostic molecular parameters.We propose a model of simultaneous analysis of KRAS gene mutation status and p16INK4a and p14ARF gene promoter methylation status in pre-treatment tumor biopsies.The simultaneous presence of p14ARF methylation and KRAS mutation was associated with more aggressive tumor behavior. The concurrent presence of alterations in all three examined genes was associated with shorter overall survival.Combined analysis of examined gene alterations revealed patient subgroups with a distinct pattern of tumor response and disease outcome.Abstract: Current management of locally advanced rectal carcinoma (LARC) involves preoperative chemoradiotherapy (preCRT) before surgery. Despite improved local control rate, the response to preCRT of individual patients is variable and may reflect heterogeneous biological properties among tumors of the same clinical stage. Identifying novel molecular parameters with predictive and/or prognostic value is of great clinical importance for a personalized therapeutic approach. In this study, KRAS mutation status was analyzed by direct sequencing, while methylation-specific polymerase chain reaction (MSP) was used to examine p16INK4a and p14ARF gene methylation status in pretreatment tumor biopsies of 60 patients with LARC. The examined molecular changes of KRAS, p16INK4a and p14ARF genes were mutually independent (p16INK4a/KRAS, P=0.272; p14ARF/KRAS, P=0.923; p16INK4a/p14ARF, P=0.715). However, the simultaneous presence of p14ARF methylation and KRAS mutation was associated with a more frequent appearance of local recurrences and distant metastasis (P=0.027). Moreover, patients with the simultaneous presence of p16INK4a and p14ARF methylation and KRAS mutation had significantly shorter overall survival (P=0.011). The obtained results strongly suggest th

The discovery of the Estrogen Receptor Beta (ERβ) in 1996 opened new perspectives in the diagnostics and therapy of different types of cancer. Here, we present a review of the present research knowledge about its role in endocrine-related cancers: breast, prostate, and thyroid, and colorectal cancers. We also discuss the reasons for the controversy of its role in carcinogenesis and why it is still not in use as a biomarker in clinical practice. Given that the diagnostics and therapy would benefit from the introduction of new biomarkers, we suggest ways to overcome the contradictions in elucidating the role of ERβ.

Background: Rectal cancer represents approximately 30% of cases of colorectal carcinoma and locally advanced stages of rectal cancers (LARC) remain a great clinical challenge due to chemoresistance and high local recurrence rate. The current management of LARC involves neoadjuvant chemoradiotherapy (neoCRT) before surgery. Since only a subset of patients benefit from this preoperative treatment, the development of reliable molecular biomarkers is required. In this retrospective study, we investigated the mutation status of K-ras proto-oncogene, as well as the expression level of apoptosis regulator protein, BCL2, to evaluate their potential predictive role in LARC. Patients and Methods: K-ras gene mutation status was determined by direct sequencing, while BCL2 protein expression was detected immunohistochemically (semi-quantitatively method) in pre-therapeutic and pre-operative biopsy specimens of 61 patients with LARC treated with neoCRT. Results: According to the results of this study, K-ras mutation status and BCL2 expression status were mutually independent events. In general, K-ras mutation status did not affect the response to CRT, while in the group of patients with high BCL2 expression was observed a tendency toward a worse response to the same treatment (p=0.098). However, the subgroup of patients with the simultaneous presence of K-ras mutation and high BCL2 expression showed significantly worse response to neoCRT (p=0.022). Conclusion: Obtained results strongly suggest that combined analyses of molecular aberrations in K-ras proto-oncogene and BCL2 anti-apoptotic protein expression level could have a potential predictive role and important clinical relevance in the identification of LARC patient subgroups, with a distinct pattern of response to neoCRT.

zadnje vreme proučava kao zaseban klinički entitet. Aktuelan terapijski pristup u lečenju lokalno uznapredovalih stadijuma KR podrazumeva primenu preoperativne hemioradioterapije (HRT), nakon koje sledi hirurško uklanjanje tumora. Iako se ovim tretmanom postiže bolja lokalna kontrola bolesti, individualni odgovor na primenjenu terapiju je varijabilan i ukazuje na značajnu biološku heterogenost tumora u okviru istog kliničkog stadijuma. Stoga je u cilju kreiranja i primene personalizovane terapije neophodno definisati prediktivne i prognostičke molekularne parametre. Primenom PCR metode specifične za metilaciju (MSP), ispitivan je metilacioni status gena p16INK4a i p14ARF, dok je automatskim sekvenciranjem analiziran mutacioni status gena KRAS u pre-tretmanskim biopsijama tumorskog tkiva obolelih od KR u lokalno uznapredovalom stadijumu, kao bi se utvrdio njihov potencijalni prediktivni i prognostički značaj. Rezultati ove studije su pokazali da ispitivane promene u genima p16INK4a, p14ARF i KRAS pojedinačno gledano, nisu značajni prediktivni i prognostički faktori kod obolelih od KR, mada je istovremeno prisustvo metilacije gena p14ARF i mutacije gena KRAS povezano sa agresivnijim ponašanjem tumora, dok je istovremeno prisustvo izmena u sva tri ispitivana gena povezano sa kraćim ukupnim preživljavanjem. Međutim, kombinovane analize genetičkih, epigenetičkih i imunohistohemijskih parametara (EGFR, VEGF, Bcl-2 i Ki-67), mogu imati klinički značaj u identifikaciji podgrupa obolelih, koje se međusobno razlikuju kako prema odgovoru na HRT, tako i prema toku i ishodu bolesti., nowadays studing as a distinct clinical entity. The current management of locally advanced rectal carcinoma involves neoadjuvant chemoradiotherapy (CRT) prior to surgery. Despite improved local control rate, individual patient response to CRT is variable and may reflect heterogeneous biological properties among tumors of the same clinical stage. So, there is utility to define predictive and prognostic molecular parameters of response and to personalize therapeutic approach. Methylation-specific polymerase chain reaction (MSP) was used to examine p16INK4a and p14ARF methylation status, while KRAS mutation status was analyzed by direct sequencing in pretreatment tumor biopsies of patients with locally advanced RC, in order to evaluate its potential predictive and/or prognostic role. According to the results of this study, examined molecular changes of p16INK4a, p14ARF and KRAS genes, considering separately, were not proven to be significant predictive and prognostic factors in RC patients, although simultaneous presence of p14ARF methylation and KRAS mutation was associated with more aggressive tumor behavior, while concurrent presence of genetic/epigenetic alteration in all three examined genes was correlated with shorter overall survival. However, combined analyses of genetic, epigenetic and immunohistochemical parameters (EGFR, VEGF, Bcl-2 and Ki-67), could have important clinical relevance in identification of RC patients subgroups, with distinct pattern of response to CRT and disease outcome

Methylation of p16 and p14 genes is a common event in colorectal cancers; however, their exact role in the prediction of patients' outcome is unclear. We conducted this retrospective study to evaluate their potential predictive and/or prognostic roles. Methylation-specific PCR was used to examine the methylation status of p16 and p14 in pretherapeutic and preoperative biopsy specimens of 60 patients with locally advanced rectal cancer. The methylation status of the examined genes did not affect the response to preoperative chemoradiotherapy (CRT), recurrence rate and overall survival. However, patients with a simultaneous presence of either p16 or p14 methylation and high vascular endothelial growth factor (VEGF) expression showed a significantly worse response to CRT (p=0.005 and p=0.038, respectively). Moreover, patients with both p16 methylation and high VEGF expression had significantly shorter overall survival (p=0.010), while no such association was found in patients with p14 methylation and high VEGF expression. On the other hand, a subgroup of patients with p16 methylation and low VEGF and high epidermal growth factor receptor (EGFR) expression showed a significantly better response to CRT (p=0.024). The obtained results point to the importance of p16 and p14 methylation analyses in combination with VEGF and EGFR expression, aimed at better predicting treatment response and patient outcome. © 2018 by the Serbian Biological Society.

Karcinom prostate (KP) predstavlja jedan od vodećih globalnih zdravstvenih problema, s obzirom na to da je reč o drugom najučestalijem malignom tumoru kod muškaraca. U kliničkoj praksi vezanoj za KP jedan od najvećih izazova je razlikovanje latentnog od klinički značajnog karcinoma. U tom smislu, od izuzetnog značaja je identifikacija genetičkih varijanti čija bi primena kao nestandardnih prognostičkih parametara mogla da se koristi za konstruisanje preciznih algoritama za procenu rizika za razvoj i progresiju KP. U okviru ove disertacije analizirana je potencijalna asocijacija varijanti u četiri gena za mikroRNK (rs2910164 u hsa-miR-146a, rs3746444 u hsa-miR-499, rs11614913 u hsa-miR-196a2 i rs895819 u genu za miR-27a) sa rizikom za razvoj i progresiju KP. Takođe, analizirana je asocijacija varijanti u genima za proteine utišavajućeg kompleksa (RISC) (DICER1, AGO2, TARBP2, GEMIN3 i GEMIN4) sa navedenim rizicima. Studija je obuhvatala 355 bolesnika sa KP, 360 bolesnika sa benignom hiperplazijom prostate (BPH) i 318 muškaraca koji su činili kontrolnu grupu. Genotipizacija genetičkih varijanti vršena je metodama PCR-RFLP, HRMA, alelspecifičnog PCR-a, primenom TaqMan® eseja za genotipizaciju polimorfizama i PCR-a u realnom vremenu baziranom na korišćenju specifičnih proba. Rezultati genotipizacije su statistički obrađeni u cilju ispitivanja asocijacija sa rizikom za razvoj i progresiju KP, kao i statističkih epistatičkih interakcija. Takođe, sprovedena je meta-analiza koja je uključivala naše rezultate, kao i rezultate prethodnih studija koje su analizirale asocijaciju rs2910164 sa rizikom za razvoj drugih malignih tumora. Rezultati ove studije pokazali su asocijaciju rs2910164 sa rizikom za razvoj bolesti, dok je za gotovo sve ostale analizirane genetičke varijante ustanovljena asocijacija sa vrednostima standardnih prognostičkih parametara KP i/ili rizikom za progresiju KP. Pokazanа je i asocijacija epistatičke interakcije između varijanti rs11614913 u genu hsa-miR-1, Being the second most common cancer among men worldwide, prostate cancer (PCa) is one of the major global health issues. In the clinical practice related to this malignant disease, one of the most important challenges is to distinct latent from clinically significant forms of disease. Therefore, it is of utmost importance to identify genetic variants which could be used as nonstandard prognostic parameters for constructing precise algorithms for evaluating the risk of PCa onset and progression. In this dissertation, the association of four microRNA genetic variants (rs2910164 in hsa-miR- 146a, rs3746444 in hsa-miR-499, rs11614913 in hsa-miR-196a2 and rs895819 in the gene encoding miR-27a) with PCa risk and progression was assessed. Also, the potential association with PCa was estimated for genetic variants located in genes encoding the proteins of RNA-induced silencing complex (RISC) (DICER1, AGO2, TARBP2, GEMIN3 and GEMIN4). The study involved 355 patients with PCa, 360 patients with benign prostatic hyperplasia (BPH), as well as 318 healthy controls. Genotyping was performed by using PCR-RFLP, HRMA, alelle-specific PCR, TaqMan® SNP Genotyping Assays and real-time PCR-based genotyping assay using specific probes. Results were statistically analyzed, aiming to evaluate the potential associations of genetic variants with PCa risk and progression, as well as to assess statistical epistatic interactions. Furthermore, a meta-analysis was conducted involving our results, together with the results of previous studies on association between rs2910164 and different types malignant tumors. The results of this study suggested the association of rs2910164 with PCa risk, while for most of the other analyzed genetic variants the association with the values of standard prognostic parameters and/or PCa progression was found. Also, the epistatic interaction between rs11614913 in hsa-miR- 196a2, rs7813 in GEMIN4 and rs784567 in TARBP2 was shown to be associated with the risk of PCa

Background: Preoperative chemoradiotherapy (CRT) represents the standard treatment for patients with locally advanced rectal cancer. Since only subset of patients has benefit from this preoperative treatment, development of reliable molecular biomarkers is required. In this retrospective study, we investigated methylation status of p16 and p14 tumor suppressor genes in locally advanced rectal cancer, in order to evaluate their potential predictive and prognostic role. Methods: Methylation-specific PCR was used to examine methylation status of p16 and p14 genes in pretherapeutic and preoperative biopsy specimens of 60 patients with locally advanced rectal cancer. Results: Aberrant methylation of p16 and p14 genes was detected in 43.3% (26/60) and 39.6% (23/58) of cases, respectively. In general, p16 and p14 methylation status did not affect the response to CRT, recurrences rate and overall survival. However, patients with simultaneous presence of either p16 or p14 methylation and high vascular endothelial growth factor (VEGF) expression showed significantly worse response to CRT (p = 0.005 and p = 0.038, respectively). In addition, tendency toward more frequent local recurrences and metastasis was observed in cases with concurrent presence of methylation of either p16 or p14 gene and high VEGF expression (p = 0.075 and p = 0.072, respectively), while patients with both of p16 methylation and high VEGF expression had significantly shorter overall survival (p = 0.010). Conclusion: Obtained results strongly suggest the importance of p16 and p14 methylation analyses in combination with other parameters, particularly VEGF expression, in order to better predict treatment response and patient outcome.

Breast cancer (BC) is a heterogeneous group of diseases that still represents a major cause of death in the female population. MicroRNAs (miRNAs, miRs), such as miR-221 and miR-222, have been shown to be involved in BC pathology by acting via its target genes such as tissue inhibitor of metalloproteinase 3 (TIMP3). The main goals of this study were to find differences in miR-221/222 levels of expression in BC groups based on invasiveness, and to investigate the association with estrogen receptor (ER), TIMP3 messenger RNA (mRNA) levels, and clinicopathological characteristics of patients and tumors. In this study, we measured levels of miR-221/222 in 63 breast tissue samples by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) using TaqMan(A (R)) technology and immunohistochemistry. miR-221/222 levels varied significantly across groups based on invasiveness (P LT 0.001). In in situ tumors, miR-221 and miR-222 were negatively associated with ER (P = 0.001, r = -0.714, and P = 0.013, r = -0.585, respectively). In invasive breast carcinomas associated with non-invasive tumors, miR-222 was inversely associated with ER (P = 0.039, r = -0.620). Pure invasive BCs showed a positive correlation of miR-221 and miR-222 with TIMP3 mRNA levels (P = 0.008, r = 0.508, and P = 0.010, r = 0.497, respectively). An increase in miR-221/222 might be an important event for in situ carcinoma formation, and miR-221/222 may be important molecules that highlight potential differences between invasive breast carcinomas associated with non-invasive and pure invasive BCs.

The goal of this study was to identify host and viral factors affecting the response to pegylated interferon/ribavirin (PEG-IFN/RBV) treatment in patients with chronic hepatitis C genotype 1b. Baseline characteristics of the patients and sequences within the p7 region were analyzed in pre-treatment serum samples from 53 individuals with chronic hepatitis C genotype 1b and related to the outcome of therapy. We found a significant correlation between age and response to therapy (p LT 0.001). Furthermore, the pre-treatment viral load was closely associated with the stage of liver fibrosis (p LT 0.001). The presence of fewer than 4 mutations and age above 40 were significantly associated with non-response (NR) (p LT 0.001). Our findings may be useful for estimating the likelihood of achieving a sustained virologic response (SVR) in patients who are chronically infected with hepatitis C virus genotype 1b.

In this study, we investigated the mutation status of KRAS gene in pretherapeutic and preoperative biopsies in 63 specimens of locally advanced rectal cancers in order to evaluate its potential predictive and/or prognostic role. Regions of interest of KRAS exon 2 were amplified and visualized on 2% agarose gel. Obtained PCR products were subjected to direct sequencing. KRAS mutations were detected in 35% of patients, 91% of which were located in codon 12 and 9% in codon 13. In general, KRAS mutation status did not affect the response to neoadjuvant chemoradiotherapy (CRT). However, patients harboring mutated KRAS gene, simultaneously with high vascular endothelial growth factor (VEGF) expression, exhibited a worse response to CRT (p = 0.030), a more frequent appearance of local recurrences and distant metastasis (p = 0.003), and shorter overall survival (p = 0.001) compared to all others. On the contrary, patients with GGT GT GCT KRAS mutation exhibited a significantly better response to CRT than those with any other type of KRAS mutation (p = 0.017). Moreover, the presence of GGT GT GCT mutation was associated with low VEGF and Ki67 expression (p = 0.012 in both cases), parameters related to less aggressiveness of the disease. Our results suggest that KRAS mutation status could have some predictive and prognostic importance in rectal cancer when analyzed together with other parameters, such as VEGF and Ki67 expression. In addition, it seems that not only the presence but the type of KRAS mutation is important for examining its impact on CRT response. (C) 2016 Elsevier GmbH. All rights reserved.

Invasive ductal carcinomas with a non-invasive component (IDC-DCIS) are classified as a group of invasive breast carcinomas, together with pure invasive ductal carcinomas of the breast (IDC). MicroRNA-21 (miR-21) has been characterized as a factor of breast cancer invasiveness, however the difference in miR-21 expression levels between IDC-DCIS and pure IDC tumors and the correlations with standard diagnostic and prognostic parameters inside the IDC-DCIS group are unknown. Our aim was to determine if miR-21 had the ability to distinguish these two invasive breast cancer groups. Levels of miR-21 expression were measured by a stem-loop quantitative Real-Time PCR (RT-qPCR) method. Expression levels of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (Her-2) and proliferative index Ki-67 were evaluated by immunohistochemistry. IDC-DCIS tumors had significantly lower levels of miR-21 expression in grade 2 (P=0.003, Mann-Whitney U test), ER positive (P=0.025, Mann-Whitney U test) and PR positive tumors (P=0.024, Mann-Whitney U test) than pure IDCs. miR-21 levels showed a different pattern of expression in IDC-DCIS compared to IDC tumors, which is based on the difference in miR-21 expression between Her-2 negative and Her-2 positive IDC-DCIS tumors (P=0.030, Mann-Whitney U test) and high negative correlation of miR-21 levels with PR levels (rho=-0.886, P=0.006, Spearman correlation). According to our results, IDC-DCIS breast carcinomas act in a different manner in pure IDC tumors with regard to the relations between miR-21 expression levels and the standard diagnostic and prognostic parameters, such as Her-2 status, ER and PR status and protein levels.

Methylation-specific PCR (MSP) is still the method of choice for a single gene methylation study. The proper design of the primer pairs is a prerequisite for obtaining reliable PCR results. Despite numerous protocols describing the rules for MSP primer design, none of them provide a comprehensive approach to the problem. Our aim was to depict a workflow for the primer design that is concise and easy to follow. In order to achieve this goal, adequate tools for promoter sequence retrieval, MSP primer design and subsequent in silico analysis are presented and discussed. Furthermore, a few instructive examples regarding a good versus a poor primer design are provided. Finally, primer design is demonstrated according to the proposed workflow. This article aims to provide researchers, interested in a single gene methylation studies, with useful information regarding successful primer design.

Difuzni B-krupnoćelijski limfom (DBKL) i folikularni limfom (FL), predstavljaju najučestalije entitete nehočkinovih limfoma i zajedno čine oko 50 % svih B-ćelijskih neoplazija. To su klinički i biološki heterogena oboljenja, sa visoko varijabilnim odgovorom na terapiju i uprkos značajnom napretku u terapiji, u velikom broju slučajeva ostaju neizlečiva. Aktuelni terapijski pristupi su praćeni brojnim neželjenim efektima i dovode do dugotrajne remisije i preživljavanja kod svega 50 % bolesnika. Najznačajniji klinički prediktor ishoda bolesti do danas je internacionalni prognostički indeks (IPI za DBKL, odnosno, FLIPI za FL), koji obuhvata odgovarajuće kliničke prognostičke parametre. Uprkos tome, oboleli koji pripadaju istim IPI/FLIPI kategorijama ispoljavaju značajne razlike u odgovoru na terapiju i preživljavanju, što ukazuje na postojanje značajne biološke heterogenosti u okviru svake IPI/FLIPI kategorije. Zbog toga je neophodno pronalaženje novih prognostičkih molekularnih parametara, koji bi omogućili precizniju podelu obolelih u grupe različitog stepena rizika i izbor odgovarajuće terapije. Primenom PCR metode specifične za metilaciju (MSP), ispitivan je metilacioni status p16, p15, MGMT i DAPK gena kod obolelih od DBKL i FL, kao bi se utvrdio njihov potencijalni prognostički značaj. Kod obolelih od DBKL, metilacija nijednog gena nije pokazala prognostički značaj, mada je uočena tendencija ka akumulaciji metilacije p15 gena kod ispitanika sa povoljnijim kliničko-patološkim karakteristikama. Kod obolelih od FL metilacija p16, p15 i MGMT gena bi mogla da ima određeni prognostički značaj, ukoliko se kombinuje sa FLIPI, gradusom tumora, odnosno starosnim dobom obolelihi. Istovremena metilacija MGMT i DAPK gena bi mogla da ukaže na grupu obolelih potencijalno hemorezistentnih na primenjenu hemoterapiju i sklonu recidivima bolesti., Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) are the most comon subgroups of non-Hodgkin’s lymphoma and comprise approximately 50 % of all cases. They represents clinically and biologically heterogeneous diseases, characterized with highly variable response to the treatment, and remain incurable despite the significant advances in therapy. Actual therapy regimens are agressive, and long-term remission and survival are ashieved in only 50 % of patients. The strongest clinical predictor of outcome to date in DLBCL and FL patients is the International Prognostic Index (IPI for DLBCL and FLIPI for FL) which includes several clinico-pathological prognostic parameters. However, patients with identical IPI/FLIPI still exhibit marked variability in survival, suggesting the presence of significant biological heterogenity within the same risk category. So, it is important to investigate additional molecular markers in order to further stratify patients into diferent risk groups, choose appropriate treatement strategy and improve prognosis. In this study, methylation-specific polymerase chain reaction (MSP) was used to investigate the role and potential prognostic significance of the methylation status of p16, p15, MGMT and DAPK genes in patients with DLBCL and FL. No one of four examined genes showed prognostic significance in patients with DLBCL, though we observed a tendency toward accumulation of p15 methylation with favorable clinico-pathological parameters. However, in patients with FL, our results suggest that promoter methylation of p16, p15 and MGMT genes could have some prognostic value when used in combination with the FLIPI, tumor grade and patients age, respectively. Concurrent methylation of MGMT and DAPK genes could be the marker of tumor chemoresistance and disease recurrence.

Liposarcoma represents the most frequent group of soft tissue sarcomas. The group can be divided into three different classes: (1) differentiated/undifferentiated (WDLPS/DDLPS), (2) myxoid/round cell (MLPS/RCLPS) and (3) pleomorphic liposarcoma (PLS). It has become apparent that p53-p14 and Rb-p16 pathways play important roles in the pathogenesis of various sarcoma types. Molecular studies of the genes involved in these two pathways showed wide variations between the liposarcoma subtypes or even within the same subtype. We sought to examine mutational status of p53 and methylation status of p16(INK4a)/p14(ARF) genes in primary and recurrent liposarcoma tumors. There were twelve myxoid (12/18, 66.7 %) and six pleomorphic liposarcoma (6/18, 33.3 %) samples. Immunohistochemical analysis revealed that p53 protein was overexpressed in 3/12 MLPS (25 %) and 6/6 PLS (100 %). Mutational analysis showed that 2/11 MLPS (18.2 %) and 2/6 PLS (33.3 %) contained mutated p53 gene. On the other hand, 3/18 samples (16.7 %) had methylated p16 promoter. However, the frequencies of the p14(ARF) gene methylation were 83.3 % (10/12) and 50 % (3/6) in myxoid and pleomorphic group, respectively. Overall, 15 out of 18 (83.3 %) samples had either p53 gene mutation or methylated p14(ARF) promoter. The results from the current study suggest significant impact of the p14(ARF) gene methylation on the pathogenesis and progression of myxoid and to a lesser extent pleomorphic liposarcoma. Despite the limited number of samples, our study points to necessity of further investigation of p53-p14 and Rb-p16 pathways in liposarcoma.

The aim of the study was to assess how hypermethylation of the ON promoter of the estrogen receptor beta (ER beta) gene affects its expression (at the mRNA and protein level) and to correlate these with some clinical and histopathological parameters. A total of 131 samples of frozen breast cancer tissue was analyzed. A custom-designed, two-step PCR method was used to measure the methylation index of the ER beta gene ON promoter region. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed to quantify mRNA of the ER beta 1 isoform, while ER beta 1 protein was determined using the Western blot method. There was a significant difference in the methylation index of the ER beta gene ON promoter between the groups of patients with negative and positive axillary lymph node status (P = 0.03). In addition, the methylation index of the ON promoter was positively correlated with estrogen receptor alfa (ER alpha) protein levels (q = 0.31, P = 0.02). There was a significant difference in the methylation index of the ON promoter between the progesterone receptor (PR)-negative and PR-positive groups of patients (P = 0.01). ER beta 1 protein levels were negatively correlated with ER alpha protein (q = -0.27, P LT 0.01). The methylation index of the ON promoter could be a more reliable additional parameter for prediction and/or prognosis in breast cancer than ER beta 1-mRNA and/or protein levels.

In this study, methylation-specific polymerase chain reaction was used to investigate the role and potential prognostic significance of the methylation status of p16, p15, MGMT and DAPK genes in 32 specimens of follicular lymphoma (FL). Hypermethylation of p15 gene was associated with lower hemoglobin level (P = 0.020) and MGMT/DAPK comethylation with relapsed disease (P = 0.018). Among all patients with FL, there was no significant difference in the overall survival between those with hypermethylated and unmethylated of any examined genes. Therefore, we analyzed methylation in the different groups according to FL International Prognostic Index (FLIPI) and tumor grade. In the high-risk group, patients with hypermethylated p16 gene had significant lower overall survival than those with unmethylated p16 (P = 0.006) and trend toward shorter failure-free survival (P = 0.068). In the same risk group, there was a trend toward longer overall survival for patients with hypermethylated MGMT gene, compared to those with unmethylated MGMT gene (P = 0.066). p15 methylation had impact on shorter overall survival in grade I group of patients (P = 0.013), and DAPK methylation tended to have impact on shorter failure-free survival in the whole examined group (P = 0.079). Our results suggest that promotermethylation of p16 and MGMT genes could have prognostic value when used in combination with the FLIPI and p15 methylation in combination with tumor grade. Concurrent methylation of MGMT and DAPK genes could be the marker of tumor chemoresistance and disease recurrence.

In this study, methylation-specific polymerase chain reaction (MS-PCR) was used to define the methylation status of the target promoter sequences of p15 and MGMT genes in the group of 21 adult patients with acute myeloid leukemia (AML). The incidence of aberrant hypermethylation of p15 gene (71 %) was higher comparing to MGMT gene (33 %), whereas concomitant methylation of both genes had 24 % of the patients. Although the incidence of cytogenetic abnormalities between the groups with a different methylation status of p15 and/or MGMT genes was not significantly different, we observed general trend of clustering of abnormalities with adverse prognosis into groups with concomitant hypermethylation of both genes and only p15 gene. Also, we showed that AML patients with concomitant methylation of p15/MGMT genes had a higher proportion of leukemic blast cells characterized with specific expression of individual leukocyte surface antigens (CD117(+)/CD7(+)/CD34(+)/CD15(-)), indicating leukemic cells as early myeloid progenitors. Although we could not prove that hypermethylation of p15 and/or MGMT genes is predictive parameter for response to therapy and overall survival, we noticed that AML patients with comethylated p15/MGMT genes or methylated p15 gene exhibited a higher frequency of early death, lower frequency of complete remissions as well as a trend for shorter overall survival. Assessing of the methylation status of p15 and MGMT genes may allow stratification of patients with AML into distinct groups with potentially different prognosis.

The methylation status of the target promoter sequences of p15(INK4B) (p15) and O-6-methylguanine-DNA methyltransferase (MGMT) genes was studied by methylation-specific PCR in 10 adult patients with de novo B-cell non-Hodgkin lymphoma (B-NHL). The aberrant hypermethylation of the p15 gene was more frequent (50%) compared to the hypermethylation of the MGMT gene (30%), and was detected in different types of B-NHL in both genes. Hypermethylation of the MGMT gene occurred exclusively in association with the hypermethylation of the p15 gene. All lymphoma patients with hypermethylation of the p15 and/or MGMT genes had a higher clinical stage of the disease (IV - V). We show the association of anemia and/or thrombocytopenia with the hypermethylation of the p15 gene, ascribing the p15 gene as a potential prognostic marker in B-NHL. Comethylation of MGMT with the p15 gene represents a novel finding and presents both genes as candidates for future studies of the hypermethylation profiles of B-NHL.

AIM: To investigate the significance of p16 and O-6- methylguanine-DNA methyltransferase (MGMT) genes promoter hypermethylation and K-ras mutations on colorectal tumorigenesis and progression. METHODS: p16 and MGMT methylation status was examined on 47 tumor samples, and K-ras mutational status was examined on 85 tumor samples. For methylation analysis, a methylation specific PCR (MS-PCR) method was used. RESULTS: p16 and MGMT promoter methylation was found in 51% (24/47) and 43% (20/47) of CRCS, respectively, and the K-ras mutation was found in 44% (37/85) of CRCs. Comethylation of p16 and MGMT genes was significantly associated with lower aggressiveness of the disease within a two-year period of observation. Only 27% of patients with simultaneous p16 and MGMT methylation showed the detectible occurrence of metastasis and/or death, compared to 67% of patients without double methylation or with no methylation (3/11 vs 22/33, P LT 0.05, chi(2)-test). In addition, p16 and MGMT comethylation showed a trend toward an association with longer survival in patients with CRCs (35.5 +/- 6.0 mo vs 23.1 +/- 3.2 mo, P = 0.072, Log-rank test). Progression of the disease within a two-year period was observed in 66% of patients carrying the K-ras mutation, compared to only 19% of patients with wild type K-ras (29/44 vs 7/37, P LT 0.001, chi(2)-test). The presence of the K-ras mutation significantly correlated to shortened overall survival (20.0 +/- 1.9 mo vs 37.0 +/- 1.8 mo, P LT 0.001, Log-rank test). The comethylation of p16 and MGMT genes was significantly associated with lower aggressiveness of the disease even when K-ras mutations were included in the analysis as an independent variable. CONCLUSION: Our data suggest that comethylation of promoters of p16 and MGMT genes could have a prognostic value in patients with CRC. Specifically, concurrent methylation of both genes correlates with better prognosis. (C) 2007 The WJG Press. All rights reserved.