BACKGROUND: Glioblastoma is the commonest malignant brain tumor and has a very poor prognosis. Reduced expression of the MGMT gene (10q26.3), influenced primarily by the methylation of two differentially methylated regions (DMR1 and DMR2), is associated with a good response to temozolomide treatment. However, suitable methods for detecting the methylation of the MGMT gene promoter and setting appropriate cutoff values are debated. RESULTS: A cohort of 108 patients with histologically and genetically defined glioblastoma was retrospectively examined with methylation-specific Sanger sequencing (sSeq) and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) methods. The DMR2 region was methylated in 29% of samples, whereas DMR1 was methylated in 12% of samples. Methylation detected with the MS-MLPA method using probes MGMT_215, MGMT_190, and MGMT_124 from the ME012-A1 kit (located in DMR1 and DMR2) correlated with the methylation of the corresponding CpG dinucleotides detected with sSeq (p = 0.005 for probe MGMT_215; p < 0.001 for probe MGMT_190; p = 0.016 for probe MGMT_124). The threshold for methylation detection with the MS-MLPA method was calculated with a ROC curve analysis and principal components analysis of the data obtained with the MS-MLPA and sSeq methods, yielding a weighted value of 0.362. Thus, methylation of the MGMT gene promoter was confirmed in 36% of samples. These patients had statistically significantly better overall survival (p = 0.003). CONCLUSIONS: Our results show that the threshold for methylation detection with the MS-MLPA method determined here is useful from a diagnostic perspective because it allows the stratification of patients who will benefit from specific treatment protocols, including temozolomide. Detailed analysis of the MGMT gene promoter enables the more-precise and personalized treatment of patients with glioblastoma.

• Klíčová slova
• MGMT, Glioblastoma, MS-MLPA, Methylation, Sanger sequencing, Stupp protocol,
• MeSH
• DNA modifikační methylasy genetika   MeSH
• dospělí MeSH
• enzymy opravy DNA * genetika   MeSH
• glioblastom * genetika   farmakoterapie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• metylace DNA * MeSH
• multiplexová polymerázová řetězová reakce * metody   MeSH
• nádorové supresorové proteiny genetika   MeSH
• nádory mozku * genetika   farmakoterapie   MeSH
• promotorové oblasti (genetika) MeSH
• retrospektivní studie MeSH
• sekvenční analýza DNA * metody   MeSH
• senioři MeSH
• temozolomid MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• validační studie MeSH
• Názvy látek
• DNA modifikační methylasy MeSH
• enzymy opravy DNA * MeSH
• MGMT protein, human MeSH Prohlížeč
• nádorové supresorové proteiny MeSH
• temozolomid MeSH

Epigenetic changes are considered to be a frequent event during tumour development. Hypermethylation of promoter CpG islands represents an alternative mechanism for inactivation of tumour suppressor genes, DNA repair genes, cell cycle regulators and transcription factors. The aim of this study was to investigate promoter methylation of specific genes in ovarian cancer by comparison with normal ovarian tissue. To search for epigenetic events we used methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) to compare the methylation status of 69 tissue samples of ovarian cancer with 40 control samples. Using a 15% cut-off for methylation, we observed significantly higher methylation in genes MGMT, PAX5, CDH13, WT1, THBS1, GATA5 in the ovarian cancer group, while in the ESR1 gene we observed significantly higher methylation in the control group compared with the ovarian cancer group. These findings could potentially be used in screening of ovarian cancer and may have implications for future chemotherapy based on epigenetic changes.

• MeSH
• dospělí MeSH
• geny nádorové genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• metylace DNA genetika   MeSH
• multiplexová polymerázová řetězová reakce metody   MeSH
• nádory vaječníků genetika   patologie   MeSH
• promotorové oblasti (genetika) MeSH
• studie případů a kontrol MeSH
• tumor supresorové geny * MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Epigenetic changes are considered to be a frequent event during tumor development. Hypermethylation of promoter CpG islands represents an alternative mechanism for inactivation of tumor suppressor genes, DNA repair genes, cell cycle regulators and transcription factors. The aim of this study was to investigate promoter methylation of specific genes in endometrial cancer by comparison with normal endometrial tissue. MATERIALS AND METHODS: We used MS-MLPA (Methylation-specific Multiplex ligation-dependent probe amplification) to compare the methylation status of 59 tissue samples of endometroid type of endometrial carcinoma with 20 control samples of non-neoplastic endometrium. RESULTS: Using 15% cut-off for methylation, we observed significantly higher methylation in the CDH13 gene in endometrial cancer group. We observed significantly higher methylation in both WT1 and GATA5 genes in IB stage of endometroid carcinoma. We also observed significantly higher methylation in GATA5 gene in the group of poorly differentiated endometroid carcinoma. CONCLUSION: The findings suggest the importance of hypermethylation of CDH13, WT1 and GATA5 genes in endometrial carcinogenesis and could have implications for future diagnostic and therapeutic strategies of endometrial cancer based on epigenetic changes.

• MeSH
• dospělí MeSH
• karcinom genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• metylace DNA * MeSH
• multiplexová polymerázová řetězová reakce MeSH
• nádory endometria genetika   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• tumor supresorové geny * MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Epigenetic modifications have been recognized as an important mechanism underlying carcinoma progression. DNA methylation plays an important role in cancer biology and represents potentially heritable changes in gene expression that do not involve DNA sequence. The aim of this study was to investigate promoter methylation of selected genes in sinonasal carcinoma by comparison with noncancerous sinonasal tissue. METHODS: To search for epigenetic events (methylation in 25 tumor suppressor genes) we used MS-MLPA (Methylation-specific multiplex ligation-dependent probe amplification) to compare methylation status of 59 formalin fixed, paraffin embedded tissue samples of sinonasal carcinomas with 18 control samples. The most important changes in methylation were confirmed using MSP (Methylation specific PCR). Detected alterations in methylation were compared with clinicopathological characteristics. RESULTS: Using a 20% cut-off for methylation (MS-MLPA), we found significantly higher methylation in GATA5 (P=0.0005), THSB1 (P=0.0002) and PAX5 (P=0.03) genes in the sinonasal cancer group compared to the control group. Methylation in five or more genes was associated with impaired overall survival (P=0.017). CONCLUSION: These findings provide evidence that alterations in methylation profile may be one of the major mechanisms in sinonasal carcinogenesis. In addition, changes in methylation could potentially be used as prognostic factors of sinonasal carcinoma and may have implications for future individualized therapy based on epigenetic changes.

• Klíčová slova
• DNA methylation, biomarkers, epigenetics, sinonasal carcinoma,
• MeSH
• dospělí MeSH
• epigeneze genetická genetika   MeSH
• geny nádorové genetika   MeSH
• Kaplanův-Meierův odhad MeSH
• lidé středního věku MeSH
• lidé MeSH
• metylace DNA fyziologie   MeSH
• mladý dospělý MeSH
• nádorové biomarkery fyziologie   MeSH
• nádory vedlejších dutin nosních diagnóza   genetika   mortalita   MeSH
• prognóza MeSH
• promotorové oblasti (genetika) genetika   MeSH
• retrospektivní studie MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• nádorové biomarkery MeSH

The aim of the study was detailed clinicopathological investigation of SMARCB1/INI1-deficient sinonasal carcinomas, including molecular genetic analysis of mutational status and DNA methylation of selected protooncogenes and tumor suppressor genes by means of next generation sequencing (NGS) and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). A total of 4/56 (7%) cases of SMARCB1/INI1-deficient carcinomas were detected among 56 sinonasal carcinomas diagnosed over a 19year period using immunohistochemical screening. The series comprised 3 males and 1 female, aged 27-76 years (median 64 years). All tumors arose in the nasal cavity. Three neoplasms were diagnosed in advanced stage pT4. During the follow-up period (range 14-111 months (median 72 months)), three tumors recurred locally, but none of the patients developed regional or distant metastases. Ultimately, two patients died due to the tumor. Microscopically, all tumors consisted of infiltrating nests of polygonal basaloid cells with a variable component of rhabdoid cells with eosinophilic cytoplasm. Immunohistochemically, there was almost diffuse expression of cytokeratins (CK), p16, p40 and p63 in all cases, while expression of CK5/6, CK7 and vimentin was only focal or absent. The detection of NUT gave negative results. In three cases, the absence of SMARCB1/INI1 expression was due to deletion of SMARCB1/INI1 gene. Methylation of SMARCB1/INI1 gene was not found. One tumor harbored HPV18 E6/E7 mRNA. All 12 genes (BRAF, BRCA1, BRCA2, KIT, EGFR, KRAS, NRAS, PDGFRA, PIK3CA, PTEN, RET, and ROS1) tested for mutations using NGS were wild-type. Regarding DNA methylation, all four SMARCB1/INI1-deficient tumors showed methylation of RASSF1 gene by means of MS-MLPA. There was a statistically significant difference in RASSF1 gene methylation between SMARCB1/INI1-deficient and SMARCB1/INI1-positive tumors (p=0.0095). All other examined genes (ATM, BRCA1, BRCA2, CADM1, CASP8, CD44, CDKN1B, CDKN2A, CDKN2B, CHFR, DAPK1, ESR1, FHIT, GSTP1, HIC1, KLLN, MLH1a, MLH1b, RARB, and VLH) were unmethylated. In summary, we described four cases of SMARCB1/INI1-deficient sinonasal carcinoma with detailed clinicopathological data indicating that these tumors can be regarded as a distinct entity with aggressive behaviour. For the first time, we performed analysis of DNA methylation in SMARCB1/INI1-deficient sinonasal carcinomas, reporting on significantly higher methylation of RASSF1 gene in this neoplasm.

• Klíčová slova
• DNA methylation, RASSF1, Rhabdoid, SMARCB1/INI1, Sinonasal tract, Squamous cell carcinoma,
• MeSH
• dospělí MeSH
• gen SMARCB1 genetika   metabolismus   MeSH
• imunohistochemie MeSH
• karcinom genetika   metabolismus   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• metylace DNA * MeSH
• nádorové biomarkery genetika   metabolismus   MeSH
• nádorové supresorové proteiny genetika   metabolismus   MeSH
• nádory sinu maxillaris genetika   metabolismus   patologie   MeSH
• senioři MeSH
• staging nádorů MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• gen SMARCB1 MeSH
• nádorové biomarkery MeSH
• nádorové supresorové proteiny MeSH
• RASSF1 protein, human MeSH Prohlížeč

INTRODUCTION: Among children born small for gestational age, 10-15% fail to catch up and remain short (SGA-SS). The underlying mechanisms are mostly unknown. We aimed to decipher genetic aetiologies of SGA-SS within a large single-centre cohort. METHODS: Out of 820 patients treated with growth hormone (GH), 256 were classified as SGA-SS (birth length and/or birth weight <-2 SD for gestational age and life-minimum height <-2.5 SD). Those with the DNA triplet available (child and both parents) were included in the study (176/256). Targeted testing (karyotype/FISH/MLPA/specific Sanger sequencing) was performed if a specific genetic disorder was clinically suggestive. All remaining patients underwent MS-MLPA to identify Silver-Russell syndrome, and those with unknown genetic aetiology were subsequently examined using whole-exome sequencing or targeted panel of 398 growth-related genes. Genetic variants were classified using ACMG guidelines. RESULTS: The genetic aetiology was elucidated in 74/176 (42%) children. Of these, 12/74 (16%) had pathogenic or likely pathogenic (P/LP) gene variants affecting pituitary development (LHX4, OTX2, PROKR2, PTCH1, POU1F1), the GH-IGF-1 or IGF-2 axis (GHSR, IGFALS, IGF1R, STAT3, HMGA2), 2/74 (3%) the thyroid axis (TRHR, THRA), 17/74 (23%) the cartilaginous matrix (ACAN, various collagens, FLNB, MATN3), and 7/74 (9%) the paracrine chondrocyte regulation (FGFR3, FGFR2, NPR2). In 12/74 (16%), we revealed P/LP affecting fundamental intracellular/intranuclear processes (CDC42, KMT2D, LMNA, NSD1, PTPN11, SRCAP, SON, SOS1, SOX9, TLK2). SHOX deficiency was found in 7/74 (9%), Silver-Russell syndrome in 12/74 (16%) (11p15, UPD7), and miscellaneous chromosomal aberrations in 5/74 (7%) children. CONCLUSIONS: The high diagnostic yield sheds a new light on the genetic landscape of SGA-SS, with a central role for the growth plate with substantial contributions from the GH-IGF-1 and thyroid axes and intracellular regulation and signalling.

• Klíčová slova
• GH-IGF-1 axis, Genetics, Growth plate, Short stature, Small for gestational age,
• MeSH
• dítě MeSH
• gestační stáří MeSH
• hypotrofický novorozenec MeSH
• insulinu podobný růstový faktor I MeSH
• lidé MeSH
• lidský růstový hormon * genetika   MeSH
• nanismus * MeSH
• novorozenec MeSH
• poruchy růstu genetika   diagnóza   MeSH
• protein SHOX MeSH
• Silverův-Russellův syndrom * genetika   MeSH
• tělesná výška genetika   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• novorozenec MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• insulinu podobný růstový faktor I MeSH
• lidský růstový hormon * MeSH
• protein SHOX MeSH
• SHOX protein, human MeSH Prohlížeč

Epigenetic changes are considered to be a frequent event during tumour development. Hypermethylation of promoter CpG islands represents an alternative mechanism for inactivation of tumour suppressor genes, DNA repair genes, cell cycle regulators and transcription factors. The aim of this study was to investigate promoter methylation of specific genes in samples of sinonasal carcinoma by comparison with normal sinonasal tissue. To search for epigenetic events we used methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) to compare the methylation status of 64 tissue samples of sinonasal carcinomas with 19 control samples. We also compared the human papilloma virus (HPV) status with DNA methylation. Using a 20% cut-off for methylation, we observed significantly higher methylation in RASSF1, CDH13, ESR1 and TP73 genes in the sinonasal cancer group compared with the control group. HPV positivity was found in 15/64 (23.4 %) of all samples in the carcinoma group and in no sample in the control group. No correlation was found between DNA methylation and HPV status. In conclusion, our study showed that there are significant differences in promoter methylation in the RASSF1, ESR 1, TP73 and CDH13 genes between sinonasal carcinoma and normal sinonasal tissue, suggesting the importance of epigenetic changes in these genes in carcinogenesis of the sinonasal area. These findings could be used as prognostic factors and may have implications for future individualised therapies based on epigenetic changes.

• MeSH
• aktivace enzymů MeSH
• dlaždicobuněčné karcinomy hlavy a krku MeSH
• DNA-(cytosin-5-)methyltransferasa genetika   metabolismus   MeSH
• DNA-(cytosin-5)-methyltransferasa 1 MeSH
• epigenomika MeSH
• kadheriny genetika   metabolismus   MeSH
• lidé MeSH
• metylace DNA * MeSH
• nádory hlavy a krku diagnóza   genetika   patofyziologie   virologie   MeSH
• Papillomaviridae izolace a purifikace   MeSH
• prognóza MeSH
• promotorové oblasti (genetika) genetika   MeSH
• spinocelulární karcinom diagnóza   genetika   patofyziologie   virologie   MeSH
• tumor supresorové geny * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA-(cytosin-5-)methyltransferasa MeSH
• DNA-(cytosin-5)-methyltransferasa 1 MeSH
• H-cadherin MeSH Prohlížeč
• kadheriny MeSH

BACKGROUND AND AIMS: Genetic and epigenetic alterations play an important role in urothelial cancer pathogenesis. Deeper understanding of these processes could help us achieve better diagnosis and management of this life-threatening disease. The aim of this research was to evaluate the methylation status of selected tumor suppressor genes for predicting BCG response in patients with high grade non-muscle-invasive bladder tumor (NMIBC). MATERIALS AND METHODS: We retrospectively evaluated 82 patients with high grade non-muscle-invasive bladder tumor (stage Ta, T1, CIS) who had undergone BCG instillation therapy. We compared epigenetic methylation status in BCG-responsive and BCG-failure groups. We used the MS-MLPA (Methylation-Specific Multiplex Ligation-Dependent Probe Amplification probe sets ME001 and ME004. The control group was 13 specimens of normal urotel (bladder tissue)). RESULTS: Newly identified methylations in high grade NMIBC were found in MUS81a, NTRK1 and PCCA. The methylation status of CDKN2B (P=0.00312**) and MUS81a (P=0.0191*) is associated with clinical outcomes of BCG instillation therapy response. CDKN2B and MUS81a unmethylation was found in BCG failure patients. CONCLUSION: The results show that the methylation status of selected tumor suppressor genes (TSGs) has the potential for predicting BCG response in patients with NMIBC high grade tumors. Tumor suppressor genes such as CDKN2b, MUS81a, PFM-1, MSH6 and THBS1 are very promising for future research.

• Klíčová slova
• BCG, CDKN2b, bladder cancer, methylation,
• MeSH
• adjuvancia imunologická terapeutické užití   MeSH
• BCG vakcína terapeutické užití   MeSH
• dospělí MeSH
• imunoterapie MeSH
• lidé středního věku MeSH
• lidé MeSH
• lokální recidiva nádoru farmakoterapie   MeSH
• metylace MeSH
• nádory močového měchýře farmakoterapie   genetika   MeSH
• protinádorové látky terapeutické užití   MeSH
• retrospektivní studie MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• tumor supresorové geny * MeSH
• výsledek terapie MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• adjuvancia imunologická MeSH
• BCG vakcína MeSH
• protinádorové látky MeSH

Cervical cancer affects women worldwide, especially in developing countries. Approximately 500,000 cases of this disease are diagnosed per year. The method of choice in the treatment of advanced cervical cancers (in accordance with the International Federation of Gynecology and Obstetrics staging system (FIGO) starting from stage IIB) is combined radiotherapy with concomitant chemotherapy. This treatment provides good tumour control, but it carries a risk of late complications in the irradiated area in 10-15 % of cases. Methylation is one of the methods of epigenetic control, which has an important role in gene expression. Aberrant methylation of normal CpG islands in promoters of tumour suppressor genes such as RB, p53 or DNA reparation genes ATM, BRCA1,2, and RAD51 gene family causes silencing of their function and cell cycle deregulation, which is one of the efficient ways of neoplastic transformation. The significantly decreased expression of molecules involved in DNA response may cause facilitated radiosensitivity in predisposed individuals. We looked for the relationship between hypermethylation of 18 DNA reparation genes and late toxicity occurrence in cervical cancer patients treated by chemoradiotherapy using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). The cut-off value for the hypermethylation was set at 10 %. We confirmed significant association between promoter hypermethylation in the XRCC2 gene and occurrence of late grade III-IV toxicity in cervical cancer patients (P = 0.0357). This finding could be useful in the late toxicity prediction in radiotherapy-treated patients.

• MeSH
• chemoradioterapie MeSH
• DNA vazebné proteiny genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• metylace DNA genetika   MeSH
• nádory děložního čípku genetika   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• RAD51D protein, human MeSH Prohlížeč
• XRCC2 protein, human MeSH Prohlížeč

Gliomas are a heterogeneous group of tumours varying in prognosis, treatment approach, and overall survival. Recently, novel markers have been identified which are linked to patient prognosis and therapeutic response. Especially the mutation of the enzyme isocitrate dehydrogenase 1 or 2 (IDH1/2) gene and the O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status seem to be the most important predictors of survival. From 2012 to 2015, 94 Czech patients with primary brain tumours were enrolled into the study. The IDH1/2 mutation was detected by denaturing capillary electrophores.The methylation status of the MGMT gene and other 46 genes was revealed by MS-MLPA. In all 94 patients, the clinical data were correlated with molecular markers by Kaplan-Meier analyses and Cox regression model. The MGMT promoter methylation status was established and compared to clinical data. In our study eight different probes were used to elucidate the MGMT methylation status; hypermethylation was proclaimed if four and more probes were positive. This 3 : 5 ratio was tested and confirmed by Kaplan-Meier and Cox analyses. The study confirmed the importance of the IDH1/2 mutation and hypermethylation of the MGMT gene promoter being present in tumour tissue. Both markers are independent positive survival predictors; in the Cox model the IDH hazard ratio was 0.10 and in the case of MGMT methylation it reached 0.32. The methylation analysis of the panel of additional 46 genes did not reveal any other significant epigenetic markers; none of the candidate genes have been confirmed in the Cox regression analyses as an independent prognostic factor.

• MeSH
• DNA modifikační methylasy genetika   MeSH
• enzymy opravy DNA genetika   MeSH
• epigeneze genetická MeSH
• gliom enzymologie   genetika   MeSH
• isocitrátdehydrogenasa genetika   MeSH
• Kaplanův-Meierův odhad MeSH
• lidé středního věku MeSH
• lidé MeSH
• metylace DNA genetika   MeSH
• mutace genetika   MeSH
• nádorové supresorové proteiny genetika   MeSH
• nádory mozku enzymologie   genetika   MeSH
• přežití bez známek nemoci MeSH
• promotorové oblasti (genetika) * MeSH
• regresní analýza MeSH
• ROC křivka MeSH
• senzitivita a specificita MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• DNA modifikační methylasy MeSH
• enzymy opravy DNA MeSH
• IDH1 protein, human MeSH Prohlížeč
• IDH2 protein, human MeSH Prohlížeč
• isocitrátdehydrogenasa MeSH
• MGMT protein, human MeSH Prohlížeč
• nádorové supresorové proteiny MeSH