Methylation-specific Multiplex Ligation-dependent Probe Amplification Research Articles

Monitoring of methylation changes in 9p21 region in patients with myelodysplastic syndromes and acute myeloid leukemia

Epigenetic de novo methylation of CpG islands is an important event in malignant transformation. Two genes are frequently methylated: cyclin-dependent kinase inhibitor 2B (CDKN2B) and cyclin-dependent kinase inhibitor 2A (CDKN2A). In our study methylation of these genes was studied in 63 patients with myelodysplastic syndromes (MDS), 2 with myelodysplastic/myeloproliferative neoplasms (MDS/MPN) and 13 with acute myeloid leukemia (AML). Five patients were monitored during 5-azacytidine treatment. Twenty-six healthy donors were tested in a control group. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) method with all associated techniques was used for detection. Aberrant methylation was present in the CDKN2A gene in 38% and in the CDKN2B gene in 77% of the patients in MDS group. The level of methylation was higher in the group of AML patients - 77% in CDKN2A gene and 100% in CDKN2B gene. In MDS patients, an aberrant methylation was associated with a tendency to disease progression towards more advanced forms according to the World Health Organization (WHO) classification and the International Prognostic Scoring System (IPSS). Significant differences in methylation level were observed between early and advanced forms of MDS in CDKN2B gene (P value < 0.05) but not for CDKN2A gene. The trend of methylation in patients treated with azacitidine was analyzed in CDKN2B gene and correlated with the course of the disease. Increased methylation was connected with disease progression. We concluded that the methylation level of CDKN2B gene might be used as a marker of leukemic transformation in MDS. Our study indicates the role of hypermethylation as an important event in the progression of MDS to AML.

Promoter hypermethylation patterns in fallopian tube epithelium of BRCA1 and BRCA2 germ line mutation carriers

BRCA1/2 germ line mutation carriers have a high risk of developing fallopian tube carcinoma (FTC), thought to occur through different early (p53 signatures) and later (dysplasia, intra-epithelial carcinoma) premalignant stages. Promoter hypermethylation of tumour suppressor genes is known to play a key role in (early) carcinogenesis. However, little is known about methylation in normal and (pre)malignant fallopian tube tissue. We identified 14 areas of p53 accumulation in the fallopian tubes of BRCA mutation carriers. Cells from these areas were harvested together with cells from adjacent benign appearing areas. An age-matched non-BRCA sporadic control group (n=13) and eight sporadic FTCs were included as negative and positive controls respectively. Methylation-specific multiplex ligation-dependent probe amplification was used to assess promoter methylation of 70 tumour suppressor genes in all samples. We observed a gradual increase in methylation from sporadic control tissue (median cumulative methylation index (CMI) 568.19) through normal tissue and from areas of p53 accumulation in BRCA carriers (median CMI 687.54 and 676.72) to FTC (median CMI 780.97). Furthermore, the methylation percentage of many individual tumour suppressor genes differed significantly between these groups, gradually increasing as for CMI. Between areas with and without p53 accumulation in BRCA mutation carriers no significant differences were found. In this paper, we have shown that BRCA mutation carriers display increased methylation of tumour suppressor genes in their non-malignant fallopian tube epithelium, closer to methylation levels in FTC than to normal sporadic tissue. Methylation could, therefore, play an important role in the increased risk of gynaecological malignancies in BRCA mutation carriers.

Altered Methylation of the DNA Repair Gene MGMT Is Associated with Neural Tube Defects

DNA repair is critical for proper embryogenesis, and altered expression of DNA repair genes has been associated with neural tube defects (NTDs). The expression of DNA repair enzymes may be controlled, in part, by methylation of the promoter region. To assess whether disturbed promoter methylation pattern increases the incidence of NTDs, we employed methylation specific-multiplex ligation-dependent probe amplification (MS-MLPA) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to quantify the methylation levels of multiple promoter CpG sites in normal human embryos and embryos with NTDs. Of the total seven DNA repair genes, two genes (MGMT, MSH6) were examined as having disturbed methylation levels by MS-MLPA kit, while only one gene was confirmed with a significant alternated methylation pattern by MALDI-TOF MS. In our research, methylation profiling of the DNA repair gene O (6)-methylguanine-DNA methyltransferase (MGMT) showed gender difference in embryogenesis. Comparison of MGMT promoter methylation revealed that hypomethylation was associated with an increased risk for cephalic malformations, especially with female embryos (Adjusted Odds Ratio = 8.250). The majority of individual CpG units within the promoter demonstrated hypomethylation. Meanwhile, the expression of MGMT was proven to increase significant in female cases. These results underscore the role of stable promoter methylation in the DNA repair enzymes MGMT for proper embryogenesis.

Unique and atypical deletions in Prader–Willi syndrome reveal distinct phenotypes

Prader-Willi syndrome (PWS) is a multisystem, contiguous gene disorder caused by an absence of paternally expressed genes within the 15q11.2-q13 region via one of the three main genetic mechanisms: deletion of the paternally inherited 15q11.2-q13 region, maternal uniparental disomy and imprinting defect. The deletion class is typically subdivided into Type 1 and Type 2 based on their proximal breakpoints (BP1-BP3 and BP2-BP3, respectively). Despite PWS being a well-characterized genetic disorder the role of the specific genes contributing to various aspects of the phenotype are not well understood. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) is a recently developed technique that detects copy number changes and aberrant DNA methylation. In this study, we initially applied MS-MLPA to elucidate the deletion subtypes of 88 subjects. In our cohort, 32 had a Type 1 and 49 had a Type 2 deletion. The remaining seven subjects had unique or atypical deletions that were either smaller (n=5) or larger (n=2) than typically described and were further characterized by array-based comparative genome hybridization. In two subjects both the PWS region (15q11.2) and the newly described 15q13.3 microdeletion syndrome region were deleted. The subjects with a unique or an atypical deletion revealed distinct phenotypic features. In conclusion, unique or atypical deletions were found in ∼8% of the deletion subjects with PWS in our cohort. These novel deletions provide further insight into the potential role of several of the genes within the 15q11.2 and the 15q13.3 regions.

Methylation-Specific Multiplex Ligation-Dependent Probe Amplification and Identification of Deletion Genetic Subtypes in Prader-Willi Syndrome

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are complex neurodevelopmental disorders caused by loss of expression of imprinted genes from the 15q11-q13 region depending on the parent of origin. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) kits from MRC-Holland (Amsterdam, The Netherlands) were used to detect PWS and AS deletion subtypes. We report our experience with two versions of the MS-MLPA-PWS/AS kit (original A1 and newer B1) in determining methylation status and deletion subtypes in individuals with PWS. MS-MLPA analysis was performed on DNA isolated from a large cohort of PWS subjects with the MS-MLPA-PWS/AS-A1 and -B1 probe sets. Both MS-MLPA kits will identify deletions in the 15q11-q13 region but the original MS-MLPA-A1 kit has a higher density of probes at the telomeric end of the 15q11-q13 region, which is more useful for identifying individuals with atypical deletions. The newer B1 kit contains more probes in the imprinting center (IC) and adjoining small noncoding RNAs useful in identifying small microdeletions. The A1 kit identified the typical deletions and smaller atypical deletions, whereas the B1 kit was more informative for identifying microdeletions including the IC and SNORD116 regions. Both kits should be made available for accurate characterization of PWS/AS deletion subtypes as well as evaluating for IC and SNORD116 microdeletions.

Detection of p16INK4a Promoter Methylation Status &amp;1QJ;in Non-small Cell Lung Cancer by a Fluorescence Polarization Assay

The detection of the p16INK4a promoter methylation status has a good value for the prognosis, early detection, and individualized management of patients with non-small cell lung cancer. A novel method detecting the p16INK4a promoter methylation status of primary carcinoma tissue samples by a fluorescence polarization assay was developed in this research. A pair of general primers was used to amplify a 305-basepair fragment in the promoter region of p16INK4a. Two probes specific for either methylated p16INK4a or unmethylated p16INK4a DNA labeled with either tetramethyl 6-carboxyrhodamine or 6-carboxy-fluorescein hybridized, respectively, with their target amplicons, and the hybridization increased the fluorescence polarization values. The p16INK4a promoter methylation status was determined by the analysis of the fluorescence polarization values. One hundred and twenty-nine non-small cell lung cancer samples were analyzed in parallel with a fluorescence polarization assay and a gel-based methylation-specific polymerase chain reaction (PCR) assay. There was no significant difference between the results of the p16INK4a promoter methylation status obtained with the fluorescence polarization assay and the results obtained with the gel-based methylation-specific PCR assay. The minimum detection level of the fluorescence polarization assay was 25 copies/μL. The fluorescence polarization assay allowed the semiautomated detection of the methylated p16INK4a and unmethylated p16INK4a promoters directly in the solution with 1 PCR cycle, and it was much simpler than methylation-specific PCR and methylation-specific multiplex ligation-dependent probe amplification assays.

Using MS‐MLPA as an efficient screening tool for detecting 9p21 abnormalities in pediatric acute lymphoblastic leukemia

Characterization of recurrent genetic lesions in childhood acute lymphoblastic leukemia (ALL) has enabled therapeutic stratification with improved outcomes. The tumor suppressor genes, CDKN2A and CDKN2B, encoding p16(INK4a) , p14(ARF) , and p15(INK4b) have been localized to 9p21. Abnormalities of 9p21 have been reported in 10-30% of childhood ALL using conventional cytogenetics and fluorescence in situ hybridization (FISH). The incidence of 9p21 using more sensitive techniques, such as methylation specific multiplex ligation-dependent probe amplification (MS-MLPA), remains uncertain, and thus also the prognostic significance. We investigated the incidence and prognostic importance of 9p21 abnormalities in pediatric ALL patients using MS-MLPA and compared these results to FISH. In total, MS-MLPA or FISH detected aberrations (both dosage and methylation abnormalities) at 9p21 in a remarkable 32/48 (67%) patients in contrast to a much lower rate of only 8% of patients identified to have deletions by standard G banding cytogenetics. MS-MLPA identified five deletions not found by FISH. Aberrant methylation at CDKN2B was found in 19 (46%) patients. 9p21 abnormalities were associated with National Cancer Institute (NCI) high-risk criteria (P = 0.04) and were present in all five patients with T-cell disease. Four pre-B-cell ALL patients relapsed, three of whom had prior 9p21 abnormalities. MS-MLPA had a higher detection rate for 9p21 abnormalities than previously reported for other techniques. Given the ease of processing, minimal equipment and low cost of MS-MLPA, our results suggest that previous reports may have underestimated the true frequency of 9p21 abnormalities and their potential impact upon ALL outcome.

MGMT promoter gene methylation in pediatric glioblastoma: analysis using MS-MLPA

Promoter methylation of the O⁶-methylguanine-DNA-methyltransferase (MGMT) gene is widely recognized as an important predictive factor in the treatment of glioblastoma (GBM) patients with temozolomide. However, data regarding the methylation status of the MGMT promoter in pediatric GBM are yet to be elucidated. Nineteen tissue samples of pediatric GBM were evaluated for the MGMT promoter methylation status using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). Methylation status was also evaluated using methylation-specific polymerase chain reaction (MSP) for 17 of the 19 patients. The correlation between MGMT promoter methylation and clinical outcome was assessed. Three of the 19 patients (16%) showed methylation of the MGMT promoter, according to MS-MLPA, as did 1 of the 17 (6%), according to MSP. The methylation status did not seem to have a definite effect on clinical outcome. Pediatric GBMs rarely have methylated MGMT promoters. With a better clinical outcome and lower methylation rate than their adult counterparts, it may be suggested that, for pediatric GBM, MGMT promoter methylation does not play a significant role as a prognostic factor.

A methylation assay for the detection of non‐muscle‐invasive bladder cancer (NMIBC) recurrences in voided urine

To develop a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay for the detection of non-muscle invasive bladder cancer (NMIBC) recurrences in voided urine. Genes frequently methylated in NMIBC tumours (n= 37) were selected to develop a BC-specific MS-MLPA assay. Genes methylated in blood from patients with BC (n= 29) and genes methylated in urine from patients with no history of BC (n= 46) were excluded. A four-gene panel with the highest predictive value was selected from the initial assay. This four-gene panel was tested and validated on urine from patients with a histologically confirmed recurrence (n= 68 test set; n= 49 validation set) and urine samples from patients without BC (n= 91, test set) and urine from recurrence-free BC (rec-free BC) patients (n= 60, validation set). A model was developed to predict the probability of having a recurrence based on methylation of the four-gene panel and a threshold probability with the highest sensitivity and specificity was determined. The outcome of the model was validated on BC urine samples (n= 65) and on urine samples from rec-free BC patients (n= 29). The BC MS-MLPA assay consisted of 23 methylation probes. The selected four-gene panel included: APC_a, TERT_a, TERT_b, and EDNRB. This panel reached an area under the receiver operating characteristic curve (AUC) of 0.82 (test set) and AUC 0.69 (validation set). Sensitivity and specificity for the detection of a concomitant tumour were 63.3% and 58.3% respectively (test set) and 72.3% and 55.2%, respectively (validation set). We have developed a methylation detection assay specifically for the detection of recurrences in patients with NMIBC in voided urine. The findings are promising and improvement of this test could eventually contribute to a more individualized patient friendly surveillance.

Clinical characterization and identification of two novel mutations of the GNAS gene in patients with pseudohypoparathyroidism and pseudopseudohypoparathyroidism

Pseudohypoparathyroidism (PHP) and pseudopseudohypoparathyroidism (PPHP) are rare disorders resulting from genetic and epigenetic aberrations in the GNAS locus. Investigation of clinical characteristics and molecular analysis in PHP and PPHP. Fourteen subjects from 13 unrelated families including subjects with PPHP (n = 1), PHP-Ia (n = 6) and PHP-Ib (n = 7) were enrolled. Clinical data, including age at presentation, presenting symptom, auxological findings, family history, presence of Albright hereditary osteodystrophy (AHO) features and hormonal and biochemical findings, were analysed. The GNAS locus was subjected to direct sequencing and methylation analysis using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). Of the 13 PHP subjects, 10 (three PHP-Ia and seven PHP-Ib) presented with hypocalcemic tetany at ages ranging from 7 to 14·8 years. Subcutaneous calcification was observed as an early manifestation of AHO in one PHP-Ia patient (age, 2·9 years) and one PPHP patient (age, 7 months). Six PHP-Ia and one PPHP harboured four different heterozygous mutations within the coding region of GNAS, p.Asp189_Tyr190delinsMetfxX14, p.Val117fsX23, p.Tyr190CysfsX19, and a splicing mutation (c.659 + 1G>A), of which the latter two were novel. Five subjects with PHP-Ib exhibited complete loss of the maternal-specific methylation pattern. The remaining two PHP-Ib showed a loss of methylation of exon 1A on the maternal allele as a consequence of heterozygous 3-kb microdeletions within the STX16 gene. GNAS mutation analyses and MS-MLPA assays are useful molecular tools for understanding the molecular bases and confirming the diagnosis of PHP and PPHP.

Testing of Buccal Swab DNA Does Not Increase the Detection Rate for Imprinting Control Region 1 Hypomethylation in Silver-Russell Syndrome

Silver-Russell syndrome (SRS) is a congenital imprinting disorder mainly characterized by growth restriction, a triangular shaped face, a relative macrocephaly, and asymmetry of the body and the limbs. In 7%-10% of the patients a maternal uniparental disomy of chromosome 7 (upd(7)mat) can be observed; a hypomethylation of the imprinting control region 1 (ICR1) in 11p15 is present in >38% of patients. Methylation-specific multiplex ligation-dependent probe amplification is a well-established method for the detection of (epi)mutations in 11p15. In routine diagnostics, DNA samples derived from leukocytes are used for this testing approach. We now analyzed buccal smear DNA taken from both cheek sides of 8 carriers of an ICR1 hypomethylation and 25 SRS patients without 11p15 epimutaton or upd(7)mat to check whether (i) the epimutation can be detected in other tissues and (ii) the detection rate can be increased. Indeed, the ICR1 hypomethylation diagnosed in blood cells could be confirmed in the buccal swab DNA of all 11p15 epimutation carriers, but we could not discover any further carriers among the patients without 11p15 epimutation and upd(7)mat in lymphocytes. Thus, the overall detection rate for the 11p15 epimutation could not be increased by including further tissues originating from different germ layers. We rather assume that other-so far unknown-(genetic) factors are contributing to the etiology of SRS that escape the current diagnostic procedures.

Evaluation of the Methylation Status of Tumour Suppressor Genes for Predicting Bacillus Calmette-Guérin Response in Patients With T1G3 High-Risk Bladder Tumours

BackgroundBacillus Calmette-Guérin (BCG) is a standard treatment for reducing tumour recurrence and delaying progression of high-risk non–muscle-invasive bladder tumours. However, it is not clear yet which patients are more likely to respond to BCG. ObjectiveTo evaluate the role of the methylation of 25 tumour suppressor genes (TSG) as clinical outcome predictive biomarkers in T1G3 bladder tumours treated with BCG. Design, setting, and participantsA retrospective design included 91 paraffin-embedded tumours of patients with T1G3 primary non–muscle-invasive disease undergoing nonmaintenance BCG treatment. MeasurementsThe methylation status of 25 TSGs was measured using a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay. Recurrence, progression into muscle-invasive tumours, and disease-specific survival (DSS) rates were analysed using univariate and multivariate tests. Results and limitationsThe genes most frequently methylated included STK11 (94.5%), MSH6 (81.3%), BRCA1 (72.5%), PAX5A (68.1%), MGMT (67.0%), CDH13 (62.6%), and IGSF4 (61.5%). Methylation was newly identified in T1G3 tumours for TP73, MSH6, ESR1, PAX5A, WT1, CD44, ATM, IGSF4, CHFR, BRCA2, THBS1, PYCARD, STK11, and GATA5. Methylation for several TSGs was significantly associated with multifocality and tumour size. Patients with different methylation statuses of TSGs showed differential recurrence rates (PAX6: p=0.025), progression rates (MSH6: p=0.040; RB1: p=0.042; THBS1: p=0.041; PYCARD: p=0.048; TP73: p=0.048; ESR1: p=0.036; and GATA5: p=0.019), and DSS rates (GATA5: p=0.037). Several combinations improved prediction for progression. Multivariate analyses indicated that among the combinations remaining as independent predictors, two genes—MSH6 and THBS1—already provided the most significant predictive assessment for progression (p=0.004). The major limitation of this study is related to its retrospective design. ConclusionsThe methylation status of TSGs was associated with the clinical outcome of patients with T1G3 tumours undergoing BCG treatment under three clinical end points: recurrence, progression, and DSS. The methylation status of TSGs distinguished patients responding to BCG from those who may require a more aggressive therapeutic intervention.

DNA Methylation in Thyroid Tumorigenesis

Thyroid cancer is the most common endocrine cancer with 1,690 deaths each year. There are four main types of which the papillary and follicular types together account for >90% followed by medullary cancers with 3% to 5% and anaplastic carcinomas making up <3%. Epigenetic events of DNA hypermethylation are emerging as promising molecular targets for cancer detection. Our immediate and long term goal is to identify DNA methylation markers for early detection of thyroid cancer. This pilot study comprised of 21 patients to include 11 papillary thyroid cancers (PTC), 2 follicular thyroid cancers (FTC), 5 normal thyroid cases, and 3 hyperthyroid cases. Aberrant promoter methylation was examined in 24 tumor suppressor genes using the methylation specific multiplex ligation-dependent probe amplification (MS-MLPA) assay and in the NIS gene using methylation-specific PCR (MSP). The frequently methylated genes were CASP8 (17/21), RASSF1 (16/21) and NIS (9/21). In the normal samples, CASP8, RASSF1 and NIS were methylated in 5/5, 4/5 and 1/5 respectively. In the hyperthyroid samples, CASP8, RASSF1 and NIS were methylated in 3/3, 2/3 and 1/3 respectively. In the thyroid cancers, CASP8, RASSF1, and NIS were methylated in 9/13, 10/13, and 7/13 respectively. CASP8, RASSF1 and NIS were also methylated in concurrently present normal thyroid tissue in 3/11, 4/11 and 3/11 matched thyroid cancer cases (matched for presence of both normal thyroid tissue and thyroid cancer), respectively. Our data suggests that aberrant methylation of CASP8, RASSF1, and NIS maybe an early change in thyroid tumorigenesis regardless of cell type.

Altered Methylation at MicroRNA-Associated CpG Islands in Hereditary and Sporadic Carcinomas: A Methylation-Specific Multiplex Ligation-Dependent Probe Amplification (MS-MLPA)-Based Approach

MicroRNAs (miRNAs) are small noncoding RNAs that contribute to tumorigenesis by acting as oncogenes or tumor suppressor genes and may be important in the diagnosis, prognosis and treatment of cancer. Many miRNA genes have associated CpG islands, suggesting epigenetic regulation of their expression. Compared with sporadic cancers, the role of miRNAs in hereditary or familial cancer is poorly understood. We investigated 96 colorectal carcinomas, 58 gastric carcinomas and 41 endometrial carcinomas, occurring as part of inherited DNA mismatch repair (MMR) deficiency (Lynch syndrome), familial colorectal carcinoma without MMR gene mutations or sporadically. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assays were developed for 11 miRNA loci that were chosen because all could be epigenetically regulated through the associated CpG islands and some could additionally modulate the epigenome by putatively targeting the DNA methyltransferases or their antagonist retinoblastoma-like 2 (RBL2). Compared with the respective normal tissues, the predominant alteration in tumor tissues was increased methylation for the miRNAs 1-1, 124a-1, 124a-2, 124a-3, 148a, 152 and 18b; decreased methylation for 200a and 208a; and no major change for 373 and let-7a-3. The frequencies with which the individual miRNA loci were affected in tumors showed statistically significant differences relative to the tissue of origin (colorectal versus gastric versus endometrial), MMR proficiency versus deficiency and sporadic versus hereditary disease. In particular, hypermethylation at miR-148a and miR-152 was associated with microsatellite-unstable (as opposed to stable) tumors and hypermethylation at miR-18b with sporadic disease (as opposed to Lynch syndrome). Hypermethylation at miRNA loci correlated with hypermethylation at classic tumor suppressor promoters in the same tumors. Our results highlight the importance of epigenetic events in hereditary and sporadic cancers and suggest that MS-MLPA is an excellent choice for quantitative analysis of methylation in archival formalin-fixed, paraffin-embedded samples, which pose challenges to many other techniques commonly used for methylation studies.

Interleukin‐6 promotes tumorigenesis by altering DNA methylation in oral cancer cells

Worldwide oral squamous cell carcinoma (OSCC) accounts for more than 100,000 deaths each year. Chronic inflammation constitutes one of the key risk factors for OSCC. Accumulating evidence suggests that aberrant DNA methylation may contribute to OSCC tumorigenesis. This study investigated whether chronic inflammation alters DNA methylation and expression of cancer-associated genes in OSCC. We established an in vitro model of interleukin (IL)-6 mediating chronic inflammation in OSCC cell lines. Thereafter, we measured the ability of IL-6 to induce global hypomethylation of long interspersed nuclear element-1 (LINE-1) sequences, as well as CpG methylation changes using multiple methodologies including quantitative pyrosequencing, methylation-specific multiplex ligation-dependent probe amplification and sensitive melting analysis after real-time-methylation-specific polymerase chain reaction (PCR). Gene expression was investigated by quantitative reverse transcriptase-PCR. IL-6 induced significant global LINE-1 hypomethylation (p=0.016) in our in vitro model of inflammatory stress in OSCC cell lines. Simultaneously, IL-6 induced CpG promoter methylation changes in several important putative tumor suppressor genes including CHFR, GATA5 and PAX6. Methylation changes correlated inversely with the changes in the expression of corresponding genes. Our results indicate that IL-6-induced inflammation promotes tumorigenesis in the oral cavity by altering global LINE-1 hypomethylation. In addition, concurrent hypermethylation of multiple tumor suppressor genes by IL-6 suggests that epigenetic gene silencing may be an important consequence of chronic inflammation in the oral cavity. These findings have clinical relevance, as both methylation and inflammation are suitable targets for developing novel preventive and therapeutic measures.

Comprehensive Analysis of MGMT Promoter Methylation: Correlation with MGMT Expression and Clinical Response in GBM

O6-methylguanine DNA-methyltransferase (MGMT) promoter methylation has been identified as a potential prognostic marker for glioblastoma patients. The relationship between the exact site of promoter methylation and its effect on gene silencing, and the patient's subsequent response to therapy, is still being defined. The aim of this study was to comprehensively characterize cytosine-guanine (CpG) dinucleotide methylation across the entire MGMT promoter and to correlate individual CpG site methylation patterns to mRNA expression, protein expression, and progression-free survival. To best identify the specific MGMT promoter region most predictive of gene silencing and response to therapy, we determined the methylation status of all 97 CpG sites in the MGMT promoter in tumor samples from 70 GBM patients using quantitative bisulfite sequencing. We next identified the CpG site specific and regional methylation patterns most predictive of gene silencing and improved progression-free survival. Using this data, we propose a new classification scheme utilizing methylation data from across the entire promoter and show that an analysis based on this approach, which we call 3R classification, is predictive of progression-free survival (HR = 5.23, 95% CI [2.089–13.097], p<0.0001). To adapt this approach to the clinical setting, we used a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) test based on the 3R classification and show that this test is both feasible in the clinical setting and predictive of progression free survival (HR = 3.076, 95% CI [1.301–7.27], p = 0.007). We discuss the potential advantages of a test based on this promoter-wide analysis and compare it to the commonly used methylation-specific PCR test. Further prospective validation of these two methods in a large independent patient cohort will be needed to confirm the added value of promoter wide analysis of MGMT methylation in the clinical setting.

Alterations of Copy Number of Methylation Pattern in Mismatch Repair Genes by Methylation Specific-Multiplex Ligation-Dependent Probe Amplification in Cases of Colon Cancer

Genetic alterations and changes in genomic DNA cytosine methylation patterns are associated with all types of cancer and are caused by germline mutations in DNA mismatch repair (MMR) genes, predominantly MLH1 (MutL homolog 1, 19 exons) and MSH2 (MutS homolog 2, 16 exons). Genomic DNA was extracted from tissue samples embedded in paraffin from 49 patients with adenocarcinoma and from 21 patients with carcinoma for the study group; genomic DNA was extracted from lymphocytes from 10 healthy donors for the control group. We used methylation specific multiplex ligation-dependent probe amplification (MS-ML-PA), which allows the detection of copy number changes and unusual methylation levels of 10 to 50 different sequences in one reaction by use of the methylation-sensitive restriction enzyme HhaI and sequence-specific capillary electrophoresis for the study of 24 genes.We found the mean methylation rates for MLH1 (97.14%), MSH2 (24.28%), MSH6 (MutS homolog 6) (67.14%), MSH3 (MutS homolog 3) (78.57%), MLH3 (MutL homolog 3) (75.71%), PMS2 (postmeiotic segregation increased 2) (65.71%), MGMT(O-6-methylguanine-DNA methyltransferase ) (82.85%). We conclude that the mismatch repair (MMR) system is critical for the maintenance of genomic stability.

Multiplexed Methylation Profiles of Tumor Suppressor Genes in Bladder Cancer

Changes in DNA methylation of tumor suppressors can occur early in carcinogenesis and are potentially important early indicators of cancer. The objective of this study was to assess the methylation of 25 tumor suppressor genes in bladder cancer using a methylation-specific (MS) multiplex ligation-dependent probe amplification assay (MLPA). Initial analyses in bladder cancer cell lines (n = 14) and fresh-frozen primary bladder tumor specimens (n = 31) supported the panel of genes selected being altered in bladder cancer. The process of MS-MLPA was optimized for its application in body fluids using two independent training and validation sets of urinary specimens (n = 146), including patients with bladder cancer (n = 96) and controls (n = 50). BRCA1 (71.0%), WT1 (38.7%), and RARB (38.7%) were the most frequently methylated genes in bladder tumors, with WT1 methylation being significantly associated with tumor stage (P = 0.011). WT1 and PAX5A were identified as methylated tumor suppressors. In addition, BRCA1, WT1, and RARB were the most frequently methylated genes in urinary specimens. Receiver operating characteristic curve analyses revealed significant diagnostic accuracies in both urinary sets for BRCA1, RARB, and WT1. The novelty of this report relates to applying MS-MLPA, a multiplexed methylation technique, for tumor suppressors in bladder cancer and body fluids. Methylation profiles of tumor suppressor genes were clinically relevant for histopathological stratification of bladder tumors and offered a noninvasive diagnostic strategy for the clinical management of patients affected with uroepithelial neoplasias.

Uniparentale Disomien

Zusammenfassung Der Begriff uniparentale Disomie (UPD) beschreibt eine Chromosomenaberration, bei der beide Homologen/beide Kopien eines Chromosomenabschnitts (Heterodisomie) bzw. 2 Kopien eines Homologen/eines Chromosomenabschnitts (Isodisomie) von nur einem Elternteil herrühren. Man kann UPDs ganzer Chromosomen von segmentalen und von komplexen UPDs unterscheiden. Mögliche mit einer UPD assoziierte Probleme sind Trisomiemosaike, Homozygotie autosomal-rezessiv vererbter Mutationen, Vater-Kind- und Mutter-Tochter-Übertragung X-chromosomal vererbter Mutationen und mit einem Genomic Imprinting assoziierte Erkrankungen. Letzteres beschreibt die von der elterlichen Herkunft abhängige monoallelische Expression bestimmter Gene. Bislang bekannte Imprintingerkrankungen sind ein transienter neonataler Diabetes mellitus, das Silver-Russell-Syndrom, das Beckwith-Wiedemann-Syndrom, die upd(14)mat (Temple-Syndrom), die upd(14)pat, das Prader-Willi-Syndrom und das Angelman-Syndrom. Als Entstehungsmechanismen kommen ein Trisomic oder Monosomic Rescue, eine Gametenkomplementation oder ein Postfertilisierungsfehler in Frage. Gesamtinzidenz und -prävalenz sind nicht bekannt, für einzelne mit einer Imprintingerkrankung assoziierte UPDs werden Häufigkeiten bis 1:3400 unter Geburten angegeben. Als Nachweismethoden kommen in der Routinediagnostik vor allem die Mikrosatellitenmarkeranalyse, methylierungsspezifische Polymerasekettenreaktion (PCR) und methylierungsspezifische MLPA („multiplex ligation-dependent probe amplification“) zum Einsatz.

The Incidence and Prognostic Significance of 9p21 Abnormalities In Pediatric Acute Lymphoblastic Leukemia Patients – the IWK Experience

AbstractAbstract 1705 Background:Outcomes in acute lymphoblastic leukemia (ALL), the most common childhood malignancy, have improved significantly due to risk-based multi-agent chemotherapy strategies derived from diagnostic advances in cytogenetics and molecular biology. Characterization of recurrent genetic lesions has enabled further prognostic stratification on recent clinical trials. Abnormalities involving the short arm of chromosome 9 are well-documented in pediatric ALL. Three critical tumor suppressors: CDKN2a (p16INK4a), CDKN2b (p15INK4b) and p14ARF have been localized to band 9p21. While 9p21 abnormalities have been reported in 10% of childhood ALL, their prognostic significance remains uncertain and may depend on which of these genes are lost/inactivated. Moreover, 9p deletions may be cryptic and/or not readily identified by standard G-banding techniques. We investigated the incidence and prognostic significance of 9p21 abnormalities identified retrospectively in pediatric ALL patients. Methods:A total of 76 children were diagnosed with ALL at the IWK Health Centre in Halifax, Nova Scotia from 2000–2005. Cell pellets were available for analysis on 48 patients. Chart review was conducted retrospectively. Approval was obtained by the IWK Research Ethics Board. 9p21 abnormalities were assessed by two modalities: 1) fluorescence in-situ hybridization (FISH) using a commercial p16 probe; and 2) methylation specific multiplex ligation-dependent probe amplification (MS-MLPA), using SALSA MLPA kit ME024-A1 9p21 CDKN2A/2B region (MRC Holland). Results:Of the 48 samples studied by FISH, 18 (38%) had a 9p21 deletion, only 4 of which were previously identified by G-banding. MLPA was performed on 40 samples and identified a total of 18 (45%) patients with a 9p21 deletion, 5 of which were not identified by FISH. Aberrant methylation at CDKN2b (p15INK4b) was found by MLPA in 19 patients (46%). Overall, 32 of 48 (67%) patients were determined to have an abnormal 9p21, representing a substantially greater frequency than previously reported. The detection rate for deletions at 9p21 between FISH and MLPA, was not statistically different (p=0.31). Interestingly, all patients with T-cell disease (n=5) had an abnormal 9p21, suggesting an association between this immunophenotype and 9p21 abnormalities (p=0.02). Overall, 9p21 abnormalities were associated with National Cancer Institute (NCI) high risk criteria (p=0.04), specifically white blood cell count >50,000/μL at diagnosis (p=0.019) and a higher percentage of leukemic blasts in the peripheral blood (p=0.016). Only four B-cell lineage patients in our cohort relapsed, three of which had 9p21 abnormalities and one of these ultimately died of disease. Conclusions:Using FISH and MLPA, our study has revealed a much higher incidence of 9p21 abnormalities in childhood ALL than has previously been described. Moreover, our findings demonstrate an association with high risk features and possibly inferior prognosis. It is possible that the lack of consistent reports linking 9p21 abnormalities to prognosis are related to suboptimal ascertainment. A small sample size in our study prevents the conclusive determination of an independent prognostic impact. However, our results justify the need for a larger multi-center investigation to evaluate how 9p21 abnormalities might affect ALL outcome and possibly influence future risk stratification for determining treatment. Disclosures:No relevant conflicts of interest to declare.