Methylation-specific Multiplex Ligation-dependent Probe Amplification Research Articles

Methylation-specific multiplex ligation-dependent probe amplification and its impact on clinical findings in medulloblastoma

Gain of (proto-)oncogenes and loss or promoter hypermethylation of tumor suppressor genes (TSGs) play essential roles in tumorigenesis. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) allows simultaneous detection of both these alterations. MS-MLPA was performed on 20 medulloblastoma samples (n = 12 cryoconserved; n = 8 formalin-fixed paraffin-embedded, FFPE) in order to screen for copy number changes in 77 unselected TSGs and (proto-)oncogenes as well as for promoter hypermethylation in a subset of 33 TSGs. In all specimens, determination of promoter methylation status was possible, whereas robust data concerning copy number changes could be obtained on cryopreserved material only. We found a median of 1.5 deletions and 6.5 amplifications in the 12 cryopreserved medulloblastoma and a median of 5 promoter hypermethylation per tumor. Frequent copy number changes included amplification of ASC on 16p12 (5/12) and amplification of several adjacent genes on 17q (3/12) including IGFBP4. Hypermethylation of MSH6 on 2p16 was found in 16 samples. MS-MLPA findings were also correlated with clinical and histological characteristics. The number of promoter hypermethylation was significantly associated with presence of necrosis (p = 0.004). Tumors which recurred within 1 year were more likely to show amplification of the GATA5 gene (p = 0.038), while hypermethylation of CASP8 was associated with a lower tumor recurrence rate (p = 0.036). There was also a trend towards a correlation between total number of aberrations and CSF dissemination (p = 0.055). Our findings confirm frequent presence of certain aberrations and reveal novel candidates for improving prognosis based on genetic and epigenetic tumor features. A medulloblastoma-specific MS-MLPA probe set seems a potentially valuable tool for further investigations on larger sample series.

High frequency of copy number variations (CNVs) in the chromosome 11p15 region in patients with Beckwith–Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS), an overgrowth and tumor predisposition syndrome is clinically heterogeneous. Its variable presentation makes molecular diagnosis particularly important for appropriate counseling of patients with respect to embyronal tumor risk and recurrence risk. BWS is characterized by macrosomia, omphalocele, and macroglossia. Additional clinical features can include hemihyperplasia, embryonal tumors, umbilical hernia, and ear anomalies. BWS is etiologically heterogeneous arising from dysregulation of one or both of the chromosome 11p15.5 imprinting centers (IC) and/or imprinted growth regulatory genes on chromosome 11p15.5. Most BWS cases are sporadic and result from loss of maternal methylation at imprinting center 2 (IC2), gain of maternal methylation at imprinting center 1 (IC1) or paternal uniparental disomy (UPD). Heritable forms of BWS (15 %) have been attributed mainly to mutations in the growth suppressor gene CDKN1C, but have also infrequently been identified in patients with copy number variations (CNVs) in the chromosome 11p15.5 region. Four hundred and thirty-four unrelated BWS patients referred to the molecular diagnostic laboratory were tested by methylation-specific multiplex ligation-dependent probe amplification. Molecular alterations were detected in 167 patients, where 103 (62 %) showed loss of methylation at IC2, 23 (14 %) had gain of methylation at IC1, and 41 (25 %) showed changes at both ICs usually associated with paternal UPD. In each of the three groups, we identified patients in whom the abnormalities in the chromosome 11p15.5 region were due to CNVs. Surprisingly, 14 patients (9 %) demonstrated either deletions or duplications of the BWS critical region that were confirmed using comparative genomic hybridization array analysis. The majority of these CNVs were associated with a methylation change at IC1. Our results suggest that CNVs in the 11p15.5 region contribute significantly to the etiology of BWS. We highlight the importance of performing deletion/duplication testing in addition to methylation analysis in the molecular investigation of BWS to improve our understanding of the molecular basis of this disorder, and to provide accurate genetic counseling.

The clinical and molecular genetic study of 20 Silver Russell Syndrome cases

Methods 20 SRS cases diagnosed in Beijing Children’s Hospital from 2006 to 2011 were studied retrospectively for clinical manifestations, physical signs, laboratory examinations and respond of GH treatment. We compared with 3 different diagnostic criteria and used the methylationspecific multiplex ligation dependent probe amplification (MS-MLPA) method to detect the chromosome 11p15 imprinting defects in 16 patients of them, meanwhile take 10 normal control.

Regulation of MLH1 mRNA and protein expression by promoter methylation in primary colorectal cancer: a descriptive and prognostic cancer marker study

In colorectal cancer MLH1 deficiency causes microsatellite instability, which is relevant for the patient's prognosis and treatment, and its putative heredity. Dysfunction of MLH1 is caused by sporadic gene promoter hypermethylation or by hereditary mutations as seen in Lynch Syndrome. The aim of this study was to determine in detail how DNA methylation regulates MLH1 expression and impacts clinical management. Colorectal cancer samples were collected from 210 patients. The laboratory methods used to study these samples included methylation specific multiplex ligation-dependent probe amplification (MS-MLPA), real-time quantitative PCR (qPCR), and immunohistochemistry (IHC). We found that the MLH1 mRNA and protein expression levels were highly related. MS-MLPA was successful in tumors from 195 patients. In these tumors, hypermethylation was observed in promoter regions A (n = 57), B (n = 30), C (n = 28), and D (n = 47), and in intron 1 (n = 25). The promoter region C and intron 1 methylation levels were found to be excellently suited for discriminating between low and high gene expression levels, whereas those of promoter regions A, B and D were less specific. Hypermethylation in any region (n = 77) served as an independent prognostic factor (hazard ratio 0.56, 95% confidence interval 0.36-0.89, p = 0.01). MLH1 inactivation through hypermethylation was found to be related to improved survival. Hypermethylation in promoter region C and intron 1 served as the most specific markers for this inactivation.

Methylation‐specific multiplex ligation‐dependent probe amplification: utility for prenatal diagnosis of parental origin in human triploidy

When a triploid pregnancy is diagnosed prenatally, gynaecologists have traditionally relied on the histopathological examination of the tissue from the terminated pregnancy to determine if the pregnancy is molar. However, reproducibility is poor and variability is high when diagnosing hydatidiform moles. Triploid pregnancies can have either the chromosomal constitution of two maternal and one paternal set, or two paternal and one maternal set, but only the conceptuses with two paternal sets have the potential to cause maternal complications. Therefore, it would be beneficial to introduce a method that gives the gynaecologist the parental origin of the genome of the triploid conceptus as early as possible, without delaying the process by first collecting parental samples. Using methylation-specific multiplex ligation-dependent probe amplification, we measured methylation levels at different imprinted sites. We were able to correctly determine the parental origin of the genome in all 105 triploid pregnancies analysed. We present methylation-specific multiplex ligation-dependent probe amplification as a method capable of determining the parental origin of the genome of triploid conceptuses within 24 h; it is inexpensive, simple and easy to use, and parental samples are not needed.

Gene methylation in rectal cancer: Predictive marker of response to chemoradiotherapy?

Although numerous studies have focused on the link between CpG island methylator phenotypes and the development of colorectal cancer, few studies have dealt specifically with methylation profiling in rectal cancer and its role in predicting response to neoadjuvant chemoradiotherapy (NCRT). We characterized methylation profiles in normal and neoplastic tissue samples from patients with rectal cancer and assessed the role of this molecular profile in predicting chemoradioactivity. We evaluated 74 pretreatment tumor samples and 16 apparently normal tissue biopsies from rectal cancer patients submitted to NCRT. The methylation profile of 24 different tumor suppressor genes was analyzed from FFPE samples by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). Methylation status was studied in relation to tissue type and clinical pathological parameters, in particular, pathological response evaluated by tumor regression grade (TRG). ESR1, CDH13, RARB, IGSF4, and APC genes showed high methylation levels in tumor samples (range 18.92-49.77) with respect to normal tissue. Methylation levels of the remaining genes were low and similar in both normal (range 1.91-14.56) and tumor tissue (range 1.84-11). Analysis of the association between methylation and response to therapy in tumor samples showed that only TIMP3 methylation status differed significantly within the four TRG classes (ANOVA, P < 0.05). Results from the present explorative study suggest that quantitative epigenetic classification of rectal cancer by MS-MLPA clearly distinguishes tumor tissue from apparently normal mucosa. Conversely, with the exception of TIMP3 gene, the methylation of selected genes does not seem to correlate with response to NCRT.

BRAF mutation in sporadic colorectal cancer and Lynch syndrome

The aim of the study was to detect mutations of BRAF oncogene in colorectal cancer and to use this information to identify Lynch syndrome patients. Consecutive cases of primary colorectal cancer (n = 137) were analyzed for MLH1 protein expression using immunohistochemistry (IHC). BRAF V600E mutation was detected by IHC using a specific monoclonal antibody (VE1) and by qPCR. All MLH1 protein-negative cases were subjected to microsatellite instability analysis and MLH1 promoter methylation assay. MLH1 protein expression deficiency and high microsatellite instability (MSI-H) were detected in 18 of the 137 (13.1%) consecutive colorectal cancer specimens. Detection of the BRAF V600E mutation by IHC was 100% sensitive and specific as compared to qPCR, and this mutation was frequently present in the MSI-H group (77.8%; 14/18) and less frequently in the microsatellite-stable group (7.6%; 9/118). All BRAF V600E mutated cases of the MSI-H group presented with a MLH1 promoter methylation (14/14) as detected by methylation-specific multiplex ligation-dependent probe amplification. When BRAF was wild type in the MSI-H group, only one MLH1 promoter methylation was detected (1/4), and of the remaining three cases without MLH1 methylation, two were identified to harbor an MLH1 mutation consistent with Lynch syndrome. Finally, 11 previously confirmed Lynch syndrome cases were analyzed for BRAF V600E mutation, and all of them were wild type. In conclusion, detection of BRAF V600E in colorectal cancer specimens by IHC is sensitive and specific and may help to identify Lynch syndrome patients.

Clinical and genetic features of Prader-Willi syndrome in China

We set out to delineate the phenotypic and genotypic characteristics of Prader-Willi syndrome (PWS), a congenital neurodevelopmental disorder caused by the lack of expression of the paternally inherited imprinted genes on chromosome 15q11-13 in 31 Chinese patients. They were genotyped to identify deletions using methylation-specific polymerase chain reaction analysis and subsequent methylation-specific multiplex ligation-dependent probe amplification analysis. Microsatellite linkage analysis was performed to distinguish maternal uniparental disomy (UPD) from imprinting defect. Clinical manifestations were recorded and compared between patients with paternal 15q11-13 deletion and UPD. Deletions in the 15q11-13 region were present in 26 (83.9 %) patients, and UPD was observed in 5 (16.1 %) patients. The mean maternal age at the time of childbirth for mUPD children (32.8 ± 5.1 years) was significantly higher than that of children with paternal 15q11-13 deletion (27.1±3.2 years, P < 0.05). All patients had neonatal hypotonia, feeding difficulties in infancy, and decreased fetal activity, but only 12.9 % of the patients showed short stature, 54.8 % presented typical facial features, and 35.5 % showed skin picking lesions. Significant heterogeneity in clinical phenotypes and genotypes in PWS were observed between Chinese and Western populations in this study. This suggests that ethnic differences may be relevant to the diagnostic criteria for PWS.

Quantitative analysis of methylation status at 11p15 and 7q21 for the genetic diagnosis of Beckwith–Wiedemann syndrome and Silver–Russell syndrome

Methylation-specific (MS) multiplex ligation-dependent probe amplification (MLPA) at two differentially methylated regions (DMRs) at chromosome 11p15, H19-DMR and LIT1-DMR, and microsatellite analysis for uniparental disomy (UPD) at chromosome 7 or 11, have been recommended for the genetic diagnosis of the Beckwith-Wiedemann syndrome (BWS) and the Silver-Russell syndrome (SRS). In this study, the efficacy of the MS pyrosequencing method at H19-DMR and LIT1-DMR at 11p15 and SGCE-DMR at 7q21 was evaluated for the genetic diagnosis of BWS (n=18) and SRS (n=20) patients. Epigenetic alterations or UPD were detected in 83% of BWS and 50% of SRS individuals by MS-MLPA, but the detection rate increased to 95% of BWS and 70% of SRS by MS pyrosequencing. Thirteen BWS patients (72%) harbored loss-of-methylation (LOM) at LIT1-DMR and two patients (11%) harbored gain-of-methylation (GOM) at H19-DMR, whereas two patients (11%) had both LOM at LIT1-DMR and GOM at H19-DMR, reflecting paternal UPD 11. Thirteen SRS patients (65%) harbored LOM at H19-DMR, whereas one patient (5%) had GOM at SGCE-DMR, reflecting maternal UPD 7. Birth anthropometric profiles were significantly correlated to methylation scores at either H19-DMR or LIT1-DMR. In conclusion, MS pyrosequencing enhanced the detection rate of molecular defects in BWS and SRS. Moreover, it indicates that methylation status at 11p15.5 might have an important role in fetal growth.

Double-labelling immunohistochemistry for MGMT and a “cocktail” of non-tumourous elements is a reliable, quick and easy technique for inferring methylation status in glioblastomas and other primary brain tumours

BackgroundOur aim was to develop a new protocol for MGMT immunohistochemistry with good agreement between observers and good correlation with molecular genetic tests of tumour methylation. We examined 40 primary brain tumours (30 glioblastomas and 10 oligodendroglial tumours) with our new technique, namely double-labelling immunohistochemistry for MGMT and a "cocktail" of non-tumour antigens (CD34, CD45 and CD68). We compared the results with single-labelling immunohistochemistry for MGMT and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA, a recognised molecular genetic technique which we applied as the gold-standard for the methylation status).ResultsDouble-labelling immunohistochemistry for MGMT produced a visual separation of tumourous and non-tumourous elements on the same histological slide, making it quick and easy to determine whether tumour cell nuclei were MGMT-positive or MGMT-negative (and thereby infer the methylation status of the tumour). We found good agreement between observers (kappa 0.76) and within observer (kappa 0.84). Furthermore, double-labelling showed good specificity (80%), sensitivity (73.33%), positive predictive value (PPV, 83.33%) and negative predictive value (NPV, 68.75%) compared to MS-MLPA. Double-labelling was quicker and easier to assess than single-labelling and it outperformed quantitative computerised image analysis of MGMT single-labelling in terms of sensitivity, specificity, PPV and NPV.ConclusionsDouble-labelling immunohistochemistry for MGMT and a cocktail of non-tumourous elements provides a "one look" method for determining whether tumour cell nuclei are MGMT-positive or MGMT-negative. This can be used to infer the methylation status of the tumour. There is good observer agreement and good specificity, sensitivity, PPV and NPV compared to a molecular gold-standard.

Prognostic impact of the number of methylated genes in myelodysplastic syndromes and acute myeloid leukemias treated with azacytidine

The prognostic impact of the aberrant hypermethylation in response to azacytidine (AZA) remains to be determined. Therefore, we have analyzed the influence of the methylation status prior to AZA treatment on the overall survival and clinical response of myeloid malignancies. DNA methylation status of 24 tumor suppressor genes was analyzed by methylation-specific multiplex ligation-dependent probe amplification in 63 patients with myelodysplastic syndromes and acute myeloid leukemia treated with azacytidine. Most patients (73%) showed methylation of at least one gene, but only 12% of patients displayed ≥3 methylated genes. The multivariate analysis demonstrated that the presence of a high number (≥2) of methylated genes (P = 0.022), a high WBC count (P = 0.033), or anemia (P = 0.029) were independent prognostic factors associated with shorter overall survival. The aberrant methylation status did not correlate with the response to AZA, although four of the five patients with ≥3 methylated genes did not respond. By contrast, favorable cytogenetics independently influenced the clinical response to AZA as 64.7% of patients with good-risk cytogenetic abnormalities responded (P = 0.03). Aberrant methylation status influences the survival of patients treated with AZA, being shorter in those patients with a high number of methylated genes.

Methylation analysis of tumor suppressor genes in endometroid carcinoma of endometrium using MS-MLPA

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. 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 20control samples of non-neoplastic endometrium. 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. 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.

Optimizing the identification of anaplastic oligodendroglioma patients that benefit from adjuvant PCV chemotherapy using the Illumina platform: A further report from EORTC study 26951.

2011 Background: Adjuvant PCV chemotherapy improves overall survival (OS) in 1p/19q co-deleted grade 3 gliomas. Analyses of EORTC 26951 and RTOG 9402 suggest that other molecularly defined subsets of grade III tumors benefit as well. We previously identified a CpG-Island Hypermethylated Phenotype (CIMP+) as a candidate biomarker. This was further explored in a larger series using snap frozen (SF) or formalin fixed paraffin embedded (FFPE) tumor material, and compared to the value of 1p/19q, IDH1/2 and MGMT status. Methods: Methylation profiles were assessed using the Infinium HumanMethylation 27 or 450 BeadChip (Illumina). MGMT promoter methylation was re-assessed with a logistic regression model (MGMT-STP27) using probes cg1243587 and cg12981137 of the platform as described (Bady et al, Acta Neuropathol 2012;124:547-60). These probes correspond to area’s of the promoter correlated to MGMT protein expression. Previously, MGMT promoter methylation had been assessed using methylation specific multiplex ligation-dependent probe amplification (MS-MLPA). Results: Methylation profiling was conducted in 115 patients. CIMP status correlation between FFPE and SF was excellent (R2 0.96). In multivariate analysis, 1p/19q co-deletion and CIMP status were independent prognostic factors. Although 1p/19q status and IDH mutational status identify subgroups with more benefit of PCV chemotherapy, tests for interaction remain negative (p 0.25 and 0.33 respectively); MGMTstatus (MS-MLPA) had no impact (p = 0.70). However, CIMP status was of borderline predictive significance (p= 0.07), and MGMT-STP27 was of statistical significance (p = 0.003; HR unmethylated 1.61, 95% CI [0.71, 3.66], HR methylated 0.37, 95% CI 0.23, 0.61]. Conclusions: CIMP status and MGMT promoter methylation assessed with Illumina HumanMethylation BeadChips appear the most informative tests for identifying grade III glioma patients benefitting from the addition of PCV to RT. Validation in an independent dataset is required. Clinical trial information: NCT00002840.

Identification of a founder EPCAM deletion in Spanish Lynch syndrome families

Germline deletions at the 3'-end of EPCAM have been involved in the etiology of Lynch syndrome (LS). The aim of this study was to characterize at the molecular level Spanish families harboring EPCAM deletions. Non-commercial multiplex ligation-dependent probe amplification (MLPA) probes and long-range polymerase chain reaction (PCR) amplification were used to characterize each deletion. Haplotyping was performed by analyzing eight microsatellite markers and five MSH2single nucleotide polymorphisms (SNPs). Methylation of MSH2 was analyzed by methylation specific-MLPA. Tumors diagnosed in seven Spanish families harboring EPCAM deletions were almost exclusively colorectal. Mosaicism in MSH2 methylation was observed in EPCAM deletion carrier samples, being average methylation levels higher in normal colon and colorectal tumors (27.6% and 31.1%), than in lymphocytes and oral mucosa (1.1% and 0.7%). Three families shared the deletion c.858 + 2568_*4596del, with a common haplotype comprising 9.9 Mb. In two families the novel EPCAM deletion c.858 + 2488_*7469del was identified. This study provides knowledge on the clinical and molecular characteristics of mosaic MSH2 epimutations. The identification of an EPCAM founder mutation has useful implications for the molecular diagnosis of LS in Spain.

Abstract 650: IGSF4 methylation is an independent marker of HPV positive OPSCC.

Abstract Human papilloma virus (HPV) is a known causative agent for oropharyngeal squamous cell cancer (OPSCC). The goal of our study was to determine the relationship of HPV type 16 (HPV16) infection and methylation status in an OPSCC cohort. HPV16 was detected using real-time quantitative PCR (qPCR) in a retrospective cohort of 111 primary OPSCC. A 24 tumor suppressor gene candidate panel was assessed for methylation using the methylation specific-multiplex ligation-dependent probe amplification (MS-MLPA) assay. Univariate associations with HPV and gene methylation, age, gender, race and smoking were analyzed using Fisher's exact tests and logistic regression. A final multiple logistic regression model, to include patient characteristics and genes significant on univariate analysis, was built using backwards selection of genes. Statistical significance was set at p&amp;lt;0.05 and all analyses were done using SAS 9.2. In univariate analyses, Caucasian American (CA) OPSCC were more likely to be HPV positive as compared to African American (AA) OPSCC (OR=3.01, 95% CI 1.28, 7.07, p=0.011). Also, smoking (never vs current) [OR=3.60 (1.06, 12.24), p=0.04], and TIMP3, DAPK1, ESR1, and IGSF4 were associated with HPV positive status (OR=5.03, p=0.021; OR=3.71, p=0.04; OR=5.03, p=0.021; OR=7.68, p=0.012, respectively). The final model, after controlling for patient characteristics and after backward elimination of genes, indicated IGSF4 methylation is an independent predictor of HPV positive status [OR=7.07 (1.22, 40.79), p=0.029]. HPV infection status is independently associated with methylation of IGSF4, indicating interplay of DNA methylation and HPV in OPSCC pathogenesis. Citation Format: Kang Mei Chen, Josena K. Stephen, Meredith Mahan, Shaleta Havard, George Divine, Glendon Gardner, Vanessa P. Schweitzer, Maria J. Worsham. IGSF4 methylation is an independent marker of HPV positive OPSCC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 650. doi:10.1158/1538-7445.AM2013-650

1287 METHYLATION OF A NOVEL PANEL OF TUMOR SUPPRESSOR GENES ALLOWS NON-INVASIVE DIAGNOSIS AND PROGNOSIS IN BLADDER CANCER: A TWO CENTER PROSPECTIVE STUDY

You have accessJournal of UrologyBladder Cancer: Detection & Screening1 Apr 20131287 METHYLATION OF A NOVEL PANEL OF TUMOR SUPPRESSOR GENES ALLOWS NON-INVASIVE DIAGNOSIS AND PROGNOSIS IN BLADDER CANCER: A TWO CENTER PROSPECTIVE STUDY Rodrigo Garcia-Baquero, Patricia Puerta, Manuel Beltran, Miguel Alvarez, Raquel Sacristan, Jose Luis Ossorio, and Marta Sanchez-Carbayo Rodrigo Garcia-BaqueroRodrigo Garcia-Baquero Madrid, Spain More articles by this author , Patricia PuertaPatricia Puerta Madrid, Spain More articles by this author , Manuel BeltranManuel Beltran Cadiz, Spain More articles by this author , Miguel AlvarezMiguel Alvarez Oviedo, Spain More articles by this author , Raquel SacristanRaquel Sacristan Oviedo, Spain More articles by this author , Jose Luis OssorioJose Luis Ossorio Cadiz, Spain More articles by this author , and Marta Sanchez-CarbayoMarta Sanchez-Carbayo Madrid, Spain More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.2641AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Changes in DNA methylation of tumor suppressor genes (TSGs) occurring early in carcinogenesis, represent potential indicators for cancer detection and disease evolution. Our aim was to examine the biomarker role of the methylation of TSGs in bladder cancer. METHODS Methylation of 18 TSGs was evaluated in bladder cancer cells (n=14), fresh-frozen primary bladder tumors (n=61), and two prospective independent sets of urines (training, n=120; and validation, n=128) using methylation-specific multiplex ligation-dependent probe amplification assays (MS-MLPA). Diagnostic performance was evaluated using Receiver Operating Curves. Recurrence, progression, and disease-specific survival were analyzed using univariate and multivariate Cox models. RESULTS PRDM2, HLTF, ID4, DLC1, BNIP3, H2AFX, CACNA1G, TGIF, and CACNA1A were discovered methylated in bladder cancer. In tumors, RUNX3 (p=0.026), TWIST1 (p=0.009), SFRP4 (p=0.002) and CCND2 (p= 0.027) methylation correlated to stage. Univariate analyses indicated prognostic associations for recurrence (DLC1, SFRP5, H2AFX, CACNA1G), progression (DLC1, SFRP5, CACNA1G), disease-specific (PRDM2, DLC1, SFRP5, CACNA1G, and TIMP3), and overall survival (SFRP5 and TIMP3), remaining independent prognosticators in multivariate analyses for recurrence (SFRP5, H2AFX), progression (CACNA1G), and disease-specific survival (SFRP5). CCND2, SCGB3A1 and RUNX3 were the most frequently methylated TSGs in both urinary sets. ROC analyses revealed significant diagnostic accuracies for RUNX3 in both series. Using univariate and multivariate analyses, CACNA1A methylation correlated to recurrence in the training set, while PRDM2 with recurrence and BNIP3 with disease-specific survival in the validation set. CONCLUSIONS In tumors, TSGs methylation allowed histopathologic and outcome stratification. Urinary methylation offered non-invasive utility not only for diagnosis but also for prognosis. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e526 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Rodrigo Garcia-Baquero Madrid, Spain More articles by this author Patricia Puerta Madrid, Spain More articles by this author Manuel Beltran Cadiz, Spain More articles by this author Miguel Alvarez Oviedo, Spain More articles by this author Raquel Sacristan Oviedo, Spain More articles by this author Jose Luis Ossorio Cadiz, Spain More articles by this author Marta Sanchez-Carbayo Madrid, Spain More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

1875 METHYLATION OF TUMOR SUPPRESSOR GENES PREDICT RECURRENCE AND SUBCLASSIFY NON MUSCLE-INVASIVE DISEASE: PTA LOW GRADE VERSUS PT1 LOW GRADE AND PT1 HIGH GRADE BLADDER TUMORS

INTRODUCTION AND OBJECTIVES: Genetic and epigenetic factors are involved in tumorigenesis and bladder cancer progression. The role of epigenetic factors at distinguishing pathological and clinical subgroups is not well defined. We aimed at evaluating the role of methylation of 25 tumor suppressor genes (TSG) to subclassify nonmuscle invasive (NMI) bladder tumor subtypes and as clinical outcome prognosticators. METHODS: A retrospective design included paraffin-embedded tumors (n 251) of patients with primary NMI disease: pTa Low Grade (LG)(n 79), PT1LG(n 81), and PT1 High Grade (HG) (n 91). Methylation of 25 TSG was measured using a methylation-specific multiplex ligation-dependent probe amplification assay (MS-MLPA). Recurrence, progression into muscle invasive tumors and diseasespecific survival (DSS) rates were analyzed using competing risks regression analysis. RESULTS: The TSGs most frequently methylated in the overall series were STK11 (96.8%), MGMT2 (64.5%), RARB (63.0%) and GATA5 (63.0%). TSGs methylation significantly correlated to clinicopathological variables within each specific subgroup and the overall series. Methylation of RARB (p 0.027), CD44 (p 0.002), PAX5A (p 0.018), GSTP1 (p 0.001), IGSF4 (p 0.001), PYCARD (p 0.001), CDH13 (p 0.028), TP53 (p 0.001) and GATA5 (p 0.001) allowed classification of PTa versus PT1 tumors while RARB (p 0.001), CD44 (p 0.001), GSTP1 (p 0.001), IGSF4 (p 0.005), CHFR (p 0.032), PYCARD (p 0.001), TP53 (p 0.001), STK11 (p 0.028) and GATA5 (p 0.001) distinguished LG vs HG tumors. Multivariate analyses indicated that PAX5A (p 0.019), WT1 (p 0.042) and BRCA1 (p 0.023) methylation independently predicted recurrence in pTaLG lesions, PAX6 (p 0.044), ATM (p 0.004), CHFR (p 0.035) and RB1 (p 0.007) in pT1LG tumors, and PYCARD (p 0.035) in pT1HG disease, and PAX5A (p 0.01) and RB1 (p 0.008) in the overall NMI series. CONCLUSIONS: Methylation of TSG distinguished NMI bladder tumors depending on their clinicopathological phenotypes. Methylation of TSG correlated to patient outcome and behaved as independent prognosticators of recurrence. Methylation of TSG may select NMI patients who may require closer surveillance and more aggressive therapeutic interventions.

Disomy as the Genetic Underlying Mechanisms of Loss of Heterozigosity in SDHD-Paragangliomas

Succinate dehydrogenase complex, subunit D (SDHD) mutations cause pheochromocytoma/paraganglioma syndrome. SDHD, located at chromosome 11q23, shows a parent-of-origin effect because the disease is observed almost exclusively when the mutation is transmitted from the father, although some cases of maternal transmission have been reported. Several hypotheses have been proposed for this peculiar inheritance pattern, but the underlying mechanisms have not yet been clearly elucidated. The objective of the study was to explain the parent-of-origin effect in a family, mainly affected by paternally transmitted paragangliomas, and with a maternally transmitted renal tumor. Peripheral blood DNA from 15 carriers and 7 tumor DNA samples from SDHD-p.Trp5* carriers were studied. We conducted mutation genotyping and microsatellite marker analysis in germline and tumor DNA and methylation status analysis in tumor DNA by methylation-specific multiplex ligation-dependent probe amplification. Mutation genotyping and microsatellite marker analysis demonstrated loss of heterozygosity of the wild-type allele (maternal) in all studied tumors, except the renal tumor, which lost the mutated allele (maternal), and the prostate tumor, which had no loss of heterozygosity. The methylation-specific multiplex ligation-dependent probe amplification demonstrated that the methylation profile corresponded exclusively to the paternal chromosome without genomic loss, suggesting paternal uniparental disomy as the mechanism underlying the parent-of-origin effect in this SDHD family. The paternal uniparental disomy involves the loss of maternally imprinted cell cycle regulators and the overexpression of paternally imprinted growth activators, leading to tumorigenesis in this syndrome.

Altered Expression of MGMT in High-Grade Gliomas Results from the Combined Effect of Epigenetic and Genetic Aberrations

MGMT downregulation in high-grade gliomas (HGG) has been mostly attributed to aberrant promoter methylation and is associated with increased sensitivity to alkylating agent-based chemotherapy. However, HGG harboring 10q deletions also benefit from treatment with alkylating agents. Because the MGMT gene is mapped at 10q26, we hypothesized that both epigenetic and genetic alterations might affect its expression and predict response to chemotherapy. To test this hypothesis, promoter methylation and mRNA levels of MGMT were determined by quantitative methylation-specific PCR (qMSP) or methylation-specific multiplex ligation dependent probe amplification (MS-MLPA) and quantitative RT-PCR, respectively, in a retrospective series of 61 HGG. MGMT/chromosome 10 copy number variations were determined by FISH or MS-MLPA analysis. Molecular findings were correlated with clinical parameters to assess their predictive value. Overall, MGMT methylation ratios assessed by qMSP and MS-MLPA were inversely correlated with mRNA expression levels (best coefficient value obtained with MS-MLPA). By FISH analysis in 68.3% of the cases there was loss of 10q26.1 and in 15% of the cases polysomy was demonstrated; the latter displayed the highest levels of transcript. When genetic and epigenetic data were combined, cases with MGMT promoter methylation and MGMT loss depicted the lowest transcript levels, although an impact in response to alkylating agent chemotherapy was not apparent. Cooperation between epigenetic (promoter methylation) and genetic (monosomy, locus deletion) changes affecting MGMT in HGG is required for effective MGMT silencing. Hence, evaluation of copy number alterations might add relevant prognostic and predictive information concerning response to alkylating agent-based chemotherapy.

Promoter hypermethylation contributes to TIMP3 down‐regulation in high stage endometrioid endometrial carcinomas

Expression of tissue inhibitor of metalloproteinases-3 (TIMP-3) has been found to be decreased in several types of cancer by promoter gene hypermethylation. However, little is known regarding the silencing effect of TIMP3 promoter hypermethylation on gene and protein expression in endometrial carcinomas and its prognostic significance. TIMP3 promoter hypermethylation and gene copy number variations were evaluated using a methylation-specific multiplex ligation-dependent probe amplification approach in 60 cases of endometrioid endometrial carcinomas. TIMP3 expression was also evaluated at the transcript and protein levels. Loss of TIMP-3 protein expression was found in 44 (73%) of 60 carcinomas. Promoter hypermethylation was identified in 25% (15 of 60); was more frequent in stages II-IV (55%, six of 11) than in stage I (18%, nine of 49; P=0.021); and was found more commonly in tumours with deep myometrial invasion. MLH1 and TIMP3 promoters were hypermethylated simultaneously in the same group of tumours (P<0.001). A correlation between TIMP3 methylation and microsatellite instability (MSI) was found (P=0.005). TIMP3 copy number changes were frequently a loss (35%), whereas a gain was detected in only 5%. TIMP3 promoter hypermethylation was associated with high stage endometrioid endometrial tumours with extrauterine spread. Nevertheless, promoter hypermethylation and loss of heterozygosity are not the only mechanisms for TIMP3 inactivation.