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1. Sperm imprinting integrity in seminoma patients?

Author

Magali Guilleman, Oxana Blagoskonov, Cécile Choux, Christine Binquet, Patricia Fauque, Julie Barberet, Sandrine Daniel, Benjamin Tournier, Céline Bruno, Laboratoire de Biologie de la reproduction CECOS - [CHU de Dijon], Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand (CHU Dijon), Equipe GAD (LNC - U1231), Lipides - Nutrition - Cancer [Dijon - U1231] (LNC), Université de Bourgogne (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Université de Bourgogne (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, Service de Biologie et de Médecine de la Reproduction (CHRU Besançon), Centre Hospitalier Régional Universitaire de Besançon (CHRU Besançon), Centre d'Investigation Clinique 1432 (Dijon) - Epidemiologie Clinique/Essais Cliniques (CIC-EC), Université de Bourgogne (UB)-Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand (CHU Dijon)-Institut National de la Santé et de la Recherche Médicale (INSERM), Service de Pathologie [CHU de Dijon], and Service de Gynécologie Obstétrique, Médecine Foetale et Stérilité Conjugale - Chirurgie Gynécologie et Oncologique [CHU de Dijon]

Subjects
Adult, Male, endocrine system diseases, lcsh:QH426-470, Imprinted genes, Testicular Germ Cell Tumor, lcsh:Medicine, [SDV.CAN]Life Sciences [q-bio]/Cancer, Biology, Andrology, 03 medical and health sciences, Genomic Imprinting, 0302 clinical medicine, Testicular Neoplasms, Insulin-Like Growth Factor II, Testicular germ cell tumor (TGCT), Genetics, medicine, Humans, Sperm DNA methylation, Oligozoospermia, Molecular Biology, Genetics (clinical), Testicular dysgenesis syndrome (TDS), 030219 obstetrics & reproductive medicine, Research, Intratubular germ cell neoplasia, Calcium-Binding Proteins, lcsh:R, Membrane Proteins, Nuclear Proteins, Seminoma, Oligospermia, Sequence Analysis, DNA, Middle Aged, medicine.disease, Sperm, Spermatozoa, 3. Good health, lcsh:Genetics, Differentially methylated regions, 030220 oncology & carcinogenesis, DNA methylation, Intercellular Signaling Peptides and Proteins, RNA, Long Noncoding, Genomic imprinting, Spermatogenesis, Developmental Biology
Abstract

Background Testicular germ cell tumor such as seminoma is strongly associated with male reproductive problems commonly associated with the alteration of sperm parameters as described in testicular dysgenesis syndrome. Interestingly, numerous studies have reported that the precursor of germ cell cancer, germ cell neoplasia in situ (GCNIS), present similarities to fetal gonocytes, specifically characterized by global DNA hypomethylation particularly on imprinting sequences. These disorders may have a common origin derived from perturbations of embryonal programming during fetal development. Presently, there is no available information concerning the sperm DNA methylation patterns of testicular cancer patients. For the first time, we evaluated the sperm imprinting of seminoma patients. A total of 92 cryopreserved sperm samples were included, 31 before seminoma treatment (S): 23 normozoospermic (SN) and 8 oligozoospermic (SO) and 61 sperm controls samples: 31 normozoospermic (N) and 30 oligozoospermic (O). DNA methylation levels of seven differentially methylated regions (DMRs) of imprinted genes [H19/IGF2: IG-DMR (CTCF3 and CTCF6 of H19 gene); IGF2-DMRs (DMR0 and DMR2); MEG3/DLK1:IG-DMR; SNURF:TSS-DMR; KCNQ1OT1:TSS-DMR] were assessed by pyrosequencing. All comparative analyses were adjusted for age. Results Comparisons of sperm DNA methylation levels between seminoma (S) and normozoospermic (N) samples showed a significant difference for the SNURF sequence (p = 0.017), but after taking into account the sperm parameters, no difference was observed. However, we confirmed a significant association between oligozoospermia (O) and imprinting defects for H19/IGF2-CTCF6 (p = 0.001), MEG3/DLK1 (p = 0.017), IGF2-DMR2 (p = 0.022), and SNURF (p = 0.032) in comparison with control groups (N). Conclusions This study highlights the high risk of sperm imprinting defects in cases of oligozoospermia and shows for the first time that seminoma patients with normal spermatogenesis present sperm imprinting integrity. These data suggest a low probability of the involvement of a common imprinting defect in fetal cells leading to both TGCT and subfertility. Electronic supplementary material The online version of this article (10.1186/s13148-018-0559-z) contains supplementary material, which is available to authorized users.

Published
2018
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2. Analysis of RET promoter CpG island methylation using methylation-specific PCR (MSP), pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM): impact on stage II colon cancer patient outcome

Author

Chantal Ramaekers, Manon van Engeland, Caroline Chapusot, Benjamin Tournier, Veerle Melotte, Martijn Vervoort, Matty P. Weijenberg, Kim M. Smits, Valérie Jooste, Muriel X. G. Draht, Department of patholgy [School of Oncology and Developmental Biology - Maastricht], School for Oncology and Developmental Biology [Maastricht] ( GROW ), Maastricht University [Maastricht]-Maastricht University Medical Center-Maastricht University [Maastricht]-Maastricht University Medical Center, Department of radiotherapy [School of Oncology and Developmental Biology - Maastricht], Registre Bourguignon des Cancers Digestifs, Lipides - Nutrition - Cancer (U866) ( LNC ), Université de Bourgogne ( UB ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation de Dijon ( ENSBANA ) -Université de Bourgogne ( UB ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation de Dijon ( ENSBANA ) -Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand ( CHU Dijon ), Service de Pathologie [CHU de Dijon], Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand ( CHU Dijon ), Zuyd University of Applied Sciences (Heerlen, Pays-Bas), Chemelot Innovation and Learning Labs (Geleen, Pays-Bas), Department of Epidemiology, GROW-School for Oncology and Developmental Biology, Maastricht University [Maastricht], Pathologie, RS: GROW - R2 - Basic and Translational Cancer Biology, Promovendi ODB, Epidemiologie, RS: GROW - R1 - Prevention, Radiotherapie, School for Oncology and Developmental Biology [Maastricht] (GROW), Maastricht University [Maastricht]-Maastricht University Medical Centre (MUMC), Maastricht University [Maastricht]-Maastricht University [Maastricht]-Maastricht University Medical Centre (MUMC), Lipides - Nutrition - Cancer (U866) (LNC), Université de Bourgogne (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation de Dijon (ENSBANA)-Université de Bourgogne (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation de Dijon (ENSBANA)-Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand (CHU Dijon), and Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand (CHU Dijon)

Subjects
Male, 0301 basic medicine, MESH: Sequence Analysis, DNA, Bisulfite sequencing, Analytic sensitivity, MS-HRM, MESH : Aged, MESH : Promoter Regions, Genetic, Polymerase Chain Reaction, [ SDV.CAN ] Life Sciences [q-bio]/Cancer, 0302 clinical medicine, MESH: DNA Methylation, MESH : Female, MESH : Proto-Oncogene Proteins c-ret, Promoter Regions, Genetic, MESH: CpG Islands, MESH : Polymerase Chain Reaction, Genetics (clinical), MESH: Aged, DNA methylation, MESH : Prognosis, Methylation, MESH : CpG Islands, Prognosis, pyrosequencing, 030220 oncology & carcinogenesis, MESH: Survival Analysis, Female, MESH : Colorectal Neoplasms, MESH : Sensitivity and Specificity, Colorectal Neoplasms, MESH : Male, [SDV.CAN]Life Sciences [q-bio]/Cancer, Biology, Sensitivity and Specificity, MESH: Proto-Oncogene Proteins c-ret, High Resolution Melt, MESH: Prognosis, 03 medical and health sciences, MESH: Promoter Regions, Genetic, Genetics, Humans, Molecular Biology, Aged, MESH: Humans, Research, MSP, Proto-Oncogene Proteins c-ret, MESH : Humans, MESH: Polymerase Chain Reaction, Sequence Analysis, DNA, Survival Analysis, Molecular biology, MESH: Sensitivity and Specificity, MESH: Male, 030104 developmental biology, Pyrosequencing, Illumina Methylation Assay, CpG Islands, Cancer biomarkers, Clinical sensitivity, Primer (molecular biology), MESH : Survival Analysis, RET, MESH: Female, MESH : DNA Methylation, MESH: Colorectal Neoplasms, Developmental Biology, MESH : Sequence Analysis, DNA
Abstract

Background Already since the 1990s, promoter CpG island methylation markers have been considered promising diagnostic, prognostic, and predictive cancer biomarkers. However, so far, only a limited number of DNA methylation markers have been introduced into clinical practice. One reason why the vast majority of methylation markers do not translate into clinical applications is lack of independent validation of methylation markers, often caused by differences in methylation analysis techniques. We recently described RET promoter CpG island methylation as a potential prognostic marker in stage II colorectal cancer (CRC) patients of two independent series. Methods In the current study, we analyzed the RET promoter CpG island methylation of 241 stage II colon cancer patients by direct methylation-specific PCR (MSP), nested-MSP, pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM). All primers were designed as close as possible to the same genomic region. In order to investigate the effect of different DNA methylation assays on patient outcome, we assessed the clinical sensitivity and specificity as well as the association of RET methylation with overall survival for three and five years of follow-up. Results Using direct-MSP and nested-MSP, 12.0 % (25/209) and 29.6 % (71/240) of the patients showed RET promoter CpG island methylation. Methylation frequencies detected by pyrosequencing were related to the threshold for positivity that defined RET methylation. Methylation frequencies obtained by pyrosequencing (threshold for positivity at 20 %) and MS-HRM were 13.3 % (32/240) and 13.8 % (33/239), respectively. The pyrosequencing threshold for positivity of 20 % showed the best correlation with MS-HRM and direct-MSP results. Nested-MSP detected RET promoter CpG island methylation in deceased patients with a higher sensitivity (33.1 %) compared to direct-MSP (10.7 %), pyrosequencing (14.4 %), and MS-HRM (15.4 %). While RET methylation frequencies detected by nested-MSP, pyrosequencing, and MS-HRM varied, the prognostic effect seemed similar (HR 1.74, 95 % CI 0.97–3.15; HR 1.85, 95 % CI 0.93–3.86; HR 1.83, 95 % CI 0.92–3.65, respectively). Conclusions Our results show that upon optimizing and aligning four RET methylation assays with regard to primer location and sensitivity, differences in methylation frequencies and clinical sensitivities are observed; however, the effect on the marker’s prognostic outcome is minimal. Electronic supplementary material The online version of this article (doi:10.1186/s13148-016-0211-8) contains supplementary material, which is available to authorized users.

Published
2016
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3. Why do results conflict regarding the prognostic value of the methylation status in colon cancers? The role of the preservation method

Author

Emilie Courcet, Caroline Chapusot, F. Piard, Jean Faivre, Benjamin Tournier, Côme Lepage, Laurent Martin, Lipides - Nutrition - Cancer (U866) ( LNC ), Université de Bourgogne ( UB ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation de Dijon ( ENSBANA ), Service d'anatomie et de cytologie pathologiques, Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand ( CHU Dijon ), Registre Bourguignon des Cancers Digestifs, Université de Bourgogne ( UB ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation de Dijon ( ENSBANA ) -Université de Bourgogne ( UB ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation de Dijon ( ENSBANA ) -Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand ( CHU Dijon ), Centre de ressources biologiques Ferdinand Cabanne [Dijon] ( CRB Ferdinand Cabanne ), BMC, Ed., Lipides - Nutrition - Cancer (U866) (LNC), Université de Bourgogne (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation de Dijon (ENSBANA), Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand (CHU Dijon), Université de Bourgogne (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation de Dijon (ENSBANA)-Université de Bourgogne (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation de Dijon (ENSBANA)-Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand (CHU Dijon), and Centre de ressources biologiques Ferdinand Cabanne [Dijon] (CRB Ferdinand Cabanne)

Subjects
Cancer Research, Bisulfite sequencing, [SDV.CAN]Life Sciences [q-bio]/Cancer, Adenocarcinoma, Biology, MLH1, lcsh:RC254-282, [ SDV.CAN ] Life Sciences [q-bio]/Cancer, chemistry.chemical_compound, [SDV.CAN] Life Sciences [q-bio]/Cancer, Predictive Value of Tests, Biomarkers, Tumor, Genetics, Humans, Sulfites, DNA Modification Methylases, Adaptor Proteins, Signal Transducing, Cryopreservation, Paraffin Embedding, Tumor Suppressor Proteins, Nuclear Proteins, Reproducibility of Results, DNA, Neoplasm, Methylation, DNA Methylation, Prognosis, lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens, Molecular biology, digestive system diseases, Neoplasm Proteins, Bisulfite, DNA Repair Enzymes, Long Interspersed Nucleotide Elements, Phenotype, Oncology, CpG site, chemistry, Sodium bisulfite, Colonic Neoplasms, DNA methylation, Feasibility Studies, Pyrosequencing, CpG Islands, MutL Protein Homolog 1, Research Article
Abstract

Background In colorectal carcinoma, extensive gene promoter hypermethylation is called the CpG island methylator phenotype (CIMP). Explaining why studies on CIMP and survival yield conflicting results is essential. Most experiments to measure DNA methylation rely on the sodium bisulfite conversion of unmethylated cytosines into uracils. No study has evaluated the performance of bisulfite conversion and methylation levels from matched cryo-preserved and Formalin-Fixed Paraffin Embedded (FFPE) samples using pyrosequencing. Methods Couples of matched cryo-preserved and FFPE samples from 40 colon adenocarcinomas were analyzed. Rates of bisulfite conversion and levels of methylation of LINE-1, MLH1 and MGMT markers were measured. Results For the reproducibility of bisulfite conversion, the mean of bisulfite-to-bisulfite standard deviation (SD) was 1.3%. The mean of run-to-run SD of PCR/pyrosequencing was 0.9%. Of the 40 DNA couples, only 67.5%, 55.0%, and 57.5% of FFPE DNA were interpretable for LINE-1, MLH1, and MGMT markers, respectively, after the first analysis. On frozen samples the proportion of well converted samples was 95.0%, 97.4% and 87.2% respectively. For DNA showing a total bisulfite conversion, 8 couples (27.6%) for LINE-1, 4 couples (15.4%) for MLH1 and 8 couples (25.8%) for MGMT displayed significant differences in methylation levels. Conclusions Frozen samples gave reproducible results for bisulfite conversion and reliable methylation levels. FFPE samples gave unsatisfactory and non reproducible bisulfite conversions leading to random results for methylation levels. The use of FFPE collections to assess DNA methylation by bisulfite methods must not be recommended. This can partly explain the conflicting results on the prognosis of CIMP colon cancers.

Published
2012
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