Ignite Creation Date:
2024-10-26 @ 3:37 PM
Last Modification Date:
2024-10-26 @ 3:37 PM
Study NCT ID:
NCT06538428
Status:
COMPLETED
Last Update Posted:
None
First Post:
2024-05-12
Brief Title:
Methylation Patterns of the NUPR1 MGMT NDRG2 and GLI1 Genes and Their Impact on Therapeutic Outcome of GBM Patients
Sponsor:
None
Organization:
None
Study Overview
Official Title:
Studying Some Epigenetic Modifications In Glioblastoma Multiforme and Their Impact On Clinical Outcome
Status:
COMPLETED
Status Verified Date:
2024-04
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
No
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
The study aimed to investigate the role of promoter methylation of MGMT NUPR1 NDRG2 and GLI1 genes in glioblastoma multiforme GBM patients Tissue samples were collected from GBM patients and individuals with non-neurooncological diseases NND The methylation status of the four genes was analyzed and the clinical characteristics and survival of GBM patients were evaluated
Detailed Description:
Objectives Elucidate the potential role of methylation levels of the MGMT NUPR1 NDRG2 and GLI1 genes as epigenetic markers for GBM through measuring and comparing the methylation levels of four genes in GBM and NND samples The study researchers started recruiting participants from September 2020 to October 2022 after receiving research ethical approvals from the Medical Ethical Committees at the National Research Center ID20110 and from the Research Ethics Committee of Faculty of Pharmacy Ain Shams University Cairo Egypt serial noENREC-ASU 2020-8
Informed consents were signed by patients or their first-degree relatives Study participants either patients or controls were informed with the study problem aim and objectives The study was conducted in accordance with the Declaration of Helsinki Guidelines approved in 2013
A total of 58 primary GBM treatment-naïve Egyptian patients were recruited from the Clinical Oncology Department Faculty of Medicine Ain Shams University Hospital Cairo Egypt
Patients Inclusion Criteria Adult patients age 18 years with a recent diagnosis of GBM and had a performance level of less than or equal to 2 on the Ester Clinical Oncology Group ECOG scale
Therapeutic Approaches All GBM patients were evaluated clinically through a full medical history physical and neurological examinations Brain scan was done to enable the patient to receive standardized therapeutic protocol which includes the greatest secured surgical removal if attainable followed by conventional fractionated radiotherapy aggregate dosage of 60 gray Gy provided 2 Gy per fraction for 30 fractions during six weeks or hypo-fractionated radiotherapy 45 Gy in 15 fractions during three weeks alongside concurrent TMZ as chemotherapeutic agent in dose of 75 mgm2 of body surface area daily until the completion of the radiation therapy with periodical follow-up then re-evaluated clinically and radiologically given an adjuvant therapy at total of six cycles of TMZ therapy at a dosage of 150 mgm2 of body surface area from day one to five for a total of 28 days with closely medical surveillance
Throughout standard medical monitoring patients underwent evaluation using gadolinium-enhanced magnetic resonance imaging Gd-MRI 45 days following radiotherapy and subsequently every three months or whenever medical proof of neurological deterioration emerged
The demographic characteristics of 58 GBM patients were extracted from the medical records of the hospital These included demographic attributes such as age in years 22-88 sex 35 male and 23 female clinicopathological characteristics including ECOG score date of initial GBM surgery extent of tumor resection and previously mentioned therapeutic approach and survival outcomes including progression-free survival PFS and overall survival OS
The NND group comprised 20 sex-matched individuals recruited randomly The age ranged from 4 to 54 years old with an equal male-to-female ratio of 10 to 10 Those who were not receiving any medications or suffering from any current or past malignancy
Methods Sample Proscessing Before utilizing any oncological therapeutic approaches brain specimens were surgically retrieved using open stereotactic biopsy technique then preserved in neutral buffered formalin and wrapped in paraffin stained with hematoxylin-eosin HE A panel of neuropathologists then assessed all enrolled samples to confirm the diagnosis in accordance with the 2016 CNS Tumors WHO classification
Optimizing GBM tissue quality for accurate DNA methylation profiling The following inclusion criteria were used to choose the GBM tissue chunks a histopathological confirmation of GBM tissue with a minimum of 80 viable malignant cells as well as archived paraffin-embedded tissue sections must be readily available The subsequent procedures were carried out on all GBM tissues Full slice of the formalin-fixed paraffin-embedded FFPE block of GBM tissues were cut at a thickness of 4 microns and subsequently stained with the standard HE stains to evaluate the viability of the donated malignant tissue Newly cut up to 10-um-thick FFPE slices were used to prepare samples for PCR analysis
DNA extraction DNA was isolated from FFPE samples via a QIAamp FFPE kit Cat No 56404 Valencia CA as directed by the supplier Purity and concentration were determined via a nono-drop spectrophotometer Quawell Q-500 Scribner USA through determining the intensity of absorption at 260 and 280 nm and verified on a 1 agarose gel The isolated DNA samples were kept at -20 0C for subsequent analysis to identify methylation levels of MGMT NUPR1 NDRG2 and GLI1 geneS
Detection of MGMT NUPR1 NDRG2 and GLI1 methylation patterns via Methyl II quantitative PCR Methylation patterns of MGMT NUPR1 NDRG2 and GLI1 have been detected in DNA-retrieved specimens using the EpiTect Methyl II quantitative polymerase chain reaction qPCR system Qiagen Germany This is verified by detecting the amount of extracted DNA that remains following fragmentation using methylation specific restriction enzymes Subsequently the remaining DNA was quantified by real-time PCR using specific methylation primers for the targeted genes that contain promoter regions of interest
331 Methylation-Specific Restriction Enzyme Digestion technique The procedure was conducted in two phases with certain modifications implemented in our laboratory In Phase I the EpiTect Methyl II DNA Restriction Kit cat no 335452 was utilized for the reactions Genomic DNA which had been previously extracted was aliquoted into two equivalent portions in 2 separate PCR reaction tubes one tube had no added restriction enzyme and the other tube had methylation-sensitive restriction enzyme that selectively digests unmethylated DNA The tubes were then subjected to incubation in a thermal cycler SureCycler 8800 Agilent Santa Clara CA USA at 37 C for 6 hours followed by incubation at 65 C for 20 minutes
332 Quantitative PCR qPCR Analysis The quantification of the remaining genomic DNA samples in each tube was performed in Phase II using a Max3005P qPCR system Stratagene Agilent Technologies CA USA To initiate this phase 5 μL aliquot of the remaining DNA from each tube was mixed with the qPCR master mix RT2 qPCR SYBR GreenROX Master Mix Cat no 330520 The resulting mixture was then dispensed into a PCR plate containing pre-aliquoted MGMT NUPR1 NDRG2 and GLI1 methylated and unmethylated primers
Statistical Analysis The Mann-Whitney U test was used to compare the methylation levels of the four genes across the two groups
The optimal cut-off points for promoter methylation of the four genes were determined after being plotted on ROC curves using the mean of methylation in each patient The area under the curve AUC and test accuracy were calculated
Survival data in months were calculated from the start of each chemotherapy line to the date of death or last follow-up Differences in PFS and OS were compared using Kaplan-Meier survival curves and the log-rank test was used to determine statistical significance
The Cox proportional hazards model was used for multivariate survival analysis Cox regression multivariate analysis was performed using a forward stepwise parameter with a significance of 005 for entry and 01 for removal of factors significantly associated with OS
The binary logistic regression model was used to analyze the prognostic significance of the methylation of the four genes MGMT NUPR1 GLI1 and NUPR1 sex age tumor size and ECOG with respect to clinical outcomes The goodness-of-fit of the logistic regression models was evaluated using the Hosmer-Lemeshow test
A significance level of p value less than 005 was chosen Statistical analysis was performed using Statistical package for social studies software SPSS version 270 IBM Armonk NY and R Core Team 2023
Informed consents were signed by patients or their first-degree relatives Study participants either patients or controls were informed with the study problem aim and objectives The study was conducted in accordance with the Declaration of Helsinki Guidelines approved in 2013
A total of 58 primary GBM treatment-naïve Egyptian patients were recruited from the Clinical Oncology Department Faculty of Medicine Ain Shams University Hospital Cairo Egypt
Patients Inclusion Criteria Adult patients age 18 years with a recent diagnosis of GBM and had a performance level of less than or equal to 2 on the Ester Clinical Oncology Group ECOG scale
Therapeutic Approaches All GBM patients were evaluated clinically through a full medical history physical and neurological examinations Brain scan was done to enable the patient to receive standardized therapeutic protocol which includes the greatest secured surgical removal if attainable followed by conventional fractionated radiotherapy aggregate dosage of 60 gray Gy provided 2 Gy per fraction for 30 fractions during six weeks or hypo-fractionated radiotherapy 45 Gy in 15 fractions during three weeks alongside concurrent TMZ as chemotherapeutic agent in dose of 75 mgm2 of body surface area daily until the completion of the radiation therapy with periodical follow-up then re-evaluated clinically and radiologically given an adjuvant therapy at total of six cycles of TMZ therapy at a dosage of 150 mgm2 of body surface area from day one to five for a total of 28 days with closely medical surveillance
Throughout standard medical monitoring patients underwent evaluation using gadolinium-enhanced magnetic resonance imaging Gd-MRI 45 days following radiotherapy and subsequently every three months or whenever medical proof of neurological deterioration emerged
The demographic characteristics of 58 GBM patients were extracted from the medical records of the hospital These included demographic attributes such as age in years 22-88 sex 35 male and 23 female clinicopathological characteristics including ECOG score date of initial GBM surgery extent of tumor resection and previously mentioned therapeutic approach and survival outcomes including progression-free survival PFS and overall survival OS
The NND group comprised 20 sex-matched individuals recruited randomly The age ranged from 4 to 54 years old with an equal male-to-female ratio of 10 to 10 Those who were not receiving any medications or suffering from any current or past malignancy
Methods Sample Proscessing Before utilizing any oncological therapeutic approaches brain specimens were surgically retrieved using open stereotactic biopsy technique then preserved in neutral buffered formalin and wrapped in paraffin stained with hematoxylin-eosin HE A panel of neuropathologists then assessed all enrolled samples to confirm the diagnosis in accordance with the 2016 CNS Tumors WHO classification
Optimizing GBM tissue quality for accurate DNA methylation profiling The following inclusion criteria were used to choose the GBM tissue chunks a histopathological confirmation of GBM tissue with a minimum of 80 viable malignant cells as well as archived paraffin-embedded tissue sections must be readily available The subsequent procedures were carried out on all GBM tissues Full slice of the formalin-fixed paraffin-embedded FFPE block of GBM tissues were cut at a thickness of 4 microns and subsequently stained with the standard HE stains to evaluate the viability of the donated malignant tissue Newly cut up to 10-um-thick FFPE slices were used to prepare samples for PCR analysis
DNA extraction DNA was isolated from FFPE samples via a QIAamp FFPE kit Cat No 56404 Valencia CA as directed by the supplier Purity and concentration were determined via a nono-drop spectrophotometer Quawell Q-500 Scribner USA through determining the intensity of absorption at 260 and 280 nm and verified on a 1 agarose gel The isolated DNA samples were kept at -20 0C for subsequent analysis to identify methylation levels of MGMT NUPR1 NDRG2 and GLI1 geneS
Detection of MGMT NUPR1 NDRG2 and GLI1 methylation patterns via Methyl II quantitative PCR Methylation patterns of MGMT NUPR1 NDRG2 and GLI1 have been detected in DNA-retrieved specimens using the EpiTect Methyl II quantitative polymerase chain reaction qPCR system Qiagen Germany This is verified by detecting the amount of extracted DNA that remains following fragmentation using methylation specific restriction enzymes Subsequently the remaining DNA was quantified by real-time PCR using specific methylation primers for the targeted genes that contain promoter regions of interest
331 Methylation-Specific Restriction Enzyme Digestion technique The procedure was conducted in two phases with certain modifications implemented in our laboratory In Phase I the EpiTect Methyl II DNA Restriction Kit cat no 335452 was utilized for the reactions Genomic DNA which had been previously extracted was aliquoted into two equivalent portions in 2 separate PCR reaction tubes one tube had no added restriction enzyme and the other tube had methylation-sensitive restriction enzyme that selectively digests unmethylated DNA The tubes were then subjected to incubation in a thermal cycler SureCycler 8800 Agilent Santa Clara CA USA at 37 C for 6 hours followed by incubation at 65 C for 20 minutes
332 Quantitative PCR qPCR Analysis The quantification of the remaining genomic DNA samples in each tube was performed in Phase II using a Max3005P qPCR system Stratagene Agilent Technologies CA USA To initiate this phase 5 μL aliquot of the remaining DNA from each tube was mixed with the qPCR master mix RT2 qPCR SYBR GreenROX Master Mix Cat no 330520 The resulting mixture was then dispensed into a PCR plate containing pre-aliquoted MGMT NUPR1 NDRG2 and GLI1 methylated and unmethylated primers
Statistical Analysis The Mann-Whitney U test was used to compare the methylation levels of the four genes across the two groups
The optimal cut-off points for promoter methylation of the four genes were determined after being plotted on ROC curves using the mean of methylation in each patient The area under the curve AUC and test accuracy were calculated
Survival data in months were calculated from the start of each chemotherapy line to the date of death or last follow-up Differences in PFS and OS were compared using Kaplan-Meier survival curves and the log-rank test was used to determine statistical significance
The Cox proportional hazards model was used for multivariate survival analysis Cox regression multivariate analysis was performed using a forward stepwise parameter with a significance of 005 for entry and 01 for removal of factors significantly associated with OS
The binary logistic regression model was used to analyze the prognostic significance of the methylation of the four genes MGMT NUPR1 GLI1 and NUPR1 sex age tumor size and ECOG with respect to clinical outcomes The goodness-of-fit of the logistic regression models was evaluated using the Hosmer-Lemeshow test
A significance level of p value less than 005 was chosen Statistical analysis was performed using Statistical package for social studies software SPSS version 270 IBM Armonk NY and R Core Team 2023
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None