Analysis Of Chromosome 1p/19q LOH, Status Of Methylated MGMT And Expression Of P53 Protein As Possible Diagnostic And Prognostic Markers Of Oligodendrogliomas: A Study Of 161 Chinese Cases

Gliomas are the most common malignant neoplasm in human central nervous system,which are compromised of two major subtypes:astrocytic glioma and oligodendroglioma.Oligodendrogliomas,which have different molecular markers and clinical features from astrocytic gliomas,account for 4-5%of all gliomas less than astrocytic gliomas.In 1988,Cairncross et al had reported that about 60-70%of anaplastic oligodendrogliomas showed to be chemosensitive,particularly to the combination of procarbazine,lomustine,and vincristine(PCV).Oligodendrogliomas were also associated with a longer survival than astrocytic gliomas.The histologic distinction between oligodendroglioma and astrocytic glioma based on cellular morphology was often highly subjective and with significant inter-observer variability in the diagnosis.Fortunately,mutant P53 gene was frequently identified in the tumors with astrocytic differentiation,and aberrant p53 protein expression present in astrocytic gliomas.Loss of heterozygosity(LOH) at chromosomes 1p/19q usually occurred concomitantly in 60-80%of oligodendrogliomas,but only in 10-20%of astrocytic gliomas.Now the alkylating agent,temozolomide had been adopted to the first-line drug to treat the malignant gliomas and had better effect to improve the patients' survival. DNA repair gene MGMT repairs DNA damage and removes alkyl groups from the O~6-position of guanine,the site of a number of chemotherapy induced DNA alkylations.Therefore,MGMT will interfere with the therapeutic effects of chemotherapy on alkylating agents.Methylation of the MGMT promoter CpG island was associated with silencing of the gene and became useful predicator of the responsiveness of glioblastomas to alkylating agents and those patients had longer survival.Both of the 1p/19q LOH and MGMT methylation influenced in patients' prognosis.However,the relationship between methylation of MGMT and 1p/19q LOH was less reported in LOs.In this study,we investigated LOH at chromosome1p/19q combined with methylation of MGMT,expression of MGMT protein and p53 protein and clinical course in a series of 161 Chinese patients with glioma,especially in low grade of oligodendrogliomas in the hope of improving diagnostic criteria and predicting prognosis.PartⅠStudy of correlation between 1p/19q LOH and p53 protein expression as well as their role in prognosis of oligodendrogliomasObjective The aim is to study the status of loss of heterozagosity(LOH) at chromosome 1p/19q and p53 protein expression in oligodendrogliomas compared with astrocytomas.And to explore correlation between 1p/19q LOH and p53 protein expression as well as their role in prognosis of oligodendrogliomas.Methods 191 cases of different subtypes and grades of gliomas were investigated, including 116 low grade oligodendrogliomas(LOs,86 paraffin and 30 frozen tissues), 45 anaplastic oligodendrogliomas(AOs) and 30 different grades of astrocytic gliomas paraffin-embedded tissues.LOH at chromosome 1p/19q was investigated by PCR based microsatellite analysis.The p53 protein expression was examined by immunohistochemical staining.Pearson's chi-squared test was applied to determine the correlation between variables measured.Kaplan-Meier analysis was performed to determine the correlation of variable with progression-free survival(PFS) and overall survival(OS) in univariate analysis.Statistical significance was evaluated using the log-rank test and P＜0.05 was considered to be significant.Results The rate of 1p LOH,19q LOH and 1p/19q LOH was 69.8%,64%,57.0%in 86 paraffin-embedded LOs respectively and was 71.1%,60.0%,55.6%in 45 paraffin-embedded AOs respectively.There was no statistical difference of the LOH of 1p/19q between LOs and AOs(P＞0.05).In 30 frozen LOs,the rate of 1p LOH, 19q LOH and1p/19q co-LOH was 70.0%,63.3%,60.0%respectively.The LOH of 1p/19q between paraffin-embedded and frozen samples showed no statistical significance(P＞0.05).In 30 astrocytic gliomas,1p LOH was 23.3%,19q LOH was 33.3%and 1p/19q co-LOH was 20.0%,less than oligodendrogliomas(P<0.05). Positive expression of p53 protein was identified in 21(21/131,16.0%) of 131 oligodendrogliomas and in 13(13/30,43.3%) of 30 astrocytic gliomas.The expression of p53 protein in oligodendrogliomas was much lower than astrocytic gliomas(P=0.001).The expression of p53 protein was more frequently occurring in AOs(14/45,31.1%) as compared with LOs(7/86,8.1%)(P=0.007).p53 expression was more common in patients with 1p/19q LOH in AOs.we found that the median PFS of patients suffered from oligodendroglioma with 1p/19q intact were 52.00±1.65 months,while the patients with 1p/19q LOH were 70.00±11.63 months.The median PFS in cases with1p/19q LOH was significantly longer than those with 1p/19q intact (P=0.006).While the median PFS in the cases with expression of p53 protein was shorter than those without expression of p53 protein(P=0.047).Summary The 1p/19q LOH was a common event in oligodendrogliomas,but not in astrocytic tumors.Both paraffin-embeded and frozen tissues could be used to detect 1p/19q LOH.The expression of p53 protein in oligodendrogliomas was less common than in astrocytic gliomas.There was a negative correlation between expression of p53 protein and 1p/19q LOH in patients with oligodendroglioma.Patients with 1p/19q LOH and negative expression of p53 protein had a better prognosis.PartⅡThe status of methylation of MGMT and expression of MGMT protein with correlative of 1p/19q LOH,p53 protein expression and their correlation to patients' prognosis in oligodendrogliomasObjective The purpose is to find the correlation among methylation of MGMT, 1p/19q LOH and p53 protein expression as well as their role in prognosis of oligodendrogliomasMethods The status of methylation of MGMT of 73 gliomas were detected with methylation specific PCR(MSP).The immunohistochemistry was performed to determine the MGMT protein expression.Pearson's chi-squared test was applied to explore the correlation between variables measured.Kaplan-Meier analysis was used to test the correlation of variable with progression-free survival(PFS) and overall survival(OS) in univariate analysis.Statistical significance was evaluated using the log-rank test and P＜0.05 was considered to be significant.Results MSP was performed in 73 cases of gliomas.There were 25(69.4%) out of 36 LOs,15(65.2%) out of 23 AOs and 8(8/14,57.1%) out of 14 astrocytic gliomas showing methylation of MGMT.No statistical significance could be found among them(P＞0.05).Among 10 cases of immuno-positive MGMT,6(6/36,16.7%) were out of 36 LOs,2(2/23,8.7%) out of 23 AOs,and 2(2/14,14.3%) from 14 astrocytic gliomas respectively.In those 10 immuno-positive cases,five cases revealed a methylated MGMT promoter.It seemed no correlationship between the MSP and immunohistochemistry of MGMT.Detective rate of methylated MGMT promoter was higher in 1p/19q LOH than in 1p/19q intact in oligodendrogliomas.Furthermore,we found that astrocytic gliomas with MGMT methylation had more frequent expression of p53 protein than those without MGMT methylation,although it didn't meet the statistical criteria.The relationships of PFS between methylation and unmethylation of MGMT were analyzed in LOs,AOs and combined AOs and LOs,but no statistical significance at all.Summary High frequency of methylated MGMT was detected in oligodendrogliomas and astrocytic gliomas.The methylated MGMT promoter was higher with 1p/19q LOH than 1p/19q intact in oligodendrogliomas.Methylation of MGMT was no significant relationship with oligodengroglioma patients' survival period.Thus,we suggested that detection of chromosome 1p/19q LOH combined with p53 protein immunohistochemistry might be helpful to improve pathological diagnosis and to have better prediction on prognosis of patients with oligodendrogliomas.ConclusionsThe 1p/19q LOH was a common event in oligodendrogliomas.Both paraffin-embedded and frozen tissues could be used to detect 1p/19q LOH.The expression of p53 protein in oligodendrogliomas was less than in astrocytic gliomas.There was a negative correlation between expression of p53 protein and 1p/19q LOH in patients with oligodendroglioma.Patients suffered from oligodendroglioma with 1p/19q LOH and negative expression of p53 protein had a better prognosis.High frequency of methylated MGMT was detected in oligodendrogliomas,but there was no significant relationship with patients' survival period.Thus,we suggested that chromosome 1p/19q LOH combined with p53 protein immunohistochemistry might be helpful to improve pathological diagnosis and have better prediction on prognosis of patients with oligodendrogliomas.