Alterations And Prognostic Significance Of Gene Promoter Hypermethylation And Histone Modification Pattern In Glioma Patients

Approximately22,500new cases of primary brain tumors occur annually in theUnited States, accounting for1.4%of all tumors and2.3%of cancer-related deaths.Glioma represents>70%of all brain tumors, including low-grade glioma (WHO grade1and2) and high-grade glioma (WHO grade3and4). Despite recent advances in surgery,radiotherapy, and chemotherapy, survival of glioma patients remains poor. The5-yearsurvival rates of low-grade glioma are30%to70%depending on histology, and themedian survival is only12to15months for the most frequent and malignant glioblastoma(5). Although WHO grade is the most important prognostic factor for glioma patients,there are broad differences within grades, which may be caused by differenttumor-specific biological behavior. Thus, the identification of biomarkers might help to assess more precisely the prognosis and to address more clearly the use of adjuvanttherapy.Besides genetic alterations, epigenetic modifications are critical to the developmentand progression of cancer. Epigenetics was defined as heritable changes in geneexpression that are not a result of any alteration in the DNA sequence, whose mechanismsinclude but are not limited to aberrant DNA methylation, histone modification, andmicroRNAs. The best-known epigenetic marker is DNA methylation. Hypermethylationof the CpG island promoter can induce silencing of genes affecting the cell cycle, DNArepair, metabolism of carcinogens, cell-to-cell interaction, apoptosis, and angiogenesis, allof which may occur at different stages in cancer development and interact with geneticlesions. In addition to DNA promoter hypermethylation, global alterations of histonemodification patterns have the potential to affect the structure and integrity of the genomeand to disrupt normal patterns of gene expression, which may also contribute tocarcinogenesis.The invasive and metastatic potential of gliomas are the major challenge of thetreatment, which account for the high rates of tumor relapse and poor prognosis. Gliomatumor tissues typically contain both neoplastic and stromal tissues, which lead to theirhistologic heterogeneity and thus the discrepancies between pathologic grades and clinicaloutcomes. Genetic and epigenetic studies potentially allow for better classification of thesetumors and separation of the tumors into different prognostic groups. The contribution ofepigenetic changes to glioma carcinogenesis has been broadly studied in terms of aberrantpromoter methylation–induced gene silencing. Besides glioma, aberrant DNA methylationhas been investigated in carcinogenesis and cancer development in various types of solidcancers and hematologic malignancies, and the potential targeting of DNA methylation asa therapeutic approach for cancer treatment is being evaluated. The current study analyzedthe alterations and prognostic value of gene promoter hypermethylation and global histonemodification in patients with glioma, hoping to define their potential role in clinicalapplication regarding prognosis prediction and chemotherapy selection.1Quantitative detection of multiple gene promoter hypermethylation in tumor tissue, serum, and cerebrospinal fluid predicts prognosis of malignant gliomasAberrant promoter hypermethylation of several known or putative tumor suppressorgenes occurs frequently during the malignant transformation in gliomas. Promoterhypermethylation as a potential biomarker for early diagnosis as well as prognosis hasbeen successfully used to detect neoplastic DNA in body fluids from several types ofcancer, including sputum, bronchoal-veolar lavage, serum, and plasma from lung cancerpatients; urine sediment, blood, and ejaculates from prostate cancer patients; and ductalfluid and plasma from breast cancer patients. We hypothesized that quantitative analysis ofmethylated genes in tumor tissue, serum, and cerebrospinal fluid (CSF) will provideprognostic values in malignant glioma patients.We used an immunocapturing approach followed by real-time polymerase chainreaction analysis to detect altered patterns of promoter methylation in4genes thatparticipate in different stges of carcinogenesis, including DNA repair geneO-6-methylguanine-DNA methyltransferase (MGMT), cell cycle regulator p16INK4a,tumor metastasis inhibitor gene tissue inhibitor of metalloproteinase-3(TIMP-3), andangiogenesis inhibitor gene thrombospondin1(THBS1). The tumor tissue and pairedserum as well as CSF from66patients with malignant gliomas were studied. Serum andCSF from20age-matched noncancer individuals were used as control.Promoter hypermethylation in MGMT, p16INK4a, TIMP-3, and THBS1wasdetected at high frequencies in tumor tissue, serum, and CSF. None of the control serum orCSF showed aber-rant methylation. Hypermethylation in serum and CSF DNA was allaccompanied with methylation in the corresponding tumor tissues (Pearson’s correlationcoefficient, r ranged from0.349to0.963; P <0.0001) with100%specificity. Additionally,hypermethylation of MGMT in tumor (Spearman’s correlation coefficient, r=0.422; P <0.0001) and CSF (r=0.373; P=0.002), p16INK4a in tumor (r=0.369; P=0.002), serum(r=0.297; P=0.015), and CSF (r=0.311; P=0.011), TIMP-3in tumor (r=0.338; P=0.006), THBS1in tumor (r=0.355; P=0.003) were positively correlated with increasingWHO grades.By applying a maximal χ2method, we divided the patients into good and poor prognostic subgroups with different cutoff values of the4genes detected in tumor tissue,serum, and CSF, respectively. Cutoff values representing methylation ratios bestsegregated patients into good and poor prognostic groups. Except for TIMP-3in CSF, allthe low promoter methylation levels in tumor tissue, serum, and CSF correlated with abetter survival.Highly elevated MGMT, p16INK4a, and THBS1methylation levels in gliomas serumwere the sole independent factors predicting inferior overall survival in this cohort. Forprogression-free survival, hypermethylation of MGMT and THBS1in CSF were theindependent prognostic factors.Multiple gene promoter hypermethylation analysis appears to be promising as aprognostic factor in glioma and as a mini-invasive tumor marker in serum and/or CSFDNA. Moreover, this approach has many potential clinical applications including primarydiagnosis, selecting treatment strategy, monitoring for disease progression and evolution,and measurement of therapeutic response, which need further investigation. Werecommend that microarray techniques be applied in future investigations to determine theprobable network of chemotherapeutic drug’s metabolism including MGMT, making itpossible to select the most appropriate therapies on the basis of epigenetic profiles of thepatients’ tumors.2Global histone modification patterns as prognostic markers to classify gliomapatientsThe global pattern of histone modification was identified by different study groups asa predictor of prognosis in prostate cancer, non–small cell lung cancer, and gastricadenocarcinoma patients. We further studied epigenetic changes involving multiplemodifications of histones to better predict prognosis of glioma patients.Immunohistochemistry was done to investigate global histone modificationexpression of histone3lysine4dimethylation (H3K4diMe), histone4arginine3monomethylation (H4R3monoMe), histone4lysine20trimethylation (H4K20triMe), andacetylation of histone3lysine9(H3K9Ac), histone3lysine18(H3K18Ac), histone4lysine12(H4K12Ac), and histone4lysine16(H4K16Ac) in resected tumor samples of 230glioma patients. Data were analyzed using a recursive partitioning analysis (RPA).The overall median expression of H3K4diMe, H4R3monoMe, H4K20triMe,H3K9Ac, H3K18Ac, H4K12Ac, and H4K16Ac was63%,60%,75%,74%,78%,65%,and68%, respectively. For each single tumor sample, the changes in expressions ofH3K4diMe, H4R3monoMe, H3K9Ac, H3K18Ac, H4K12Ac, and H4K16Ac were allcorrelated with each other (Pearson’s correlation coefficient, r ranged from0.205to0.528;P≤0.028), with the exception of H4K20triMe when compared respectively with theformer six modifications. H3K4diMe staining (Spearman’s correlation coefficient, r=0.328; P=0.0003), H4R3monoMe (r=0.193; P=0.039), H3K9Ac (r=0.200; P=0.032),and H4K12Ac (r=0.314; P=0.0006) had low positive correlations with increasing WHOgrades.RPA classified the patients into10distinct prognostic groups based on WHO grade,histology, and histone modifications: H3K9Ac (<88%or≥88%tumor cells), H3K4diMe(<64%or≥64%tumor cells), H3K18Ac (<74%or≥74%tumor cells), and H4K20triMe(<75%or≥75%tumor cells). The10groups were associated with significantly differentprogression-free (P <0.0001) and overall survival (P <0.0001). Cox proportional hazardsmodels including age, sex, WHO grade, histology, extent of tumor resection, Karnofskyperformance status score, and RPA groups retained age and RPA groups as the soleindependent factors significantly influencing overall survival. For progression-freesurvival, RPA grouping was the only independent prognostic factor.Multiple histone modifications seem to have prognostic relevance in glioma. Furtherevaluation of histone modifications as prognostic markers of treatment and predictors ofchemotherapy response using histone deacetylase inhibitors is warranted.Based on the existing results including ours, we suggest that histone modificationsshould be further investigated in combination with DNA methylation as predictors ofresponse to chemotherapy in the future, which may better help to select the mostappropriate therapies on the basis of epigenetic profiles of tumors. This approach mayultimately optimize treatment selection in glioma patients and improve their clinicaloutcome.