MGMT Methylation Status Research Articles

PO69THE IMPACT OF MGMT METHYLATION AND IDH-1 MUTATION ON LONG TERM OUTCOME FOR GLIOBLASTOMA TREATED WITH CHEMORADIOTHERAPY

Background Increasingly, biomarkers have been identified that correlate with improved overall and progression-free survival (OS and PFS) in glioblastoma, including MGMT methylation status and mutations in the IDH1 gene. In this study, we investigated the clinical and biological factors associated with long-term survival in glioblastoma patients treated with chemoradiotherapy.

ATPS-42INHIBITION OF STAT3 ENHANCES THE RADIOSENSITIZING EFFECT OF TEMOZOLOMIDE IN MALIGNANT GLIOMA CELLS IN VITRO AND IN VIVO

PURPOSE: Despite aggressive treatment with radiation therapy plus concurrent and adjuvant temozolomide (TMZ), the prognosis for glioblastoma remain poor. We investigated the potential of targeting signal transducer and activator of transcription-3 (STAT3) to improve the therapeutic outcome of combined radiotherapy and TMZ in glioblastoma. METHODS AND MATERIALS: We evaluated the preclinical potential of a STAT3 inhibitor, Cpd188 combined with temozolomide and radiation by in vitro clonogenic assays using two established glioblastoma cell lines (U251, U87) and two patients-derived glioblastoma cell lines (GBL12, GBL28) and in vivo studies using nude mice bearing intracranial U251 xenografts. RESULTS: Cpd188 potentiated the radiosensitizing effect of TMZ in U251 glioblastoma cell line which has high levels of p-STAT3 expression and in vitro. Increased radiosensitizing effects of TMZ were associated with the induction of apoptosis and the reversion of epithelial-mesenchymal transition (EMT). Cpd188 delayed in vivo tumor growth both alone and in combination with fractionated radiation and TMZ with a trend toward improved survival rates. Immunohistochemical staining of tumor sections showed that Cpd188 decreased the expression of CD31 (a marker of endothelial proliferation), vascular endothelial growth factor, and hypoxia-inducible factor-1α, suggesting that Cpd188 also has anti-angiogenic effects. We also confirmed the radiosensitizing effect of Cpd188 of GBL28 cell line which was originated from a patient who had a glioblastoma and also was confirmed high level of STAT3 expression and unmethylated MGMT. CONCLUSION: These data indicate that Cpd188 has the potential to improve the therapeutic outcome of combined radiotherapy and TMZ in human glioblastoma, especially in patients whose tumor has a high level of STAT3 expression regardless of MGMT methylation status. Work supported by grant (#2012-0004867 & #2013R1A1A2074531) from National Research Foundation, Korean Ministry of Future Creative Science to In Ah Kim.

MTR-09ABCB1 AS PREDICTIVE MARKER FOR POOR SURVIVAL IN PATIENTS WITH GLIOBLASTOMA TREATED WITH RADIOTHERAPY AND CONCOMITANT AND ADJUVANT TEMOZOLOMIDE

BACKGROUND: Standard treatment for newly diagnosed glioblastoma (GBM) patients is surgery followed by radiotherapy (RT) and temozolomide (TMZ) given concomitant and adjuvant. Tumor MGMT methylation status has been identified as predictive for response to TMZ. Also other factors are believed to influence TMZ effect. We examined the role of different single nucleotide variants (SNV:s) of ABCB1, a transmembrane drug transporter, for survival for GBM patients receiving RT concomitant with TMZ. PATIENTS AND METHODS: Genomic samples from patients with GBM who received RT + TMZ concomitant was analyzed for SNV:s of ABCB1, namely variants G1199A, C1236T, G2677T_A and C3435T and correlated to overall survival (OS). ABCB1 was determined using DNA from blood. RESULTS: The cohort included 101 patients, diagnosed and treated between 2004 and 2014. Median age was 60 (range 32-74). 83% of patients received RT 60 Gy, 5% 34 Gy and 12% between 40-58 Gy concomitant with TMZ. For SNV G1199A median OS for variant G/G was 18.2 months versus 11.5 months for those with G/A (p = 0.009), while the other SNV:s showed no significant correlation to survival. For 78 patients MGMT status is available. In multivariate analyses MGMT status and G1199A SNV were both independent factors for survival (p = 0.000 and 0.018 respectively). CONCLUSION: For GBM patients receiving RT concomitant with TMZ the ABCB1 SNV G1199A could be a new treatment predictive factor.

Applicability of HIN-1, MGMT and RASSF1A promoter methylation as biomarkers for detecting field cancerization in breast cancer.

IntroductionIt has been shown in some articles that genetic and epigenetic abnormalities cannot only be found in tumor tissues but also in adjacent regions that appear histologically normal. This phenomenon is metaphorically called field cancerization or field defect. Field cancerization is regarded as clinically significant because it is assumed to be an important factor in local recurrence of cancer. As the field showing these molecular abnormalities may not be removed completely by surgery, these changes might lead to neoplasms and subsequent transformation to a tumor. We aimed to investigate the applicability of the methylation status of six tumor suppressor genes as biomarkers for detecting field cancerization in breast cancer.MethodsThe promoter methylation status of CCND2, DAPK1, GSTP1, HIN-1, MGMT and RASSF1A was determined by methylation-sensitive high-resolution melting (MS-HRM) analysis. MS-HRM methods for CCND2, MGMT and RASSF1A were developed in-house, primer sequences for DAPK1, GSTP1 and HIN-1 have already been published. Biopsy samples were taken from tumor, tumor-adjacent and tumor-distant tissue from 17 breast cancer patients. Normal breast tissues of four healthy women served as controls.ResultsAll MS-HRM methods proved to be very sensitive. LODs were in the range from 0.1 to 1.5 %, LOQs ranged from 0.3 to 5.3 %. A total of 94 %, 82 % and 65 % of the tumors showed methylation of RASSF1A, HIN-1 and MGMT promoters, respectively. The methylation status of these promoters was significantly lower in tumor-distant tissues than in tumor tissues. Tumor-adjacent tissues showed higher methylation status of RASSF1A, HIN-1 and MGMT promoters than tumor-distant tissues, indicating field cancerization. The methylation status of the HIN-1 promoter in tumor-adjacent tissues was found to correlate strongly with that in the corresponding tumors (r = 0.785, p < 0.001), but not with that in the corresponding tumor-distant tissues (r = 0.312, p = 0.239).ConclusionsAmong the gene promoters investigated, the methylation status of the HIN-1 promoter can be considered the best suitable biomarker for detecting field cancerization. Further investigation is needed to test whether it can be used for defining surgical margins in order to prevent future recurrence of breast cancer.

Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer

O(6)-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and cell-free circulating DNA (cfDNA) from plasma samples using an ultra-sensitive two-step digital PCR technique (methyl-BEAMing). Results were compared with two established techniques, methylation-specific PCR (MSP) and Bs-pyrosequencing. Thresholds for MGMT methylated status for each technique were established in a training set of 98 glioblastoma (GBM) patients. The prognostic and the predictive value of MGMT methylated status was validated in a second cohort of 66 GBM patients treated with temozolomide in which methyl-BEAMing displayed a better specificity than the other techniques. Cutoff values of MGMT methylation specific for metastatic colorectal cancer (mCRC) tissue samples were established in a cohort of 60 patients treated with dacarbazine. In mCRC, both quantitative assays methyl-BEAMing and Bs-pyrosequencing outperformed MSP, providing better prediction of treatment response and improvement in progression-free survival (PFS) (P < 0.001). Ability of methyl-BEAMing to identify responding patients was validated in a cohort of 23 mCRC patients treated with temozolomide and preselected for MGMT methylated status according to MSP. In mCRC patients treated with dacarbazine, exploratory analysis of cfDNA by methyl-BEAMing showed that MGMT methylation was associated with better response and improved median PFS (P = 0.008). Methyl-BEAMing showed high reproducibility, specificity and sensitivity and was applicable to formalin-fixed paraffin-embedded tissues and cfDNA. This study supports the quantitative assessment of MGMT methylation for clinical purposes since it could refine prediction of response to alkylating agents.

MGMT methylation assessment in glioblastoma: MS-MLPA versus human methylation 450K beadchip array and immunohistochemistry.

The MGMT gene encodes a DNA repair enzyme that counteracts with chemotherapy efficiency, specifically with alkylating agents such as temozolomide (TMZ). It is well established that MGMT methylation should be screened as a predictive marker for TMZ in glioblastoma, and we thus aimed to determine a reliable and practical diagnostic method of MGMT methylation detection. 55 glioblastomas were investigated for MGMT methylation status using methylation-specific multiplexed ligation probe amplification (MS-MLPA), illumina human methylation 450K BeadChip array (HM450 K) analysis, and compared to MGMT protein expression by immunohistochemistry (IHC) staining. The methylation status of promoter, intron and all MGMT CpG targeted sites were separately correlated to patient's survival. In addition to MS-MLPA and 450 K concordance, our results showed significantly higher overall survival (OS) of patients receiving TMZ and presenting MGMT methylated promoter (mean OS = 21.5 months, p = 0.046). Including all glioblastoma cases and regardless of chemotherapy, MS-MLPA showed significant survival difference between MGMT methylated and unmethylated cases (mean OS = 13, p = 0.021). We concluded that in glioblastoma, MGMT promoter methylation predicts TMZ sensitivity. This current comparative analysis leads to consider that MS-MLPA is a valuable as HM450 K array for MGMT methylation status screening.

Clinical outcome and molecular characterization of pediatric glioblastoma treated with postoperative radiotherapy with concurrent and adjuvant temozolomide: a single institutional study of 66 children.

Glioblastoma (GBM) in children is rare. Pediatric GBM have a distinct molecular profile as compared to adult GBM. There are relatively few studies of pediatric GBMs and no standard of care on adjuvant therapy. We aimed to evaluate the clinical outcome and molecular profile of pediatric GBM. Between 2004 and 2013, 66 consecutive children with histologically proven GBM were identified from our database. The majority of the children underwent maximal safe resection followed by focal radiotherapy with concurrent and adjuvant temozolomide. Immunohistochemical staining was performed for p53, MIB-1 labeling index, MGMT overexpression, and EGFR amplification and isocitrate dehydrogenase (IDH1) R132H point mutation. Survival and impact of possible prognostic factors on outcomes were analyzed. Median survival was 15 months. The overall survival rate at 1 year was 62%, at 2 years was 30%, and at 3 years was 27%. Patients with thalamic tumors (P < .001), incompletely resected tumors (P < .00001), and tumors with MIB-1 labeling index >25% (P < .002) had poor overall survival rates. p53 was overexpressed in 74% of patients, MGMT promoter methylation was seen in 37% of patients, IDH1 mutation was seen in 4% of patients, and no patients had EGFR amplification. MGMT methylation and p53 overexpression did not impact survival. Clinical outcome of pediatric GBM is similar to that reported for adult GBM. The frequency of p53 overexpression is higher than in adult GBM, while MGMT methylation, IDH1 mutations and EGFR amplification is lower than in adult GBM. MGMT methylation and p53 expression status do not have any prognostic significance.

Abstract 3839: Investigation of epigenetic based inter-tumoral heterogeneity identifies novel ZMIZ1 gene as a biomarker of cancer patient survival in multiple tumor types

Abstract Glioblastoma is the most malignant primary brain tumor and is universally fatal despite the use of the best treatment available. However a small percentage of these patients live considerably longer. Extensive genetic and epigenetic analysis has identified IDH1 mutation, G-CIMP (Glioma-CpG Island Methylator Phenotype) phenotype and MGMT as markers of better survival in glioblastoma. These markers though can only be associated with a small fraction of long term survivors (defined as glioblastoma patients surviving &amp;gt;3 years), underlying the need for better biomarkers of improved survival. Epigenetic analysis of inter-tumoral heterogeneity in the TCGA glioblastoma cohort identified a truncated form of the novel ZMZI1 gene to be associated with improved survival in patients hypermethylated in the alternative promoter of the truncated ZMIZ1 form, in an independent manner from IDH1, G-CIMP or MGMT methylation status. A validation cohort of 31 glioblastoma long term survivors with low prevalence of the known markers IDH1, G-CIMP and MGMT showed that ZMIZ1 methylation was positive in 42% of the patients, more sensitive and specific than IDH1, G-CIMP or MGMT. Extending our analysis to lower grade gliomas as well as other central nervous system tumors showed that higher methylation of the truncated ZMIZ1 was predicting a more favorable outcome in astrocytomas but not in oligodendrogliomas. Furthermore, in non-CNS tumors including breast cancer, colorectal cancer, bladder cancer and renal cell carcinoma, lower expression of the truncated ZMIZ1 form predicted a favorable survival, statistically significant in univariate and multivariate analysis adjusting for tumor stage and grade. In vitro loss of function and gain of function studies showed that ZMIZ1 is significantly affecting migration of glioblastoma cell lines, leaving unaffected the proliferation capacity of these cells. Loss of function studies showed that decreased ZMIZ1 expression in glioblastoma is resulting in higher sensitivity of glioblastoma cells to temozolamide. These results identify a novel pan-cancer biomarker of prognosis as well as a new gene that could potentially play a key role in cancer pathogenesis and resistance to treatment. Interestingly, unlike many other key cancer genes ZMIZ1 expression is regulated via epigenetic rather than genetic alterations in the entirety of human cancers. Citation Format: Dimitrios Mathios, Taeyoung Hwang, Jillian Phallen, Chetan Bettewgoda, Patrick Ha, Peter Burger, Henry Brem, Kerrie McDonald, Chul-Kee Park, Michael Lim. Investigation of epigenetic based inter-tumoral heterogeneity identifies novel ZMIZ1 gene as a biomarker of cancer patient survival in multiple tumor types. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3839. doi:10.1158/1538-7445.AM2015-3839

Genomic predictors of patterns of progression in glioblastoma and possible influences on radiation field design.

We present a retrospective investigation of the role of genomics in the prediction of central versus marginal disease progression patterns for glioblastoma (GBM). Between August 2000 and May 2010, 41 patients with GBM and gene expression and methylation data available were treated with radiotherapy with or without concurrent temozolomide. Location of disease progression was categorized as within the high dose (60Gy) or low dose (46Gy) volume. Samples were grouped into previously described TCGA genomic groupings: Mesenchymal (m), classical (c), proneural (pn), and neural (n); and were also classified by MGMT-Methylation status and G-Cimp methylation phenotype. Genomic groupings and methylation status were investigated as a possible predictor of disease progression in the high dose region, progression in the low dose region, and time to progression. Based on TCGA category there was no difference in OS (p=0.26), 60Gy progression (PN: 71%, N: 60%, M: 89%, C: 83%, p=0.19), 46Gy progression (PN: 57%, N: 40%, M: 61%,C: 50%, p=0.8) or time to progression (PN: 9months, N:15months, M: 9months, C: 7months, p=0.58). MGMT methylation predicted for improved OS (median 25 vs. 13months, p=0.01), improved DFS (median 13 vs. 8months, p=0.007) and decreased 60Gy (p=0.003) and 46Gy (p=0.006) progression. There was a cohort of MGMT methylated patients with late marginal disease progression (4/22 patients, 18%). TCGA groups demonstrated no difference in survival or progression patterns. MGMT methylation predicted for a statistically significant decrease in in-field and marginal disease progression. There was a cohort of MGMT methylated patients with late marginal progression. Validations of these findings would have implications that could affect radiation field size.

Novel Surgical Approaches to High-Grade Gliomas.

Treatment of patients with high-grade glioma (HGG) should begin with thorough evaluation by a specialized multidisciplinary team to determine whether or not the patient is appropriate for surgery, chemotherapy and radiotherapy. Particular attention should be paid to the performance status and neurological function. Surgery is the first step in therapeutic intervention. Patients undergo either biopsy, debulking surgery or maximal resection depending on the anatomical location of the tumour and the patient's clinical condition. Extent of resection has a prognostic value. In patients who are 'fit for surgery', the aim is to remove all contrast-enhancing tumour without causing neurological deficit. If microsurgical resection is not feasible, then a biopsy, either open or stereotactic, should be performed to confirm high-grade glioma diagnosis and to perform molecular genetic analyses (MGMT methylation status, loss of heterozygosity in 1p/19q, IDH1 status) as this has treatment implications. Over the past decade, much glioma research has focussed on novel surgical approaches to improve long-term outcomes. The evidence to support the benefit of maximizing extent of resection is growing. Advances in neurosurgical techniques allow safer, more aggressive surgery to maximize tumour resection whilst minimizing neurological deficit. Surgical adjuncts including advanced neuronavigation, intraoperative magnetic resonance imaging, high-frequency ultrasonography, fluorescence-guided microsurgery using intraoperative fluorescence, functional mapping of motor and language pathways, and locally delivered therapies are extending the armamentarium of the neurosurgeon to provide patients with the best outcome. Operating on elderly patients and those with recurrent disease, although controversial, is becoming more common due to emerging neurosurgical approaches. Here, we discuss the emerging surgical techniques and comment on the future of HGG surgery.

Prognostic impact of molecular phenotype in patients with recurrent anaplastic glioma treated with prolonged administration of temozolomide

We retrospectively investigated the prognostic impact of molecular phenotypes in patients with recurrent anaplastic glioma treated with prolonged administration of temozolomide (TMZ). Malignant gliomas have a dismal prognosis despite the available multimodal treatments. We reviewed 87 patients who were diagnosed with recurrent anaplastic gliomas between March 2004 and June 2010, and 58 were enrolled for analysis. In the cohort, 21 patients had anaplastic oligodendrogliomas, 18 anaplastic oligoastrocytomas and 19 anaplastic astrocytomas. All patients were initially treated with surgical resection or biopsy followed by involved-field radiotherapy. At recurrence, patients were treated with 150–200mg/m2 of TMZ on days 1–5 in 28day cycles until disease progression. We evaluated the association of molecular phenotypes, including 1p19q deletion, O6-methylguanine DNA methyltransferase (MGMT) promoter methylation status, isocitrate dehydrogenase-1 (IDH1) mutation and other clinico-histopathological findings with treatment outcome. During the mean follow-up period of 34.6months, 33 patients were still alive (56.9%). Median survival from recurrence was 39.7months (95% confidence interval [CI] 22.7–56.7). Time to progression from administration of TMZ was 6.4months (95% CI 5.0–7.8). Univariate analysis demonstrated that the presence of the IDH1 mutation was closely associated with treatment response (8.4 versus 3.8months; p=0.015). Oligodendroglial lineage, 1p19q deletion status and MGMT promoter methylation status were not independent variables for determining the TMZ treatment outcome. In recurrent anaplastic gliomas, TMZ treatment is an effective modality regardless of MGMT methylation status or histological type. The IDH1 mutation has the most powerful prognostic impact on overall patient survival.

MB-06 * PROGNOSTIC IMPACT OF O6-METHYLGUANINE-DNA METHYLTRANSFERASE (MGMT) PROMOTER METHYLATION IN MEDULLOBLASTOMA

BACKGROUND: O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that plays an important role in tumor resistance to alkylating agents. Recent study revealed that MGMT promoter methylation is associated with favorable outcomes in malignant glioma patients. However, MGMT status in pediatric medulloblastoma and the prognostic impact of this methylation status and clinical outcomes of this tumor has not been fully evaluated. The objective of this study is to investigate the association between MGMT methylation status and clinical outcomes for medulloblastoma patients. MATERIALS AND METHODS: Patients with medulloblastoma treated at our institution between 1995 and 2012 were reviewed retrospectively. Tumor tissues were obtained from formalin-fixed paraffin-embedded tumor samples. Methylation statuses of 18 CpG sites of MGMT promoter region were determined by bisulfite sequencing analysis. RESULTS: Tumor material and clinical information were available for 22 patients. Median follow-up period was 48 months (13-190 months). Median age at diagnosis was 8.5 years (1-23 years). After the first line treatment, 10 had been in complete remission, 8 had relapse of disease, 1 had progressive disease, 1 had stable disease and 2 were lost follow-up. The patients in complete remission had higher accumulation of methylated CpG sites (median 5, range 2-9) than the relapsed patients (median 2, range 1-4) (p = 0.036). CONCLUSION: The results indicated that MGMT promoter methylation correlates with favorable outcome of medulloblastoma.

HG-01 * MGMT PROMOTER METHYLATION STATUS AND miRNA-181 FAMILY EXPRESSION AS PROGNOSTIC MARKERS IN GLIOBLASTOMA PATIENTS

BACKGROUND: The O6-methylguanine DNA methyltransferase (MGMT) enzyme is related with temozolomide (TMZ) resistance. The miRNA-181 family is the important role of radio- and chemo-resistant in glioblastoma (GBM) patients. Our research is to analyze the prognostic significance of MGMT promoter methylation status and miRNA-181 family expression in a group of GBM patients treated with radiotherapy and TMZ. The possible connection between these GBM markers was also investigated. METHODS: Sixty-three patients with GBM treated with radiotherapy combined with concomitant and adjuvant TMZ were analyzed for MGMT and miRNA-181 family. We examined methylation status of MGMT promoter by methylation-specific real-time PCR. MiRNA-181 family expression was assessed qPCR. Overall and progression-free survival was calculated according to the Kaplan–Meier method. RESULTS: The MGMT gene promoter was found to be methylated in 29 patients (46.0%) and unmethylated in 34 patients (54.0%). MGMT methylation status was significant correlated with response to concomitant chemoradiotherapy with temozolomide (RT/TMZ). MiRNA-181c and miR-181d expression were the predictive markers in GBM patients prognosis (p < 0.05). MiRNA-181c and miRNA-181d were down-regulated in patients who responded to RT/TMZ (p = 0.032; p = 0.017, respectively) in comparison to patients with poor prognosis. Analyses of possible correlation between miRNA-181d expression and MGMT promoter methylation status were positive. CONCLUSIONS: Our results support the hypothesis that MGMT promoter methylation status may be a predictive biomarker in the treatment of patients with GBM. In addition, our data indicate for the first time that expression levels of miRNA-181c and miRNA-181d could serve as a predictive marker of response to RT/TMZ therapy in GBM patients. Moreover, these data suggest that miRNA-181d is a potential role to regulate MGMT promoter methylation status.

MGMT promoter methylation and glioblastoma: a comparison of analytical methods and of tumor specimens.

It is already well known that hypermethylation of the O6-methylguanine DNA methyltransferase (MGMT) gene promoter is a predictive biomarker of response to temozolomide treatment and of favorable outcomes in terms of overall survival (OS) and progression-free survival (PFS) in glioblastoma (GBM) patients. Nevertheless, MGMT methylation status has not currently been introduced into routine clinical practice, as the choice of the ideal technique and tissue sample specimen is still controversial. The aim of this study was to compare 2 analytical methods, methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ), and their use on 2 different tissue type samples, snap-frozen and formalin-fixed paraffin-embedded (FFPE), obtained from a single-center and uniformly treated cohort of 46 GBM patients. We obtained methylation data from all frozen tissues, while no results were obtained for 5 FFPE samples. The highest concordance for methylation was found on frozen tissues (88.5%, 23/26 samples), using PSQ (76.7%, 23/30 samples). Moreover, we confirmed that OS and PFS for patients carrying methylation of the MGMT promoter were longer than for patients with an unmethylated promoter. In conclusion, we considered MSP a limited technique for FFPE tissues due to the high risk of false-positive results; in contrast, our data indicated PSQ as the most powerful method to stratify methylated/unmethylated patients as it allows reaching quantitative results with high sensitivity and specificity. Furthermore, frozen tumor tissues were shown to be the best specimens for MGMT methylation analysis, due to the low DNA degradation and homogeneity in methylation throughout the tumor.

Protein expression and methylation of MGMT, a DNA repair gene and their correlation with clinicopathological parameters in invasive ductal carcinoma of the breast.

Epigenetic mechanisms such as DNA methylation are being increasingly recognized to play an important role in cancer and may serve as a cancer biomarker. The aim of this study was to evaluate the promoter methylation status of MGMT (O6-methylguanine-DNA methyltransferase) and a possible correlation with the expression of MGMT and standard clinicopathological parameters in invasive ductal breast carcinoma patients (IDC) of Kashmir. Methylation-specific PCR was carried out to investigate the promoter methylation status of MGMT in breast tumors paired with the corresponding normal tissue samples from 128 breast cancer patients. The effect of promoter methylation on protein expression in the primary breast cancer and adjacent normal tissues was evaluated by immunohistochemistry (n = 128) and western blotting (n = 30). The frequency of tumor hypermethylation was 39.8 % and a significant difference in methylation frequency among breast tumors were found (p < 0.001) when compared with the corresponding normal tissue. Immunohistochemical analysis showed no detectable expression of MGMT in 68/128 (53.1 %) tumors. MGMT promoter methylation mediated gene silencing was associated with loss of its protein expression (rs = -0.285, p = 0.001, OR = 3.38, 95 % CI = 1.59-7.17). A significant correlation was seen between loss of MGMT and lymph node involvement (p = 0.030), tumor grade (p < 0.0001), loss of estrogen receptors (ER; p = 0.021) and progesterone receptors (PR) (p = 0.016). Also, MGMT methylation was found to be associated with tumor grade (p = 0.011), tumor stage (p = 0.009), and loss of ER (p = 0.003) and PR receptors (p = 0.009). To our knowledge, our findings, for the first time, in Kashmiri population, indicate that MGMT is aberrantly methylated in breast cancer and promoter hypermethylation could be attributed to silencing of MGMT gene expression in breast cancer. Our data suggests that MGMT promoter hypermethylation could have a potential function as molecular biomarker of breast oncogenesis. Also, based on their predictive value of response to therapy, the immunohistochemical evaluation and interpretation of MGMT may also help in future to establish therapeutic strategies for patients with breast cancer.

HIN-1: a New Epigenetic Biomarker Crucial for Therapy Selection in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most common brain tumor in adults. The role of high in normal-1 (HIN-1) as a potential biomarker in combating this disease is being described for the first time in this study. A combination of O6-methylguanine DNA methyltransferase (MGMT) and HIN-1 methylation could be a possible biomarker in therapy choice. Interestingly, survival data shows a similar trend for the methylation of MGMT and for unmethylation of HIN-1 and vice versa. Eighty-eight paraffin-embedded brain tumors were analyzed to screen methylation rates of different genes and evaluate the association between genes methylation and clinicopathologic variables. Our study is the first of its kind to indicate that MGMT and HIN-1 methylation status are inverted (97.7% of methylated ones) and could be new markers in the study of GBM prognosis, especially in the therapy selection.

Variation over time and interdependence between disease progression and death among patients with glioblastoma on RTOG 0525.

We assessed the longitudinal hazard characteristics for death and progression in patients with glioblastoma, evaluated the impact of prognostic factors and treatment on the hazard within different time intervals to determine if effects are time varying, and quantified the influence of progression on survival. Among patients randomized to Radiation Therapy Oncology Group trial 0525, which compared dose-dense with standard-dose temozolomide, we estimated the hazards of death and treatment failure (death or progression) over time and their interdependence. The peak hazard of death was reached at around 16 months with a slow decline after that; the hazard of progression/death reached a peak at around 6 months and decreased dramatically thereafter. The survival advantages for patients with MGMT gene promoter methylation and recursive partitioning analysis class III were substantial in the first 2 years, but lessened thereafter. The progression-free survival benefit of dose-dense over standard-dose temozolomide occurred in the first 6 months (hazard ratio: 0.70; 95% CI: 0.58-0.86; P < .001), although it diminished thereafter. After adjusting for recursive partitioning analysis class and MGMT methylation status, the hazard ratio of death for patients who had progressed over nonprogressors was 6.59 (95% CI: 5.15-8.43; P < .001). After the peak hazard of death, a consistently high hazard remains, but it is lower than in the peak period. The progression hazard peak is earlier, and then hazard consistently declines. The rate of dying after disease progression is about 6.59 times the rate for nonprogressors, suggesting that progression-free survival may be a relevant clinical endpoint.

End of the road: confounding results of the CORE trial terminate the arduous journey of cilengitide for glioblastoma

End of the road: confounding results of the CORE trial terminate the arduous journey of cilengitide for glioblastoma

Prognostic value of MGMT promoter status in non-resectable glioblastoma after adjuvant therapy

BackgroundMethylation of MGMT promoter has been identified as a favourable predictive factor of benefit from XRT/TMZ→TMZ. Patients with non-resectable glioblastoma (GBM) generally exhibit a poor prognosis, even after XRT/TMZ. Few data are available concerning the predictive value of MGMT promoter methylation in this population. MethodsThis is an observational retrospective study in patients with malignant brain glioma, treated between June 2008 and October 2011 and followed up until April 2012 at the Neurosurgery-Neurotraumatology Unit of the University Hospital of Parma and at the Neurosurgery Unit of IRCCS “ASMN” of Reggio Emilia, Italy. The medical records of an overall number of 174 patients with a newly diagnosed GBM were reviewed. Volumetry analysis of the lesions was performed on pre- and post-operative neuroimaging by Voxar 3D Ebit AET software. The genetic characterization was performed on paraffin embedded tissue from all resected tumours. Isolation of nucleic acids, bisulfite modification of DNA, methylation-specific PCR and sequencing analyses were done mainly on fresh tissue from biopsy withdrawals. Within 3–4 weeks after either biopsy or surgery, patients were assigned to receive XRT/TMZ→TMZ: treatment included XRT (60Gy in 30 fractions)/TMZ (daily dose of 75mg/m2)/TMZ (150–200mg/m2 per day for 5 days of every 28-day cycle). Results and discussionA total of 55 consecutive patients (23 men, 22 women) fulfilled inclusion criteria consisting of age over 18 years, supratentorial histologically proven primary malignant glioma, complete determination of the MGMT methylation status, no prior history of surgery, XRT and/or chemotherapy, adequate clinical and radiological follow-up no lesser than 6 months. Twenty-three patients underwent neuronavigation needle biopsy (B Group) and thirty-two patients were operated with craniotomy for tumour resection (R Group). The pre-operative mean age was similar between groups (61.7±10.7 vs 60.3±11.8 years in the B and R groups respectively; p>0.05). The B groups showed a slightly lower KPS than the R Group (82.1±17.3 vs 90.3±14.1 respectively; p>0.05). The mean pre-operative volume of the tumour did not differ between groups (46.2±40.2cm3 vs 44.1±33.2cm3 in the R Group and B Group respectively; p>0.05). The MGMT promoter was methylated in 12 patients (51.2%) of B Group and in 17 patients (53.1%) of R Group. XRT/TMZ→TMZ was accomplished in 11 patients (47.8%) of B Group and in 24 patients (75%) of R Group; in 24/29 methylated patients (82.8%) and in 11/26 unmethylated patients (42.3%). Survival analysis of methylated vs unmethylated tumours was statistically significant (Log Rank Mantel Cox: 0.019 in B Group and 0.023 in R Group). In B Group the mean overall survival (OS) of methylated patients was 11.4 months (IC 95% 6.5–16.4) vs 4.8 months (95% IC, 2.6–7.0) of unmethylated patients. In R Group the mean OS was 21.7 months (95% IC, 16.9–26.6) for methylated patients and 14.0 months (95% IC, 8.5–19.4) for unmethylated patients. At the multivariate Cox regression analysis conducted on the total population (55 patients), XRT and TMZ were found to be predictive of OS. In the R Group, KPS, XRT and TMZ correlated with a better outcome. In the B Group, XRT and MGMT promoter methylation were favourably related with OS. ConclusionMGMT promoter unmethylation has a predominant unfavourable impact on clinical outcomes even in the subpopulation of patients with non-resectable GBM. The unmethylated MGMT promoter status could be considered the main predictor of poor prognosis in biopsied GBM, due to the greater probability of patients not having benefits from adjuvant therapies and not being able to accomplish XRT/TMZ→TMZ. The frameless neuronavigation biopsy technique is safe and effective for predictive evaluation and could help in treatment decision making.

Prognostic value of MGMT methylation in colorectal cancer: a meta-analysis and literature review.

The development of colorectal cancer (CRC) spans about 5-10 years, making early detection and prevention beneficial to the survival of CRC patients. To address inconsistencies in evidence regarding O(6)-methylguanine-DNA-methyltransferase (MGMT) methylation as a potential prognostic factor in CRC, we conducted a meta-analysis to evaluate MGMT methylation in CRC patients. Fourteen studies were included in the meta-analysis after screening 120 articles. The following items were collected from each study: author, published year, country, patient gender, MGMT methylation status, and patients' disease progression. Pooled hazard ratios and odd ratios with 95% confidence intervals (CIs) were calculated using fixed or random effect models depending on the heterogeneity between studies. The overall survival of CRC patients was found not to be significantly associated with MGMT methylation. Further subgroup analysis showed that the frequency of MGMT methylation was significantly higher in CRC than in normal tissues (p < 0.00001). MGMT promoter in CRC patients was more frequently methylated than in adenoma patients. In addition, MGMT methylation was significantly increased in adenoma than in normal tissues (p < 0.0001). In conclusion, MGMT methylation is central to the development of cancer that involves a stepwise carcinogenesis of normal adenoma carcinoma cascade. However, MGMT methylation is not associated with the prognosis of CRC.