The Detection Of Isocitrate Dehydrogenase Gene Mutation In Gliomas And Its Effect On Chemotherapy

Part I:Clinical siginficance of IDH1and IDH2mutations in Chinese glioma patientsBackground and Objective:Gliomas are the most frequent and lethal tumors of the central nervous system(CNS) and show wide diversity with location, morphology, genetic status, and response to therapy. This group of tumors includes specific histologic subtypes, the most common of which are astrocytomas, oligodendrogliomas, and ependymomas. These tumors have been classified as grade I to grade IV based on histopathological and clinical criteria established by the World Health Organization (WHO). Despite intensive therapies, including surgery, radiotherapy, and chemotherapy, the outcome of glioma patients remains depressing. The median survival is only12to15months for patients with glioblastomas and2to5years for patients with anaplastic gliomas. Recently, there has been important progress in the treatment of malignant gliomas and in our understanding of the molecular pathogenesis of these tumors and the critical role that stem cells play in their development and resistance to treatment. As our understanding of the molecular correlates of response improves, it may be possible to select the most appropriate therapies on the basis of the patient’s tumor genotype. These advances provide real opportunities for the development of effective therapies for malignant gliomas.The latest breakthrough came in2008, when the genes encoding isocitrate dehydrogenase1(IDH1)(and to a lesser extent IDH2) were found to be mutated in lower grade gliomas and a subset of glioblastomas (those of the proneural type or having evolved from lower grade tumors). Interestingly, only1copy of the gene is mutated in the tumors, suggesting that the mutations do not result in a simple loss of function. The mutation is very specific and leads to a single amino acid change (arginine132usually becomes histidine) in the IDH1active site, whereby the enzyme loses its ability to catalyze conversion of isocitrate to a-ketoglutarate. Balss J et al. analyzed the genomic region spanning wild type R132of IDH1by direct sequencing in685brain tumors including41pilocytic astrocytomas,12subependymal giant cell astrocytomas,7pleomorphic xanthoastrocytomas,93diffuse astrocytomas,120adult glioblastomas,14pediatric glioblastomas,105oligodendrogliomas,83oligoastrocytomas,31ependymomas,58medulloblastomas,9supratentorial primitive neuroectodermal tumors,17schwannomas,72meningiomas and23pituitary adenomas. A total of221somatic IDH1mutations were detected and the highest frequencies occurred in diffuse astrocytomas (68%), oligodendrogliomas (69%), oligoastrocytomas (78%) and secondary glioblastomas (88%). Primary glioblastomas and other entities were characterized by a low frequency or absence of mutations in amino acid position132of IDH1. Yan H et al. determined the sequence of the IDH1gene and the related IDH2gene in445central nervous system (CNS) tumors and494non-CNS tumors, and identified mutations that affected amino acid132of IDH1in more than70%of WHO grade Ⅱand Ⅲastrocytomas and oligodendrogliomas and in glioblastomas that developed from these lower-grade lesions. Tumors without mutations in IDH1often had mutations affecting the analogous amino acid (R172) of the IDH2gene. Tumors with IDH1or IDH2mutations had distinctive genetic and clinical characteristics, and patients with such tumors had a better outcome than those with wild-type IDH genes. The impact of IDH1/2mutations on clinical outcome and the correlation between IDH1/2mutations and other molecular markers such as TP53mutations, EGFR amplification, MGMT promoter mthylation and BRAF fusion gene, had also been demonstrated in prospective clinical studies as well as in other various retrospective studies.However, no report is currently available regarding the IDH1/2mutations in Chinese glioma patients and the predictive value of IDH mutations in patients treated with concomitant chemoradiotherapy following surgery. In the present study, we retrospectively analyzed a cohort of203Chinese glioma samples for IDH1/2mutations by high resolution melting (HRM) analysis combined with direct sequencing. This study aimed:i) to discern whether IDH mutations are common in Chinese glioma patients and ii) whether the mutations predict response to CCRT in anaplastic gliomas.Methods and materialsThis retrospective study was conducted by a single university hospital centre (Nanfang Gliomas Centre, Nanfang Hospital, Southern Medical University). All patients provided written informed consent for molecular studies of their tumor and the protocol was approved by the Ethics Committee of Nanfang Hospital. Clinical data were retrieved either from the case report forms for those patients participating in a clinical trial or from the hospital patient records. Clinical data included age, sex, location of tumors, pathological diagnosis, WHO performance scale(WHO-PS), date of final diagnosis, extent of surgery, adjuvant therapy after surgery (radiotherapy, chemotherapy), date of surgical resection, date of last follow-up or last contact, date of relapse, date of death and patient’s status at this time. All patients who received concomitant chemoradiotherapy(CCRT) after surgery, took orally TMZ200mg/m2/d for5consecutive days of every28days(i.e. standard schedule) concurrent with radiotherapy(RT). In the absence of unacceptable toxicity or of disease progression, patients continued to receive TMZ for at least12cycles and up to30cycles or until progression. Formalin-fixed and paraffin-embedded archival tumour specimens were centralised at the Department of Pathology of Nanfang Hospital. After consensus pathological review by two independent neuropathologists, the tissue sections with the highest proportion of malignant cells was cut and sufficient for analysis of IDH mutations. Statistical methodsAll statistical analyses were done with SPSS13.0for Windows. All patients were evaluated by clinical examination and by gadolinium-enhanced magnetic resonance imaging (Gd-MRI) of the brain for every3months to6months. Responses of brain tumors are assessed using Macdonald criteria and disease progression is defined as greater than25%increase in T2hypersignal or contrast enhancement, or tumor-related neurologic deterioration apart from pseudoprogression and pseudoresponse that easily confuse the assessment of outcome. Frequency distribution and summary statistics were calculated for all clinical, histological, and molecular variables. The Chi-square test was used to assess the genotype distribution. The Independent-Samples T test was used to compare data acquired in each group for the patient age. Progression-free survival (PFS) and overall survival(OS)were both used to study the prognostic impact of the analyzed variables. Progression-free survival (PFS) was calculated from the start of the surgery until the first unequivocal clinical or radiologic sign of progressive disease or last follow-up (for censored cases). The overall survival was defined as the time between the first surgery and death or last follow-up (for censored cases). Survival distributions were estimated by Kaplan-Meier method and compared among patient subsets using log-rank tests. All statistical tests were two-sided, and the threshold for statistical significance was P=0.05. Patients who died within2weeks for HGG and2months for LGG after surgery were excluded from survival analyses to avoid the inclusion of cases in which death may have been attributable to surgical complications.ResultsIDH1mutations were present in75of the203cases (36.9%) while IDH2mutations in5of the203cases (2.5%). No tumor was mutated in both IDH1and IDH2. IDH1/2mutations were associated with prolonged overall survival in the whole series of patients exclusive of pilocytic astrocytoma (P﹤0.001), WHO grade II patients who received no adjuvant therapy after surgery(P=0.014), and WHO grade III patients who received concomitant chemoradiotherapy (standard schedule) after surgery(P=0.033). Furthermore, there was no correlation between IDH1/2 mutations and reponse to concomitant chemoradiotherapy in anaplastic gliomas.ConclusionOur results suggest that IDHl mutations also occur freuqently in Chinese glioma patients but the frequency of IDH1mutations is below the findings reported by North American and European groups. Furthermore, we confirm the prognostic significance of IDH1/2mutations in gliomas. but the mutations cannot predict a favorable response to concomitant chemoradiotherapy in anaplastic gliomas. Part II:IDH mutations predict longer survival and response to temozolomide in secondary glioblastomaBackground and Objective:Glioblastoma(GBM) is the most common malignant primary brain tumor in adults and among the most aggressive of all tumors, accounting for approximately50%of gliomas. The yearly incidence is3to5newly diagnosed cases per100.000population. Although glioblastoma occurs more frequently in the elderly (median age,64years), it may present at any age, but is rare in children. In younger patients, sec-ondary glioblastoma is more frequent (median age,45years). There is a slight preponderance of glioblastoma in males, with a male to female ratio of approximately1.3to1. The overall median progression-free and overall survival times for patients treated with the current standard chemoradiotherapy within large clinical trials are approximately7and15months, respectively. Glioblastoma is a diffusely growing malignant brain neoplasm with characteristic histological and imaging features. Most cases of glioblastoma (>90%, primary glioblastoma, pGBM) develop rapidly with a clinical history of only a few days or weeks (de novo). Some glioblastomas (10%) progress gradually from a lower grade glioma (grade Ⅱ or Ⅲ) and are also referred to as secondary glioblastoma(sGBM). Although molecularly distinct, primary and secondary glioblastoma are histologically indistinguishable. Recently, mutations of the IDH1and IDH2genes have been identified in>80%of all secondary glioblastoma cases.In GBM the clinical value of O(6)-methylguanine DNA methyltransferase (MGMT) promotor methylation status in predicting benefit from alkylating agents has been validated by several clinical trials, both in patients treated with nitrosourea and in those with temozolomide (TMZ). In the European Organization for Research and Treatment of Cancer (EORTC)/National Cancer Institute of Canada (NCIC)26981/22981trial evaluating the effect of radiotherapy plus concomitant and adjuvant temozolomide versus radiotherapy alone in GBM, methylation of MGMT promotor methylation emerged as the strongest predictor for outcome and benefit from chemotherapy. However, all these studies were based on newly diagnosed GBMs, most of which were primary GBMs. So far there is no clear evidence that MGMT promotor methylation plays the same role in secondary GBMs since these two subtypes constitute distinct disease entities and develop through different genetic pathways. Similar to MGMT promotor methylation, patients with IDH1mutations are also associated with a longer survival than patients without in GBM. However, no data are currently available regarding the predictive value of IDH mutation in patients with secondary GBM treated with TMZ who have received surgery and radiotherapy due to a precursor lesion.Therefore, in this study we investigated IDH mutation and MGMT promoter methylation in sGBM and their sensitivity to TMZ treatment.Methods and materialsSearch for IDH1and IDH2mutations,1p19q codeletion, O(6)-methylguanine DNA methyltransferase (MGMT) promoter methylation, and p53expression was performed in a series of86secondary glioblastomas and correlated with progression-free survival and overall survival. Response to temozolomide was evaluated by progression-free survival, as well as by tumor size on successive MR1scans, and then correlated with molecular alterations. The following inclusion criteria were selected:1) age18years or above at time of surgery;2) histologic diagnosis of sGBM (histologic evidence of a preceding low-grade glioma [LGG] or anaplastic glioma) according to the WHO classification[1].3) detailed clinical information at diagnosis and during follow-up;4) availability of tumor samples for molecular analysis;5) treated with TMZ in the period of January2003to December2009; and6) at least2cycles of TMZ after which time they were evaluated for response (clinical and Gd-MRI).Statistical methodsAll statistical analyses were done with SPSS13.0for Windows. The Chi-square test was used to test the association between molecular alterations and between radiologic response to chemotherapy and molecular alterations (Bonferroni test was used for multiple comparisons). The Independent-Samples T test was used to compare data for the patient age. Progression-free survival (PFS) and overall survival (OS) were both used to study the prognostic impact of the analyzed variables. PFS and OS were calculated from the date of the first day of TMZ administration until disease progression, death or last follow-up (for censored cases). Survival distributions were estimated by Kaplan-Meier method and compared among patient subsets using log-rank tests. Multivariate analysis was performed with the multivariate Cox proportional hazard regression model analysis. The significant variables in univariate analysis were included in the multivariate model:age (>55vs <55years), Karnofsky performance status (KPS)(>80vs≤80), IDH status and MGMT promoter status, as well as1p19q codeletion. All statistical tests were two-sided, and the threshold for statistical significance was p=0.05.ResultsIDH (IDH1or IDH2) mutations were found in58/79patients (73.4%). IDH mutation, MGMT promoter methylation, and1p19q codeletion were associated with prolonged progression-free survival in univariate (P<0.001, P<0.001, and P=0.003) and multivariate analysis (P<0.001, P<0.001and P=0.035). IDH mutation (P=0.001) and MGMT promoter methylation (P=0.011) were correlated with a higher rate of objective response to temozolomide. Further analysis of response to temozolomide showed that patients with both IDH mutation and MGMT promoter methylation had the best response rate to temozolomide. ConclusionIDH mutation appears to be a significant marker of positive chemosensitivity in secondary glioblastoma. Use of IDH status combined with MGMT promoter status as a stratification factor in future clinical trials involving temozolomide for the treatment of patients with secondary glioblastoma seems appropriate.