Current classification of gliomas is based on histological criteria according to the World Health Organization (WHO) classification of tumors of the central nervous system. Over the past years, ...characteristic genetic profiles have been identified in various glioma types. These can refine tumor diagnostics and provide important prognostic and predictive information. We report on the establishment and validation of gene panel next generation sequencing (NGS) for the molecular diagnostics of gliomas. We designed a glioma‐tailored gene panel covering 660 amplicons derived from 20 genes frequently aberrant in different glioma types. Sensitivity and specificity of glioma gene panel NGS for detection of DNA sequence variants and copy number changes were validated by single gene analyses. NGS‐based mutation detection was optimized for application on formalin‐fixed paraffin‐embedded tissue specimens including small stereotactic biopsy samples. NGS data obtained in a retrospective analysis of 121 gliomas allowed for their molecular classification into distinct biological groups, including (i) isocitrate dehydrogenase gene (IDH) 1 or 2 mutant astrocytic gliomas with frequent α‐thalassemia/mental retardation syndrome X‐linked (ATRX) and tumor protein p53 (TP53) gene mutations, (ii) IDH mutant oligodendroglial tumors with 1p/19q codeletion, telomerase reverse transcriptase (TERT) promoter mutation and frequent Drosophila homolog of capicua (CIC) gene mutation, as well as (iii) IDH wildtype glioblastomas with frequent TERT promoter mutation, phosphatase and tensin homolog (PTEN) mutation and/or epidermal growth factor receptor (EGFR) amplification. Oligoastrocytic gliomas were genetically assigned to either of these groups. Our findings implicate gene panel NGS as a promising diagnostic technique that may facilitate integrated histological and molecular glioma classification.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Epigenetic silencing of the O6‐methylguanine‐DNA methyltransferase (MGMT) gene promoter is associated with prolonged survival in glioblastoma patients treated with temozolomide (TMZ). We investigated ...whether glioblastoma recurrence is associated with changes in the promoter methylation status and the expression of MGMT and the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 in pairs of primary and recurrent glioblastomas of 80 patients, including 64 patients treated with radiotherapy and TMZ after the first operation. Among the primary tumors, the MGMT promoter was methylated in 31 patients and unmethylated in 49 patients. In 71 patients (89%), the MGMT promoter methylation status of the primary tumor was retained at recurrence. MGMT promoter methylation, but not MGMT protein expression, was associated with longer progression‐free survival, overall survival and postrecurrence survival (PRS). Moreover, PRS was increased under salvage chemotherapy. Investigation of primary and recurrent glioblastomas of 43 patients did not identify promoter methylation in any of the four MMR genes. However, recurrent glioblastomas demonstrated significantly lower MSH2, MSH6 and PMS2 protein expression as detected by immunohistochemistry. In conclusion, reduced expression of MMR proteins, but not changes in MGMT promoter methylation, is characteristic of glioblastomas recurring after the current standards of care.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
O6‐methylguanine‐DNA‐methyltransferase (MGMT) promoter methylation identifies a subpopulation of glioblastoma patients with more favorable prognosis and predicts a benefit from alkylating agent ...chemotherapy (CT). Little is known about its prevalence and clinical significance in older glioblastoma patients. We studied 233 glioblastoma patients aged 70 years or more (144 males, 89 females, median age: 74 years, range: 70.0–86.6 years), who were prospectively enrolled in the German Glioma Network, for MGMT promoter methylation by methylation‐specific PCR (MSP) in all patients and DNA pyrosequencing in 166 patients. MGMT data were correlated with patient outcome. Median progression‐free survival (PFS) was 4.8 months (95% CI: 4.3–5.3) and median overall survival (OS) was 7.7 months (95% CI: 6.3–9.0). MGMT promoter methylation was detected by MSP in 134 patients (57.5%). For the whole cohort, PFS was 5.2 versus 4.7 months (p = 0.207) and OS was 8.4 versus 6.4 months (p = 0.031) in patients with versus without MGMT promoter methylation. Patients with MGMT methylated tumors had longer PFS when treated with radiotherapy (RT) plus CT or CT alone compared to patients treated with RT alone. Patients with MGMT unmethylated tumors appeared to derive no survival benefit from CT, regardless of whether given at diagnosis together with RT or as a salvage treatment. Patients treated with RT plus CT or CT alone demonstrated longer OS when pyrosequencing revealed >25% MGMT methylated alleles. Taken together, MGMT promoter methylation may be a useful biomarker to stratify elderly glioblastoma patients for treatment with versus without alkylating agent CT.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
We studied how intratumoral genetic heterogeneity shapes tumor growth and therapy response for isocitrate dehydrogenase (IDH)-wild-type glioblastoma, a rapidly regrowing tumor. We inferred the ...evolutionary trajectories of matched pairs of primary and relapsed tumors based on deep whole-genome-sequencing data. This analysis suggests both a distant origin of de novo glioblastoma, up to 7 years before diagnosis, and a common path of early tumorigenesis, with one or more of chromosome 7 gain, 9p loss, or 10 loss, at tumor initiation. TERT promoter mutations often occurred later as a prerequisite for rapid growth. In contrast to this common early path, relapsed tumors acquired no stereotypical pattern of mutations and typically regrew from oligoclonal origins, suggesting sparse selective pressure by therapeutic measures.
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•We inferred evolutionary trajectories of pairs of primary/relapsed glioblastomas•Chromosome 7 gain, 9p loss, or 10 loss commonly occurred at tumor initiation•TERT promoter mutations often occurred later as a prerequisite for rapid growth•Relapsed tumors typically regrew from oligoclonal origins
By analyzing 21 paired primary and locally relapsed IDH-wild-type glioblastomas (GBM), Körber et al. show that most GBM initiate by gains and losses of specific chromosomes; TERT promoter mutations often occur later as a prerequisite for rapid growth, and relapsed GBM acquire few stereotypical mutations.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Glioblastoma frequently exhibits therapy-associated subtype transitions to mesenchymal phenotypes with adverse prognosis. Here, we perform multi-omic profiling of 60 glioblastoma primary tumours and ...use orthogonal analysis of chromatin and RNA-derived gene regulatory networks to identify 38 subtype master regulators, whose cell population-specific activities we further map in published single-cell RNA sequencing data. These analyses identify the oligodendrocyte precursor marker and chromatin modifier SOX10 as a master regulator in RTK I-subtype tumours. In vitro functional studies demonstrate that SOX10 loss causes a subtype switch analogous to the proneural-mesenchymal transition observed in patients at the transcriptomic, epigenetic and phenotypic levels. SOX10 repression in an in vivo syngeneic graft glioblastoma mouse model results in increased tumour invasion, immune cell infiltration and significantly reduced survival, reminiscent of progressive human glioblastoma. These results identify SOX10 as a bona fide master regulator of the RTK I subtype, with both tumour cell-intrinsic and microenvironmental effects.
Rechallenge with temozolomide (TMZ) at first progression of glioblastoma after temozolomide chemoradiotherapy (TMZ/RT→TMZ) has been studied in retrospective and single-arm prospective studies, ...applying temozolomide continuously or using 7/14 or 21/28 days schedules. The DIRECTOR trial sought to show superiority of the 7/14 regimen.
Patients with glioblastoma at first progression after TMZ/RT→TMZ and at least two maintenance temozolomide cycles were randomized to Arm A one week on (120 mg/m(2) per day)/one week off or Arm B 3 weeks on (80 mg/m(2) per day)/one week off. The primary endpoint was median time-to-treatment failure (TTF) defined as progression, premature temozolomide discontinuation for toxicity, or death from any cause. O(6)-methylguanine DNA methyltransferase (MGMT) promoter methylation was prospectively assessed by methylation-specific PCR.
Because of withdrawal of support, the trial was prematurely closed to accrual after 105 patients. There was a similar outcome in both arms for median TTF A: 1.8 months; 95% confidence intervals (CI), 1.8-3.2 vs. B: 2.0 months; 95% CI, 1.8-3.5 and overall survival A: 9.8 months (95% CI, 6.7-13.0) vs. B: 10.6 months (95% CI, 8.1-11.6). Median TTF in patients with MGMT-methylated tumors was 3.2 months (95% CI, 1.8-7.4) versus 1.8 months (95% CI, 1.8-2) in MGMT-unmethylated glioblastoma. Progression-free survival rates at 6 months (PFS-6) were 39.7% with versus 6.9% without MGMT promoter methylation.
Temozolomide rechallenge is a treatment option for MGMT promoter-methylated recurrent glioblastoma. Alternative strategies need to be considered for patients with progressive glioblastoma without MGMT promoter methylation.
The prognostic value of genetic alterations characteristic of glioblastoma in patients treated according to present standards of care is unclear.
Three hundred one patients with glioblastoma were ...prospectively recruited between October 2004 and December 2006 at the clinical centers of the German Glioma Network. Two hundred fifty-eight patients had radiotherapy, 199 patients had temozolomide, 189 had both, and seven had another chemotherapy as the initial treatment. The tumors were investigated for TP53 mutation, p53 immunoreactivity, epidermal growth factor receptor, cyclin-dependent kinase CDK 4 or murine double minute 2 amplification, CDKN2A homozygous deletion, allelic losses on chromosome arms 1p, 9p, 10q, and 19q, O(6)-methylguanine methyltransferase (MGMT) promoter methylation, and isocitrate dehydrogenase 1 (IDH1) mutations.
Median progression-free (PFS) and overall survival (OS) were 6.8 and 12.5 months. Multivariate analysis revealed younger age, higher performance score, MGMT promoter methylation, and temozolomide radiochemotherapy as independent factors associated with longer OS. MGMT promoter methylation was associated with longer PFS (relative risk RR, 0.5; 95% CI, 0.38 to 0.68; P < .001) and OS (RR, 0.39; 95% CI, 0.28 to 0.54; P < .001) in patients receiving temozolomide. IDH1 mutations were associated with prolonged PFS (RR, 0.42; 95% CI, 0.19 to 0.91; P = .028) and a trend for prolonged OS (RR, 0.43; 95% CI, 0.15 to 1.19; P = .10). No other molecular factor was associated with outcome.
Molecular changes associated with gliomagenesis do not predict response to therapy in glioblastoma patients managed according to current standards of care. MGMT promoter methylation and IDH1 mutational status allow for stratification into prognostically distinct subgroups.
Somatic mutations in the
IDH1
gene encoding cytosolic NADP+-dependent isocitrate dehydrogenase have been shown in the majority of astrocytomas, oligodendrogliomas and oligoastrocytomas of WHO grades ...II and III.
IDH2
encoding mitochondrial NADP+-dependent isocitrate dehydrogenase is also mutated in these tumors, albeit at much lower frequencies. Preliminary data suggest an importance of
IDH1
mutation for prognosis showing that patients with anaplastic astrocytomas, oligodendrogliomas and oligoastrocytomas harboring
IDH1
mutations seem to fare much better than patients without this mutation in their tumors. To determine mutation types and their frequencies, we examined 1,010 diffuse gliomas. We detected 716
IDH1
mutations and 31
IDH2
mutations. We found 165
IDH1
(72.7%) and 2
IDH2
mutations (0.9%) in 227 diffuse astrocytomas WHO grade II, 146
IDH1
(64.0%) and 2
IDH2
mutations (0.9%) in 228 anaplastic astrocytomas WHO grade III, 105
IDH1
(82.0%) and 6
IDH2
mutations (4.7%) in 128 oligodendrogliomas WHO grade II, 121
IDH1
(69.5%) and 9
IDH2
mutations (5.2%) in 174 anaplastic oligodendrogliomas WHO grade III, 62
IDH1
(81.6%) and 1
IDH2
mutations (1.3%) in 76 oligoastrocytomas WHO grade II and 117
IDH1
(66.1%) and 11
IDH2
mutations (6.2%) in 177 anaplastic oligoastrocytomas WHO grade III. We report on an inverse association of
IDH1
and
IDH2
mutations in these gliomas and a non-random distribution of the mutation types within the tumor entities.
IDH1
mutations of the R132C type are strongly associated with astrocytoma, while
IDH2
mutations predominantly occur in oligodendroglial tumors. In addition, patients with anaplastic glioma harboring
IDH1
mutations were on average 6 years younger than those without these alterations.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Survival of glioblastoma patients has been linked to the completeness of surgical resection. Available data, however, were generated with adjuvant radiotherapy. Data confirming that extensive ...cytoreduction remains beneficial to patients treated with the current standard, concomitant temozolomide radiochemotherapy, are limited. We therefore analyzed the efficacy of radiochemotherapy for patients with little or no residual tumor after surgery. In this prospective, non-interventional multicenter cohort study, entry criteria were histological diagnosis of glioblastoma, small enhancing or no residual tumor on post-operative MRI, and intended temozolomide radiochemotherapy. The primary study objective was progression-free survival; secondary study objectives were survival and toxicity. Furthermore, the prognostic value of O6-methylguanine-DNA methyltransferase (
MGMT
) promoter methylation was investigated in a subgroup of patients. One-hundred and eighty patients were enrolled. Fourteen were excluded by patient request or failure to initiate radiochemotherapy. Twenty-three patients had non-evaluable post-operative imaging. Thus, 143 patients qualified for analysis, with 107 patients having residual tumor diameters ≤1.5 cm. Median follow-up was 24.0 months. Median survival or patients without residual enhancing tumor exceeded the follow-up period. Median survival was 16.9 months for 32 patients with residual tumor diameters >0 to ≤1.5 cm (95% CI: 13.3–20.5,
p
= 0.039), and 13.9 months (10.3–17.5, overall
p
< 0.001) for 36 patients with residual tumor diameters >1.5 cm. Patient age at diagnosis and extent of resection were independently associated with survival. Patients with
MGMT
promoter methylated tumors and complete resection made the best prognosis. Completeness of resection acts synergistically with concomitant and adjuvant radiochemotherapy, especially in patients with
MGMT
promoter methylation.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The standard of care for anaplastic gliomas is surgery followed by radiotherapy. The NOA-04 phase III trial compared efficacy and safety of radiotherapy followed by chemotherapy at progression with ...the reverse sequence in patients with newly diagnosed anaplastic gliomas.
Patients (N = 318) were randomly assigned 2:1:1 (A:B1:B2) to receive conventional radiotherapy (arm A); procarbazine, lomustine (CCNU), and vincristine (PCV; arm B1); or temozolomide (arm B2) at diagnosis. At occurrence of unacceptable toxicity or disease progression, patients in arm A were treated with PCV or temozolomide (1:1 random assignment), whereas patients in arms B1 or B2 received radiotherapy. The primary end point was time to treatment failure (TTF), defined as progression after radiotherapy and one chemotherapy in either sequence.
Patient characteristics in the intention-to-treat population (n = 274) were balanced between arms. All histologic diagnoses were centrally confirmed. Median TTF (hazard ratio HR = 1.2; 95% CI, 0.8 to 1.8), progression-free survival (PFS; HR = 1.0; 95% CI, 0.7 to 1.3, and overall survival (HR = 1.2; 95% CI, 0.8 to 1.9) were similar for arms A and B1/B2. Extent of resection was an important prognosticator. Anaplastic oligodendrogliomas and oligoastrocytomas share the same, better prognosis than anaplastic astrocytomas. Hypermethylation of the O(6)-methylguanine DNA-methyltransferase (MGMT) promoter (HR = 0.59; 95% CI, 0.36 to 1.0), mutations of the isocitrate dehydrogenase (IDH1) gene (HR = 0.48; 95% CI, 0.29 to 0.77), and oligodendroglial histology (HR = 0.33; 95% CI, 0.2 to 0.55) reduced the risk of progression. Hypermethylation of the MGMT promoter was associated with prolonged PFS in the chemotherapy and radiotherapy arm.
Initial radiotherapy or chemotherapy achieved comparable results in patients with anaplastic gliomas. IDH1 mutations are a novel positive prognostic factor in anaplastic gliomas, with a favorable impact stronger than that of 1p/19q codeletion or MGMT promoter methylation.
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