Stefanie Brehmer

DNA methylation-based classification and grading system for meningioma: a multicentre, retrospective analysis

BACKGROUND The WHO classification of brain tumours describes 15 subtypes of meningioma. Nine of these subtypes are allotted to WHO grade I, and three each to grade II and grade III. Grading is based solely on histology, with an absence of molecular markers. Although the existing classification and grading approach is of prognostic value, it harbours shortcomings such as ill-defined parameters for subtypes and grading criteria prone to arbitrary judgment. In this study, we aimed for a comprehensive characterisation of the entire molecular genetic landscape of meningioma to identify biologically and clinically relevant subgroups. METHODS In this multicentre, retrospective analysis, we investigated genome-wide DNA methylation patterns of meningiomas from ten European academic neuro-oncology centres to identify distinct methylation classes of meningiomas. The methylation classes were further characterised by DNA copy number analysis, mutational profiling, and RNA sequencing. Methylation classes were analysed for progression-free survival outcomes by the Kaplan-Meier method. The DNA methylation-based and WHO classification schema were compared using the Brier prediction score, analysed in an independent cohort with WHO grading, progression-free survival, and disease-specific survival data available, collected at the Medical University Vienna (Vienna, Austria), assessing methylation patterns with an alternative methylation chip. FINDINGS We retrospectively collected 497 meningiomas along with 309 samples of other extra-axial skull tumours that might histologically mimic meningioma variants. Unsupervised clustering of DNA methylation data clearly segregated all meningiomas from other skull tumours. We generated genome-wide DNA methylation profiles from all 497 meningioma samples. DNA methylation profiling distinguished six distinct clinically relevant methylation classes associated with typical mutational, cytogenetic, and gene expression patterns. Compared with WHO grading, classification by individual and combined methylation classes more accurately identifies patients at high risk of disease progression in tumours with WHO grade I histology, and patients at lower risk of recurrence among WHO grade II tumours (p=0·0096) from the Brier prediction test). We validated this finding in our independent cohort of 140 patients with meningioma. INTERPRETATION DNA methylation-based meningioma classification captures clinically more homogenous groups and has a higher power for predicting tumour recurrence and prognosis than the WHO classification. The approach presented here is potentially very useful for stratifying meningioma patients to observation-only or adjuvant treatment groups. We consider methylation-based tumour classification highly relevant for the future diagnosis and treatment of meningioma. FUNDING German Cancer Aid, Else Kröner-Fresenius Foundation, and DKFZ/Heidelberg Institute of Personalized Oncology/Precision Oncology Program.

TERT Promoter Mutations and Risk of Recurrence in Meningioma

The World Health Organization (WHO) classification and grading system attempts to predict the clinical course of meningiomas based on morphological parameters. However, because of high interobserver variation of some criteria, more reliable prognostic markers are required. Here, we assessed the TERT promoter for mutations in the hotspot regions C228T and C250T in meningioma samples from 252 patients. Mutations were detected in 16 samples (6.4% across the cohort, 1.7%, 5.7%, and 20.0% of WHO grade I, II, and III cases, respectively). Data were analyzed by t test, Fishers exact test, log-rank test, and Cox proportional hazard model. All statistical tests were two-sided. Within a mean follow-up time in surviving patients of 68.1 months, TERT promoter mutations were statistically significantly associated with shorter time to progression (P < .001). Median time to progression among mutant cases was 10.1 months compared with 179.0 months among wild-type cases. Our results indicate that the inclusion of molecular data (ie, analysis of TERT promoter status) into a histologically and genetically integrated classification and grading system for meningiomas increases prognostic power. Consequently, we propose to incorporate the assessment of TERT promoter status in upcoming grading schemes for meningioma.

Bevacizumab Plus Irinotecan Versus Temozolomide in Newly Diagnosed O6-Methylguanine–DNA Methyltransferase Nonmethylated Glioblastoma: The Randomized GLARIUS Trial

PURPOSE In patients with newly diagnosed glioblastoma that harbors a nonmethylated O(6)-methylguanine-DNA methyltransferase promotor, standard temozolomide (TMZ) has, at best, limited efficacy. The GLARIUS trial thus explored bevacizumab plus irinotecan (BEV+IRI) as an alternative to TMZ. PATIENTS AND METHODS In this phase II, unblinded trial 182 patients in 22 centers were randomly assigned 2:1 to BEV (10 mg/kg every 2 weeks) during radiotherapy (RT) followed by maintenance BEV (10 mg/kg every 2 weeks) plus IRI(125 mg/m(2) every 2 weeks) or to daily TMZ (75 mg/m(2)) during RT followed by six courses of TMZ (150-200 mg/m(2)/d for 5 days every 4 weeks). The primary end point was the progression-free survival rate after 6 months (PFS-6). RESULTS In the modified intention-to-treat (ITT) population, PFS-6 was increased from 42.6% with TMZ (95% CI, 29.4% to 55.8%) to 79.3% with BEV+IRI (95% CI, 71.9% to 86.7%; P <.001). PFS was prolonged from a median of 5.99 months (95% CI, 2.7 to 7.3 months) to 9.7 months (95% CI, 8.7 to 10.8 months; P < .001). At progression, crossover BEV therapy was given to 81.8% of all patients who received any sort of second-line therapy in the TMZ arm. Overall survival (OS) was not different in the two arms: the median OS was 16.6 months (95% CI, 15.4 to 18.4 months) with BEV+IRI and was 17.5 months (95% CI, 15.1 to 20.5 months) with TMZ. The time course of quality of life (QOL) in six selected domains of the European Organisation for Research and Treatment of Cancer Quality-of-Life Questionnaire (QLQ) -C30 and QLQ-BN20 (which included cognitive functioning), of the Karnofsky performance score, and of the Mini Mental State Examination score was not different between the treatment arms. CONCLUSION BEV+IRI resulted in a superior PFS-6 rate and median PFS compared with TMZ. However, BEV+IRI did not improve OS, potentially because of the high crossover rate. BEV+IRI did not alter QOL compared with TMZ.

AKT1E17K mutations cluster with meningothelial and transitional meningiomas and can be detected by SFRP1 immunohistochemistry

The activating E17K mutation in the AKT1 gene has been detected in several tumor entities. Currently several clinical studies with specific AKT1 inhibitors are under way. To determine whether AKT1 mutations are involved in human tumors of the nervous system, we examined a series of 1,437 tumors including 391 primary intracranial brain tumors and 1,046 tumors of the coverings of the central and peripheral nervous system. AKT1E17K mutations were exclusively seen in meningiomas and occurred in 65 of 958 of these tumors. A strong preponderance was seen in the variant of meningothelial meningioma WHO grade I of basal and spinal localization. In contrast, AKT1E17K mutations were rare in WHO grade II and absent in WHO grade III meningiomas. In order to more effectively detect this mutation, we tested for immunohistochemical markers associated with this alteration. We observed strong up-regulation of SFRP1 expression in all meningiomas with AKT1E17K mutation and in HEK293 cells after transfection with mutant AKT1E17K, but not in meningiomas and HEK293 cells lacking this mutation.

Mutational analysis of D2HGDH and L2HGDH in brain tumours without IDH1 or IDH2 mutations

Heterozygous mutations in the highly homologous genes encoding cytosolic isocitrate dehydrogenase 1 (IDH1) and mitochondrial isocitrate dehydrogenase 2 (IDH2) have been identified in the majority of diffusely infiltrating astrocytomas and oligodendrogliomas of WHO grades II and III, in secondary glioblastomas WHO grade IV [1,2] and in up to 30% of acute myeloid leukaemia [3]. IDH1 and IDH2 decarboxylate isocitrate to a-ketoglutarate (aKG) and reduce NADP+ to NADPH. Missense mutations lead to an exchange of distinct arginine residues essential for binding isocitrate to the catalytic domain of IDH1 or IDH2. Instead of decarboxylating isocitrate to aKG the mutated enzymes catalyse the conversion of aKG to D-2hydroxyglutarate (2HG) [4]. This leads to approximately 100-fold raise of 2HG levels in tumour cells harbouring IDH1 or IDH2 mutations. While it is assumed that excessive 2HG levels in cells with IDH1 or IDH2 mutations contribute to tumour induction, the mechanisms involved are not known. One of the rationales for a role of 2HG in tumour formation is derived from the observation that patients suffering from hereditary L-2Hydroxyglutaric aciduria resulting in a strong intracellular increase of 2HG carry an increased risk for developing brain tumours [5]. These patients are deficient for L-2hydroxyglutarate dehydrogenase (L2HGDH). The function of L2HGDH is oxidation of L-2-HG to aKG. Patients with deficiency of D-2-hydroxyglutarate dehydrogenase (D2HGDH) responsible for oxidation of D-2-HG to aKG have not been identified to carry an increased risk for developing brain tumours. Nevertheless D2HGDH deficiency also results in strongly raised 2HG levels and therefore, deficiency of either of these enzymes L2HGDH or D2HGDH may mimic the metabolic situation in cells with IDH1 or IDH2 mutations. We therefore analysed D2HGDH or L2HGDH for inactivating mutations in patients with sporadic brain tumours not exhibiting IDH1 or IDH2 mutations. However, as discussed above, patients suffering from hereditary L-2-Hydroxyglutaric aciduria who have an increased risk of brain tumours are deficient for L2HGDH, but IDH1 and IDH2 mutations increase the levels of D-2-HG. Our series consisted of 66 brain tumours including 11 astrocytomas WHO grade II and III, four oligodendrogliomas WHO grade II and III, two oligoastrocytomas WHO grade II and III, 17 glioblastomas WHO grade IV, 16 ependymomas WHO grade II and III, 12 medulloblastomas WHO grade IV and 4 primitive neuroectodermal tumours WHO grade IV diagnosed at the Department of Neuropathology of the University Heidelberg. All samples were analysed in an anonymized manner as approved by the local institutional ethics boards. All diffusely infiltrating gliomas were analysed for IDH1 and IDH2 mutations, all other tumours for IDH1 mutations only. DNA extraction, PCR amplification and direct sequencing were described elsewhere [1]. We analysed all exons encoding the catalytic domains of D2HGDH (exons 2, 6, 8, 9, 10) and of L2HGDH (exons 1, 2) by direct sequencing. PCR conditions and primer sequences are available on request. Corresponding constitutive DNA was sequenced for each case if a sequence alteration was determined in the tumour DNA. While we detected several sequence alterations in these tumours, none of them turned out as somatic mutations. Among the alterations were six known polymorphisms seven polymorphisms not previously described (Table 1). In order to test whether distinct alleles of the polymorphisms characterized by amino acid exchanges were enriched in patients with brain tumours we analysed D2HGDH exon 8 and L2HGDH exon 2 in 88 unaffected controls. The allelic distributions of the polymorphisms in tumour patients and control individuals were comparable. We did not find evidence for mutations in the genes D2HGDH and L2HGDH as an alternative mechanism for raised 2HG levels in brain tumours and we did not find evidence for distinct alleles of these genes conferring an increased risk for development of brain tumours. Furthermore, epigenetic mechanism like promoter methylation may reduce expression of D2HGDH and L2HGDH and, thereby, deactivate their function. Both genes carry large CpG islands around exon 1. However, when we analysed both genes in an array data set (http://www. ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE18166) [6] for expression differences between gliomas with and

Intraoperative radiotherapy (IORT)—a resurrected option for treating glioblastoma?

Despite surgical resection, radiochemotherapy and adjuvant chemotherapy, glioblastoma multiforme (GBM) is still associated with an extremely poor prognosis. As almost all tumors recur locally and evidence for a radiation dose-dependent effect on survival exists, intraoperative radiotherapy (IORT) could be an approach to avoid tumor cell proliferation between surgery and radiochemotherapy while sparing healthy tissue. The majority of previous studies used forward-scattering electron tubes (resembling intraoperative electron radiotherapy, IOERT) and suffered from technical and geometrical limitations. Consequently, the outcomes in previous studies range from highly beneficial to merely effective. This review shall give an overview on past, present and future applications of IORT for GBM and shall discuss what prospective steps are to be taken to thoroughly assess whether the approach has the ability to prolong patient survival.

Intraoperative Radiotherapy in Newly Diagnosed Glioblastoma (INTRAGO): An Open-Label, Dose-Escalation Phase I/II Trial

BACKGROUND The median time to recurrence of glioblastoma (GB) following multimodal treatment is ∼7 mo. Nearly all cancers recur locally, suggesting that augmenting local treatments may improve outcomes. OBJECTIVE To investigate whether intraoperative radiotherapy (IORT) to the resection cavity is safe and effective. METHODS INTRAGO was a phase I/II trial to evaluate the safety and tolerability of IORT with 20 to 40 Gy of low-energy photons in addition to standard radiochemotherapy (ClinicalTrials.gov ID, NCT02685605). The primary endpoint was safety as per occurrence of dose-limiting toxicities. Secondary endpoints were progression-free survival (PFS) and overall survival (OS). We also performed an exploratory analysis of the local PFS (L-PFS), defined as recurrence within 1 cm of the treated margin. RESULTS Fifteen patients were treated at 3 dose levels. Of these, 13 underwent incomplete resection, 6 had unresected satellites, and 3 did not receive per-protocol treatment (PPT). The MGMT promoter was unmethylated in 10 patients. The median follow-up was 13.8 mo. The majority of grade 3 to 5 adverse events were deemed unrelated to IORT. Five cases of radionecrosis were observed, 2 were classified as grade 3 events. Other grade 3 events judged related to radiotherapy (external-beam radiotherapy and/or IORT) were wound dehiscence (n = 1), CSF leakage (n = 1), cyst formation (n = 1). No IORT-related deaths occurred. The median PFS was 11.2 mo (95% confidence interval [CI]: 5.4-17.0) for all patients and 11.3 mo (95% CI: 10.9-11.6) for those receiving PPT. The median L-PFS was 14.3 mo (95% CI: 8.4-20.2) for all patients and 17.8 mo (95% CI: 9.7-25.9) for those receiving PPT. The median OS was 16.2 mo (95% CI: 11.1-21.4) for all patients and 17.8 mo (95% CI: 13.9-21.7) for those receiving PPT. CONCLUSION These data suggest that IORT is associated with manageable toxicity. Considering the limitations of a 15-patient phase I/II trial, further studies aimed at assessing an outcome benefit are warranted.

Study Protocol: Early Stereotactic Gamma Knife Radiosurgery to Residual Tumor After Surgery of Newly Diagnosed Glioblastoma (Gamma-GBM)

BACKGROUND Glioblastoma (GBM) is the most common malignant brain tumor in adult patients. Tumor recurrence commonly occurs around the resection cavity, especially after subtotal resection (STR). Consequently, the extent of resection correlates with overall survival (OS), suggesting that depletion of postoperative tumor remnants will improve outcome. OBJECTIVE To assess safety and efficacy of adding stereotactic radiosurgery (SRS) to the standard treatment of GBM in patients with postoperative residual tumor. METHODS Gamma-GBM is a single center, open-label, prospective, single arm, phase II study that includes patients with newly diagnosed GBM (intraoperative via frozen sections) who underwent STR (residual tumor will be identified by native and contrast enhanced T1-weighted magnetic resonance imaging scans). All patients will receive SRS with 15 Gy (prescribed to the 50% isodose enclosing all areas of residual tumor) early (within 24-72 h) after surgery. Thereafter, all patients undergo standard-of-care therapy for GBM (radiochemotherapy with 60 Gy external beam radiotherapy [EBRT] plus concomitant temozolomide and 6 cycles of adjuvant temozolomide chemotherapy). The primary outcome is median progression-free survival, secondary outcomes are median OS, occurrence of radiation induced acute (<3 wk), early delayed (<3 mo), and late (>3 mo post-SRS) neurotoxicity and incidence of symptomatic radionecrosis. EXPECTED OUTCOMES We expect to detect efficacy and safety signals by the immediate application of SRS to standard-of-care therapy in newly diagnosed GBM. DISCUSSION Early postoperative SRS to areas of residual tumor could bridge the therapeutic gap between surgery and adjuvant therapies.

Molecular transition of an adult low-grade brain tumor to an atypical teratoid/rhabdoid tumor over a time-course of 14 years

Atypical teratoid/rhabdoid tumor (AT/RT) of the central nervous system is a highly malignant, pediatric brain tumor typically arising de novo. Inactivation of SMARCB1 is a defining molecular event. We present here a rare case of an adult (35 years) low-grade SMARCB1-deleted brain tumor with transition into prototypical AT/RT over 14 years. Molecular analysis was performed for 3 tumor presentations including copy number analysis, DNA methylation analysis (450k), and whole exome sequencing. We detected the identical somatic SMARCB1 deletion at all 3 time-points. In an unsupervised hierarchical clustering of methylation data together with 127 reference cases comprising 9 brain tumor classes all 3 manifestations clustered with AT/RT. Exome sequencing revealed an increase of mutational burden over time. The acquired mutations and additional copy number changes did not affect known cancer genes. In conclusion, we demonstrate molecular changes associated with histological and clinical transition of a low-grade brain tumor to an adult AT/RT. Our observation of a stable disease course for nearly 10 years in a tumor with SMARCB1 loss and an AT/RT-like DNA methylation profile indicates that caution may be required in the diagnostic interpretation of such findings in adult patients.