David E. Reuss

ATRX and IDH1-R132H immunohistochemistry with subsequent copy number analysis and IDH sequencing as a basis for an "integrated" diagnostic approach for adult astrocytoma, oligodendroglioma and glioblastoma.

Diffuse gliomas are represented in the 2007 WHO classification as astrocytomas, oligoastrocytomas and oligodendrogliomas of grades II and III and glioblastomas WHO grade IV. Molecular data on these tumors have a major impact on prognosis and therapy of the patients. Consequently, the inclusion of molecular parameters in the WHO definition of brain tumors is being planned and has been forwarded as the “ISN-Haarlem” consensus. We, here, analyze markers of special interest including ATRX, IDH and 1p/19q codeletion in a series of 405 adult patients. Among the WHO 2007 classified tumors were 152 astrocytomas, 61 oligodendrogliomas, 63 oligoastrocytomas and 129 glioblastomas. Following the concepts of the “ISN-Haarlem”, we rediagnosed the series to obtain “integrated” diagnoses with 155 tumors being astrocytomas, 100 oligodendrogliomas and 150 glioblastomas. In a subset of 100 diffuse gliomas from the NOA-04 trial with long-term follow-up data available, the “integrated” diagnosis had a significantly greater prognostic power for overall and progression-free survival compared to WHO 2007. Based on the “integrated” diagnoses, loss of ATRX expression was close to being mutually exclusive to 1p/19q codeletion, with only 2 of 167 ATRX-negative tumors exhibiting 1p/19q codeletion. All but 4 of 141 patients with loss of ATRX expression and diffuse glioma carried either IDH1 or IDH2 mutations. Interestingly, the majority of glioblastoma patients with loss of ATRX expression but no IDH mutations exhibited an H3F3A mutation. Further, all patients with 1p/19 codeletion carried a mutation in IDH1 or IDH2. We present an algorithm based on stepwise analysis with initial immunohistochemistry for ATRX and IDH1-R132H followed by 1p/19q analysis followed by IDH sequencing which reduces the number of molecular analyses and which has a far better association with patient outcome than WHO 2007.

Meningeal hemangiopericytoma and solitary fibrous tumors carry the NAB2-STAT6 fusion and can be diagnosed by nuclear expression of STAT6 protein

Non-central nervous system hemangiopericytoma (HPC) and solitary fibrous tumor (SFT) are considered by pathologists as two variants of a single tumor entity now subsumed under the entity SFT. Recent detection of frequent NAB2-STAT6 fusions in both, HPC and SFT, provided additional support for this view. On the other hand, current neuropathological practice still distinguishes between HPC and SFT. The present study set out to identify genes involved in the formation of meningeal HPC. We performed exome sequencing and detected the NAB2-STAT6 fusion in DNA of 8/10 meningeal HPC thereby providing evidence of close relationship of these tumors with peripheral SFT. Due to the considerable effort required for exome sequencing, we sought to explore surrogate markers for the NAB2-STAT6 fusion protein. We adopted the Duolink proximity ligation assay and demonstrated the presence of NAB2-STAT6 fusion protein in 17/17 HPC and the absence in 15/15 meningiomas. More practical, presence of the NAB2-STAT6 fusion protein resulted in a strong nuclear signal in STAT6 immunohistochemistry. The nuclear reallocation of STAT6 was detected in 35/37 meningeal HPC and 25/25 meningeal SFT but not in 87 meningiomas representing the most important differential diagnosis. Tissues not harboring the NAB2-STAT6 fusion protein presented with nuclear expression of NAB2 and cytoplasmic expression of STAT6 proteins. In conclusion, we provide strong evidence for meningeal HPC and SFT to constitute variants of a single entity which is defined by NAB2-STAT6 fusion. In addition, we demonstrate that this fusion can be rapidly detected by STAT6 immunohistochemistry which shows a consistent nuclear reallocation. This immunohistochemical assay may prove valuable for the differentiation of HPC and SFT from other mesenchymal neoplasms.

Farewell to oligoastrocytoma: in situ molecular genetics favor classification as either oligodendroglioma or astrocytoma

Astrocytoma and oligodendroglioma are histologically and genetically well-defined entities. The majority of astrocytomas harbor concurrent TP53 and ATRX mutations, while most oligodendrogliomas carry the 1p/19q co-deletion. Both entities share high frequencies of IDH mutations. In contrast, oligoastrocytomas (OA) appear less clearly defined and, therefore, there is an ongoing debate whether these tumors indeed constitute an entity or whether they represent a mixed bag containing both astrocytomas and oligodendrogliomas. We investigated 43 OA diagnosed in different institutions employing histology, immunohistochemistry and in situ hybridization addressing surrogates for the molecular genetic markers IDH1R132H, TP53, ATRX and 1p/19q loss. In all but one OA the combination of nuclear p53 accumulation and ATRX loss was mutually exclusive with 1p/19q co-deletion. In 31/43 OA, only alterations typical for oligodendroglioma were observed, while in 11/43 OA, only indicators for mutations typical for astrocytomas were detected. A single case exhibited a distinct pattern, nuclear expression of p53, ATRX loss, IDH1 mutation and partial 1p/19q loss. However, this was the only patient undergoing radiotherapy prior to surgery, possibly contributing to the acquisition of this uncommon combination. In OA with oligodendroglioma typical alterations, the portions corresponding to astrocytic part were determined as reactive, while in OA with astrocytoma typical alterations the portions corresponding to oligodendroglial differentiation were neoplastic. These data provide strong evidence against the existence of an independent OA entity.

Quercetin promotes degradation of survivin and thereby enhances death-receptor–mediated apoptosis in glioma cells

The flavonoid quercetin has been reported to inhibit the proliferation of cancer cells, whereas it has no effect on nonneoplastic cells. U87-MG, U251, A172, LN229, and U373 malignant glioma cells were treated with quercetin (50-200 microM). Quercetin did not cause cytotoxicity 24 h after treatment. Combining quercetin with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) strongly augmented TRAIL-mediated apoptosis in U87-MG, U251, A172, and LN229 glioma cells; U373 cells could not be sensitized by quercetin to TRAIL-mediated apoptosis. TRAIL-induced apoptosis was enhanced by quercetin-induced reduction of survivin protein levels. Upon treatment with quercetin, the protein level of survivin was strongly suppressed in U87-MG, U251, and A172 but not in U373 glioma cells. Quercetin exposure resulted in proteasomal degradation of survivin. TRAIL-quercetin-induced apoptosis was markedly reduced by overexpression of survivin. In addition, upon treatment with quercetin, downregulation of survivin was also regulated by the Akt pathway. Taken together, the results of the present study suggest that quercetin sensitizes glioma cells to death-receptor-mediated apoptosis by suppression of inhibitor of the apoptosis protein survivin.

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.

The flavonoid kaempferol sensitizes human glioma cells to TRAIL-mediated apoptosis by proteasomal degradation of survivin

Resistance to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL/Apo2L) limits its potential as a drug for cancer therapy. Here, we report that kaempferol, a bioactive plant flavonoid, sensitizes U251 and U87 glioma cells to TRAIL-mediated apoptosis. In contrast, U373 cells are not affected by kaempferol treatment. Treatment of kaempferol alone for 24 h did not induce apoptosis in the cell lines. We provide evidence that TRAIL-induced apoptosis is partially driven by kaempferol-mediated reduction of survivin protein levels. On kaempferol treatment, proteasomal degradation of survivin was observed. Inhibition of proteasomal degradation with MG132 in kaempferol-treated cells restored survivin protein levels in both glial cell lines. Consequently, overexpression of survivin attenuated TRAIL-kaempferol–induced apoptosis. In addition, we show that kaempferol mediates down-regulation of phosphorylated Akt, thereby further reducing survivin protein level. Furthermore, the blockage of the serine/threonine kinase Akt activity by kaempferol is important for inhibition of survivin because active phosphorylated Akt enhances the stability of survivin. However, we also show that the combined treatment of TRAIL and kaempferol induces cleavage (activation) of caspase-8, thereby exerting a proapoptotic effect independent of survivin known not to inhibit caspase-8 activation. Other effects induced by kaempferol were suppression of X-linked inhibitor of apoptosis proteins as the antiapoptotic members of the Bcl-2 family, Bcl-2, Bcl-xL, and Mcl-1 in a concentration-dependent manner. In summary, we showed that suppression of survivin is an essential mechanism in TRAIL-kaempferol–mediated apoptosis. [Mol Cancer Ther 2008;7(11):3566–74]

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.

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.

Morphologic and immunohistochemical features of malignant peripheral nerve sheath tumors and cellular schwannomas.

Cellular schwannoma is an uncommon, but well-recognized, benign peripheral nerve sheath tumor, which can be misdiagnosed as malignant peripheral nerve sheath tumor. To develop consensus diagnostic criteria for cellular schwannoma, we reviewed 115 malignant peripheral nerve sheath tumor and 26 cellular schwannoma cases from two institutions. Clinical data were retrieved from the electronic medical records, and morphologic features, maximal mitotic counts, Ki67 labeling indices, and immunohistochemical profiles (SOX10, SOX2, p75NTR, p16, p53, EGFR, and neurofibromin) were assessed. Several features distinguish cellular schwannoma from malignant peripheral nerve sheath tumor. First, in contrast to patients with malignant peripheral nerve sheath tumor, no metastases or disease-specific deaths were found in patients with cellular schwannoma. More specifically, 5-year progression-free survival rates were 100 and 18%, and 5-year disease-specific survival rates were 100 and 32% for cellular schwannoma and malignant peripheral nerve sheath tumor, respectively. Second, the presence of Schwannian whorls, a peritumoral capsule, subcapsular lymphocytes, macrophage-rich infiltrates, and the absence of fascicles favored the diagnosis of cellular schwannoma, while the presence of perivascular hypercellularity, tumor herniation into vascular lumens, and necrosis favor malignant peripheral nerve sheath tumor. Third, complete loss of SOX10, neurofibromin or p16 expression, or the presence of EGFR immunoreactivity was specific for malignant peripheral nerve sheath tumor (P<0.001 for each). Expression of p75NTR was observed in 80% of malignant peripheral nerve sheath tumors compared with 31% of cellular schwannomas (P<0.001). Fourth, Ki-67 labeling indices ≥20% were highly predictive of malignant peripheral nerve sheath tumor (87% sensitivity and 96% specificity). Taken together, the combinations of these histopathological and immunohistochemical features provide useful criteria to distinguish between malignant peripheral nerve sheath tumor and cellular schwannoma with high sensitivity and specificity. Additional retrospective and prospective multicenter studies with larger data sets will be required to validate these findings.

Nuclear relocation of STAT6 reliably predicts NAB2-STAT6 fusion for the diagnosis of solitary fibrous tumour.

Nuclear relocation of STAT6 has been shown in tumours with NAB2–STAT6 fusion, and has been proposed as an ancillary marker for the diagnosis of solitary fibrous tumours (SFTs). The aim of this study was to verify the utility of STAT6 immunohistology in diagnosing SFT.