Benjamin Brokinkel

Molecular Classification of Low-Grade Diffuse Gliomas

The current World Health Organization classification recognizes three histological types of grade II low-grade diffuse glioma (diffuse astrocytoma, oligoastrocytoma, and oligodendroglioma). However, the diagnostic criteria, in particular for oligoastrocytoma, are highly subjective. The aim of our study was to establish genetic profiles for diffuse gliomas and to estimate their predictive impact. In this study, we screened 360 World Health Organization grade II gliomas for mutations in the IDH1, IDH2, and TP53 genes and for 1p/19q loss and correlated these with clinical outcome. Most tumors (86%) were characterized genetically by TP53 mutation plus IDH1/2 mutation (32%), 1p/19q loss plus IDH1/2 mutation (37%), or IDH1/2 mutation only (17%). TP53 mutations only or 1p/19q loss only was rare (2 and 3%, respectively). The median survival of patients with TP53 mutation ± IDH1/2 mutation was significantly shorter than that of patients with 1p/19q loss ± IDH1/2 mutation (51.8 months vs. 58.7 months, respectively; P = 0.0037). Multivariate analysis with adjustment for age and treatment confirmed these results (P = 0.0087) and also revealed that TP53 mutation is a significant prognostic marker for shorter survival (P = 0.0005) and 1p/19q loss for longer survival (P = 0.0002), while IDH1/2 mutations are not prognostic (P = 0.8737). The molecular classification on the basis of IDH1/2 mutation, TP53 mutation, and 1p/19q loss has power similar to histological classification and avoids the ambiguity inherent to the diagnosis of oligoastrocytoma.

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.

The impact of lesion location and lesion size on poststroke infection frequency

Objectives Infections in patients with stroke are common and significantly affect outcome. Various predictors of poststroke infections were determined, such as degree of neurological impairment and implementation of therapeutic interventions. The authors investigated whether stroke location and stroke size are independent risk factors for poststroke infections. Methods 591 patients with acute stroke who were treated on our stroke unit were included in a prospective observational study. Predefined endpoints were pneumonia, urinary-tract infection (UTI) and other infections. The OR of infections was calculated for various stroke locations, stroke lateralisation and three categories of stroke size. Logistic regression models were used to adjust for factors significantly associated with poststroke infections in a single-factor analysis. Results In the single-factor analysis, the left anterior cerebral artery territory was associated with pneumonia. After adjustment for relevant covariates, this association was no longer statistically significant. Stroke lateralisation showed no association with infection frequency. The largest stroke size was positively associated with pneumonia (OR 3.5, p<0.001). The smallest lesion size was significantly less associated with the occurrence of UTI (OR 0.4, p<0.01). Conclusion In this study, lesion size is an independent risk factor for the development of poststroke infection. Particular brain regions associated with infections could not be determined.

Transcriptional Factors for Epithelial–Mesenchymal Transition Are Associated with Mesenchymal Differentiation in Gliosarcoma

Gliosarcoma is a rare variant of glioblastoma characterized by a biphasic pattern of glial and mesenchymal differentiation. It is unclear whether mesenchymal differentiation in gliosarcomas is because of extensive genomic instability and/or to a mechanism similar to epithelial–mesenchymal transition (EMT). In the present study, we assessed 40 gliosarcomas for immunoreactivity of Slug, Twist, matrix metalloproteinase‐2 (MMP‐2) and matrix metalloproteinase‐9 (MMP‐9), which are involved in EMT in epithelial tumors. Nuclear Slug expression was observed in >50% of neoplastic cells in mesenchymal tumor areas of 33 (83%) gliosarcomas, but not in glial areas (P < 0.0001). Nuclear Twist expression was observed in >50% of neoplastic cells in mesenchymal tumor areas of 35 (88%) gliosarcomas, but glial tumor areas were largely negative except in four cases (P < 0.0001). Expression of MMP‐2 and MMP‐9 was also significantly more extensive in mesenchymal than in glial tumor areas. None of 20 ordinary glioblastomas showed Slug or Twist expression in >10% neoplastic cells. Thus, expression of Slug, Twist, MMP‐2 and MMP‐9 was characteristic of mesenchymal tumor areas of gliosarcomas, suggesting that mechanisms involved in the EMT in epithelial neoplasms may play roles in mesenchymal differentiation in gliosarcomas.

Alterations in the RB1 pathway in low-grade diffuse gliomas lacking common genetic alterations.

We recently reported that the vast majority (>90%) of low‐grade diffuse gliomas (diffuse astrocytoma, oligoastrocytoma and oligodendroglioma) carry at least one of the following genetic alterations: IDH1/2 mutation, TP53 mutation or 1p/19q loss. Only 7% of cases were triple‐negative (ie, lacking any of these alterations). In the present study, array comparative genomic hybridization (CGH) in 15 triple‐negative WHO grade II gliomas (eight diffuse astrocytomas and seven oligodendrogliomas) showed loss at 9p21 (p14ARF, p15INK4b, p16INK4a loci) and 13q14–13q32 (containing the RB1 locus) in three and two cases, respectively. Further analyses in 31 triple‐negative cases as well as a total of 160 non‐triple‐negative cases revealed that alterations in the RB1 pathway (homozygous deletion and promoter methylation of the p15INK4b, p16INK4a and RB1 genes) were significantly more frequent in triple‐negative (26%) than in non‐triple‐negative cases (11%; P = 0.0371). Multivariate analysis after adjustment for age, histology and treatment showed that RB1 pathway alterations were significantly associated with unfavorable outcome for patients with low‐grade diffuse glioma [hazard ratio, 3.024 (1.279–6.631); P = 0.0057]. These results suggest that a fraction of low‐grade diffuse gliomas lacking common genetic alterations may develop through a distinct genetic pathway, which may include loss of cell‐cycle control regulated by the RB1 pathway.

Fluorescence in neurosurgery: Its diagnostic and therapeutic use. Review of the literature.

Fluorescent agents, e.g. 5-aminolevulinic acid (5-ALA), fluorescein and indocyanine green (ICG) are in common use in neurosurgery for tumor resection and neurovascular surgery. Protoporphyrine IX (PPIX) as major metabolite of 5-ALA is a strong fluorescent substance accumulated within malignant glioma tissue and a very sensitive and specific tool for visualizing high grade glioma tissue during surgery. Furthermore, 5-ALA or rather PPIX also offers an intratumoral therapeutic option stimulated by laser light in specific wavelength. Fluorescein was demonstrated to show similar fluorescent reactions in neurosurgery, but is controversial in its use, especially in high grade tumor surgery. Intraoperative angiography during resection of arterio-venous malformations, extracranial-intracranial-bypass or aneurysm surgery is supported by ICG fluorescence. Generally ICG will provide beneficial information for both, exposure of the pathology and illustration of healthy structures. This manuscript shows an overview of the literature focussing fluorescence in neurosurgery.

Frequent BRAF Gain in Low‐Grade Diffuse Gliomas with 1p/19q Loss

Chromosomal 7q34 duplication and BRAF‐KIAA1549 fusion is a characteristic genetic alteration in pilocytic astrocytomas. 7q34 gain appears to be common in diffuse astrocytomas, but its significance is unclear. We assessed BRAF gain and BRAF mutations in 123 low‐grade diffuse gliomas, including 55 diffuse astrocytomas, 18 oligoastrocytomas and 50 oligodendrogliomas. Quantitative polymerase chain reaction (PCR) revealed BRAF gain in 17/50 (34%) oligodendrogliomas, a significantly higher frequency than in diffuse astrocytomas (7/55; 13%; P = 0.0112). BRAF gain was common in low‐grade diffuse gliomas with 1p/19q loss (39%) and those lacking any of the genetic alterations analyzed (31%), but was rare in those with TP53 mutations (2%). Logistic regression analysis showed a significant positive association between 1p/19q loss and BRAF gain (P = 0.0032) and a significant negative association between TP53 mutations and BRAF gain (P = 0.0042). Fluorescence in situ hybridization (FISH) analysis of 26 low‐grade diffuse gliomas with BRAF gain additionally revealed BRAF‐KIAA1549 fusion in one oligodendroglioma. Sequencing of cDNA in 17 low‐grade diffuse gliomas showed BRAF‐KIAA1549 fusion in another oligodendroglioma. A BRAFV600E mutation was also detected in one oligodendroglioma, and a BRAFA598V in one diffuse astrocytoma. These results suggest that low‐grade diffuse gliomas with 1p/19q loss have frequent BRAF gains, and a small fraction of oligodendrogliomas may show BRAF‐KIAA1549 fusion.

Alterations in the NF2/LATS1/LATS2/YAP Pathway in Schwannomas.

Abstract Schwannomas are benign nerve sheath tumors composed of well-differentiated Schwann cells. Other than frequent NF2 (neurofibromatosis type 2) mutations (50%–60%), their molecular pathogenesis is not fully understood. LATS1 and LATS2 are downstream molecules of NF2 and are negative regulators of the yes-associated protein (YAP) oncogene in the Hippo signaling pathway. We assessed mutations of the NF2, LATS1, and LATS2 genes, promoter methylation of LATS1 and LATS2, and expression of YAP and phosphorylated YAP in 82 cases of sporadic schwannomas. Targeted sequencing using the Ion Torrent Proton instrument revealed NF2 mutations in 45 cases (55%), LATS1 mutations in 2 cases (2%), and LATS2 mutations in 1 case (1%) of schwannoma. Methylation-specific polymerase chain reaction showed promoter methylation of LATS1 and LATS2 in 14 cases (17%) and 25 cases (30%), respectively. Overall, 62 cases (76%) had at least 1 alteration in the NF2, LATS1, and/or LATS2 genes. Immunohistochemistry revealed nuclear YAP expression in 18 of 42 cases of schwannoma (43%) and reduced cytoplasmic phosphorylated YAP expression in 15 of 49 cases of schwannoma (31%), all of which had at least 1 alteration in the NF2, LATS1, and/or LATS2 genes. These results suggest that an abnormal Hippo signaling pathway is involved in the pathogenesis of most sporadic schwannomas.

MET Gain in Diffuse Astrocytomas is Associated with Poorer Outcome

Glioblastoma may develop rapidly without evidence for precursor lesions (primary glioblastomas), or progress from diffuse or anaplastic astrocytomas (secondary glioblastomas). Despite having distinct genetic profiles, these glioblastoma subtypes have similar histological features. We hypothesized that the highly malignant phenotype of glioblastoma may be attributable to genetic alterations that are common to both glioblastoma subtypes. In the present study, we first searched for commonly (>35%) amplified genes in glioblastomas with IDH1 mutation (a hallmark of secondary glioblastoma) and those without IDH1 mutation (typical for primary glioblastoma) in data from The Cancer Genome Atlas (TCGA). A total of 25 genes were identified, of which 21 were located at 7q31‐34. We then screened 264 gliomas (70 glioblastomas, 112 diffuse astrocytomas, 82 oligodendrogliomas) for gain of the MET at 7q31.2 with quantitative polymerase chain reaction (PCR). MET gain was detected in primary glioblastomas (47%) and secondary glioblastomas (44%), suggesting that this genetic alteration plays a role in the pathogenesis of both glioblastoma subtypes. MET gain was also common in diffuse astrocytomas (38%), but less frequent in oligodendrogliomas (16%). MET gain in diffuse astrocytomas was associated with shorter survival (median, 43.0 vs. 70.7 months; P = 0.004), suggesting that MET gain is a useful prognostic marker for diffuse astrocytomas.

Amplification of the STOML3, FREM2, and LHFP Genes Is Associated with Mesenchymal Differentiation in Gliosarcoma

Gliosarcoma is a rare glioblastoma variant characterized by a biphasic tissue pattern with alternating areas that display either glial (glial fibrillary acidic protein-positive) or mesenchymal (reticulin-positive) differentiation. Previous analyses have shown identical genetic alterations in glial and mesenchymal tumor areas, suggesting that gliosarcomas are genetically monoclonal, and mesenchymal differentiation was considered to reflect the elevated genomic instability of glioblastomas. In the present study, we compared genome-wide chromosomal imbalances using array comparative genomic hybridization in glial and mesenchymal tumor areas of 13 gliosarcomas. The patterns of gain and loss were similar, except that the gain at 13q13.3-q14.1 (log(2) ratio >3.0), containing the STOML3, FREM2, and LHFP genes, which was restricted to the mesenchymal tumor area of a gliosarcoma. Further analyses of 64 cases of gliosarcoma using quantitative PCR showed amplification of the STOML3, FREM2, and LHFP genes in 14 (22%), 10 (16%), and 7 (11%) mesenchymal tumor areas, respectively, but not in glial tumor areas. Results of IHC analysis confirmed that overexpression of STOML3 and FREM2 was more extensive in mesenchymal than in glial tumor areas. These results suggest that the mesenchymal components in a small fraction of gliosarcomas may be derived from glial cells with additional genetic alterations.