The mitogen-activated protein kinase (MAPK) pathway is known to play a key role in the initiation and maintenance of many tumors as well as normal development. This often occurs through mutation of the genes encoding RAS and RAF proteins which are involved in signal transduction in this pathway. BRAF is one of three RAF kinases which act as downstream effectors of growth factor signaling leading to cell cycle progression, proliferation, and survival. Initially reported as a point mutation (V600E) in the majority of metastatic melanomas, other alterations in the BRAF gene have now been reported in a variety of human cancers including papillary thyroid cancer, colon carcinomas, hairy cell leukemia, and more recently in gliomas. The identification of oncogenic mutations in the BRAF gene have led to a revolution in the treatment of metastatic melanoma using targeted molecular therapies that affect the MAPK pathway either directly through BRAF inhibition or downstream through inhibition of MEK. This review describes the molecular biology of BRAF in the context of pediatric low-grade gliomas, the role of BRAF as a diagnostic marker, the prognostic implications of BRAF, and evidence for therapeutic targeting of BRAF.

BACKGROUND The DNA repair protein O6 methylguanine-DNA methyltransferase (MGMT) causes resistance of tumour cells to alkylating agents. It is a predictive biomarker in high grade gliomas treated with temozolomide, however there is no consensus on which test method, methylation sites, and cut-off values to use. METHODS We performed a Cochrane Review to examine studies using different techniques to measure MGMT and predict survival in glioblastoma patients treated with temozolomide. Eligible longitudinal studies included adults with glioblastoma treated with temozolomide with or without radiotherapy, or surgery; where MGMT status was determined in tumour tissue, and assessed by one or more technique; and where overall survival was an outcome parameter, with sufficient information to estimate hazard ratios. Two or more methods were compared in 32 independent cohorts with 3474 patients. RESULTS Methylation-specific PCR (MSP) and pyrosequencing (PSQ) techniques were more prognostic than immunohistochemistry for MGMT protein, and PSQ is a slightly better predictor than MSP. CONCLUSIONS We cannot draw strong conclusions about use of frozen tissue versus formalin-fixed paraffin embedded in MSP and PSQ. Also, our meta-analysis does not provide strong evidence about the best CpG sites or threshold. MSP has been studied mainly for CpG sites 76-80 and 84-87 and Pyrosequencing at CpG sites ranging from 72 to 95. A cut-off threshold of 9% for CpG sites 74-78 performed better than higher thresholds of 28% or 29% in two of three good-quality studies. 190 studies were identified presenting hazard ratios from survival analysis in patients in which MGMT methylation was measured by one technique only.

Replicating the human genome efficiently and accurately is a daunting challenge involving the duplication of upward of three billion base pairs. At the core of the complex machinery that achieves this task are three members of the B family of DNA polymerases: DNA polymerases a, d, and ε. Collectively these multimeric polymerases ensure DNA replication proceeds at optimal rates approaching 2 3 10 3 nucleotides/min with an error rate of less than one per million nucleotides polymerized. The majority of DNA replication of undamaged DNA is conducted by DNA polymerases d and ε. The DNA polymerase a-primase complex performs limited synthesis to initiate the replication process, along with Okazaki-fragment synthesis on the discontinuous lagging strand. An increasing number of human disorders caused by defects in different components of the DNA-replication apparatus have been described to date. These are clinically diverse and involve a wide range of features, including variable combinations of growth delay, immunodeficiency, endocrine insufficiencies, lipodystrophy, and cancer predisposition. Here, by using various complementary approaches, including classical linkage analysis, targeted next-generation sequencing, and whole-exome sequencing, we describe distinct missense and splice-impacting mutations in POLA1 in five unrelated families presenting with an X-linked syndrome involving intellectual disability, proportionate short stature, microcephaly, and hypogonadism. POLA1 encodes the p180 catalytic subunit of DNA polymerase a-primase. A range of replicative impairments could be demonstrated in lymphoblastoid cell lines derived from affected individuals. Our findings describe the presentation of pathogenic mutations in a catalytic component of a B family DNA polymerase member, DNA polymerase a.

Pilocytic astrocytomas (PAs) are increasingly tested for KIAA1549-BRAF fusions. We used reverse transcription polymerase chain reaction for the 3 most common KIAA1549-BRAF fusions, together with BRAF V600E and histone H3.3 K27M analyses to identify relationships of these molecular characteristics with clinical features in a cohort of 32 PA patients. In this group, the overall BRAF fusion detection rate was 24 (75%). Ten (42%) of the 24 had the 16-9 fusion, 8 (33%) had only the 15-9 fusion, and 1 (4%) of the patients had only the 16-11 fusion. In the PAs with only the 15-9 fusion, 1 PA was in the cerebellum and 7 were centered in the midline outside of the cerebellum, that is, in the hypothalamus (n = 4), optic pathways (n = 2), and brainstem (n = 1). Tumors within the cerebellum were negatively associated with fusion 15-9. Seven (22%) of the 32 patients had tumor-related deaths and 25 of the patients (78%) were alive between 2 and 14 years after initial biopsy. Age, sex, tumor location, 16-9 fusion, and 15-9 fusion were not associated with overall survival. Thus, in this small cohort, 15-9 KIAA1549-BRAF fusion was associated with midline PAs located outside of the cerebellum; these tumors, which are generally difficult to resect, are prone to recurrence.

The oncogene epidermal growth factor receptor variant III (EGFRvIII) is frequently expressed in glioblastomas (GBM) but its impact on therapy response is still under controversial debate. Here we wanted to test if EGFRvIII influences the sensitivity towards the alkylating agent temozolomide (TMZ). Therefore, we retrospectively analyzed the survival of 336 GBM patients, demonstrating that under standard treatment, which includes TMZ, EGFRvIII expression is associated with prolonged survival, but only in patients with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylated tumors. Using isogenic GBM cell lines with endogenous EGFRvIII expression we could demonstrate that EGFRvIII increases TMZ sensitivity and results in enhanced numbers of DNA double-strand breaks and a pronounced S/G2-phase arrest after TMZ treatment. We observed a higher expression of DNA mismatch repair (MMR) proteins in EGFRvIII+ cells and patient tumor samples, which was most pronounced for MSH2 and MSH6. EGFRvIII-specific knockdown reduced MMR protein expression thereby increasing TMZ resistance. Subsequent functional kinome profiling revealed an increased activation of p38-and ERK1/2-dependent signaling in EGFRvIII expressing cells, which regulates MMR protein expression downstream of EGFRvIII. In summary, our results demonstrate that the oncoprotein EGFRvIII sensitizes a fraction of GBM to current standard of care treatment through the upregulation of DNA MMR.