Theo Nicolaides

Targeted Therapy for BRAFV600E Malignant Astrocytoma

Purpose: Malignant astrocytomas (MA) are aggressive central nervous system tumors with poor prognosis. Activating mutation of BRAF (BRAFV600E) has been reported in a subset of these tumors, especially in children. We have investigated the incidence of BRAFV600E in additional pediatric patient cohorts and examined the effects of BRAF blockade in preclinical models of BRAFV600E and wild-type BRAF MA. Experimental Design: BRAFV600E mutation status was examined in two pediatric MA patient cohorts. For functional studies, BRAFV600E MA cell lines were used to investigate the effects of BRAF shRNA knockdown in vitro, and to investigate BRAF pharmacologic inhibition in vitro and in vivo. Results: BRAFV600E mutations were identified in 11 and 10% of MAs from two distinct series of tumors (six of 58 cases total). BRAF was expressed in all MA cell lines examined, among which BRAFV600E was identified in four instances. Using the BRAFV600E-specific inhibitor PLX4720, pharmacologic blockade of BRAF revealed preferential antiproliferative activity against BRAFV600E mutant cells in vitro, in contrast to the use of shRNA-mediated knockdown of BRAF, which inhibited cell growth of glioma cell lines regardless of BRAF mutation status. Using orthotopic MA xenografts, we show that PLX4720 treatment decreases tumor growth and increases overall survival in mice-bearing BRAFV600E mutant xenografts, while being ineffective, and possibly tumor promoting, against xenografts with wild-type BRAF. Conclusions: Our results indicate a 10% incidence of activating BRAFV600E among pediatric MAs. With regard to implications for therapy, our results support evaluation of BRAFV600E-specific inhibitors for treating BRAFV600E MA patients. Clin Cancer Res; 17(24); 7595–604. ©2011 AACR.

High rate of concurrent BRAF-KIAA1549 gene fusion and 1p deletion in disseminated oligodendroglioma-like leptomeningeal neoplasms (DOLN)

Disseminated oligodendroglioma-like leptomeningeal neoplasm (DOLN) is a recently described entity that predominantly affects children, is slowly progressive, and exhibits little, if any, parenchymal involvement. Studies to date have demonstrated some similarities between DOLNs and adult oligodendrogliomas with respect to morphology (infiltrative, monotonous cells with round, regular nuclei and perinuclear clearing), immunohistochemistry (synaptophysin, GFAP, Olig-2 expression), and genetics (high rate of chromosome 1p deletions and some 1p19q co-deletions) [5-8]. In contrast however, no DOLNs have been shown to harbor the isocitrate dehydrogenase-1 (IDH1) R132H mutation. BRAF abnormalities are common in pediatric low-grade CNS tumors, with the BRAF-KIAA1549 fusion/tandem duplication at 7q34 identified in approximately, 60 % of cerebellar and optic pathway pilocytic astrocytomas [4]. Others, like pleomorphic xanthoastrocytoma (PXA), ganglioglioma, and dysembryoplastic neuroepithelial tumor (DNET), have shown variable frequencies of activating BRAF V600E point mutations [1]. Because DOLNs partially overlap with these neoplasms, we assessed a series of 20 cases for BRAF alterations. We examined 23 archival DOLNs by FISH for the BRAF-KIAA1549 fusion, and 17 cases for deletions of 1p and 19q (Abbott, North Chicago, IL, USA). One was additionally interrogated by SNP-array. Testing for BRAF V600E was also performed on nine cases. Of 15 informative cases for BRAF-KIAA1549 by FISH, 11 were fusion positive (Fig. 1). Another case was non-informative by FISH, yet harbored a duplication (ch7:138550993-140509923) by SNP-array consistent with gene fusion, for a total of 12 of 16 cases positive for BRAF-KIAA1549 (75 %). FISH revealed loss of 1p in 10/17 cases (59 %), with 3 of those being co-deleted for 19q (18 %). Of the 12 cases with BRAF fusions, 9 had 1p deletion (75 %) and 2 had 19q co-deletion (17 %). None of 9 tested specimens were positive for BRAF-V600E mutation. Fig. 1 DOLN nuclei with FITC-labeled probe RP11-355D18 corresponding to KIAA1549 (green) and digoxigenin-labeled probe 726N20 corresponding to BRAF (red). Yellow signals indicate fusion These findings indicate a high rate of concurrent BRAF-KIAA1549 gene fusions and 1p deletions in DOLNs. Although BRAF fusions are typical of pilocytic astrocytomas, 1p deletions are not, confirming that DOLNs are pathologically and genetically distinct in most cases. These findings also further separate these oligodendroglioma-like tumors from adult oligodendrogliomas, although some overlap remains since 1p19q co-deletion is occasionally found in DOLN and rare BRAF fusions have been recently reported in otherwise classic, 1p19q co-deleted oligoden-droglioma [3]. DOLNs have also displayed occasional ganglion cell differentiation and areas of richly myxoid stroma, raising related possible link to gangliogliomas or DNETs, yet none of our 9 tested cases showed evidence of a BRAF V600E mutation, suggesting that DOLNs are genetically distinct from those entities as well. First-generation RAF inhibitors have been demonstrated in vitro to paradoxically activate the BRAF-KIAA1549 fusion protein [9]. Instead, MEK or mTOR inhibitors could be considered in the treatment of DOLNs, as in other fusion-positive tumors [2]. This report examines the role of common BRAF abnormalities in DOLNs and establishes a high frequency of concurrent BRAF-KIAA1549 fusions and 1p deletions. Although DOLNs are already clinicopathologically distinct, these findings further demonstrate fundamental genetic differences from other entities and implicate a new potential therapeutic target for patients with these otherwise challenging disseminated tumors.

Pediatric high-grade glioma: biologically and clinically in need of new thinking

Abstract High-grade gliomas in children are different from those that arise in adults. Recent collaborative molecular analyses of these rare cancers have revealed previously unappreciated connections among chromatin regulation, developmental signaling, and tumorigenesis. As we begin to unravel the unique developmental origins and distinct biological drivers of this heterogeneous group of tumors, clinical trials need to keep pace. It is important to avoid therapeutic strategies developed purely using data obtained from studies on adult glioblastoma. This approach has resulted in repetitive trials and ineffective treatments being applied to these children, with limited improvement in clinical outcome. The authors of this perspective, comprising biology and clinical expertise in the disease, recently convened to discuss the most effective ways to translate the emerging molecular insights into patient benefit. This article reviews our current understanding of pediatric high-grade glioma and suggests approaches for innovative clinical management.

Pediatric low-grade gliomas: implications of the biologic era

For the past decade, it has been recognized that pediatric low-grade gliomas (LGGs) and glial-neuronal tumors carry distinct molecular alterations with resultant aberrant intracellular signaling in the Ras-mitogen-activated protein kinase pathway. The conclusions and recommendations of a consensus conference of how best to integrate the growing body of molecular genetic information into tumor classifications and, more importantly, for future treatment of pediatric LGGs are summarized here. There is uniform agreement that molecular characterization must be incorporated into classification and is increasingly critical for appropriate management. Molecular-targeted therapies should be integrated expeditiously, but also carefully into the management of these tumors and success measured not only by radiographic responses or stability, but also by functional outcomes. These trials need to be carried out with the caveat that the long-term impact of molecularly targeted therapy on the developing nervous system, especially with long duration treatment, is essentially unknown.