Clonal expansion and epigenetic reprogramming following deletion or amplification of mutant IDH1.

IDH1 mutation is the earliest genetic alteration in low-grade gliomas (LGGs), but its role in tumor recurrence is unclear. Mutant IDH1 drives overproduction of the oncometabolite d-2-hydroxyglutarate (2HG) and a CpG island (CGI) hypermethylation phenotype (G-CIMP). To investigate the role of mutant IDH1 at recurrence, we performed a longitudinal analysis of 50 IDH1 mutant LGGs. We discovered six cases with copy number alterations (CNAs) at the IDH1 locus at recurrence. Deletion or amplification of IDH1 was followed by clonal expansion and recurrence at a higher grade. Successful cultures derived from IDH1 mutant, but not IDH1 wild type, gliomas systematically deleted IDH1 in vitro and in vivo, further suggestive of selection against the heterozygous mutant state as tumors progress. Tumors and cultures with IDH1 CNA had decreased 2HG, maintenance of G-CIMP, and DNA methylation reprogramming outside CGI. Thus, while IDH1 mutation initiates gliomagenesis, in some patients mutant IDH1 and 2HG are not required for later clonal expansions.

Mutant IDH1 Expression Drives TERT Promoter Reactivation as Part of the Cellular Transformation Process.

Mutations in the isocitrate dehydrogenase gene IDH1 are common in low-grade glioma, where they result in the production of 2-hydroxyglutarate (2HG), disrupted patterns of histone methylation, and gliomagenesis. IDH1 mutations also cosegregate with mutations in the ATRX gene and the TERT promoter, suggesting that IDH mutation may drive the creation or selection of telomere-stabilizing events as part of immortalization/transformation process. To determine whether and how this may occur, we investigated the phenotype of pRb-/p53-deficient human astrocytes engineered with IDH1 wild-type (WT) or R132H-mutant (IDH1mut) genes as they progressed through their lifespan. IDH1mut expression promoted 2HG production and altered histone methylation within 20 population doublings (PD) but had no effect on telomerase expression or telomere length. Accordingly, cells expressing either IDH1WT or IDH1mut entered a telomere-induced crisis at PD 70. In contrast, only IDH1mut cells emerged from crisis, grew indefinitely in culture, and formed colonies in soft agar and tumors in vivo Clonal populations of postcrisis IDH1mut cells displayed shared genetic alterations, but no mutations in ATRX or the TERT promoter were detected. Instead, these cells reactivated telomerase and stabilized their telomeres in association with increased histone lysine methylation (H3K4me3) and c-Myc/Max binding at the TERT promoter. Overall, these results show that although IDH1mut does not create or select for ATRX or TERT promoter mutations, it can indirectly reactivate TERT, and in doing so contribute to astrocytic immortalization and transformation. Cancer Res; 76(22); 6680-9. ©2016 AACR.

Glioma Groups Based on 1p/19q, IDH, and TERT Promoter Mutations in Tumors.

BACKGROUND: The prediction of clinical behavior, response to therapy, and outcome of infiltrative glioma is challenging. On the basis of previous studies of tumor biology, we defined five glioma molecular groups with the use of three alterations: mutations in the TERT promoter, mutations in IDH, and codeletion of chromosome arms 1p and 19q (1p/19q codeletion). We tested the hypothesis that within groups based on these features, tumors would have similar clinical variables, acquired somatic alterations, and germline variants. METHODS: We scored tumors as negative or positive for each of these markers in 1087 gliomas and compared acquired alterations and patient characteristics among the five primary molecular groups. Using 11,590 controls, we assessed associations between these groups and known glioma germline variants. RESULTS: Among 615 grade II or III gliomas, 29% had all three alterations (i.e., were triple-positive), 5% had TERT and IDH mutations, 45% had only IDH mutations, 7% were triple-negative, and 10% had only TERT mutations; 5% had other combinations. Among 472 grade IV gliomas, less than 1% were triple-positive, 2% had TERT and IDH mutations, 7% had only IDH mutations, 17% were triple-negative, and 74% had only TERT mutations. The mean age at diagnosis was lowest (37 years) among patients who had gliomas with only IDH mutations and was highest (59 years) among patients who had gliomas with only TERT mutations. The molecular groups were independently associated with overall survival among patients with grade II or III gliomas but not among patients with grade IV gliomas. The molecular groups were associated with specific germline variants. CONCLUSIONS: Gliomas were classified into five principal groups on the basis of three tumor markers. The groups had different ages at onset, overall survival, and associations with germline variants, which implies that they are characterized by distinct mechanisms of pathogenesis. (Funded by the National Institutes of Health and others.).

CDKN2A loss is associated with shortened overall survival in lower-grade (World Health Organization Grades II-III) astrocytomas.

Lower-grade (World Health Organization Grades II and III) gliomas vary widely in clinical behavior and are classified as astrocytic, oligodendroglial, or mixed. Anaplasia depends greatly on mitotic activity, with CDKN2A loss considered as the most common mechanism for cell cycle dysregulation. We investigated whether loss of the CDKN2A gene is associated with overall survival across pathologically and genetically defined glioma subtypes. After adjustment for IDH mutation, sex, and age, CDKN2A deletion was strongly associated with poorer overall survival in astrocytomas but not in oligodendrogliomas or oligoastrocytomas. Molecular classification of astrocytomas by IDH mutation, TP53 mutation, and /or ATRX loss of expression revealed that CDKN2A loss in IDH/TP53 mutated tumors was strongly associated with worse overall survival. CDKN2A loss in IDH mutated tumors with ATRX loss was only weakly associated with worse overall survival. These findings suggest that CDKN2A testing may provide further clinical aid in lower-grade glioma substratification beyond IDH mutation and 1p19q codeletion status, particularly in IDH/TP53 mutated astrocytomas.

The differential diagnosis of pilocytic astrocytoma with atypical features and malignant glioma: an analysis of 16 cases with emphasis on distinguishing molecular features.

Rare pilocytic astrocytomas (PA) have atypical histologic and clinicoradiologic features that raise the differential diagnosis of glioblastoma. Whether ancillary studies can supplement histopathologic examination in placing these cases accurately on the spectrum of WHO Grade I PA to higher-grade glioma is not always clear, partly because these cases are not common. Here, ten PAs with atypical clinicoradiologic and histologic features and six pediatric glioblastoma multiforme (pGBMs) were analyzed for BRAF V600E, IDH1, IDH2, and TP53 mutations. Ki-67, p53, and p16 protein expression were also examined by immunohistochemistry. BRAF-KIAA1549 fusion status was assessed in the PA subgroup. The rate of BRAF-KIAA1549 fusion was high in these PAs (5/7 tumors) including four extracerebellar examples. A single BRAF V600E mutation was identified in the fusion-negative extracerebellar PA of a very young child who succumbed to the disease. TP53 mutations were present only in malignant gliomas, including three pGBMs and one case designated as PA with anaplastic features (with consultation opinion of pGBM). IDH1 and IDH2 were wild type in all cases, consistent with earlier findings that IDH mutations are not typical in high-grade gliomas of patients ≤14 years of age. Immunohistochemical studies showed substantial overlap in Ki-67 labeling indices, an imperfect correlation between p53 labeling and TP53 mutation status, and complete p16 loss in only two pGBMs but in no PAs. These results suggest that (a) BRAF-KIAA1549 fusion may be common in PAs with atypical clinicoradiologic and histologic features, including those at extracerebellar sites, (b) BRAF V600E mutation is uncommon in extracerebellar PAs, and (c) TP53 mutation analysis remains a valuable tool in identifying childhood gliomas that will likely behave in a malignant fashion.

The alternative lengthening of telomere phenotype is significantly associated with loss of ATRX expression in high-grade pediatric and adult astrocytomas: a multi-institutional study of 214 astrocytomas.

Loss-of-function of alpha thalassemia/mental retardation syndrome X-linked (ATRX) protein leads to a phenotype called alternative lengthening of telomeres (ALT) in some tumors. High-grade astrocytomas comprise a heterogeneous group of central nervous system tumors. We examined a large cohort of adult (91) and pediatric (n=88) high-grade astrocytomas as well as lower grade forms (n=35) for immunohistochemical loss of ATRX protein expression and the presence of ALT using telomere-specific fluorescence in situ hybridization, with further correlation to other known genetic alterations. We found that in pediatric high-grade astrocytomas, 29.6% of tumors were positive for ALT and 24.5% were immunonegative for the ATRX protein, these two alterations being highly associated with one another (P<0.0001). In adult high-grade astrocytomas, 26.4% of tumors were similarly positive for ALT, including 80% of ATRX protein immunonegative cases (P<0.0001). Similar frequencies were found in 11 adult low-grade astrocytomas, whereas all 24 pilocytic astrocytomas were negative for ALT. We did not find any significant correlations between isocitrate dehydrogenase status and either ALT positivity or ATRX protein expression in our adult high-grade astrocytomas. In both cohorts, however, the ALT positive high-grade astrocytomas showed more frequent amplification of the platelet-derived growth factor receptor alpha gene (PDGFRA; 45% and 50%, respectively) than the ALT negative counterparts (18% and 26%; P=0.03 for each). In summary, our data show that the ALT and ATRX protein alterations are common in both pediatric and adult high-grade astrocytomas, often with associated PDGFRA gene amplification.

PDGFRA amplification is common in pediatric and adult high-grade astrocytomas and identifies a poor prognostic group in IDH1 mutant glioblastoma.

High-grade astrocytomas (HGAs), corresponding to World Health Organization grades III (anaplastic astrocytoma) and IV (glioblastoma; GBM), are biologically aggressive, and their molecular classification is increasingly relevant to clinical management. PDGFRA amplification is common in HGAs, although its prognostic significance remains unclear. Using fluorescence in situ hybridization (FISH), the most sensitive technique for detecting PDGFRA copy number gains, we determined PDGFRA amplification status in 123 pediatric and 263 adult HGAs. A range of PDGFRA FISH patterns were identified and cases were scored as non-amplified (normal and polysomy) or amplified (low-level and high-level). PDGFRA amplification was frequent in pediatric (29.3%) and adult (20.9%) tumors. Amplification was not prognostic in pediatric HGAs. In adult tumors diagnosed initially as GBM, the presence of combined PDGFRA amplification and isocitrate dehydrogenase 1 (IDH1)(R132H) mutation was a significant independent prognostic factor (P = 0.01). In HGAs, PDGFRA amplification is common and can manifest as high-level and focal or low-level amplifications. Our data indicate that the latter is more prevalent than previously reported with copy number averaging techniques. To our knowledge, this is the largest survey of PDGFRA status in adult and pediatric HGAs and suggests PDGFRA amplification increases with grade and is associated with a less favorable prognosis in IDH1 mutant de novo GBMs.

Gene expression profile identifies tyrosine kinase c-Met as a targetable mediator of antiangiogenic therapy resistance.

PURPOSE: To identify mediators of glioblastoma antiangiogenic therapy resistance and target these mediators in xenografts. EXPERIMENTAL DESIGN: We conducted microarray analysis comparing bevacizumab-resistant glioblastomas (BRG) with pretreatment tumors from the same patients. We established novel xenograft models of antiangiogenic therapy resistance to target candidate resistance mediator(s). RESULTS: BRG microarray analysis revealed upregulation versus pretreatment of receptor tyrosine kinase c-Met, which underwent further investigation because of its prior biologic plausibility as a bevacizumab resistance mediator. BRGs exhibited increased hypoxia versus pretreatment in a manner correlating with their c-Met upregulation, increased c-Met phosphorylation, and increased phosphorylation of c-Met-activated focal adhesion kinase and STAT3. We developed 2 novel xenograft models of antiangiogenic therapy resistance. In the first model, serial bevacizumab treatment of an initially responsive xenograft generated a xenograft with acquired bevacizumab resistance, which exhibited upregulated c-Met expression versus pretreatment. In the second model, a BRG-derived xenograft maintained refractoriness to the MRI tumor vasculature alterations and survival-promoting effects of bevacizumab. Growth of this BRG-derived xenograft was inhibited by a c-Met inhibitor. Transducing these xenograft cells with c-Met short hairpin RNA inhibited their invasion and survival in hypoxia, disrupted their mesenchymal morphology, and converted them from bevacizumab-resistant to bevacizumab-responsive. Engineering bevacizumab-responsive cells to express constitutively active c-Met caused these cells to form bevacizumab-resistant xenografts. CONCLUSION: These findings support the role of c-Met in survival in hypoxia and invasion, features associated with antiangiogenic therapy resistance, and growth and therapeutic resistance of xenografts resistant to antiangiogenic therapy. Therapeutically targeting c-Met could prevent or overcome antiangiogenic therapy resistance.