Prognostic stratification of gliomatosis cerebri by IDH1 R132H and INA expression.

Gliomatosis cerebri (GC) constitutes a heterogeneous group of conditions involving diffuse neoplastic glial cell infiltration of the brain. Management is difficult and an obvious challenge is to identify prognostic factors. Alpha-internexin (INA) expression, which is closely related to the 1p19q codeletion, is a strong prognostic marker in oligodendroglial tumors. Similarly, the R132H isocitrate dehydrogenase 1 IDH1 mutation, which can now be detected by use of a specific antibody, predicts better outcome in gliomas. In a retrospective series of 40 GC treated with up-front chemotherapy, we analyzed IDH1(R132H) mutant protein and INA immunohistochemical expression and correlated it with outcome; 17/40 GC expressed IDH1(R132H) and 10/40 GC expressed INA. IDH1(R132H) staining was strongly related to progression-free survival (42.3 vs. 15.5 months for positive IDH1(R132H) vs. negative tumors; P < 0.0001) and overall survival (73.9 vs. 23.6 months; P < 0.0001). This effect was independent of grade, histologic subtype, and INA expression (P < 0.001). Combined expression of IDH1(R132H) and INA was strongly associated with response to chemotherapy (100% vs. 36%; P = 0.003). These data strongly suggest that INA and IDH1(R132H) mutant protein immunohistochemical analysis is of a great prognostic value in biopsied GC.

Diagnostic and prognostic value of alpha internexin expression in a series of 409 gliomas.

The neuronal intermediate filament alpha internexin (INA) is expressed in most gliomas with 1p19q codeletion and could represent a valuable prognostic marker in clinical routine. INA expression was analysed on 409 gliomas and correlated with histology, progression free survival (PFS), overall survival (OS), genomic profile assessed by CGH-array, IDH1/IDH2 mutation and p53 expression. INA was expressed in 59% of grade II oligodendrogliomas (n=73), 45% of grade III oligodendrogliomas (n=133), 15% of grade II oligoastrocytomas (n=61), 12% of grade III oligoastrocytomas (n=41), 23% of glioblastomas with oligodendroglial component (n=31), 0% of grade I astrocytomas (n=3), 0% of grade II astrocytomas (n=14), 6% of grade III astrocytomas (n=17) and 0% of glioblastomas (n=36). INA expression was detected in 85% of gliomas with complete 1p19q codeletion ('true 1p19q signature') (n=85) versus 15% of gliomas without 1p19q codeletion (n=245), including 14% of gliomas with variable/partial 1p19q deletion ('false 1p19q signature') (n=72) (p<0.0001). INA was expressed by 43% of gliomas with IDH1 mutation (n=197) versus 12% of gliomas without IDH1 mutation (n=156) (p<0.0001). In oligodendroglial gliomas (n=240), INA expression specificity for 1p19q codeletion was 80%, sensitivity 85%, positive predictive value 70%, and negative predictive value was 91%. Combining INA and p53 expressions improved INA predictive accuracy for 1p19q codeletion. In grade III gliomas, INA expression was associated with longer PFS (42.1 versus 10.2 months, p=0.0007) and longer OS (124.6 versus 20.6 months, p=0.0001). In conclusion, INA expression is a fast, cheap and reliable prognostic marker, and represents a surrogate marker for 1p19q complete codeletion.

Methylation profiling identifies 2 groups of gliomas according to their tumorigenesis.

Extensive genomic and gene expression studies have been performed in gliomas, but the epigenetic alterations that characterize different subtypes of gliomas remain largely unknown. Here, we analyzed the methylation patterns of 807 genes (1536 CpGs) in a series of 33 low-grade gliomas (LGGs), 36 glioblastomas (GBMs), 8 paired initial and recurrent gliomas, and 9 controls. This analysis was performed with Illumina's Golden Gate Bead methylation arrays and was correlated with clinical, histological, genomic, gene expression, and genotyping data, including IDH1 mutations. Unsupervised hierarchical clustering resulted in 2 groups of gliomas: a group corresponding to de novo GBMs and a group consisting of LGGs, recurrent anaplastic gliomas, and secondary GBMs. When compared with de novo GBMs and controls, this latter group was characterized by a very high frequency of IDH1 mutations and by a hypermethylated profile similar to the recently described glioma CpG island methylator phenotype. MGMT methylation was more frequent in this group. Among the LGG cluster, 1p19q codeleted LGG displayed a distinct methylation profile. A study of paired initial and recurrent gliomas demonstrated that methylation profiles were remarkably stable across glioma evolution, even during anaplastic transformation, suggesting that epigenetic alterations occur early during gliomagenesis. Using the Cancer Genome Atlas data set, we demonstrated that GBM samples that had an LGG-like hypermethylated profile had a high rate of IDH1 mutations and a better outcome. Finally, we identified several hypermethylated and downregulated genes that may be associated with LGG and GBM oncogenesis, LGG oncogenesis, 1p19q codeleted LGG oncogenesis, and GBM oncogenesis.

Chromosome 9p and 10q losses predict unfavorable outcome in low-grade gliomas.

The loss of chromosomes 1p-19q is the only prognostic molecular alteration identified in low-grade gliomas (LGGs) to date. Search for loss of heterozygosity (LOH) on chromosomes 1p, 9p, 10q, and 19q was performed in a series of 231 LGGs. Loss of chromosomes 1p-19q was strongly correlated with prolonged progression-free survival (PFS) and overall survival (OS) in univariate and multivariate analyses. LOH on 9p and 10q were associated with shortened PFS (P = .01 and .03, respectively) on univariate analysis. On multivariate analysis, LOH on 9p remained significant for PFS (P = .05), whereas LOH on 10q had a significant effect on OS (P = .02). Search for LOH 9p and 10q appears to be a useful complement to analysis of chromosomes 1p-19q in LGGs.

Specific chromosomal imbalances as detected by array CGH in ependymomas in association with tumor location, histological subtype and grade.

Ependymomas are glial neoplasms originating from the wall of the ventricles or from the spinal canal. The significance of histopathological features in accurately predicting biological behavior is still debated. Moreover, key molecular events in the pathogenesis of ependymoma are yet to be defined. The main objective of the present study was to identify specific patterns of chromosomal aberrations that correlate with tumor location, histological subtype and grade. Forty-five ependymoma samples were analyzed by 1-megabase resolution array comparative genomic hybridization (CGH). Association between clinical or histopathological parameters and the genomic alterations identified was evaluated. The most frequently detected chromosome (chr) abnormalities were gain of chr 7, 9, 12 and 15 and loss of chr 22. Co-occurrence of those five alterations characterized spinal ependymomas (P = 0.01). Myxopapillary ependymomas displayed a specific genomic profile defined by concurrent gain of chr 5, 7, 9, 16 and 18 (P = 0.0007). Overall, the number of chromosomal abnormalities detected was inversely correlated with the malignancy grade. Gain of chr 1q correlated with intracranial high-grade tumors (P = 0.002). Loss of chr 6q was mainly observed in infratentorial (P = 0.02) and World Health Organization (WHO) grade III (P = 0.04) lesions. Chr 10q loss was associated with high-grade ependymomas (P = 0.01). The +7/+9/+12/+15/-22 genomic profile is significantly associated with WHO grade II spinal ependymomas, whereas the +5/+7/+9/+16/+18 genomic pattern is specific of myxopapillary ependymomas. Identification of specific genomic imbalances at a given tumor location suggests that ependymomas from different central nervous system (CNS) regions represent genetically distinct diseases. Detecting genomic alterations associated with aggressive biological behavior may help stratify patients in high- and low-risk disease.

Array-based genomics in glioma research.

Over the years, several relevant biomarkers with a potential clinical interest have been identified in gliomas using various techniques, such as karyotype, microsatellite analysis, fluorescent in situ hybridization and chromosome comparative genomic hybridization. Despite their pivotal contribution to our understanding of gliomas biology, clinical application of these approaches has been limited by technological and clinical complexities. In contrast, genomic arrays (array-based comparative genomic hybridization and single nucleotide polymorphisms array) have emerged as promising technologies for clinical use in the setting of gliomas. Indeed, their feasibility and reliability have been rigorously assessed in gliomas and are discussed in this review. The well-known genomic biomarkers in gliomas are in fact readily and reliably identified using genomic arrays. Moreover, it detects a multitude of new cryptic genomic markers, with potential biological and/or clinical significances. The main studies dedicated to genomic characterization of gliomas using genomic arrays are reviewed here. Interestingly, several recurrent genomic signatures have been reported by different teams, suggesting the validity of these genomic patterns. In light of this, genomic arrays are relatively simple and cost-effective techniques whose implementation in molecular diagnostic laboratories should be encouraged as a valuable clinical tool for management of glioma patients.

Identification of regions correlating MGMT promoter methylation and gene expression in glioblastomas.

The O(6)-methylguanine-DNA methyltransferase gene (MGMT) is methylated in several cancers, including gliomas. However, the functional role of cysteine-phosphate-guanine (CpG) island (CGI) methylation in MGMT silencing is still controversial. The aim of this study was to investigate whether MGMT CGI methylation correlates inversely with RNA expression of MGMT in glioblastomas and to determine the CpG region whose methylation best reflects the level of expression. The methylation level of CpG sites that are potentially related to expression was investigated in 54 glioblastomas by pyrosequencing, a highly quantitative method, and analyzed with respect to their MGMT mRNA expression status. Three groups of patients were identified according to the methylation pattern of all 52 analyzed CpG sites. Overall, an 85% rate of concordance was observed between methylation and expression (p < 0.0001). When analyzing each CpG separately, six CpG sites were highly correlated with expression (p < 0.0001), and two CpG regions could be used as surrogate markers for RNA expression in 81.5% of the patients. This study indicates that there is good statistical agreement between MGMT methylation and expression, and that some CpG regions better reflect MGMT expression than do others. However, if transcriptional repression is the key mechanism in explaining the higher chemosensitivity of MGMT-methylated tumors, a substantial rate of discordance should lead clinicians to be cautious when deciding on a therapeutic strategy based on MGMT methylation status alone.

NOTCH2 is neither rearranged nor mutated in t(1;19) positive oligodendrogliomas.

The combined deletion of 1p and 19q chromosomal arms is frequent in oligodendrogliomas (OD) and has recently been shown to be mediated by an unbalanced t(1;19) translocation. Recent studies of 1p/19q co-deleted OD suggest that the NOTCH2 gene is implicated in oligodendrocyte differentiation and may be involved in this rearrangement. The objective of the present study was to analyze the NOTCH2 locus either as a chromosomal translocation locus that may be altered by the 1p/19q recurrent rearrangement or as a gene that may be inactivated by a two hit process. We performed an array-CGH analysis of 15 ODs presenting 1p/19q co-deletion using a high-density oligonucleotide microarray spanning 1p and 19q pericentromeric regions with 377 bp average probe spacing. We showed that the 1p deletion extends to the centromere of chromosome 1 and includes the entire NOTCH2 gene. No internal rearrangement of this gene was observed. This strongly suggests that the t(1;19) translocation does not lead to an abnormal NOTCH2 structure. The analysis of the entire NOTCH2 coding sequence was performed in four cases and did not reveal any mutation therefore indicating that NOTCH2 does not harbor genetic characteristics of a tumor suppressor gene. Finally, the detailed analysis of chromosome 19 pericentromeric region led to the identification of two breakpoint clusters at 19p12 and 19q11-12. Interestingly, these two regions share a large stretch of homology. Together with previous observations of similarities between chromosome 1 and 19 alphoid sequences, this suggests that the t(1;19) translocation arises from complex intra and interchromosomal rearrangements.This is the first comprehensive deletion mapping by high density oligo-array of the 1p/19q co-deletion in oligodendroglioma tumors using a methodological approach superior to others previously applied. As such this paper provides clear evidence that the NOTCH2 gene is not physically rearranged by t(1;19) translocation of oligodendroglioma tumors.

Genomic changes in progression of low-grade gliomas.

Using a one-megabase BAC-based array comparative genomic hybridization technique (aCGH), we have investigated a series of 16 low-grade gliomas (LGGs) and their subsequent progression to higher-grade malignancies. The most frequent chromosome imbalances in primary tumors were gains of chromosomes 7q, 8q, and 22q, and losses of chromosomes 1p, 13q, and 19q. In tumor progression, gains of chromosomes 11q, 7q, 20q, and 21q, and losses of chromosomes 9p, including CDKN2A locus, 19q, 14q, 1p, and 6q were the most frequent genomic disequilibria. Progressive tumors were more imbalanced than primary tumors in terms of altered chromosomal arms (3.8 vs. 6.6 in mean abnormal chromosomal arm) and altered BACs (17 vs. 21%). Interestingly, putative novel candidate genes associated with glioma progression were identified, in particular DOCK8, PTPRD, CER1, TPHO, DHFR, MSH3, ETS1, ACACA, and CSE1L.

Gene amplification is a poor prognostic factor in anaplastic oligodendrogliomas.

Various gene amplifications have been observed in gliomas. Prognostic-genomic correlations testing simultaneously all these amplified genes have never been conducted in anaplastic oligodendrogliomas. A set of 38 genes that have been reported to be amplified in gliomas and investigated as the main targets of amplicons were studied in a series of 52 anaplastic oligodendrogliomas using bacterial artificial chromosome-array based comparative genomic hybridization and quantitative polymerase chain reaction. Among the 38 target genes, 15 were found to be amplified in at least one tumor. Overall, 27% of anaplastic oligodendrogliomas exhibited at least one gene amplification. The most frequently amplified genes were epidermal growth factor receptor (EGFR) and cyclin-dependent kinase 4/sarcoma amplified sequence (CDK4/SAS) in 17% and 8% of anaplastic oligodendrogliomas, respectively. Gene amplification and codeletion of chromosome arms 1p/19q were perfectly exclusive (p = 0.005). In uni- and multivariate analyses, gene amplification was a negative prognostic factor for progression-free survival and overall survival in anaplastic oligodendrogliomas, providing complementary information to the classic prognostic factors identified in anaplastic oligodendrogliomas (extent of surgery, KPS, and chromosome arms 1p/19q status).

Influence of MDM2 SNP309 alone or in combination with the TP53 R72P polymorphism in oligodendroglial tumors.

The transcription factor p53 and its negative regulator MDM2 are pivotal in normal and cancer cells biology. Recently, a functional single-nucleotide polymorphism in the promoter region of MDM2 (MDM2 SNP309), alone or in combination with TP53 R72P, was shown to be associated with the risk, prognosis, age at onset, molecular markers, and response to chemotherapy of various cancers. This SNP has never been specifically investigated in a large series of oligodendroglial tumors. In a comparison with 232 healthy controls, we retrospectively analyzed blood samples of 293 oligodendroglial tumor patients for MDM2 SNP309. In addition, the TP53 R72P polymorphism and chromosome 1p/19q status, a major biomarker in oligodendroglial tumors, were investigated. The frequencies of T/T, T/G, and G/G genotypes in patients and controls did not suggest an increased risk of oligodendroglial tumor formation correlating with MDM2 SNP309. A borderline association was found between MDM2 SNP309 and overall survival (p=0.05), but in multivariate analysis, MDM2 SNP309 did not provide prognostic information complementary to age, tumor phenotype, grade, and 1p/19q status in oligodendroglial tumors. Finally, MDM2 SNP309, alone or in combination with TP53 R72P, was not associated with oligodendroglial tumors.

TP53 codon 72 polymorphism, p53 expression, and 1p/19q status in oligodendroglial tumors.

The functional single-nucleotide polymorphism (SNP) in codon 72 of TP53 has been shown to be both a risk factor and a prognostic biomarker in various cancers. Such results were also reported in brain tumors, notably in astrocytomas. This SNP has never been precisely investigated in oligodendroglial tumors. We retrospectively analyzed blood samples of 275 oligodendroglial tumor patients for the TP53 codon 72 polymorphism and compared them with a series of 144 healthy controls. Arg/Arg, Arg/Pro, and Pro/Pro genotypes were found in 54.2 versus 60.4%, 39.3 versus 34.0%, and 7.3 versus 5.6% of patients and controls, respectively. This suggests no association between oligodendroglial tumors and the SNP in codon 72 of TP53. Similarly, no correlation was found among the TP53 codon 72 polymorphism and prognosis, p53 expression, and chromosomes 1p and 19q status.

Tumor genomic profiling and TP53 germline mutation analysis of first-degree relative familial gliomas.

About 5% of gliomas occur in a familial context, which suggests a genetic origin, but the predisposing molecular factors remain unknown in most cases. A series of nine familial gliomas were characterized with 1-megabase resolution BAC array-based comparative genomic hybridization (aCGH) together with germline sequence analysis of TP53. This series was compared with a literature series of familial gliomas and a personal series of sporadic gliomas, analyzed by chromosome CGH and aCGH, respectively. No significant difference was noted between the three populations in terms of clinical characteristics, pathologic features, and the most frequent chromosomal alterations, including loss of 1p, 10p, 10q, 13q, and 19q, and gain of 7p, 7q, 16p, 18q, 19p, 19q, 20p, and 22q. However, a genomic region located in 6q was more frequently gained in our series of familial as compared to sporadic gliomas (P=0.028). A germline TP53 mutation was observed in 1/9 cases, which suggests Li-Fraumeni syndrome. Interestingly, the Pro allele in the codon 72 of TP53 was observed in 5/9 tumors. Although familial and sporadic gliomas share very similar cytogenetic quantitative patterns, aCGH is a promising technique for the detection of small genomic differences of potential significance.

MGMT prognostic impact on glioblastoma is dependent on therapeutic modalities.

MGMT promoter methylation, which has been correlated with the response to alkylating agents, was investigated in a retrospective series of 219 glioblastomas (GBMs) treated with various modalities. MGMT methylation had no impact on survival for the whole group, but showed a significant advantage (17.1 months vs. 13.1) for patients treated with RT+ adjuvant chemotherapy (relative risk of death (RR) = 0.53; P = 0.041), particularly when patients received CT during the course of RT (MS = 19.9 months vs. 12.5 months; RR = 0.227, P = 0.001). This suggests that the prognostic impact of MGMT methylation is dependent on therapeutic modalities and schedules. MGMT methylation was not correlated with the main molecular alterations, such as 10q loss and p53 expression.

MGMT methylation: a marker of response to temozolomide in low-grade gliomas.

The methylation status of the O6-methylguanine-methyltransferase promoter (MGMTP) was evaluated in 68 low-grade gliomas treated by neoadjuvant temozolomide. Methylated MGMTP was detected in 63 of 68 (92.6 %) patients and was a favorable predictor of progression-free survival as compared with unmethylated MGMTP tumors (p < 0.0001). Assessment of MGMTP status could help identifying low-grade gliomas patients more likely to respond to chemotherapy or to benefit from MGMT depletion strategies.

Prognostic impact of molecular markers in a series of 220 primary glioblastomas.

BACKGROUND: In contrast to oligodendrogliomas, molecular predictors of prognosis have not been consistently found in glioblastomas. However, genetic studies show that glioblastomas consist of several genetic subtypes and raise the possibility that molecular alterations could be predictive of survival. METHODS: A search for loss of heterozygosity (LOH) on chromosome 1p, 9p, 10q, 19q, EGFR (epidermal growth factor receptor), CDK4, and MDM2 (mouse double minute) amplifications, CDKN2A (INK4A/ARF) homozygous deletions, p53 expression, was performed in a series of 220 primary glioblastomas. The molecular alterations were then correlated with each other to identify distinct molecular pathways and with clinical parameters and the course of the disease to identify prognostic markers. RESULTS: Nonrandom associations were found between EGFR amplification and LOH10q, LOH9p, and INK4A/ARF deletion, LOH1p and LOH19q, and MDM2 and CDK4 amplification, whereas mutual exclusions were found between p53 expression and EGFR amplification, LOH 9p/INK4A/ARF homozygous deletion, and MDM2 and CDK4 amplification. Age (P = 4.10(-5)) and performance status (P = .003) were the main predictors of outcome. In contrast, molecular markers were of limited impact: MDM2 amplification correlated with poor outcome on both univariate and multivariate analysis (P = .01) and EGFR amplification with good prognosis on multivariate analysis (P = .02). CONCLUSION: Despite their limited prognostic impact, the genetic markers investigated here outline distinct molecular pathways involved in glioblastoma tumorigenesis and warrant broader molecular screening.

A uniform activated B-cell-like immunophenotype might explain the poor prognosis of primary central nervous system lymphomas: analysis of 83 cases.

Most primary central nervous system lymphomas (PCNSLs) in immunocompetent patients are diffuse large B-cell lymphomas (DLBCLs), characterized by poor prognosis, compared with systemic forms. A germinal center B-cell-like (GCB) origin of PCNSL was hypothesized on the basis of BCL-6 expression and ongoing mutational activity. Our goal herein was to determine, for 83 PCNSLs, the percentages of GCB and activated B-cell-like (ABC) phenotypes and their prognostic significance. CD10, BCL-6, MUM1, BCL-2, and CD138 antigens were immunohistochemically labeled on paraffin-embedded sections; the first 4 were positive in 2.4%, 55.5%, 92.6%, and 55.5% of the tumors, respectively. None of the 56 tested samples expressed CD138. Among the 82 patients with complete information, 79 (96.3%) were classified as ABC; 42 (51.2%) expressed BCL-6+ MUM1+, suggesting an "activated GCB" origin; 33 (40.2%) were exclusively MUM1+, and the remaining 4 (4.9%) were negative for all markers tested. These findings provide new insights into interpreting the poor PCNSL prognostic, which may, in part, be due to biologic aggressiveness associated with its activated B-cell-like pattern. We postulate assigning PCNSL a histogenetic "time-slot," overlapping late GC and early post-GC, that could explain the predominant ABC phenotype observed.

Loss of 22q chromosome is related to glioma progression and loss of 10q.

Loss of heterozygosity (LOH) of chromosome 22q has been investigated in 160 gliomas. LOH at one or more microsatellite increased with increasing grade of the tumor (P < 0.01). LOH22q was more frequent in astrocytic tumors (37%) compared to mixed or oligodendroglial tumors (21%) (P = 0.02). LOH22q was correlated to 10q loss but not to 1p or 9p loss. Taken together, these data suggest that LOH22q is an alteration associated with malignant progression of gliomas.

Chromosome 1p loss: a favorable prognostic factor in low-grade gliomas.

Search for loss of heterozygosity on chromosomes 1p, 9p, 10q, and 19q, epidermal growth factor receptor (EGFR) gene amplification, and p53 expression was performed in a series of 131 low-grade gliomas. The profile of molecular changes, clinical findings, and histology were subsequently correlated with the course of the disease, mainly progression-free survival. When these parameters were considered as candidate variables in a multivariate analysis, only loss of heterozygosity on chromosome 1p was associated with increased progression-free survival (hazard ratio, 0.521), indicating a major favorable prognostic role of this genetic alteration in low-grade gliomas.

Distinct responses of xenografted gliomas to different alkylating agents are related to histology and genetic alterations.

A series of 12 human gliomas was established as xenografts in nude mice and used to evaluate the relationship between histology, genetic parameters, and response to alkylating agents. Eight were high-grade oligodendroglial tumors, and four were glioblastoma. They were characterized for their genetic alterations, including those considered as "early" alterations, namely loss of chromosome 1 +/- loss of chromosome 19q, TP53 mutation, and those considered as "late" alterations, namely loss of chromosome 10, loss of chromosome 9p, EGFR genomic amplification, PTEN mutation, CDKN2A homozygous deletion, and telomerase reactivation. Chemosensitivity of xenografts to four alkylating agents, temozolomide (42 mg/kg, days 1-5, p.o.), 1,3-bis(2-chloroethyl)-1-nitrosourea (5 mg/kg, day 1, i.p.), Ifosfamide (90 mg/kg, days 1-3, i.p.), and carboplatin (66 mg/kg, day 1, i.p.) was tested by administration of drugs to tumor-bearing mice. Although each tumor presented an individual response pattern, glioblastoma had a lower chemosensitivity than oligodendrogliomas, and temozolomide was the most effective drug. Deletion of 1p +/- 19q was associated with higher chemosensitivity, whereas late molecular alterations, particularly EGFR amplification, were associated with chemoresistance. These results suggest that the combined use of histology and molecular markers should eventually be helpful selecting the most appropriate agents for treatment of malignant oligodendrogliomas and astrocytomas.