Anjan Misra

Integrated Array-Comparative Genomic Hybridization and Expression Array Profiles Identify Clinically Relevant Molecular Subtypes of Glioblastoma

Glioblastoma, the most aggressive primary brain tumor in humans, exhibits a large degree of molecular heterogeneity. Understanding the molecular pathology of a tumor and its linkage to behavior is an important foundation for developing and evaluating approaches to clinical management. Here we integrate array-comparative genomic hybridization and array-based gene expression profiles to identify relationships between DNA copy number aberrations, gene expression alterations, and survival in 34 patients with glioblastoma. Unsupervised clustering on either profile resulted in similar groups of patients, and groups defined by either method were associated with survival. The high concordance between these separate molecular classifications suggested a strong association between alterations on the DNA and RNA levels. We therefore investigated relationships between DNA copy number and gene expression changes. Loss of chromosome 10, a predominant genetic change, was associated not only with changes in the expression of genes located on chromosome 10 but also with genome-wide differences in gene expression. We found that CHI3L1/YKL-40 was significantly associated with both chromosome 10 copy number loss and poorer survival. Immortalized human astrocytes stably transfected with CHI3L1/YKL-40 exhibited changes in gene expression similar to patterns observed in human tumors and conferred radioresistance and increased invasion in vitro. Taken together, the results indicate that integrating DNA and mRNA-based tumor profiles offers the potential for a clinically relevant classification more robust than either method alone and provides a basis for identifying genes important in glioma pathogenesis.

Angiogenesis-independent tumor growth mediated by stem-like cancer cells

In this work, highly infiltrative brain tumors with a stem-like phenotype were established by xenotransplantation of human brain tumors in immunodeficient nude rats. These tumors coopted the host vasculature and presented as an aggressive disease without signs of angiogenesis. The malignant cells expressed neural stem cell markers, showed a migratory behavior similar to normal human neural stem cells, and gave rise to tumors in vivo after regrafting. Serial passages in animals gradually transformed the tumors into an angiogenesis-dependent phenotype. This process was characterized by a reduction in stem cells markers. Gene expression profiling combined with high throughput immunoblotting analyses of the angiogenic and nonangiogenic tumors identified distinct signaling networks in the two phenotypes. Furthermore, proinvasive genes were up-regulated and angiogenesis signaling genes were down-regulated in the stem-like tumors. In contrast, proinvasive genes were down-regulated in the angiogenesis-dependent tumors derived from the stem-like tumors. The described angiogenesis-independent tumor growth and the uncoupling of invasion and angiogenesis, represented by the stem-like cancer cells and the cells derived from them, respectively, point at two completely independent mechanisms that drive tumor progression. This article underlines the need for developing therapies that specifically target the stem-like cell pools in tumors.

Integrated genomic and epigenomic analyses pinpoint biallelic gene inactivation in tumors

Aberrant methylation of CpG islands and genomic deletion are two predominant mechanisms of gene inactivation in tumorigenesis, but the extent to which they interact is largely unknown. The lack of an integrated approach to study these mechanisms has limited the understanding of tumor genomes and cancer genes. Restriction landmark genomic scanning (RLGS; ref. 1) is useful for global analysis of aberrant methylation of CpG islands, but has not been amenable to alignment with deletion maps because the identity of most RLGS fragments is unknown. Here, we determined the nucleotide sequence and exact chromosomal position of RLGS fragments throughout the genome using the whole chromosome of origin of the fragments and in silico restriction digestion of the human genome sequence. To study the interaction of these gene-inactivation mechanisms in primary brain tumors, we integrated RLGS-based methylation analysis with high-resolution deletion maps from microarray-based comparative genomic hybridization (array CGH; ref. 3). Certain subsets of gene-associated CpG islands were preferentially affected by convergent methylation and deletion, including genes that exhibit tumor-suppressor activity, such as CISH1 (encoding SOCS1; ref. 4), as well as genes such as COE3 that have been missed by traditional non-integrated approaches. Our results show that most aberrant methylation events are focal and independent of deletions, and the rare convergence of these mechanisms can pinpoint biallelic gene inactivation without the use of positional cloning.

Identification of IGF2 signaling through phosphoinositide-3-kinase regulatory subunit 3 as a growth-promoting axis in glioblastoma.

Amplification or overexpression of growth factor receptors is a frequent occurrence in malignant gliomas. Using both expression profiling and in situ hybridization, we identified insulin-like growth factor 2 (IGF2) as a marker for a subset of glioblastomas (GBMs) that lack amplification or overexpression of EGF receptor. Among 165 primary high-grade astrocytomas, 13% of grade IV tumors and 2% of grade III tumors expressed IGF2 mRNA levels >50-fold the sample population median. IGF2-overexpressing tumors frequently displayed PTEN loss, were highly proliferative, exhibited strong staining for phospho-Akt, and belonged to a subclass of GBMs characterized by poor survival. Using a serum-free culture system, we discovered that IGF2 can substitute for EGF to support the growth of GBM-derived neurospheres. The growth-promoting effects of IGF2 were mediated by the insulin-like growth factor receptor 1 and phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3), a regulatory subunit of phosphoinositide 3-kinase that shows genomic gains in some highly proliferative GBM cases. PIK3R3 knockdown inhibited IGF2-induced growth of GBM-derived neurospheres. The current results provide evidence that the IGF2–PIK3R3 signaling axis is involved in promoting the growth of a subclass of highly aggressive human GBMs that lack EGF receptor amplification. Our data underscore the importance of the phosphoinositide 3-kinase/Akt pathway for growth of high-grade gliomas and suggest that multiple molecular alterations that activate this signaling cascade may promote tumorigenesis. Further, these findings highlight the parallels between growth factors or receptors that are overexpressed in GBMs and those that support in vitro growth of tumor-derived stem-like cells.

Chromosomal imbalances detected by array comparative genomic hybridization in human oligodendrogliomas and mixed oligoastrocytomas.

Loss of heterozygosity and fluorescence in situ hybridization (FISH) studies have shown that deletions of 1p and 19q are highly prevalent in oligodendroglioma. However, these tumors have not been comprehensively screened for other alterations in chromosomal dosage. In this study, we used array‐based comparative genomic hybridization (CGHa) of mapped BAC DNA to screen for such alterations in 31 oligodendrogliomas (20 grade II, 9 grade III, and 2 grade IV) and 4 mixed oligoastrocytomas (1 grade I, 1 grade II, and 2 grade IV). The most frequent aberrations were loss of 1p (17 cases; 49%) and 19q (15 cases; 43%) and combined loss of 1p/19q (13 cases; 37%). In addition, deletion of 4q, 5p, 9p, 10q, 11p, and 13q was observed in 10, 4, 8, 4, 4, and 13 cases, respectively; loss of whole chromosomes 4, 9, and 13 in 4, 1, and 7 cases, respectively; gain of 7p, 8q, 10p, and 11q in 6, 6, 5, and 10 cases, respectively, and gain of whole chromosomes 7 and 11 in 2 patients each. Minimally altered regions detected by CGHa involved chromosome bands 1p36.32, 4q33, 5p15, 8q24, 11p15, and 19q13.3. Univariate analysis of all 35 cases suggested that combined deletion of 1p and 19q is associated with better survival (P = 0.03). In addition, 8q gain in the oligodendrogliomas was strongly associated with poor outcome (P = 0.002). Also associated with poor disease outcome were alterations that had low prevalence in the pure oligodendrogliomas, including loss of 3q, 9q, and 12q and gain of 1p, 8p, and 10q. In summary, in oligodendrogliomas, CGHa was able to detect novel small alterations in chromosomal dosage that had not been previously detected by other methods. In addition, our findings support the hypotheses that oligodendroglioma can be classified into several groups by CGHa analysis and that specific alterations in genetic dosage may have biologic or clinical significance.

Isochromosome 17q Is a Negative Prognostic Factor in Poor-Risk Childhood Medulloblastoma Patients

Background: Medulloblastomas are the most common primary malignant childhood intracranial neoplasms. Patients are currently sorted into three risk groups based on clinical criteria: standard, poor, and infant (<18 months old). We hypothesized that genetic copy number aberrations (CNA) predict prognosis and would provide improved criteria for predicting outcome. Methods: DNA from 35 medulloblastoma patients from four Childrens Cancer Group trials was analyzed by comparative genomic hybridization to determine CNAs. The genetic alterations were evaluated using statistical and cluster analyses. Results: The most frequent CNAs were gains on 17q, 7, 1q, and 7q and losses on 17p, 10q, X, 16q, and 11q. Amplification at 5p15.1-p15.3 was also detected. Isochromosome 17q (i(17)(q10)) was associated with poor overall survival (P = 0.03) and event-free survival (P = 0.04) independent of poor risk group classification. Age <3 tended to be associated with <3 CNAs (P = 0.06). Unsupervised cluster analysis sorted the study patients into four subgroups based on CNAs. Supervised analysis using the program Significance Analysis of Microarrays (SAM) quantitatively validated those CNAs identified by unsupervised clustering that significantly distinguished among the four subgroups. Conclusions: Medulloblastomas are genetically heterogeneous and can be categorized into separate genetic subgroups by their CNAs using unsupervised cluster analysis and SAM. i(17)(q10) was a significant independent negative prognostic factor. Infant medulloblastomas may be a distinct genetic subset from those of older patients.

Array Comparative Genomic Hybridization Identifies Genetic Subgroups in Grade 4 Human Astrocytoma

Alterations of DNA copy number are believed to be important indicators of tumor progression in human astrocytoma. We used an array of bacterial artificial chromosomes to map relative DNA copy number in 50 primary glioblastoma multiforme tumors at ∼1.4-Mb resolution. We identified 33 candidate sites for amplification and homozygous deletion in these tumors. We identified three major genetic subgroups within these glioblastoma multiforme tumors: tumors with chromosome 7 gain and chromosome 10 loss, tumors with only chromosome 10 loss in the absence of chromosome 7 gain, and tumors without copy number change in chromosomes 7 or 10. The significance of these genetic groups to therapeutics needs further study.

Functional inactivation of the KLF6 tumor suppressor gene by loss of heterozygosity and increased alternative splicing in glioblastoma

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor and possesses a high incidence of 10p loss. The KLF6 (Kruppel‐like transcription factor) tumor suppressor gene on 10p15 is inactivated by loss of heterozygosity (LOH) and/or somatic mutation in a number of human cancers and forced expression of KLF6 in GBM lines inhibits their growth and transformation. In addition, increased expression of its alternatively spliced, cytoplasmic isoform KLF6‐SV1 has now been shown to play a role in cancer pathogenesis. On the basis of these findings we examined the role of KLF6 and KLF6‐SV1 in the development and progression of GBM. LOH analysis of 17 primary GBM patient samples using KLF6‐specific microsatellite markers revealed that 88.2% (15/17) had LOH of the KLF6 locus. Interestingly, no KLF6 somatic mutations were identified. RNA analysis revealed concomitant decreases in all primary GBM tumors (n = 11) by ∼80% in KLF6 expression (p < 0.001) coupled with increased KLF6‐SV1 expression (p < 0.001) when compared to normal astrocytes. To determine the biological relevance of these findings, we examined the effect of KLF6 expression and KLF6‐SV1 knockdown in A235 and CRL2020 cell lines. Reconstitution of KLF6 decreased cell proliferation by almost 50%, whereas targeted KLF6 reduction increased cell proliferation 2.5–4.5 fold. Conversely, targeted KLF6‐SV1 reduction decreased cell proliferation by 50%. Taken together, our findings demonstrate that KLF6 allelic imbalance and decreased KLF6 and increased KLF6‐SV1 expression are common findings in primary GBM tumors, and these changes have antagonistic effects on the regulation of cellular proliferation in GBM cell lines.

Novel risk stratification of patients with neuroblastoma by genomic signature, which is independent of molecular signature

Human neuroblastoma remains enigmatic because it often shows spontaneous regression and aggressive growth. The prognosis of advanced stage of sporadic neuroblastomas is still poor. Here, we investigated whether genomic and molecular signatures could categorize new therapeutic risk groups in primary neuroblastomas. We conducted microarray-based comparative genomic hybridization (array-CGH) with a DNA chip carrying 2464 BAC clones to examine genomic aberrations of 236 neuroblastomas and used in-house cDNA microarrays for gene-expression profiling. Array-CGH demonstrated three major genomic groups of chromosomal aberrations: silent (GGS), partial gains and/or losses (GGP) and whole gains and/or losses (GGW), which well corresponded with the patterns of chromosome 17 abnormalities. They were further classified into subgroups with different outcomes. In 112 sporadic neuroblastomas, MYCN amplification was frequent in GGS (22%) and GGP (53%) and caused serious outcomes in patients. Sporadic tumors with a single copy of MYCN showed the 5-year cumulative survival rates of 89% in GGS, 53% in GGP and 85% in GGW. Molecular signatures also segregated patients into the favorable and unfavorable prognosis groups (P=0.001). Both univariate and multivariate analyses revealed that genomic and molecular signatures were mutually independent, powerful prognostic indicators. Thus, combined genomic and molecular signatures may categorize novel risk groups and confer new clues for allowing tailored or even individualized medicine to patients with neuroblastoma.

The Wnt inhibitory factor 1 (WIF1) is targeted in glioblastoma and has a tumor suppressing function potentially by induction of senescence

Gene expression-based prediction of genomic copy number aberrations in the chromosomal region 12q13 to 12q15 that is flanked by MDM2 and CDK4 identified Wnt inhibitory factor 1 (WIF1) as a candidate tumor suppressor gene in glioblastoma. WIF1 encodes a secreted Wnt antagonist and was strongly downregulated in most glioblastomas as compared with normal brain, implying deregulation of Wnt signaling, which is associated with cancer. WIF1 silencing was mediated by deletion (7/69, 10%) or epigenetic silencing by promoter hypermethylation (29/110, 26%). Co-amplification of MDM2 and CDK4 that is present in 10% of glioblastomas was associated in most cases with deletion of the whole genomic region enclosed, including the WIF1 locus. This interesting pathogenetic constellation targets the RB and p53 tumor suppressor pathways in tandem, while simultaneously activating oncogenic Wnt signaling. Ectopic expression of WIF1 in glioblastoma cell lines revealed a dose-dependent decrease of Wnt pathway activity. Furthermore, WIF1 expression inhibited cell proliferation in vitro, reduced anchorage-independent growth in soft agar, and completely abolished tumorigenicity in vivo. Interestingly, WIF1 overexpression in glioblastoma cells induced a senescence-like phenotype that was dose dependent. These results provide evidence that WIF1 has tumor suppressing properties. Downregulation of WIF1 in 75% of glioblastomas indicates frequent involvement of aberrant Wnt signaling and, hence, may render glioblastomas sensitive to inhibitors of Wnt signaling, potentially by diverting the tumor cells into a senescence-like state.