V. P. Collins

Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics

Glioblastoma (GB) is the most common and aggressive primary brain malignancy, with poor prognosis and a lack of effective therapeutic options. Accumulating evidence suggests that intratumor heterogeneity likely is the key to understanding treatment failure. However, the extent of intratumor heterogeneity as a result of tumor evolution is still poorly understood. To address this, we developed a unique surgical multisampling scheme to collect spatially distinct tumor fragments from 11 GB patients. We present an integrated genomic analysis that uncovers extensive intratumor heterogeneity, with most patients displaying different GB subtypes within the same tumor. Moreover, we reconstructed the phylogeny of the fragments for each patient, identifying copy number alterations in EGFR and CDKN2A/B/p14ARF as early events, and aberrations in PDGFRA and PTEN as later events during cancer progression. We also characterized the clonal organization of each tumor fragment at the single-molecule level, detecting multiple coexisting cell lineages. Our results reveal the genome-wide architecture of intratumor variability in GB across multiple spatial scales and patient-specific patterns of cancer evolution, with consequences for treatment design.

A monoclonal antibody recognizing human cancers with amplification/overexpression of the human epidermal growth factor receptor.

Epidermal growth factor receptor (EGFR) has attracted considerable attention as a target for cancer therapy. Wild-type (wt)EGFR is amplified/overexpressed in a number of tumor types, and several mutant forms of the coding gene have been found, with ΔEGFR, a deletion mutation lacking exons 2–7 of the external domain, being the most common and particularly associated with glioblastoma. We generated monoclonal antibodies (mAbs) against NR6ΔEGFR (mouse fibroblast line NR6 transfected with ΔEGFR). mAb 806 with selective reactivity for NR6ΔEGFR in mixed hemadsorption assays, fluorescence-activated cell sorting, Western blot, and immunohistochemistry was analyzed in detail and compared with mAbs 528 (anti-wtEGFR) and DH8.3 (anti-ΔEGFR). In xenograft tumors and molecularly pretyped glioblastomas, the reactivity pattern was as follows: 528 reactive with amplified and nonamplified wtEGFR; DH8.3 reactive with ΔEGFR; and 806 reactive with amplified/overexpressed wtEGFR (with or without ΔEGFR). In normal tissues, 528 but not DH8.3 or 806 was widely reactive with many organs, e.g., liver expressing high EGFR levels. In glioblastoma and non-CNS tumor panels, 806 was reactive with a high proportion of glioblastomas and a substantial number of epithelial cancers of lung and of head and neck. DH8.3 reactivity was restricted to ΔEGFR-positive glioblastoma. Thus, 806 represents a category of mAbs that recognizes tumors with EGFR amplification/overexpression but not normal tissues or tumors with normal EGFR levels. Our study also indicates that ΔEGFR is restricted to glioblastoma, in contrast to other reports that this mutation is found in tumors outside the brain.

A20 deletion is associated with copy number gain at the TNFA/B/C locus and occurs preferentially in translocation-negative MALT lymphoma of the ocular adnexa and salivary glands

The genetic basis of MALT lymphoma is largely unknown. Characteristic chromosomal translocations are frequently associated with gastric and pulmonary cases, but are rare at other sites. We compared the genetic profiles of 33 ocular adnexal and 25 pulmonary MALT lymphomas by 1 Mb array–comparative genomic hybridization (CGH) and revealed recurrent 6q23 losses and 6p21.2–6p22.1 gains exclusive to ocular cases. High‐resolution chromosome 6 tile‐path array–CGH identified NF‐κB inhibitor A20 as the target of 6q23.3 deletion and TNFA/B/C locus as a putative target of 6p21.2–22.1 gain. Interphase fluorescence in situ hybridization showed that A20 deletion occurred in MALT lymphoma of the ocular adnexa (8/42 = 19%), salivary gland (2/24 = 8%), thyroid (1/9 = 11%) and liver (1/2), but not in the lung (26), stomach (45) and skin (13). Homozygous deletion was observed in three cases. A20 deletion and TNFA/B/C gain were significantly associated (p < 0.001) and exclusively found in cases without characteristic translocation. In ocular cases, A20 deletion was associated with concurrent involvement of different adnexal tissues or extraocular sites at diagnosis (p = 0.007), a higher proportion of relapse (67% versus 37%) and a shorter relapse‐free survival (p = 0.033). A20 deletion and gain at TNFA/B/C locus may thus play an important role in the development of translocation‐negative MALT lymphoma. Copyright

High-resolution array-based comparative genomic hybridization of medulloblastomas and supratentorial primitive neuroectodermal tumors

Medulloblastomas and supratentorial primitive neuroectodermal tumors are aggressive childhood tumors. We report our findings using array comparative genomic hybridization (CGH) on a whole-genome BAC/PAC/cosmid array with a median clone separation of 0.97 Mb to study 34 medulloblastomas and 7 supratentorial primitive neuroectodermal tumors. Array CGH allowed identification and mapping of numerous novel, small regions of copy number change to genomic sequence in addition to the large regions already known from previous studies. Novel amplifications were identified, some encompassing oncogenes MYCL1, PDGFRA, KIT, and MYB not previously reported to show amplification in these tumors. In addition, one supratentorial primitive neuroectodermal tumor had lost both copies of the tumor-suppressor genes CDKN2A and CDKN2B. Ten medulloblastomas had findings suggestive of isochromosome 17q. In contrast to previous reports using conventional CGH, array CGH identified 3 distinct breakpoints in these cases: Ch 17: 17940393-19251679 (17p11.2, n = 6), Ch 17: 20111990-23308272 (17p11.2-17q11.2, n = 4), and Ch 17: 38425359-39091575 (17q21.31, n = 1). Significant differences were found in the patterns of copy number change between medulloblastomas and supratentorial primitive neuroectodermal tumors, providing further evidence that these tumors are genetically distinct despite their morphologic and behavioral similarities.

Mutational profile of the PTEN gene in primary human astrocytic tumors and cultivated xenografts

The genetic abnormality most frequently identified in glioblastomas is loss of alleles on chromosome 10. We have performed a comprehensive study of the PTEN tumor suppressor gene on 10q23, including loss of heterozygosity (LOH) analysis, multiplex PCR, mutation analysis, and reverse transcription PCR (RT-PCR). In total, 151 glioblastomas, 41 anaplastic astrocytomas, 15 astrocytomas, and 13 glioma cell lines were analyzed as well as 23 xenografts derived from primary glioblastomas, which allows a comparison of the PTEN gene status in primary tumors versus xenografts. Homozygous deletions were found in 7% of the glioblastomas and 40% showed mutation of a single retained allele. This mutation frequency is higher than reported previously. The large number of mutations identified allows the presentation of a mutational profile along the coding sequence. The majority of mutations appear to affect conserved residues or structurally conserved regions. PTEN alterations were selected for in xenografts, and there is evidence that they may even facilitate establishment of xenografts. No alterations were found in astrocytomas and only 5% of anaplastic astrocytomas had mutations. Thus, loss of wild type PTEN represents one of the major abnormalities associated with astrocytic tumor progression to glioblastoma and provides a strong selective growth advantage when cultivating glioblastoma tissue in xenografts. No correlation with EGFR amplification was evident.

A distinct region of the MGMT CpG island critical for transcriptional regulation is preferentially methylated in glioblastoma cells and xenografts.

O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl DNA adducts such as those induced by alkylating agents. Loss of MGMT expression through transcriptional silencing by hypermethylation of its CpG island (CGI) is found in diverse human cancers including glioblastomas. Glioblastomas that have MGMT methylation respond to temozolomide, an alkylating agent, resulting in improved survival. Consequently, assessment of MGMT methylation has become a therapy response and prognostic indicator. However, it is not clear whether the region of the MGMT CGI commonly analysed is the critical region involved in transcriptional control. We measured methylation levels at each CpG site for the entire MGMT CGI using bisulfite modification and pyrosequencing, and compared them with MGMT mRNA expression in glioblastoma cell lines, xenografts and normal brain tissues (41 samples). Two critical regions were identified (DMR1 and DMR2). DMR2 encompasses the commonly analysed region and was always methylated when DMR1 was methylated. A luciferase reporter assay showed that substitutions of several specific CpG sites within DMR2 significantly attenuated the promoter activity of the MGMT CGI. Our results indicate that several CpG sites within DMR2 play a critical role in the transcriptional control of MGMT, making DMR2 the optimal target for methylation testing. However, given the highly variable patterns of MGMT methylation associated with transcriptional silencing observed in this region among the tumours in this study, methylation levels need to be measured at a number of individual CpGs within DMR2 to confidently predict transcriptional silencing and thus sensitivity to alkylating agents.

Fluorescence-guided surgical sampling of glioblastoma identifies phenotypically distinct tumour-initiating cell populations in the tumour mass and margin

Background:Acquiring clinically annotated, spatially stratified tissue samples from human glioblastoma (GBM) is compromised by haemorrhage, brain shift and subjective identification of ‘normal’ brain. We tested the use of 5-aminolevulinic acid (5-ALA) fluorescence to objective tissue sampling and to derive tumour-initiating cells (TICs) from mass and margin.Methods:The 5-ALA was administered to 30 GBM patients. Samples were taken from the non-fluorescent necrotic core, fluorescent tumour mass and non-fluorescent margin. We compared the efficiency of isolating TICs from these areas in 5-ALA versus control patients. HRMAS 1H NMR was used to reveal metabolic alterations due to 5-ALA. We then characterised TICs for self-renewal in vitro and tumorigenicity in vivo.Results:The derivation of TICs was not compromised by 5-ALA and the metabolic profile was similar between tumours from 5-ALA patients and controls. The TICs from the fluorescent mass were self-renewing in vitro and tumour-forming in vivo, whereas TICs from non-fluorescent margin did not self-renew in vitro but did form tumours in vivo.Conclusion:Our data show that 5-ALA does not compromise the derivation of TICs. It also reveals that the margin contains TICs, which are phenotypically different from those isolated from the corresponding mass.

Multiple deleted regions on the long arm of chromosome 6 in astrocytic tumours

Chromosome 6 deletions are common in human neoplasms including gliomas. In order to study the frequency and identify commonly deleted regions of chromosome 6 in astrocytomas, 159 tumours (106 glioblastomas, 39 anaplastic astrocytomas and 14 astrocytomas malignancy grade II) were analysed using 31 microsatellite markers that span the chromosome. Ninety-five per cent of cases with allelic losses had losses affecting 6q. Allelic losses were infrequent in astrocytomas malignancy grade II (14%) but more usual in anaplastic astrocytomas (38%) and glioblastomas (37%). Evidence for clonal heterogeneity in the astrocytomas and anaplastic astrocytomas was frequently observed (i.e. co-existence of subpopulations with and without chromosome 6 deletions). Clonal heterogeneity was less common in glioblastomas. Five commonly deleted regions were identified on 6q. These observations suggest that a number of tumour suppressor genes are located on 6q and that these genes may be involved in the progression of astrocytic tumours.

Mutations in Rb1 pathway-related genes are associated with poor prognosis in Anaplastic Astrocytomas

Anaplastic astrocytoma (AA, WHO grade III) is, second to Glioblastoma, the most common and most malignant type of adult CNS tumour. Since survival for patients with AA varies markedly and there are no known useful prognostic or therapy response indicators, the primary purpose of this study was to examine whether knowledge of the known genetic abnormalities found in AA had any clinical value. The survival data on 37 carefully sampled AA was correlated with the results of a detailed analysis of the status of nine genes known to be involved in the development of astrocytic tumours. These included three genes coding for proteins in the p53 pathway (TP53, p14ARFand MDM2), four in the Rb1 pathway (CDKN2A, CDKN2B, RB1 and CDK4) and PTEN and EGFR. We found that loss of both wild-type copies of any of the three tumour suppressor genes CDKN2A, CDKN2B and RB1 or gene amplification of CDK4, disrupting the Rb1 pathway, were associated with shorter survival (P=0.009). This association was consistent in multivariate analysis, including adjustment for age (P=0.013). The findings suggest that analysis of the genes coding for Rb1 pathway components provides additional prognostic information in AA patients receiving conventional therapy.

Chromosome 7 rearrangements in glioblastomas; loci adjacent to EGFR are independently amplified.

The first gene found to be amplified in human glioblastomas was EGFR at 7p12. More recently the MET gene at 7q31 was also reported amplified. We have studied chromosome 7 in a series of 47 glioblastomas by FISH, RFLP and microsatellite analysis. Four per cent (2/47) had 1 centromere, 26% (12/47) 2, 32% (15/47) 3, 4% (2/47) 4, and 34% (16/47) had subpopulations with variable numbers of chromosome 7 centromeres. In 25 of the 47 tumors (53%) the pattern of allelic imbalance observed at each informative locus was similar and in accord with the FISH data, indicating loss or gain of complete chromosome copies. In 32% of tumors (15/47) varying allelic imbalance was seen at different loci along the chromosome indicative of loss or gain of parts of chromosome 7 on a background of disomy, trisomy, tetrasomy, or polysomy. Amplification was studied in an extended series of 121 glioblastomas, and was seen at the 7p12 region in 47 tumors (39%). Forty-two tumors showed amplification of EGFR and 12 of these had extensive amplicons including a number of adjacent loci, always involving only 1 allele. The amplicons of 5 tumors (11%) did not include EGFR, indicating that other unidentified genes in the region are targeted for amplification. Amplification of MET was not found. The findings show that copy number changes of chromosome 7 are common and that a number of genes may be targeted for amplification at 7p12 in glioblastomas.