Markus J. Riemenschneider

Epidermal Growth Factor Receptor Mutations and Gene Amplification in Non–Small-Cell Lung Cancer: Molecular Analysis of the IDEAL/INTACT Gefitinib Trials

PURPOSE Most cases of non-small-cell lung cancer (NSCLC) with dramatic responses to gefitinib have specific activating mutations in the epidermal growth factor receptor (EGFR), but the predictive value of these mutations has not been defined in large clinical trials. The goal of this study was to determine the contribution of molecular alterations in EGFR to response and survival within the phase II (IDEAL) and phase III (INTACT) trials of gefitinib. PATIENTS AND METHODS We analyzed the frequency of EGFR mutations in lung cancer specimens from both the IDEAL and INTACT trials and compared it with EGFR gene amplification, another genetic abnormality in NSCLC. RESULTS EGFR mutations correlated with previously identified clinical features of gefitinib response, including adenocarcinoma histology, absence of smoking history, female sex, and Asian ethnicity. No such association was seen in patients whose tumors had EGFR amplification, suggesting that these molecular markers identify different biologic subsets of NSCLC. In the IDEAL trials, responses to gefitinib were seen in six of 13 tumors (46%) with an EGFR mutation, two of seven tumors (29%) with amplification, and five of 56 tumors (9%) with neither mutation nor amplification (P = .001 for either EGFR mutation or amplification v neither abnormality). Analysis of the INTACT trials did not show a statistically significant difference in response to gefitinib plus chemotherapy according to EGFR genotype. CONCLUSION EGFR mutations and, to a lesser extent, amplification appear to identify distinct subsets of NSCLC with an increased response to gefitinib. The combination of gefitinib with chemotherapy does not improve survival in patients with these molecular markers.

Histological classification and molecular genetics of meningiomas.

Meningiomas account for up to 30% of all primary intracranial tumours. They are histologically classified according to the World Health Organization (WHO) classification of tumours of the nervous system. Most meningiomas are benign lesions of WHO grade I, whereas some meningioma variants correspond with WHO grades II and III and are associated with a higher risk of recurrence and shorter survival times. Mutations in the NF2 gene and loss of chromosome 22q are the most common genetic alterations associated with the initiation of meningiomas. With increase in tumour grade, additional progression-associated molecular aberrations can be found; however, most of the relevant genes are yet to be identified. High-throughput techniques of global genome and transcriptome analyses and new meningioma models provide increasing insight into meningioma biology and will help to identify common pathogenic pathways that may be targeted by new therapeutic approaches.

Molecular diagnostics of gliomas: state of the art.

Modern neuropathology serves a key function in the multidisciplinary management of brain tumor patients. Owing to the recent advancements in molecular neurooncology, the neuropathological assessment of brain tumors is no longer restricted to provide information on a tumor’s histological type and malignancy grade, but may be complemented by a growing number of molecular tests for clinically relevant tissue-based biomarkers. This article provides an overview and critical appraisal of the types of genetic and epigenetic aberrations that have gained significance in the molecular diagnostics of gliomas, namely deletions of chromosome arms 1p and 19q, promoter hypermethylation of the O6-methylguanine-methyl-transferase (MGMT) gene, and the mutation status of the IDH1 and IDH2 genes. In addition, the frequent oncogenic aberration of BRAF in pilocytic astrocytomas may serve as a novel diagnostic marker and therapeutic target. Finally, this review will summarize recent mechanistic insights into the molecular alterations underlying treatment resistance in malignant gliomas and outline the potential of genome-wide profiling approaches for increasing our repertoire of clinically useful glioma markers.

Prognostic Value of O-(2-18F-Fluoroethyl)-l-Tyrosine PET and MRI in Low-Grade Glioma

In glioma of World Health Organization (WHO) grade II (low-grade glioma), the natural course of a particular patient is not predictable and the treatment strategy is controversial. We determined prognostic factors in adult patients with untreated, nonenhancing, supratentorial low-grade glioma with special regard to PET using the amino acid O-(2-18F-fluoroethyl)-l-tyrosine (18F-FET) and MRI. Methods: In a prospective study, baseline 18F-FET PET and MRI analyses were performed on 33 consecutive patients with histologically confirmed low-grade glioma. None of the patients had radiation or chemotherapy. Clinical, histologic, therapeutic (initial cytoreduction vs. biopsy), 18F-FET uptake, and MRI morphologic parameters were analyzed for their prognostic significance. Statistical endpoints were clinical or radiologic tumor progression, malignant transformation to glioma of WHO grade III or IV (high-grade glioma), and death. Results: Baseline 18F-FET uptake and a diffuse versus circumscribed tumor pattern on MRI were highly significant predictors of prognosis (P < 0.01). By the combination of these prognostically significant variables, 3 major prognostic subgroups of low-grade glioma patients could be identified. The first of these subgroups was patients with circumscribed low-grade glioma on MRI without 18F-FET uptake (n = 11 patients, progression in 18%, no malignant transformation and no death). The second subgroup was patients with circumscribed low-grade glioma with 18F-FET uptake (n = 13 patients, progression in 46%, malignant transformation to a high-grade glioma in 15%, and death in 8%). The third subgroup was patients with diffuse low-grade glioma with 18F-FET uptake (n = 9 patients, progression in 100%, malignant transformation to a high-grade glioma in 78%, and death in 56%). Conclusion: We conclude that baseline amino acid uptake on 18F-FET PET and a diffuse versus circumscribed tumor pattern on MRI are strong predictors for the outcome of patients with low-grade glioma.

Downregulation of RUNX3 and TES by hypermethylation in glioblastoma

Glioblastoma, the most aggressive and least treatable form of malignant glioma, is the most common human brain tumor. Although many regions of allelic loss occur in glioblastomas, relatively few tumor suppressor genes have been found mutated at such loci. To address the possibility that epigenetic alterations are an alternative means of glioblastoma gene inactivation, we coupled pharmacological manipulation of methylation with gene profiling to identify potential methylation-regulated, tumor-related genes. Duplicates of three short-term cultured glioblastomas were exposed to 5 μM 5-aza-dC for 96 h followed by cRNA hybridization to an oligonucleotide microarray (Affymetrix U133A). We based candidate gene selection on bioinformatics, reverse transcription-polymerase chain reaction (RT–PCR), bisulfite sequencing, methylation-specific PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Two genes identified in this manner, RUNX3 and Testin (TES), were subsequently shown to harbor frequent tumor-specific epigenetic alterations in primary glioblastomas. This overall approach therefore provides a powerful means to identify candidate tumor-suppressor genes for subsequent evaluation and may lead to the identification of genes whose epigenetic dysregulation is integral to glioblastoma tumorigenesis.

AKT Activation in Human Glioblastomas Enhances Proliferation via TSC2 and S6 Kinase Signaling

Aberrant AKT (protein kinase B) signaling is common in many cancers, including glioblastoma. Current models suggest that AKT acts directly, or indirectly via the TSC complex, to activate the mammalian target of rapamycin (mTOR) as the main downstream mediator of AKT signaling. mTOR activation results in subsequent activation of S6K and STAT3, as well as suppression (i.e., phosphorylation) of 4E-BP1, leading to cell cycle progression and inhibition of apoptosis. Most studies of this pathway have used in vitro systems or tumor lysate-based approaches. We aimed to delineate these pathways in a primarily in situ manner using immunohistochemistry in a panel of 29 glioblastomas, emphasizing the histologic distribution of molecular changes. Within individual tumors, increased expression levels of p-TSC2, p-mTOR, p-4E-BP1, p-S6K, p-S6, and p-STAT3 were found in regions defined by elevated AKT activation. However, only TSC2, S6K, and S6 activation levels correlated significantly with AKT activation and clustered together in multidimensional scaling analyses. Ki-67 proliferation indices were significantly elevated in p-AKT-overexpressing regions, whereas expression of the apoptosis marker cleaved caspase 3 was generally low and not significantly different between the regions. These findings provide the first in vivo evidence for a close correlation between AKT and TSC2 phosphorylation levels in glioblastoma. Moreover, they suggest that downstream p-AKT effects are primarily mediated by S6 kinase signaling, thus enhancing proliferation rather than inhibiting apoptosis.

Refined mapping of 1q32 amplicons in malignant gliomas confirms MDM4 as the main amplification target

We previously reported on the amplification and overexpression of the mouse double minute 4 homolog gene (MDM4) from 1q32 in a subset of malignant gliomas (Riemenschneider et al., Cancer Res 1999;59:6091–6). More recently, amplification and overexpression of the neighboring contactin 2 gene (CNTN2) was reported in individual malignant gliomas without MDM4 amplification (Rickman et al., Cancer Res 2001;61:2162–8). To address the question of whether 1q32 carries 2 independent amplification targets or a common target other than MDM4 and CNTN2, we analyzed primary malignant gliomas for amplification and overexpression of 17 different genes from this region. Our results indicate a single region of amplification that comprises the genes MDM4, GAC1, PIK3C2B and PEPP3, with only MDM4 amplification being invariably associated with overexpression. CNTN2 was found to be coamplified with MDM4 in 3 malignant gliomas but overexpressed in only 1 of these tumors. No CNTN2 amplification was detected in any of 102 malignant gliomas without MDM4 amplification. Our data therefore corroborate the notion that MDM4 is the main amplification target on 1q32 in malignant gliomas. However, coamplification and overexpression of adjacent genes may provide an additional growth advantage in some malignant gliomas with MDM4 amplification.

Frequent promoter hypermethylation and transcriptional downregulation of the NDRG2 gene at 14q11.2 in primary glioblastoma.

The N‐myc downstream‐regulated gene 2 (NDRG2) at 14q11.2 has been reported to be downregulated in glioblastoma, and NDRG2 overexpression represses glioblastoma cell proliferation in vitro (Deng et al., Int J Cancer 2003;106;342–7). To further address the role of NDRG2 as a candidate tumor suppressor in human gliomas, we analyzed 67 astrocytic tumors (10 diffuse astrocytomas, 11 anaplastic astrocytomas, 34 primary glioblastomas and 12 secondary glioblastomas) for NDRG2 gene mutation, promoter methylation and expression at the mRNA and protein levels. Using real‐time reverse transcription PCR analysis, we found decreased NDRG2 mRNA levels in primary glioblastomas as compared to diffuse and anaplastic astrocytomas. Similarly, immunohistochemistry revealed low or absent NDRG2 protein expression in primary glioblastomas. Mutational analysis of the entire NDRG2 coding sequence did not reveal any tumor‐associated DNA sequence alterations. However, sequencing of sodium bisulfite‐modified DNA identified hypermethylation of the NDRG2 promoter region in 21 of 34 primary glioblastomas (62%). Moreover, NDRG2 promoter hypermethylation was associated with decreased NDRG2 mRNA expression. In contrast to primary glioblastomas, NDRG2 promoter hypermethylation was detected in only 1 of 11 anaplastic astrocytomas (9%) and was absent in 10 diffuse astrocytomas and 12 secondary glioblastomas. Taken together, our data support NDRG2 as a candidate tumor suppressor gene that is epigenetically silenced in the majority of primary glioblastomas, but not in lower grade astrocytomas and secondary glioblastomas.

In situ analysis of integrin and growth factor receptor signaling pathways in human glioblastomas suggests overlapping relationships with focal adhesion kinase activation.

Deregulated integrin signaling is common in cancers, including glioblastoma. Integrin binding and growth factor receptor signaling activate focal adhesion kinase (FAK) and subsequently up-regulate extracellular regulated kinases (ERK-1/2), leading to cell-cycle progression and cell migration. Most studies of this pathway have used in vitro systems or tumor lysate-based approaches. We examined these pathways primarily in situ using a panel of 30 glioblastomas and gene expression arrays, immunohistochemistry, and fluorescence in situ hybridization, emphasizing the histological distribution of molecular changes. Within individual tumors, increased expression of FAK, p-FAK, paxillin, ERK-1/2, and p-ERK-1/2 occurred in regions of elevated EGFR and/or PDGFRA expression. Moreover, FAK activation levels correlated with EGFR and PDGFRA expression, and p-FAK and EGFR expression co-localized at the single-cell level. In addition, integrin expression was enriched in EGFR/PDGFRA-overexpressing areas but was more regionally confined than FAK, p-FAK, and paxillin. Integrins beta8 and alpha5beta1 were most commonly expressed, often in a perinecrotic or perivascular pattern. Taken together, our data suggest that growth factor receptor overexpression facilitates alterations in the integrin signaling pathway. Thus, FAK may act in glioblastoma as a downstream target of growth factor signaling, with integrins enhancing the impact of such signaling in the tumor microenvironment.

Expression of oligodendrocyte lineage genes in oligodendroglial and astrocytic gliomas

The oligodendrocyte lineage genes OLIG1 and OLIG2 have been reported as potential diagnostic markers for oligodendrogliomas [Lu et al. (2001) Proc Natl Acad Sci USA 98:10851–10856; Marie et al. (2001) Lancet 358:298–300]. We investigated the mRNA expression of OLIG1 and OLIG2, as well as four other genes involved in oligodendrocyte development (E2A, HEB, NKX2.2, and PDGFRA) in a panel of 70 gliomas, including 9 oligodendrogliomas, 11 anaplastic oligodendrogliomas, 5 oligoastrocytomas, 10 anaplastic oligoastrocytomas, 10 diffuse astrocytomas, 10 anaplastic astrocytomas, and 15 glioblastomas. Most tumors demonstrated higher transcript levels of these genes as compared to non-neoplastic adult brain tissue. Four glioblastomas showed markedly increased PDGFRA mRNA expression due to PDGFRA gene amplification. Statistical analyses revealed no significant expression differences between oligodendroglial and astrocytic tumors. In oligodendroglial tumors, expression of the six genes was not significantly correlated to loss of heterozygosity on chromosome arms 1p and 19q. Thus, expression of the investigated oligodendrocyte lineage genes is up-regulated relative to non-neoplastic brain tissue in the majority of oligodendroglial and astrocytic tumors, suggesting that glioma cells are arrested in or recapitulate molecular phenotypes corresponding to early stages of glial development. However, the determination of mRNA expression of these genes by means of reverse transcription-PCR does not appear to be diagnostically useful as a marker for oligodendrogliomas.