A Novel Volume-Age-KPS (VAK) Glioblastoma Classification Identifies a Prognostic Cognate microRNA-Gene Signature.

BACKGROUND: Several studies have established Glioblastoma Multiforme (GBM) prognostic and predictive models based on age and Karnofsky Performance Status (KPS), while very few studies evaluated the prognostic and predictive significance of preoperative MR-imaging. However, to date, there is no simple preoperative GBM classification that also correlates with a highly prognostic genomic signature. Thus, we present for the first time a biologically relevant, and clinically applicable tumor Volume, patient Age, and KPS (VAK) GBM classification that can easily and non-invasively be determined upon patient admission. METHODS: We quantitatively analyzed the volumes of 78 GBM patient MRIs present in The Cancer Imaging Archive (TCIA) corresponding to patients in The Cancer Genome Atlas (TCGA) with VAK annotation. The variables were then combined using a simple 3-point scoring system to form the VAK classification. A validation set (N = 64) from both the TCGA and Rembrandt databases was used to confirm the classification. Transcription factor and genomic correlations were performed using the gene pattern suite and Ingenuity Pathway Analysis. RESULTS: VAK-A and VAK-B classes showed significant median survival differences in discovery (P = 0.007) and validation sets (P = 0.008). VAK-A is significantly associated with P53 activation, while VAK-B shows significant P53 inhibition. Furthermore, a molecular gene signature comprised of a total of 25 genes and microRNAs was significantly associated with the classes and predicted survival in an independent validation set (P = 0.001). A favorable MGMT promoter methylation status resulted in a 10.5 months additional survival benefit for VAK-A compared to VAK-B patients. CONCLUSIONS: The non-invasively determined VAK classification with its implication of VAK-specific molecular regulatory networks, can serve as a very robust initial prognostic tool, clinical trial selection criteria, and important step toward the refinement of genomics-based personalized therapy for GBM patients.

Epigenetic modification of the FMR1 gene in fragile X syndrome is associated with differential response to the mGluR5 antagonist AFQ056.

Fragile X syndrome (FXS) is an X-linked condition associated with intellectual disability and behavioral problems. It is caused by expansion of a CGG repeat in the 5' untranslated region of the fragile X mental retardation 1 (FMR1) gene. This mutation is associated with hypermethylation at the FMR1 promoter and resultant transcriptional silencing. FMR1 silencing has many consequences, including up-regulation of metabotropic glutamate receptor 5 (mGluR5)-mediated signaling. mGluR5 receptor antagonists have shown promise in preclinical FXS models and in one small open-label study of FXS. We examined whether a receptor subtype-selective inhibitor of mGluR5, AFQ056, improves the behavioral symptoms of FXS in a randomized, double-blind, two-treatment, two-period, crossover study of 30 male FXS patients aged 18 to 35 years. We detected no significant effects of treatment on the primary outcome measure, the Aberrant Behavior Checklist-Community Edition (ABC-C) score, at day 19 or 20 of treatment. In an exploratory analysis, however, seven patients with full FMR1 promoter methylation and no detectable FMR1 messenger RNA improved, as measured with the ABC-C, significantly more after AFQ056 treatment than with placebo (P < 0.001). We detected no response in 18 patients with partial promoter methylation. Twenty-four patients experienced an adverse event, which was mostly mild to moderately severe fatigue or headache. If confirmed in larger and longer-term studies, these results suggest that blockade of the mGluR5 receptor in patients with full methylation at the FMR1 promoter may show improvement in the behavioral attributes of FXS.

A nucleosome-dependent static loop potentiates estrogen-regulated transcription from the Xenopus vitellogenin B1 promoter in vitro.

We describe the transcriptional potentiation in estrogen responsive transcription extracts of the Xenopus vitellogenin B1 gene promoter through the formation of a positioned nucleosome. Nuclease digestion and hydroxyl radical cleavage indicate that strong, DNA sequence-directed positioning of a nucleosome occurs between -300 and -140 relative to the start site of transcription. Deletion of this DNA sequence abolishes the potentiation of transcription due to nucleosome assembly. The wrapping of DNA around the histone core of the nucleosome positioned between -300 and -140 creates a static loop in which distal estrogen receptor binding sites are brought close to proximal promoter elements. This might facilitate interactions between the trans-acting factors themselves and/or RNA polymerase. Such a nucleosome provides an example of how chromatin structure might have a positive effect on the transcription process.

Genomic roles of the HCF transcriptional regulator in "Drosophila"

Abstract : Transcriptional regulation is the result of a combination of positive and negative effectors, such as transcription factors, cofactors and chromatin modifiers. During my thesis project I studied chromatin association, and transcriptional and cell cycle regulatory functions of dHCF, the Drosophila homologue of the human protein HCF-1 (host cell factor-1). The human and Drosophila HCF proteins are synthesized as large polypeptides that are cleaved into two subunits (HCFN and HCFC), which remain associated with one another by non covalent interactions. Studies in mammalian cells over the past 20 years have been devoted to understanding the cellular functions of HCF-1 and have revealed that it is a key regulator of transcription and cell cycle regulation. In human cells, HCF-1 interacts with the histone methyltransferase Set1/Ash2 and MLL/Ash2 complexes and the histone deacetylase Sin3 complex, which are involved in transcriptional activation and repression, respectively. HCF-1 is also recruited to promoters to regulate G1 -to-S phase progression during the cell cycle by the activator transcription factors E2F1 and E2F3, and by the repressor transcription factor E2F4. HCF-1 protein structure and these interactions between HCP-1 and E2F transcriptional regulator proteins are also conserved in Drosophila. In this doctoral thesis, I use proliferating Drosophila SL2 cells to study both the genomic-binding sites of dHCF, using a combination of chromatin immunoprecipitation and ultra high throughput sequencing (ChIP-seq) analysis, and dHCF regulated genes, employing RNAi and microarray expression analysis. I show that dHCF is bound to over 7500 chromosomal sites in proliferating SL2 cells, and is located at +-200 bp relative to the transcriptional start sites of about 30% of Drosophila genes. There is also a direct relationship between dHCF promoter association and promoter- associated transcriptional activity. Thus, dHCF binding levels at promoters correlated directly with transcriptional activity. In contrast, expression studies showed that dHCF appears to be involved in both transcriptional activation and repression. Analysis of dHCF-binding sites identified nine dHCF-associated motifs, four of them linked dHCF to (i) two insulator proteins, GAGA and BEAF, (ii) the E-box motif, and (iii) a degenerated TATA-box. The dHCF-associated motifs allowed the organization of the dHCF-bound genes into five biological processes: differentiation, cell cycle and gene expression, regulation of endocytosis, and cellular localization. I further show that different mechanisms regulate dHCF association with chromatin. Despite that after dHCF cleavage the dHCFN and dHCFC subunits remain associated, the two subunits showed different affinities for chromatin and differential binding to a set of tested promoters, suggesting that dHCF could target specific promoters through each of the two subunits. Moreover, in addition to the interaction between dHCF and E2F transcription factors, the dHCF binding pattern is correlated with dE2F2 genomic 4 distribution. I show that dE2F factors are necessary for recruitment of dHCF to the promoter of a set of dHCF regulated genes. Therefore dHCF, as in mammals, is involved in regulation of G1 to S phase progression in collaboration with the dE2Fs transcription factors. In addition, gene expression arrays reveal that dHCF could indirectly regulate cell cycle progression by promoting expression of genes involved in gene expression and protein synthesis, and inhibiting expression of genes involved in cell-cell adhesion. Therefore, dHCF is an evolutionary conserved protein, which binds to many specific sites of the Drosophila genome via interaction with DNA of chromatin-binding proteins to regulate the expression of genes involved in many different cellular functions. Résumé : La regulation de la transcription est le résultat des effets positifs et négatifs des facteurs de transcription, cofacteurs et protéines effectrices qui modifient la chromatine. Pendant mon projet de thèse, j'ai étudié l'association a la chromatine, ainsi que la régulation de la transcription et du cycle cellulaire par dHCF, l'homologue chez la drosophile de la protéine humaine HCF-1 (host cell factor-1). Chez 1'humain et la V drosophile, les deux protéines HCF sont synthétisées sous la forme d'un long polypeptide, qui est ensuite coupé en deux sous-unités au centre de la protéine. Les deux sous-unités restent associées ensemble grâce a des interactions non-covalentes. Des études réalisées pendant les 20 dernières années ont permit d'établir que HCF-l et un facteur clé dans la régulation de la transcription et du cycle cellulaire. Dans les cellules humaines, HCF-1 active et réprime la transcription en interagissant avec des complexes de protéines qui activent la transcription en méthylant les histones (HMT), comme par Set1/Ash2 et MLL/Ash2, et d'autres complexes qui répriment la transcription et sont responsables de la déacétylation des histones (HDAC) comme la protéine Sin3. HCF-l est aussi recruté aux promoteurs par les activateurs de la transcription E2F l et E2F3a, et par le répresseur de la transcription E2F4 pour réguler la transition entre les phases G1 et S du cycle cellulaire. La structure de HCF-1 et les interactions entre HCF-l et les régulateurs de la transcription sont conservées chez la drosophile. Pendant ma these j'ai utilisé les cellules de la drosophile, SL2 en culture, pour étudier les endroits de liaisons de HCF-l à la chromatine, grâce a immunoprecipitation de la chromatine et du séquençage de l'ADN massif ainsi que les gènes régulés par dHCF 3 grâce a la technique de RNAi et des microarrays. Mes résultats on montré que dHCF se lie à environ 7565 endroits, et estimé a 1200 paire de bases autour des sites d'initiation de la transcription de 30% des gènes de la drosophile. J 'ai observe une relation entre dHCF et le niveau de la transcription. En effet, le niveau de liaison dHCF au promoteur corrèle avec l'activité de la transcription. Cependant, mes études d'expression ont montré que dHCF est implique dans le processus d'activation et mais aussi de répression de la transcription. L'analyse des séquences d'ADN liées par dHCF a révèle neuf motifs, quatre de ces motifs ont permis d'associer dl-ICF a deux protéines isolatrices GAGA et BEAF, au motif pour les E-boxes et a une TATA-box dégénérée. Les neuf motifs associes à dHCF ont permis d'associer les gènes lies par dHCF au promoteur a cinq processus biologiques: différentiation, cycle cellulaire, expression de gènes, régulation de l'endocytosis et la localisation cellulaire, J 'ai aussi montré qu'il y a plusieurs mécanismes qui régulent l'association de dHCF a la chromatine, malgré qu'après clivage, les deux sous-unites dHCFN and dHCFC, restent associées, elles montrent différentes affinités pour la chromatine et lient différemment un group de promoteurs, les résultats suggèrent que dHCF peut se lier aux promoteurs en utilisant chacune de ses sous-unitées. En plus de l'association de dHCF avec les facteurs de transcription dE2F s, la distribution de dHCF sur le génome corrèle avec celle du facteur de transcription dE2F2. J'ai aussi montré que les dE2Fs sont nécessaires pour le recrutement de dHCF aux promoteurs d'un sous-groupe de gènes régules par dHCF. Mes résultats ont aussi montré que chez la drosophile comme chez les humains, dl-ICF est implique dans la régulation de la progression de la phase G1 a la phase S du cycle cellulaire en collaboration avec dE2Fs. D'ailleurs, les arrays d'expression ont suggéré que dHCF pourrait réguler le cycle cellulaire de façon indirecte en activant l'expression de gènes impliqués dans l'expression génique et la synthèse de protéines, et en inhibant l'expression de gènes impliqués dans l'adhésion cellulaire. En conclusion, dHCF est une protéine, conservée dans l'évolution, qui se lie spécifiquement a beaucoup d'endroits du génome de Drosophile, grâce à l'interaction avec d'autres protéines, pour réguler l'expression des gènes impliqués dans plusieurs fonctions cellulaires.

Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial.

BACKGROUND: In 2004, a randomised phase III trial by the European Organisation for Research and Treatment of Cancer (EORTC) and National Cancer Institute of Canada Clinical Trials Group (NCIC) reported improved median and 2-year survival for patients with glioblastoma treated with concomitant and adjuvant temozolomide and radiotherapy. We report the final results with a median follow-up of more than 5 years. METHODS: Adult patients with newly diagnosed glioblastoma were randomly assigned to receive either standard radiotherapy or identical radiotherapy with concomitant temozolomide followed by up to six cycles of adjuvant temozolomide. The methylation status of the methyl-guanine methyl transferase gene, MGMT, was determined retrospectively from the tumour tissue of 206 patients. The primary endpoint was overall survival. Analyses were by intention to treat. This trial is registered with Clinicaltrials.gov, number NCT00006353. FINDINGS: Between Aug 17, 2000, and March 22, 2002, 573 patients were assigned to treatment. 278 (97%) of 286 patients in the radiotherapy alone group and 254 (89%) of 287 in the combined-treatment group died during 5 years of follow-up. Overall survival was 27.2% (95% CI 22.2-32.5) at 2 years, 16.0% (12.0-20.6) at 3 years, 12.1% (8.5-16.4) at 4 years, and 9.8% (6.4-14.0) at 5 years with temozolomide, versus 10.9% (7.6-14.8), 4.4% (2.4-7.2), 3.0% (1.4-5.7), and 1.9% (0.6-4.4) with radiotherapy alone (hazard ratio 0.6, 95% CI 0.5-0.7; p<0.0001). A benefit of combined therapy was recorded in all clinical prognostic subgroups, including patients aged 60-70 years. Methylation of the MGMT promoter was the strongest predictor for outcome and benefit from temozolomide chemotherapy. INTERPRETATION: Benefits of adjuvant temozolomide with radiotherapy lasted throughout 5 years of follow-up. A few patients in favourable prognostic categories survive longer than 5 years. MGMT methylation status identifies patients most likely to benefit from the addition of temozolomide. FUNDING: EORTC, NCIC, Nélia and Amadeo Barletta Foundation, Schering-Plough.

Functional interaction between the estrogen receptor and CTF1: analysis of the vitellogenin gene B1 promoter in yeast.

Eukaryotic gene expression depends on a complex interplay between the transcriptional apparatus and chromatin structure. We report here a yeast model system for investigating the functional interaction between the human estrogen receptor (hER) and CTF1, a member of the CTF/NFI transcription factor family. We show that a CTF1-fusion protein and the hER transactivate a synthetic promoter in yeast in a synergistic manner. This interaction requires the proline-rich transactivation domain of CTF1. When the natural estrogen-dependent vitellogenin B1 promoter is tested in yeast, CTF1 and CTF1-fusion proteins are unable to activate transcription, and no synergy is observed between hER, which activates the B1 promoter, and these factors. Chromatin structure analysis on this promoter reveals positioned nucleosomes at -430 to -270 (+/-20 bp) and at -270 to - 100 (+/-20 bp) relative to the start site of transcription. The positions of the nucleosomes remain unchanged upon hormone-dependent transcriptional activation of the promoter, and the more proximal nucleosome appears to mask the CTF/NFI site located at - 101 to -114. We conclude that a functional interaction of hER with the estrogen response element located upstream of a basal promoter occurs in yeast despite the nucleosomal organization of this promoter, whereas the interaction of CTF1 with its target site is apparently precluded by a nucleosome.

Species selectivity of mixed-lineage leukemia/trithorax and HCF proteolytic maturation pathways.

Site-specific proteolytic processing plays important roles in the regulation of cellular activities. The histone modification activity of the human trithorax group mixed-lineage leukemia (MLL) protein and the cell cycle regulatory activity of the cell proliferation factor herpes simplex virus host cell factor 1 (HCF-1) are stimulated by cleavage of precursors that generates stable heterodimeric complexes. MLL is processed by a protease called taspase 1, whereas the precise mechanisms of HCF-1 maturation are unclear, although they are known to depend on a series of sequence repeats called HCF-1(PRO) repeats. We demonstrate here that the Drosophila homologs of MLL and HCF-1, called Trithorax and dHCF, are both cleaved by Drosophila taspase 1. Although highly related, the human and Drosophila taspase 1 proteins display cognate species specificity. Thus, human taspase 1 preferentially cleaves MLL and Drosophila taspase 1 preferentially cleaves Trithorax, consistent with coevolution of taspase 1 and MLL/Trithorax proteins. HCF proteins display even greater species-specific divergence in processing: whereas dHCF is cleaved by the Drosophila taspase 1, human and mouse HCF-1 maturation is taspase 1 independent. Instead, human and Xenopus HCF-1PRO repeats are cleaved in vitro by a human proteolytic activity with novel properties. Thus, from insects to humans, HCF proteins have conserved proteolytic maturation but evolved different mechanisms.

Retinoid X receptor and peroxisome proliferator-activated receptor activate an estrogen responsive gene independent of the estrogen receptor.

Estrogen receptors regulate transcription of genes essential for sexual development and reproductive function. Since the retinoid X receptor (RXR) is able to modulate estrogen responsive genes and both 9-cis RA and fatty acids influenced development of estrogen responsive tumors, we hypothesized that estrogen responsive genes might be modulated by RXR and the fatty acid receptor (peroxisome proliferator-activated receptor, PPAR). To test this hypothesis, transfection assays in CV-1 cells were performed with an estrogen response element (ERE) coupled to a luciferase reporter construct. Addition of expression vectors for RXR and PPAR resulted in an 11-fold increase in luciferase activity in the presence of 9-cis RA. Furthermore, mobility shift assays demonstrated binding of RXR and PPAR to the vitellogenin A2-ERE and an ERE in the oxytocin promoter. Methylation interference assays demonstrated that specific guanine residues required for RXR/PPAR binding to the ERE were similar to residues required for ER binding. Moreover, RXR domain-deleted constructs in transfection assays showed that activation required RXR since an RXR delta AF-2 mutant completely abrogated reporter activity. Oligoprecipitation binding studies with biotinylated ERE and (35)S-labeled in vitro translated RXR constructs confirmed binding of delta AF-2 RXR mutant to the ERE in the presence of baculovirus-expressed PPAR. Finally, in situ hybridization confirmed RXR and PPAR mRNA expression in estrogen responsive tissues. Collectively, these data suggest that RXR and PPAR are present in reproductive tissues, are capable of activating estrogen responsive genes and suggest that the mechanism of activation may involve direct binding of the receptors to estrogen response elements.

Alkylpurine-DNA-N-glycosylase confers resistance to temozolomide in xenograft models of glioblastoma multiforme and is associated with poor survival in patients.

Glioblastoma multiforme (GBM) is the most common and lethal of all gliomas. The current standard of care includes surgery followed by concomitant radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). O⁶-methylguanine-DNA methyltransferase (MGMT) repairs the most cytotoxic of lesions generated by TMZ, O⁶-methylguanine. Methylation of the MGMT promoter in GBM correlates with increased therapeutic sensitivity to alkylating agent therapy. However, several aspects of TMZ sensitivity are not explained by MGMT promoter methylation. Here, we investigated our hypothesis that the base excision repair enzyme alkylpurine-DNA-N-glycosylase (APNG), which repairs the cytotoxic lesions N³-methyladenine and N⁷-methylguanine, may contribute to TMZ resistance. Silencing of APNG in established and primary TMZ-resistant GBM cell lines endogenously expressing MGMT and APNG attenuated repair of TMZ-induced DNA damage and enhanced apoptosis. Reintroducing expression of APNG in TMZ-sensitive GBM lines conferred resistance to TMZ in vitro and in orthotopic xenograft mouse models. In addition, resistance was enhanced with coexpression of MGMT. Evaluation of APNG protein levels in several clinical datasets demonstrated that in patients, high nuclear APNG expression correlated with poorer overall survival compared with patients lacking APNG expression. Loss of APNG expression in a subset of patients was also associated with increased APNG promoter methylation. Collectively, our data demonstrate that APNG contributes to TMZ resistance in GBM and may be useful in the diagnosis and treatment of the disease.

dKDM5/LID regulates H3K4me3 dynamics at the transcription-start site (TSS) of actively transcribed developmental genes

H3K4me3 is a histone modification that accumulates at the transcription-start site (TSS) of active genes and is known to be important for transcription activation. The way in which H3K4me3 is regulated at TSS and the actual molecular basis of its contribution to transcription remain largely unanswered. To address these questions, we have analyzed the contribution of dKDM5/LID, the main H3K4me3 demethylase in Drosophila, to the regulation of the pattern of H3K4me3. ChIP-seq results show that, at developmental genes, dKDM5/LID localizes at TSS and regulates H3K4me3. dKDM5/LID target genes are highly transcribed and enriched in active RNApol II and H3K36me3, suggesting a positive contribution to transcription. Expression-profiling show that, though weakly, dKDM5/LID target genes are significantly downregulated upon dKDM5/LID depletion. Furthermore, dKDM5/LID depletion results in decreased RNApol II occupancy, particularly by the promoter-proximal Pol lloser5 form. Our results also show that ASH2, an evolutionarily conserved factor that locates at TSS and is required for H3K4me3, binds and positively regulates dKDM5/LID target genes. However, dKDM5/LID and ASH2 do not bind simultaneously and recognize different chromatin states, enriched in H3K4me3 and not, respectively. These results indicate that, at developmental genes, dKDM5/LID and ASH2 coordinately regulate H3K4me3 at TSS and that this dynamic regulation contributes to transcription.

Functional dissection of the ash2 and ash1 transcriptomes provides insights into the transcriptional basis of wing phenotypes and reveals conserved protein interactions

Background: The trithorax group (trxG) genes absent, small or homeotic discs 1 (ash1) and 2 (ash2) were isolated in a screen for mutants with abnormal imaginal discs. Mutations in either gene cause homeotic transformations but Hox genes are not their only targets. Although analysis of double mutants revealed that ash2 and ash1 mutations enhance each other's phenotypes, suggesting they are functionally related, it was shown that these proteins are subunits of distinct complexes.Results: The analysis of wing imaginal disc transcriptomes from ash2 and ash1 mutants showed that they are highly similar. Functional annotation of regulated genes using Gene Ontology allowed identification of severely affected groups of genes that could be correlated to the wing phenotypes observed. Comparison of the differentially expressed genes with those from other genome-wide analyses revealed similarities between ASH2 and Sin3A, suggesting a putative functional relationship. Coimmunoprecipitation studies and immunolocalization on polytene chromosomes demonstrated that ASH2 and Sin3A interact with HCF (host-cell factor). The results of nucleosome western blots and clonal analysis indicated that ASH2 is necessary for trimethylation of the Lys4 on histone 3 (H3K4).Conclusion: The similarity between the transcriptomes of ash2 and ash1 mutants supports a model in which the two genes act together to maintain stable states of transcription. Like in humans, both ASH2 and Sin3A bind HCF. Finally, the reduction of H3K4 trimethylation in ash2 mutants is the first evidence in Drosophila regarding the molecular function of this trxG gene.

Role of salt-inducible kinase 1 in the activation of MEF2-dependent transcription by BDNF.

Substantial evidence supports a role for myocyte enhancer factor 2 (MEF2)-mediated transcription in neuronal survival, differentiation and synaptic function. In developing neurons, it has been shown that MEF2-dependent transcription is regulated by neurotrophins. Despite these observations, little is known about the cellular mechanisms by which neurotrophins activate MEF2 transcriptional activity. In this study, we examined the role of salt-inducible kinase 1 (SIK1), a member of the AMP-activated protein kinase (AMPK) family, in the regulation of MEF2-mediated transcription by the neurotrophin brain-derived neurotrophic factor (BDNF). We show that BDNF increases the expression of SIK1 in primary cultures of rat cortical neurons through the extracellular signal-regulated kinase 1/2 (ERK1/2)-signaling pathway. In addition to inducing SIK1 expression, BDNF triggers the phosphorylation of SIK1 at Thr182 and its translocation from the cytoplasm to the nucleus of cortical neurons. The effects of BDNF on the expression, phosphorylation and, translocation of SIK1 are followed by the phosphorylation and nuclear export of histone deacetylase 5 (HDAC5). Blockade of SIK activity with a low concentration of staurosporine abolished BDNF-induced phosphorylation and nuclear export of HDAC5 in cortical neurons. Importantly, stimulation of HDAC5 phosphorylation and nuclear export by BDNF is accompanied by the activation of MEF2-mediated transcription, an effect that is suppressed by staurosporine. Consistent with these data, BDNF induces the expression of the MEF2 target genes Arc and Nur77, in a staurosporine-sensitive manner. In further support of the role of SIK1 in the regulation of MEF2-dependent transcription by BDNF, we found that expression of wild-type SIK1 or S577A SIK1, a mutated form of SIK1 which is retained in the nucleus of transfected cells, is sufficient to enhance MEF2 transcriptional activity in cortical neurons. Together, these data identify a previously unrecognized mechanism by which SIK1 mediates the activation of MEF2-dependent transcription by BDNF.

Comprehensive molecular profiling of lung adenocarcinoma.

Adenocarcinoma of the lung is the leading cause of cancer death worldwide. Here we report molecular profiling of 230 resected lung adenocarcinomas using messenger RNA, microRNA and DNA sequencing integrated with copy number, methylation and proteomic analyses. High rates of somatic mutation were seen (mean 8.9 mutations per megabase). Eighteen genes were statistically significantly mutated, including RIT1 activating mutations and newly described loss-of-function MGA mutations which are mutually exclusive with focal MYC amplification. EGFR mutations were more frequent in female patients, whereas mutations in RBM10 were more common in males. Aberrations in NF1, MET, ERBB2 and RIT1 occurred in 13% of cases and were enriched in samples otherwise lacking an activated oncogene, suggesting a driver role for these events in certain tumours. DNA and mRNA sequence from the same tumour highlighted splicing alterations driven by somatic genomic changes, including exon 14 skipping in MET mRNA in 4% of cases. MAPK and PI(3)K pathway activity, when measured at the protein level, was explained by known mutations in only a fraction of cases, suggesting additional, unexplained mechanisms of pathway activation. These data establish a foundation for classification and further investigations of lung adenocarcinoma molecular pathogenesis.