An integrated genomic analysis of lung cancer reveals loss of DUSP4 in EGFR-mutant tumors.

To address the biological heterogeneity of lung cancer, we studied 199 lung adenocarcinomas by integrating genome-wide data on copy number alterations and gene expression with full annotation for major known somatic mutations in this cancer. This showed non-random patterns of copy number alterations significantly linked to EGFR and KRAS mutation status and to distinct clinical outcomes, and led to the discovery of a striking association of EGFR mutations with underexpression of DUSP4, a gene within a broad region of frequent single-copy loss on 8p. DUSP4 is involved in negative feedback control of EGFR signaling, and we provide functional validation for its role as a growth suppressor in EGFR-mutant lung adenocarcinoma. DUSP4 loss also associates with p16/CDKN2A deletion and defines a distinct clinical subset of lung cancer patients. Another novel observation is that of a reciprocal relationship between EGFR and LKB1 mutations. These results highlight the power of integrated genomics to identify candidate driver genes within recurrent broad regions of copy number alteration and to delineate distinct oncogenetic pathways in genetically complex common epithelial cancers

Spectral imaging in preclinical research and clinical pathology.

Spectral imaging methods are attracting increased interest from researchers and practitioners in basic science, pre-clinical and clinical arenas. A combination of better labeling reagents and better optics creates opportunities to detect and measure multiple parameters at the molecular and cellular level. These tools can provide valuable insights into the basic mechanisms of life, and yield diagnostic and prognostic information for clinical applications. There are many multispectral technologies available, each with its own advantages and limitations. This chapter will present an overview of the rationale for spectral imaging, and discuss the hardware, software and sample labeling strategies that can optimize its usefulness in clinical settings

Distinctive Patterns of MicroRNA Expression Associated with Karyotype in Acute Myeloid Leukaemia

Acute myeloid leukaemia (AML) is the most common acute leukaemia in adults; however, the genetic aetiology of the disease is not yet fully understood. A quantitative expression profile analysis of 157 mature miRNAs was performed on 100 AML patients representing the spectrum of known karyotypes common in AML. The principle observation reported here is that AMLs bearing a t(15;17) translocation had a distinctive signature throughout the whole set of genes, including the up regulation of a subset of miRNAs located in the human 14q32 imprinted domain. The set included miR-127, miR-154, miR-154*, miR-299, miR-323, miR-368, and miR-370. Furthermore, specific subsets of miRNAs were identified that provided molecular signatures characteristic of the major translocation-mediated gene fusion events in AML. Analysis of variance showed the significant deregulation of 33 miRNAs across the leukaemic set with respect to bone marrow from healthy donors. Fluorescent in situ hybridisation analysis using miRNA-specific locked nucleic acid (LNA) probes on cryopreserved patient cells confirmed the results obtained by real-time PCR. This study, conducted on about a fifth of the miRNAs currently reported in the Sanger database (microrna.sanger.ac.uk), demonstrates the potential for using miRNA expression to sub-classify cancer and suggests a role in the aetiology of leukaemia

Comprehensive molecular analysis of immortalization hallmarks in thyroid cancer reveals new prognostic markers

TERT promoter mutation; Subtelomeric gene expression; Telomere shorteningMutació del promotor TERT; Expressió gènica subtelomèrica; Escurçament dels telòmersMutación del promotor TERT; Expresión génica subtelomérica; Acortamiento de los telómerosBackground Comprehensive molecular studies on tumours are needed to delineate immortalization process steps and identify sensitive prognostic biomarkers in thyroid cancer. Methods and Results In this study, we extensively characterize telomere-related alterations in a series of 106 thyroid tumours with heterogeneous clinical outcomes. Using a custom-designed RNA-seq panel, we identified five telomerase holoenzyme-complex genes upregulated in clinically aggressive tumours compared to tumours from long-term disease-free patients, being TERT and TERC denoted as independent prognostic markers by multivariate regression model analysis. Characterization of alterations related to TERT re-expression revealed that promoter mutations, methylation and/or copy gains exclusively co-occurred in clinically aggressive tumours. Quantitative-FISH (fluorescence in situ hybridization) analysis of telomere lengths showed a significant shortening in these carcinomas, which matched with a high proliferative rate measured by Ki-67 immunohistochemistry. RNA-seq data analysis indicated that short-telomere tumours exhibit an increased transcriptional activity in the 5-Mb-subtelomeric regions, site of several telomerase-complex genes. Gene upregulation enrichment was significant for specific chromosome-ends such as the 5p, where TERT is located. Co-FISH analysis of 5p-end and TERT loci showed a more relaxed chromatin configuration in short telomere-length tumours compared to normal telomere-length tumours. Conclusions Overall, our findings support that telomere shortening leads to a 5p subtelomeric region reorganization, facilitating the transcription and accumulation of alterations at TERT-locus.This work was supported by Projects PI17/01796 and PI20/01169 [Instituto de Salud Carlos III (ISCIII), Acción Estratégica en Salud, cofinanciado a través del Fondo Europeo de Desarrollo Regional (FEDER)] and Comunidad de Madrid (S2017/BMD-3724; TIRONET2-CM) to MR. CM-C was partially supported by a grant from the Fundación Científica Asociación Española Contra el Cáncer (AIO15152858 MONT). LJL-G was supported both by the Banco Santander Foundation – CNIO Fellowship and by ‘la Caixa’ Foundation (ID 100010434), under agreement LCF/BQ/PI20/11760011. AMM-M was supported by CAM (S2017/BMD-3724; TIRONET2-CM). MM was supported by the Ministerio de Ciencia, Innovación y Universidades (Spain; ‘Formación del Profesorado Universitario – FPU’ fellowship (FPU18/00064)). We thank CNIO Biobank for their support with the frozen specimens processing. We thank the Spanish National Tumor Bank Network (RD09/0076/00047) for the support in obtaining tumour samples, and all patients, physicians and tumour biobanks involved in the study

Genome position and gene amplification

Genomic analyses of human cells expressing dihydrofolate reductase provide insight into the effects of genome position on the propensity for a drug-resistance gene to amplify in human cells

The molecular basis for central nervous system primitive neuroectodermal tumour development

PhD ThesisCentral nervous system primitive neuroectodermal tumours (CNS-PNETs) are highly aggressive tumours with similar histopathological features to other intracranial PNETs (medulloblastomas). These two tumours have accordingly been treated using unified approaches, but CNS-PNETs have a dismal prognosis. Few studies have investigated the genetic features of CNS-PNETs. The molecular basis of CNS-PNET was therefore investigated in a cohort containing CNS-PNETs from children (n=33) and adults (n=5), to aid improvements in disease classification and treatment. The common medulloblastoma molecular defects were investigated in CNS-PNETs, and showed RASSF1A promoter hypermethylation is a frequent event (18/22, 82%), and MYC family gene amplification occurs in a subgroup (MYCN: 3/25 (12%), MYCC: 0/25 (0%)). In contrast and in distinction to medulloblastoma, chromosome 17p loss is not a common feature (2/23, 9%), whilst p53 pathway signalling appears to play a major role (20/22, 91%), and associated with TP53 mutations (4/22, 18%). Aberrant Wnt signalling was identified in 2 cases (2/22, 9%) and coupled with CTNNB1 mutation in a single case. IDH1 mutations (2/25, 8%) however, appear to occur in adult but not childhood CNS-PNETs or medulloblastoma. Subsequent genome-wide investigations of the CNS-PNET DNA methylome aimed at a wider characterisation of the molecular features of CNS-PNETs and its relationships to other childhood tumours identified CNS-PNETs as a heterogenous disease group without defined sub-clusters, which were predominantly distinct from medulloblastomas, but exhibited overlap with high-grade gliomas. A panel of 76 tumour-specific methylation events were identified as disease markers. The combination of either RASSF1A hypermethylation or HLA-DPB1 hypomethylation discerned normal brain from CNS-PNET in 94% of cases (64/68). In addition, hypermethylation of TAL1, MAP3K1 and IGFBP1 is associated with non-infant disease. In conclusion, this study has shown CNS-PNETs are a heterogenous group of tumours that are molecularly distinct from medulloblastomas, and has implicated developmental pathways and genetic events in their tumorigenesis. The associations between molecular events identified and clinical features warrant further investigation to aid classification and treatment advancements.North of England Children’s Cancer Research Fund (NECCR), Clic Sargent and the Samantha Dickinson Brain Tumour Trust (SDBTT