Siu Tsan Yuen

Mutations of the BRAF gene in human cancer

Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer. The RAS–RAF–MEK–ERK–MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma.

Patterns of somatic mutation in human cancer genomes

Cancers arise owing to mutations in a subset of genes that confer growth advantage. The availability of the human genome sequence led us to propose that systematic resequencing of cancer genomes for mutations would lead to the discovery of many additional cancer genes. Here we report more than 1,000 somatic mutations found in 274 megabases (Mb) of DNA corresponding to the coding exons of 518 protein kinase genes in 210 diverse human cancers. There was substantial variation in the number and pattern of mutations in individual cancers reflecting different exposures, DNA repair defects and cellular origins. Most somatic mutations are likely to be ‘passengers’ that do not contribute to oncogenesis. However, there was evidence for ‘driver’ mutations contributing to the development of the cancers studied in approximately 120 genes. Systematic sequencing of cancer genomes therefore reveals the evolutionary diversity of cancers and implicates a larger repertoire of cancer genes than previously anticipated.

MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma.

CONTEXT MicroRNAs have potential as diagnostic biomarkers and therapeutic targets in cancer. No study has evaluated the association between microRNA expression patterns and colon cancer prognosis or therapeutic outcome. OBJECTIVE To identify microRNA expression patterns associated with colon adenocarcinomas, prognosis, or therapeutic outcome. DESIGN, SETTING, AND PATIENTS MicroRNA microarray expression profiling of tumors and paired nontumorous tissues was performed on a US test cohort of 84 patients with incident colon adenocarcinoma, recruited between 1993 and 2002. We evaluated associations with tumor status, TNM staging, survival prognosis, and response to adjuvant chemotherapy. Associations were validated in a second, independent Chinese cohort of 113 patients recruited between 1991 and 2000, using quantitative reverse transcription polymerase chain reaction assays. The final date of follow-up was December 31, 2005, for the Maryland cohort and August 16, 2004, for the Hong Kong cohort. MAIN OUTCOME MEASURES MicroRNAs that were differentially expressed in tumors and microRNA expression patterns associated with survival using cancer-specific death as the end point. RESULTS Thirty-seven microRNAs were differentially expressed in tumors from the test cohort. Selected for validation were miR-20a, miR-21, miR-106a, miR-181b, and miR-203, and all 5 were enriched in tumors from the validation cohort (P < .001). Higher miR-21 expression was present in adenomas (P = .006) and in tumors with more advanced TNM staging (P < .001). In situ hybridization demonstrated miR-21 to be expressed at high levels in colonic carcinoma cells. The 5-year cancer-specific survival rate was 57.5% for the Maryland cohort and was 49.5% for the Hong Kong cohort. High miR-21 expression was associated with poor survival in both the training (hazard ratio, 2.5; 95% confidence interval, 1.2-5.2) and validation cohorts (hazard ratio, 2.4; 95% confidence interval, 1.4-3.9), independent of clinical covariates, including TNM staging, and was associated with a poor therapeutic outcome. CONCLUSIONS Expression patterns of microRNAs are systematically altered in colon adenocarcinomas. High miR-21 expression is associated with poor survival and poor therapeutic outcome.

Kirsten ras mutations in patients with colorectal cancer: The 'RASCAL II' study

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes’ C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes’ B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

Somatic mutations of the histone H3K27 demethylase gene UTX in human cancer

Somatically acquired epigenetic changes are present in many cancers. Epigenetic regulation is maintained via post-translational modifications of core histones. Here, we describe inactivating somatic mutations in the histone lysine demethylase gene UTX, pointing to histone H3 lysine methylation deregulation in multiple tumor types. UTX reintroduction into cancer cells with inactivating UTX mutations resulted in slowing of proliferation and marked transcriptional changes. These data identify UTX as a new human cancer gene.

Heritable somatic methylation and inactivation of MSH2 in families with Lynch syndrome due to deletion of the 3' exons of TACSTD1.

Lynch syndrome patients are susceptible to colorectal and endometrial cancers owing to inactivating germline mutations in mismatch repair genes, including MSH2 (ref. 1). Here we describe patients from Dutch and Chinese families with MSH2-deficient tumors carrying heterozygous germline deletions of the last exons of TACSTD1, a gene directly upstream of MSH2 encoding Ep-CAM. Due to these deletions, transcription of TACSTD1 extends into MSH2. The MSH2 promoter in cis with the deletion is methylated in Ep-CAM positive but not in Ep-CAM negative normal tissues, thus revealing a correlation between activity of the mutated TACSTD1 allele and epigenetic inactivation of the corresponding MSH2 allele. Gene silencing by transcriptional read-through of a neighboring gene in either sense, as demonstrated here, or antisense direction, could represent a general mutational mechanism. Depending on the expression pattern of the neighboring gene that lacks its normal polyadenylation signal, this may cause either generalized or mosaic patterns of epigenetic inactivation.

Exome sequencing identifies frequent mutation of ARID1A in molecular subtypes of gastric cancer

Gastric cancer is a heterogeneous disease with multiple environmental etiologies and alternative pathways of carcinogenesis. Beyond mutations in TP53, alterations in other genes or pathways account for only small subsets of the disease. We performed exome sequencing of 22 gastric cancer samples and identified previously unreported mutated genes and pathway alterations; in particular, we found genes involved in chromatin modification to be commonly mutated. A downstream validation study confirmed frequent inactivating mutations or protein deficiency of ARID1A, which encodes a member of the SWI-SNF chromatin remodeling family, in 83% of gastric cancers with microsatellite instability (MSI), 73% of those with Epstein-Barr virus (EBV) infection and 11% of those that were not infected with EBV and microsatellite stable (MSS). The mutation spectrum for ARID1A differs between molecular subtypes of gastric cancer, and mutation prevalence is negatively associated with mutations in TP53. Clinically, ARID1A alterations were associated with better prognosis in a stage-independent manner. These results reveal the genomic landscape, and highlight the importance of chromatin remodeling, in the molecular taxonomy of gastric cancer.

Whole-genome sequencing and comprehensive molecular profiling identify new driver mutations in gastric cancer

Gastric cancer is a heterogeneous disease with diverse molecular and histological subtypes. We performed whole-genome sequencing in 100 tumor-normal pairs, along with DNA copy number, gene expression and methylation profiling, for integrative genomic analysis. We found subtype-specific genetic and epigenetic perturbations and unique mutational signatures. We identified previously known (TP53, ARID1A and CDH1) and new (MUC6, CTNNA2, GLI3, RNF43 and others) significantly mutated driver genes. Specifically, we found RHOA mutations in 14.3% of diffuse-type tumors but not in intestinal-type tumors (P < 0.001). The mutations clustered in recurrent hotspots affecting functional domains and caused defective RHOA signaling, promoting escape from anoikis in organoid cultures. The top perturbed pathways in gastric cancer included adherens junction and focal adhesion, in which RHOA and other mutated genes we identified participate as key players. These findings illustrate a multidimensional and comprehensive genomic landscape that highlights the molecular complexity of gastric cancer and provides a road map to facilitate genome-guided personalized therapy.

Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors

Human colonic epithelial cell renewal, proliferation, and differentiation are stringently controlled by numerous regulatory pathways. To identify genetic programs of human colonic epithelial cell differentiation in vivo as well as candidate marker genes that define colonic epithelial stem/progenitor cells and the stem cell niche, we applied gene expression analysis of normal human colon tops and basal crypts by using expression microarrays with 30,000 genes. Nine hundred and sixty-nine cDNA clones were found to be differentially expressed between human colon crypts and tops. Pathway analysis revealed the differential expression of genes involved in cell cycle maintenance and apoptosis, as well as genes in bone morphogenetic protein (BMP), Notch, Wnt, EPH, and MYC signaling pathways. BMP antagonists gremlin 1, gremlin 2, and chordin-like 1 were found to be expressed by colon crypts. In situ hybridization and RT-PCR confirmed that these BMP antagonists are expressed by intestinal cryptal myofibroblasts and smooth muscle cells at the colon crypt. In vitro analysis demonstrated that gremlin 1 partially inhibits Caco-2 cell differentiation upon confluence and activates Wnt signaling in normal rat intestinal epithelial cells. Collectively, the expression data set provides a comprehensive picture of human colonic epithelial cell differentiation. Our study also suggests that BMP antagonists are candidate signaling components that make up the intestinal epithelial stem cell niche.

A screen of the complete protein kinase gene family identifies diverse patterns of somatic mutations in human breast cancer

We examined the coding sequence of 518 protein kinases, ∼1.3 Mb of DNA per sample, in 25 breast cancers. In many tumors, we detected no somatic mutations. But a few had numerous somatic mutations with distinctive patterns indicative of either a mutator phenotype or a past exposure.