Sung-Min Ahn

Landscape of somatic mutations in 560 breast cancer whole-genome sequences

We analysed whole genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. 93 protein-coding cancer genes carried likely driver mutations. Some non-coding regions exhibited high mutation frequencies but most have distinctive structural features probably causing elevated mutation rates and do not harbour driver mutations. Mutational signature analysis was extended to genome rearrangements and revealed 12 base substitution and six rearrangement signatures. Three rearrangement signatures, characterised by tandem duplications or deletions, appear associated with defective homologous recombination based DNA repair: one with deficient BRCA1 function; another with deficient BRCA1 or BRCA2 function; the cause of the third is unknown. This analysis of all classes of somatic mutation across exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operative, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer.

The first Korean genome sequence and analysis: Full genome sequencing for a socio-ethnic group

We present the first Korean individual genome sequence (SJK) and analysis results. The diploid genome of a Korean male was sequenced to 28.95-fold redundancy using the Illumina paired-end sequencing method. SJK covered 99.9% of the NCBI human reference genome. We identified 420,083 novel single nucleotide polymorphisms (SNPs) that are not in the dbSNP database. Despite a close similarity, significant differences were observed between the Chinese genome (YH), the only other Asian genome available, and SJK: (1) 39.87% (1,371,239 out of 3,439,107) SNPs were SJK-specific (49.51% against Venters, 46.94% against Watsons, and 44.17% against the Yoruba genomes); (2) 99.5% (22,495 out of 22,605) of short indels (< 4 bp) discovered on the same loci had the same size and type as YH; and (3) 11.3% (331 out of 2920) deletion structural variants were SJK-specific. Even after attempting to map unmapped reads of SJK to unanchored NCBI scaffolds, HGSV, and available personal genomes, there were still 5.77% SJK reads that could not be mapped. All these findings indicate that the overall genetic differences among individuals from closely related ethnic groups may be significant. Hence, constructing reference genomes for minor socio-ethnic groups will be useful for massive individual genome sequencing.

Genomic portrait of resectable hepatocellular carcinomas: implications of RB1 and FGF19 aberrations for patient stratification.

Hepatic resection is the most curative treatment option for early‐stage hepatocellular carcinoma, but is associated with a high recurrence rate, which exceeds 50% at 5 years after surgery. Understanding the genetic basis of hepatocellular carcinoma at surgically curable stages may enable the identification of new molecular biomarkers that accurately identify patients in need of additional early therapeutic interventions. Whole exome sequencing and copy number analysis was performed on 231 hepatocellular carcinomas (72% with hepatitis B viral infection) that were classified as early‐stage hepatocellular carcinomas, candidates for surgical resection. Recurrent mutations were validated by Sanger sequencing. Unsupervised genomic analyses identified an association between specific genetic aberrations and postoperative clinical outcomes. Recurrent somatic mutations were identified in nine genes, including TP53, CTNNB1, AXIN1, RPS6KA3, and RB1. Recurrent homozygous deletions in FAM123A, RB1, and CDKN2A, and high‐copy amplifications in MYC, RSPO2, CCND1, and FGF19 were detected. Pathway analyses of these genes revealed aberrations in the p53, Wnt, PIK3/Ras, cell cycle, and chromatin remodeling pathways. RB1 mutations were significantly associated with cancer‐specific and recurrence‐free survival after resection (multivariate Pu2009=u20090.038 and Pu2009=u20090.012, respectively). FGF19 amplifications, known to activate Wnt signaling, were mutually exclusive with CTNNB1 and AXIN1 mutations, and significantly associated with cirrhosis (Pu2009=u20090.017). Conclusion: RB1 mutations can be used as a prognostic molecular biomarker for resectable hepatocellular carcinoma. Further study is required to investigate the potential role of FGF19 amplification in driving hepatocarcinogenesis in patients with liver cirrhosis and to investigate the potential of anti‐FGF19 treatment in these patients. (Hepatology 2014;60:1971–1981)

Proteomic Analysis of Exosomes from Human Neural Stem Cells by Flow Field-Flow Fractionation and Nanoflow Liquid Chromatography-Tandem Mass Spectrometry

Exosomes, small membrane vesicles secreted by a multitude of cell types, are involved in a wide range of physiological roles such as intercellular communication, membrane exchange between cells, and degradation as an alternative to lysosomes. Because of the small size of exosomes (30-100 nm) and the limitations of common separation procedures including ultracentrifugation and flow cytometry, size-based fractionation of exosomes has been challenging. In this study, we used flow field-flow fractionation (FlFFF) to fractionate exosomes according to differences in hydrodynamic diameter. The exosome fractions collected from FlFFF runs were examined by transmission electron microscopy (TEM) to morphologically confirm their identification as exosomes. Exosomal lysates of each fraction were digested and analyzed using nanoflow LC-ESI-MS-MS for protein identification. FIFFF, coupled with mass spectrometry, allows nanoscale size-based fractionation of exosomes and is more applicable to primary cells and stem cells since it requires much less starting material than conventional gel-based separation, in-gel digestion and the MS-MS method.

Integrated Expression Profiling and Genome-Wide Analysis of ChREBP Targets Reveals the Dual Role for ChREBP in Glucose-Regulated Gene Expression

The carbohydrate response element binding protein (ChREBP), a basic helix-loop-helix/leucine zipper transcription factor, plays a critical role in the control of lipogenesis in the liver. To identify the direct targets of ChREBP on a genome-wide scale and provide more insight into the mechanism by which ChREBP regulates glucose-responsive gene expression, we performed chromatin immunoprecipitation-sequencing and gene expression analysis. We identified 1153 ChREBP binding sites and 783 target genes using the chromatin from HepG2, a human hepatocellular carcinoma cell line. A motif search revealed a refined consensus sequence (CABGTG-nnCnG-nGnSTG) to better represent critical elements of a functional ChREBP binding sequence. Gene ontology analysis shows that ChREBP target genes are particularly associated with lipid, fatty acid and steroid metabolism. In addition, other functional gene clusters related to transport, development and cell motility are significantly enriched. Gene set enrichment analysis reveals that ChREBP target genes are highly correlated with genes regulated by high glucose, providing a functional relevance to the genome-wide binding study. Furthermore, we have demonstrated that ChREBP may function as a transcriptional repressor as well as an activator.

Smad3 regulates E-cadherin via miRNA-200 pathway

To identify potential microRNA (miRNA) links between Smad3, a mediator of TGF-β (transforming growth factor-β) signaling, and E-cadherin, we characterized the miRNA profiles of two gastric cancer cell lines: SNU484-LPCX, which does not express Smad3, and SNU484-Smad3, in which Smad3 is overexpressed. We found that among differentially expressed miRNAs, miR-200 family members are overexpressed in SNU484-Smad3 cells. Subsequent studies, including analysis of the effects of silencing Smad3 in SNU484-Smad3 cells and a luciferase reporter assay, revealed that Smad3 directly binds to a Smad-binding element located in the promoter region of miR-200b/a, where it functions as a transcriptional activator. TGF-β did not affect the regulatory role of Smad3 in transcription of miR-200 and expression of epithelial–mesenchymal transition markers. We conclude that Smad3 regulates, at the transcriptional level, miR-200 family members, which themselves regulate ZEB1 and ZEB2, known transcriptional repressors of E-cadherin, at the posttranscriptional level in a TGF-β-independent manner. This represents a novel link between Smad3 and posttranscriptional regulation by miRNAs in epithelial–mesenchymal transition in gastric cancer cells.

Body fluid proteomics: Prospects for biomarker discovery.

Many diseases are caused by perturbations of cellular signaling pathways and related pathway networks as a result of genetic aberrations. These perturbations are manifested by altered cellular protein profiles in the fluids bathing tissue/organs (i.e., the tissue interstitial fluid, TIF). A major challenge of clinical chemistry is to quantitatively map these perturbed protein profiles – the so‐called “signatures of disease” – using modern proteomic technologies. This information can be utilized to design protein biomarkers for the early detection of disease, monitoring disease progression and efficacy of drug action. Here, we discuss the use of body fluids in the context of prospective biomarker discovery, and the marked 1000–1500‐fold dilution of body fluid proteins, during their passage from TIF to the circulatory system. Further, we discuss proteomics strategies aimed at depleting major serum proteins, especially albumin, in order to focus on low‐abundance protein/peptides in plasma. A major limitation of depletion strategies is the removal of low‐molecular weight protein/peptides which specifically bind major plasma proteins. We present a prototype model, using albumin, for understanding the multifaceted nature of biomarker research, highlighting the involvement of albumin in Alzheimers disease. This model underscores the need for a system‐level understanding for biomarker research and personalized medicine.

Human Microglial Cells Synthesize Albumin in Brain

Albumin, an abundant plasma protein with multifunctional properties, is mainly synthesized in the liver. Albumin has been implicated in Alzheimers disease (AD) since it can bind to and transport amyloid beta (Aβ), the causative agent of AD; albumin is also a potent inhibitor of Aβ polymerization. Despite evidence of non-hepatic transcription of albumin in many tissues including kidney and pancreas, non-hepatic synthesis of albumin at the protein level has been rarely confirmed. In a pilot phase study of Human Brain Proteome Project, we found evidence that microglial cells in brain may synthesize albumin. Here we report, for the first time, the de novo synthesis of albumin in human microglial cells in brain. Furtherore, we demonstrate that the synthesis and secretion of albumin from microglial cells is enhanced upon microgial activation by Aβ1–42- or lipopolysaccharide (LPS)-treatment. These data indicate that microglial cells may play a beneficial role in AD by secreting albumin that not only inhibits Aβ polymerization but also increases its clearance.

Monogenic and polygenic determinants of sarcoma risk: an international genetic study

BACKGROUNDnSarcomas are rare, phenotypically heterogeneous cancers that disproportionately affect the young. Outside rare syndromes, the nature, extent, and clinical significance of their genetic origins are not known. We aimed to investigate the genetic basis for bone and soft-tissue sarcoma seen in routine clinical practice.nnnMETHODSnIn this genetic study, we included 1162 patients with sarcoma from four cohorts (the International Sarcoma Kindred Study [ISKS], 966 probands; Project GENESIS, 48 probands; Asan Bio-Resource Center, 138 probands; and kConFab, ten probands), who were older than 15 years at the time of consent and had a histologically confirmed diagnosis of sarcoma, recruited from specialist sarcoma clinics without regard to family history. Detailed clinical, pathological, and pedigree information was collected, and cancer diagnoses in probands and relatives were independently verified. Targeted exon sequencing using blood (n=1114) or saliva (n=48) samples was done on 72 genes (selected due to associations with increased cancer risk) and rare variants were stratified into classes approximating the International Agency for Research on Cancer (IARC) clinical classification for genetic variation. We did a case-control rare variant burden analysis using 6545 Caucasian controls included from three cohorts (ISKS, 235 controls; LifePool, 2010 controls; and National Heart, Lung, and Blood Institute Exome Sequencing Project [ESP], 4300 controls).nnnFINDINGSnThe median age at cancer diagnosis in 1162 sarcoma probands was 46 years (IQR 29-58), 170 (15%) of 1162 probands had multiple primary cancers, and 155 (17%) of 911 families with informative pedigrees fitted recognisable cancer syndromes. Using a case-control rare variant burden analysis, 638 (55%) of 1162 sarcoma probands bore an excess of pathogenic germline variants (combined odds ratio [OR] 1·43, 95% CI 1·24-1·64, p<0·0001), with 227 known or expected pathogenic variants occurring in 217 individuals. All classes of pathogenic variants (known, expected, or predicted) were associated with earlier age of cancer onset. In addition to TP53, ATM, ATR, and BRCA2, an unexpected excess of functionally pathogenic variants was seen in ERCC2. Probands were more likely than controls to have multiple pathogenic variants compared with the combined control cohort group and the LifePool control cohort (OR 2·22, 95% CI 1·57-3·14, p=1·2u2008×u200810(-6)) and the cumulative burden of multiple variants correlated with earlier age at cancer diagnosis (Mantel-Cox log-rank test for trend, p=0·0032). 66 of 1162 probands carried notifiable variants following expert clinical review (those recognised to be clinically significant to health and about which patients should be advised), whereas 293 (25%) probands carried variants with potential therapeutic significance.nnnINTERPRETATIONnAbout half of patients with sarcoma have putatively pathogenic monogenic and polygenic variation in known and novel cancer genes, with implications for risk management and treatment.nnnFUNDINGnRainbows for Kate Foundation, Johanna Sewell Research Foundation, Australian National Health and Medical Research Council, Cancer Australia, Sarcoma UK, National Cancer Institute, Liddy Shriver Sarcoma Initiative.

Comparative genomic analysis of primary and synchronous metastatic colorectal cancers.

Approximately 50% of patients with primary colorectal carcinoma develop liver metastases. Understanding the genetic differences between primary colon cancer and their metastases to the liver is essential for devising a better therapeutic approach for this disease. We performed whole exome sequencing and copy number analysis for 15 triplets, each comprising normal colorectal tissue, primary colorectal carcinoma, and its synchronous matched liver metastasis. We analyzed the similarities and differences between primary colorectal carcinoma and matched liver metastases in regards to somatic mutations and somatic copy number alterationss. The genomic profiling demonstrated mutations in APC(73%), KRAS (33%), ARID1A and PIK3CA (6.7%) genes between primary colorectal and metastatic liver tumors. TP53 mutation was observed in 47% of the primary samples and 67% in liver metastatic samples. The grouped pairs, in hierarchical clustering showed similar somatic copy number alteration patterns, in contrast to the ungrouped pairs. Many mutations (including those of known key cancer driver genes) were shared in the grouped pairs. The ungrouped pairs exhibited distinct mutation patterns with no shared mutations in key driver genes. Four ungrouped liver metastasis samples had mutations in DNA mismatch repair genes along with hypermutations and a substantial number of copy number alterations. Our results suggest that about half of the metastatic colorectal carcinoma had the same clonal origin with their primary colorectal carcinomas, whereas remaining cases were genetically distinct from their primary carcinomas. These findings underscore the need to evaluate metastatic lesions separately for optimized therapy, rather than to extrapolate from primary tumor data.