Young Seok Ju

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.

A transforming KIF5B and RET gene fusion in lung adenocarcinoma revealed from whole-genome and transcriptome sequencing

The identification of the molecular events that drive cancer transformation is essential to the development of targeted agents that improve the clinical outcome of lung cancer. Many studies have reported genomic driver mutations in non-small-cell lung cancers (NSCLCs) over the past decade; however, the molecular pathogenesis of >40% of NSCLCs is still unknown. To identify new molecular targets in NSCLCs, we performed the combined analysis of massively parallel whole-genome and transcriptome sequencing for cancer and paired normal tissue of a 33-yr-old lung adenocarcinoma patient, who is a never-smoker and has no familial cancer history. The cancer showed no known driver mutation in EGFR or KRAS and no EML4-ALK fusion. Here we report a novel fusion gene between KIF5B and the RET proto-oncogene caused by a pericentric inversion of 10p11.22-q11.21. This fusion gene overexpresses chimeric RET receptor tyrosine kinase, which could spontaneously induce cellular transformation. We identified the KIF5B-RET fusion in two more cases out of 20 primary lung adenocarcinomas in the replication study. Our data demonstrate that a subset of NSCLCs could be caused by a fusion of KIF5B and RET, and suggest the chimeric oncogene as a promising molecular target for the personalized diagnosis and treatment of lung cancer.

The transcriptional landscape and mutational profile of lung adenocarcinoma

All cancers harbor molecular alterations in their genomes. The transcriptional consequences of these somatic mutations have not yet been comprehensively explored in lung cancer. Here we present the first large scale RNA sequencing study of lung adenocarcinoma, demonstrating its power to identify somatic point mutations as well as transcriptional variants such as gene fusions, alternative splicing events, and expression outliers. Our results reveal the genetic basis of 200 lung adenocarcinomas in Koreans including deep characterization of 87 surgical specimens by transcriptome sequencing. We identified driver somatic mutations in cancer genes including EGFR, KRAS, NRAS, BRAF, PIK3CA, MET, and CTNNB1. Candidates for novel driver mutations were also identified in genes newly implicated in lung adenocarcinoma such as LMTK2, ARID1A, NOTCH2, and SMARCA4. We found 45 fusion genes, eight of which were chimeric tyrosine kinases involving ALK, RET, ROS1, FGFR2, AXL, and PDGFRA. Among 17 recurrent alternative splicing events, we identified exon 14 skipping in the proto-oncogene MET as highly likely to be a cancer driver. The number of somatic mutations and expression outliers varied markedly between individual cancers and was strongly correlated with smoking history of patients. We identified genomic blocks within which gene expression levels were consistently increased or decreased that could be explained by copy number alterations in samples. We also found an association between lymph node metastasis and somatic mutations in TP53. These findings broaden our understanding of lung adenocarcinoma and may also lead to new diagnostic and therapeutic approaches.

Discovery of common Asian copy number variants using integrated high-resolution array CGH and massively parallel DNA sequencing

Copy number variants (CNVs) account for the majority of human genomic diversity in terms of base coverage. Here, we have developed and applied a new method to combine high-resolution array comparative genomic hybridization (CGH) data with whole-genome DNA sequencing data to obtain a comprehensive catalog of common CNVs in Asian individuals. The genomes of 30 individuals from three Asian populations (Korean, Chinese and Japanese) were interrogated with an ultra-high-resolution array CGH platform containing 24 million probes. Whole-genome sequencing data from a reference genome (NA10851, with 28.3× coverage) and two Asian genomes (AK1, with 27.8× coverage and AK2, with 32.0× coverage) were used to transform the relative copy number information obtained from array CGH experiments into absolute copy number values. We discovered 5,177 CNVs, of which 3,547 were putative Asian-specific CNVs. These common CNVs in Asian populations will be a useful resource for subsequent genetic studies in these populations, and the new method of calling absolute CNVs will be essential for applying CNV data to personalized medicine.

Mutational signatures associated with tobacco smoking in human cancer

Assessing smoke damage in cancer genomes We have known for over 60 years that smoking tobacco is one of the most avoidable risk factors for cancer. Yet the detailed mechanisms by which tobacco smoke damages the genome and creates the mutations that ultimately cause cancer are still not fully understood. Alexandrov et al. examined mutational signatures and DNA methylation changes in over 5000 genome sequences from 17 different cancer types linked to smoking (see the Perspective by Pfeifer). They found a complex pattern of mutational signatures. Only cancers originating in tissues directly exposed to smoke showed a signature characteristic of the known tobacco carcinogen benzo[a]pyrene. One mysterious signature was shared by all smoking-associated cancers but is of unknown origin. Smoking had only a modest effect on DNA methylation. Science, this issue p. 618; see also p. 549 Tobacco smoke causes cancer through mutational processes that are more complex than previously thought. Tobacco smoking increases the risk of at least 17 classes of human cancer. We analyzed somatic mutations and DNA methylation in 5243 cancers of types for which tobacco smoking confers an elevated risk. Smoking is associated with increased mutation burdens of multiple distinct mutational signatures, which contribute to different extents in different cancers. One of these signatures, mainly found in cancers derived from tissues directly exposed to tobacco smoke, is attributable to misreplication of DNA damage caused by tobacco carcinogens. Others likely reflect indirect activation of DNA editing by APOBEC cytidine deaminases and of an endogenous clocklike mutational process. Smoking is associated with limited differences in methylation. The results are consistent with the proposition that smoking increases cancer risk by increasing the somatic mutation load, although direct evidence for this mechanism is lacking in some smoking-related cancer types.

Extensive transduction of nonrepetitive DNA mediated by L1 retrotransposition in cancer genomes

Introduction The human genome is peppered with mobile repetitive elements called long interspersed nuclear element–1 (L1) retrotransposons. Propagating through RNA and cDNA intermediates, these molecular parasites copy and insert themselves throughout the genome, with potentially disruptive effects on neighboring genes or regulatory sequences. In the germ line, unique sequence downstream of L1 elements can also be retrotransposed if transcription continues beyond the repeat, a process known as 3′ transduction. There has been growing interest in retrotransposition and 3′ transduction as a possible source of somatic mutations during tumorigenesis. The activity of individual L1 elements fluctuates during tumor evolution. In a lung tumor, hundreds of 3′ transductions arose from a small number of active L1 source elements (colored circles on outer rim of circle). As the tumor evolved from the preinvasive common ancestor to invasive cancer, individual elements exhibited variable activity over time. Rationale To explore whether 3′ transductions are frequent in cancer, we developed a bioinformatic algorithm for identifying somatically acquired retrotranspositions in cancer genomes. We applied our algorithm to 290 cancer samples from 244 patients across 12 tumor types. The unique downstream sequence mobilized with 3′ transductions effectively fingerprints the L1 source element, providing insights into the activity of individual L1 loci across the genome. Results Across the 290 samples, we identified 2756 somatic L1 retrotranspositions. Tumors from 53% of patients had at least one such event, with colorectal and lung cancers being most frequently affected (93% and 75% of patients, respectively). Somatic 3′ transductions comprised 24% of events, half of which represented mobilizations of unique sequence alone, without any accompanying L1 sequence. Overall, 95% of 3′ transductions identified derived from only 72 germline L1 source elements, with as few as four loci accounting for 50% of events. In a given sample, the same source element could generate 50 or more somatic transductions, scattered extensively across the genome. About 5% of somatic transductions arose from L1 source elements that were themselves somatic retrotranspositions. In three of the cases in which we sequenced more than one sample from a patient’s tumor, we were able to place 3′ transductions on the phylogenetic tree. We found that the activity of individual source elements fluctuated during tumor evolution, with different subclones exhibiting much variability in which elements were “on” and which were “off.” The ability to identify the individual L1 source elements active in a given tumor enabled us to study the promoter methylation of those elements specifically. We found that 3′ transduction activity in a patient’s tumor was always associated with hypomethylation of that element. Overall, 2.3% of transductions distributed exons or entire genes to other sites in the genome, and many more mobilized deoxyribonuclease I (DNAse-I) hypersensitive sites or transcription factor binding sites identified by the ENCODE project. Occasionally, somatic L1 insertions inserted near coding sequence and redistributed these exons elsewhere in the genome. However, we found no general effects of retrotranspositions on transcription levels of genes at the insertion points and no evidence for aberrant RNA species resulting from somatically acquired transposable elements. Indeed, as with germline retrotranspositions, somatic insertions exhibited a strong enrichment in heterochromatic, gene-poor regions of the genome. Conclusion Somatic 3′ transduction occurs frequently in human tumors, and in some cases transduction events can scatter exons, genes, and regulatory elements widely across the genome. Dissemination of these sequences appears to be due to a small number of highly active L1 elements, whose activity can wax and wane during tumor evolution. The majority of the retrotransposition events are likely to be harmless “passenger” mutations. Hitchhiking through the tumor genome Retrotransposons are DNA repeat sequences that are constantly on the move. By poaching certain cellular enzymes, they copy and insert themselves at new sites in the genome. Sometimes they carry along adjacent DNA sequences, a process called 3′ transduction. Tubio et al. found that 3′ transduction is a common event in human tumors. Because this process can scatter genes and regulatory sequences across the genome, it may represent yet another mechanism by which tumor cells acquire new mutations that help them survive and grow. Science, this issue p. 10.1126/science.1251343 Tumor genomes are peppered with mobile repeat sequences that carry along adjacent DNA when they insert into new genomic sites. Long interspersed nuclear element–1 (L1) retrotransposons are mobile repetitive elements that are abundant in the human genome. L1 elements propagate through RNA intermediates. In the germ line, neighboring, nonrepetitive sequences are occasionally mobilized by the L1 machinery, a process called 3′ transduction. Because 3′ transductions are potentially mutagenic, we explored the extent to which they occur somatically during tumorigenesis. Studying cancer genomes from 244 patients, we found that tumors from 53% of the patients had somatic retrotranspositions, of which 24% were 3′ transductions. Fingerprinting of donor L1s revealed that a handful of source L1 elements in a tumor can spawn from tens to hundreds of 3′ transductions, which can themselves seed further retrotranspositions. The activity of individual L1 elements fluctuated during tumor evolution and correlated with L1 promoter hypomethylation. The 3′ transductions disseminated genes, exons, and regulatory elements to new locations, most often to heterochromatic regions of the genome.

Origins and functional consequences of somatic mitochondrial DNA mutations in human cancer.

Recent sequencing studies have extensively explored the somatic alterations present in the nuclear genomes of cancers. Although mitochondria control energy metabolism and apoptosis, the origins and impact of cancer-associated mutations in mtDNA are unclear. In this study, we analyzed somatic alterations in mtDNA from 1675 tumors. We identified 1907 somatic substitutions, which exhibited dramatic replicative strand bias, predominantly C > T and A > G on the mitochondrial heavy strand. This strand-asymmetric signature differs from those found in nuclear cancer genomes but matches the inferred germline process shaping primate mtDNA sequence content. A number of mtDNA mutations showed considerable heterogeneity across tumor types. Missense mutations were selectively neutral and often gradually drifted towards homoplasmy over time. In contrast, mutations resulting in protein truncation undergo negative selection and were almost exclusively heteroplasmic. Our findings indicate that the endogenous mutational mechanism has far greater impact than any other external mutagens in mitochondria and is fundamentally linked to mtDNA replication. DOI: http://dx.doi.org/10.7554/eLife.02935.001

Extensive genomic and transcriptional diversity identified through massively parallel DNA and RNA sequencing of eighteen Korean individuals

Massively parallel sequencing technologies have identified a broad spectrum of human genome diversity. Here we deep sequenced and correlated 18 genomes and 17 transcriptomes of unrelated Korean individuals. This has allowed us to construct a genome-wide map of common and rare variants and also identify variants formed during DNA-RNA transcription. We identified 9.56 million genomic variants, 23.2% of which appear to be previously unidentified. From transcriptome sequencing, we discovered 4,414 transcripts not previously annotated. Finally, we revealed 1,809 sites of transcriptional base modification, where the transcriptional landscape is different from the corresponding genomic sequences, and 580 sites of allele-specific expression. Our findings suggest that a considerable number of unexplored genomic variants still remain to be identified in the human genome, and that the integrated analysis of genome and transcriptome sequencing is powerful for understanding the diversity and functional aspects of human genomic variants.

Somatic mutations reveal asymmetric cellular dynamics in the early human embryo

Somatic cells acquire mutations throughout the course of an individual’s life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and their contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. This study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.

Clonal History and Genetic Predictors of Transformation Into Small-Cell Carcinomas From Lung Adenocarcinomas.

Purpose Histologic transformation of EGFR mutant lung adenocarcinoma (LADC) into small-cell lung cancer (SCLC) has been described as one of the major resistant mechanisms for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). However, the molecular pathogenesis is still unclear. Methods We investigated 21 patients with advanced EGFR-mutant LADCs that were transformed into EGFR TKI-resistant SCLCs. Among them, whole genome sequencing was applied for nine tumors acquired at various time points from four patients to reconstruct their clonal evolutionary history and to detect genetic predictors for small-cell transformation. The findings were validated by immunohistochemistry in 210 lung cancer tissues. Results We identified that EGFR TKI-resistant LADCs and SCLCs share a common clonal origin and undergo branched evolutionary trajectories. The clonal divergence of SCLC ancestors from the LADC cells occurred before the first EGFR TKI treatments, and the complete inactivation of both RB1 and TP53 were observed from the early LADC stages in sequenced tumors. We extended the findings by immunohistochemistry in the early-stage LADC tissues of 75 patients treated with EGFR TKIs; inactivation of both Rb and p53 was strikingly more frequent in the small-cell-transformed group than in the nontransformed group (82% v 3%; odds ratio, 131; 95% CI, 19.9 to 859). Among patients registered in a predefined cohort (n = 65), an EGFR mutant LADC that harbored completely inactivated Rb and p53 had a 43× greater risk of small-cell transformation (relative risk, 42.8; 95% CI, 5.88 to 311). Branch-specific mutational signature analysis revealed that apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC)-induced hypermutation was frequent in the branches toward small-cell transformation. Conclusion EGFR TKI-resistant SCLCs are branched out early from the LADC clones that harbor completely inactivated RB1 and TP53. The evaluation of RB1 and TP53 status in EGFR TKI-treated LADCs is informative in predicting small-cell transformation.