Clarence Lee

Detection of Circulating Tumor DNA in Early- and Late-Stage Human Malignancies

Circulating tumor DNA can be used in a variety of clinical and investigational settings across tumor types and stages for screening, diagnosis, and identifying mutations responsible for therapeutic response and drug resistance. Circulating Tumor DNA for Early Detection and Managing Resistance Cancer evolves over time, without any warning signs. Similarly, the development of resistance to therapy generally becomes apparent only when there are obvious signs of tumor growth, at which point the patient may have lost valuable time. Although a repeat biopsy may be able to identify drug-resistant mutations before the tumor has a chance to regrow, it is usually not feasible to do many repeat biopsies. Now, two studies are demonstrating the utility of monitoring the patients’ blood for tumor DNA to detect cancer at the earliest stages of growth or resistance. In one study, Bettegowda and coauthors showed that sampling a patient’s blood may be sufficient to yield information about the tumor’s genetic makeup, even for many early-stage cancers, without a need for an invasive procedure to collect tumor tissue, such as surgery or endoscopy. The authors demonstrated the presence of circulating DNA from many types of tumors that had not yet metastasized or released detectable cells into the circulation. They could detect more than 50% of patients across 14 tumor types at the earliest stages, when these cancers may still be curable, suggesting that a blood draw could be a viable screening approach to detecting most cancers. They also showed that in patients with colorectal cancer, the information derived from circulating tumor DNA could be used to determine the optimal course of treatment and identify resistance to epidermal growth factor receptor (EGFR) blockade. Meanwhile, Misale and colleagues illustrated a way to use this information to overcome treatment resistance. These authors also found that mutations associated with EGFR inhibitor resistance could be detected in the blood of patients with colorectal cancer. In addition, they demonstrated that adding MEK inhibitors, another class of anticancer drugs, can successfully overcome resistance when given in conjunction with the EGFR inhibitors. Thus, the studies from Bettegowda and Misale and their colleagues show the effectiveness of analyzing circulating DNA from a variety of tumors and highlight the potential investigational and clinical applications of this novel technology for early detection, monitoring resistance, and devising treatment plans to overcome resistance. The development of noninvasive methods to detect and monitor tumors continues to be a major challenge in oncology. We used digital polymerase chain reaction–based technologies to evaluate the ability of circulating tumor DNA (ctDNA) to detect tumors in 640 patients with various cancer types. We found that ctDNA was detectable in >75% of patients with advanced pancreatic, ovarian, colorectal, bladder, gastroesophageal, breast, melanoma, hepatocellular, and head and neck cancers, but in less than 50% of primary brain, renal, prostate, or thyroid cancers. In patients with localized tumors, ctDNA was detected in 73, 57, 48, and 50% of patients with colorectal cancer, gastroesophageal cancer, pancreatic cancer, and breast adenocarcinoma, respectively. ctDNA was often present in patients without detectable circulating tumor cells, suggesting that these two biomarkers are distinct entities. In a separate panel of 206 patients with metastatic colorectal cancers, we showed that the sensitivity of ctDNA for detection of clinically relevant KRAS gene mutations was 87.2% and its specificity was 99.2%. Finally, we assessed whether ctDNA could provide clues into the mechanisms underlying resistance to epidermal growth factor receptor blockade in 24 patients who objectively responded to therapy but subsequently relapsed. Twenty-three (96%) of these patients developed one or more mutations in genes involved in the mitogen-activated protein kinase pathway. Together, these data suggest that ctDNA is a broadly applicable, sensitive, and specific biomarker that can be used for a variety of clinical and research purposes in patients with multiple different types of cancer.

mRNA-Seq whole-transcriptome analysis of a single cell.

Next-generation sequencing technology is a powerful tool for transcriptome analysis. However, under certain conditions, only a small amount of material is available, which requires more sensitive techniques that can preferably be used at the single-cell level. Here we describe a single-cell digital gene expression profiling assay. Using our mRNA-Seq assay with only a single mouse blastomere, we detected the expression of 75% (5,270) more genes than microarray techniques and identified 1,753 previously unknown splice junctions called by at least 5 reads. Moreover, 8–19% of the genes with multiple known transcript isoforms expressed at least two isoforms in the same blastomere or oocyte, which unambiguously demonstrated the complexity of the transcript variants at whole-genome scale in individual cells. Finally, for Dicer1−/− and Ago2−/− (Eif2c2−/−) oocytes, we found that 1,696 and 1,553 genes, respectively, were abnormally upregulated compared to wild-type controls, with 619 genes in common.

Stem cell transcriptome profiling via massive-scale mRNA sequencing

We developed a massive-scale RNA sequencing protocol, short quantitative random RNA libraries or SQRL, to survey the complexity, dynamics and sequence content of transcriptomes in a near-complete fashion. This method generates directional, random-primed, linear cDNA libraries that are optimized for next-generation short-tag sequencing. We surveyed the poly(A)+ transcriptomes of undifferentiated mouse embryonic stem cells (ESCs) and embryoid bodies (EBs) at an unprecedented depth (10 Gb), using the Applied Biosystems SOLiD technology. These libraries capture the genomic landscape of expression, state-specific expression, single-nucleotide polymorphisms (SNPs), the transcriptional activity of repeat elements, and both known and new alternative splicing events. We investigated the impact of transcriptional complexity on current models of key signaling pathways controlling ESC pluripotency and differentiation, highlighting how SQRL can be used to characterize transcriptome content and dynamics in a quantitative and reproducible manner, and suggesting that our understanding of transcriptional complexity is far from complete.

A small-cell lung cancer genome with complex signatures of tobacco exposure

Cancer is driven by mutation. Worldwide, tobacco smoking is the principal lifestyle exposure that causes cancer, exerting carcinogenicity through >60 chemicals that bind and mutate DNA. Using massively parallel sequencing technology, we sequenced a small-cell lung cancer cell line, NCI-H209, to explore the mutational burden associated with tobacco smoking. A total of 22,910 somatic substitutions were identified, including 134 in coding exons. Multiple mutation signatures testify to the cocktail of carcinogens in tobacco smoke and their proclivities for particular bases and surrounding sequence context. Effects of transcription-coupled repair and a second, more general, expression-linked repair pathway were evident. We identified a tandem duplication that duplicates exons 3–8 of CHD7 in frame, and another two lines carrying PVT1–CHD7 fusion genes, indicating that CHD7 may be recurrently rearranged in this disease. These findings illustrate the potential for next-generation sequencing to provide unprecedented insights into mutational processes, cellular repair pathways and gene networks associated with cancer.SUMMARY Cancer is driven by mutation. Worldwide, tobacco smoking is the major lifestyle exposure that causes cancer, exerting carcinogenicity through >60 chemicals that bind and mutate DNA. Using massively parallel sequencing technology, we sequenced a small cell lung cancer cell line, NCI-H209, to explore the mutational burden associated with tobacco smoking. 22,910 somatic substitutions were identified, including 132 in coding exons. Multiple mutation signatures testify to the cocktail of carcinogens in tobacco smoke and their proclivities for particular bases and surrounding sequence context. Effects of transcription-coupled repair and a second, more general expression-linked repair pathway were evident. We identified a tandem duplication that duplicates exons 3-8 of CHD7 in-frame, and another two lines carrying PVT1-CHD7 fusion genes, suggesting that CHD7 may be recurrently rearranged in this disease. These findings illustrate the potential for next-generation sequencing to provide unprecedented insights into mutational processes, cellular repair pathways and gene networks associated with cancer.

Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding

We describe the genome sequencing of an anonymous individual of African origin using a novel ligation-based sequencing assay that enables a unique form of error correction that improves the raw accuracy of the aligned reads to >99.9%, allowing us to accurately call SNPs with as few as two reads per allele. We collected several billion mate-paired reads yielding approximately 18x haploid coverage of aligned sequence and close to 300x clone coverage. Over 98% of the reference genome is covered with at least one uniquely placed read, and 99.65% is spanned by at least one uniquely placed mate-paired clone. We identify over 3.8 million SNPs, 19% of which are novel. Mate-paired data are used to physically resolve haplotype phases of nearly two-thirds of the genotypes obtained and produce phased segments of up to 215 kb. We detect 226,529 intra-read indels, 5590 indels between mate-paired reads, 91 inversions, and four gene fusions. We use a novel approach for detecting indels between mate-paired reads that are smaller than the standard deviation of the insert size of the library and discover deletions in common with those detected with our intra-read approach. Dozens of mutations previously described in OMIM and hundreds of nonsynonymous single-nucleotide and structural variants in genes previously implicated in disease are identified in this individual. There is more genetic variation in the human genome still to be uncovered, and we provide guidance for future surveys in populations and cancer biopsies.

Carrier Testing for Severe Childhood Recessive Diseases by Next-Generation Sequencing

Carrier testing for 448 severe childhood recessive diseases by next-generation sequencing has good predictive value and suggests that every individual carries about three disease mutations. Shining a Light on Comprehensive Carrier Screening Although diseases inherited in a Mendelian fashion are rare, together they account for about 20% of deaths in infancy. For Mendelian diseases that are recessive (of which there are more than 1000), screening before pregnancy (preconception screening) together with genetic counseling of those carrying a mutant allele could reduce the incidence of these diseases and the suffering that they incur. In the case of Tay-Sachs disease, an incurable neurodegenerative disease of infancy, preconception screening for disease gene mutations and genetic counseling among individuals of Ashkenazi descent has reduced the incidence of this tragic disease by 90%. However, simultaneous testing for many recessive childhood diseases is costly, so, to date, screening has included just a few diseases such as Tay-Sachs disease, cystic fibrosis, and familial dysautonomia. In a new study, Kingsmore and his colleagues have combined target gene capture and enrichment, next-generation sequencing, and sophisticated bioinformatic analysis to develop a platform capable of screening several hundred DNA samples simultaneously for 448 severe recessive diseases of childhood. They demonstrate that their method is sensitive, specific, and scalable in a research setting and that it should be straightforward to automate the process. The authors report that individuals in the general population carry an average of three recessive childhood disease mutations. They also discovered that about 10% of disease mutations in commonly used databases are incorrect, suggesting that disease mutation annotations in such databases should be carefully scrutinized. The authors predict that their screening test could be made faster and more cost-effective with the advent of microdroplet polymerase chain reaction and third-generation sequencing technologies. Their study provides a proof of concept that it should be possible to introduce preconception carrier screening for many recessive pediatric disease mutations as long as the disease genes are known. Many social, legal, and societal issues need to be addressed before preconception carrier screening can be made available for the general population, and cost is still a big consideration. However, this methodology could also be applied for comprehensive screening of newborns and would allow early diagnosis and intervention for a variety of Mendelian diseases. Although it may be some time before preconception carrier testing enters the community setting, physicians, patients, parents, and genetic counselors need to discuss the impact and implications of this new technology. Of 7028 disorders with suspected Mendelian inheritance, 1139 are recessive and have an established molecular basis. Although individually uncommon, Mendelian diseases collectively account for ~20% of infant mortality and ~10% of pediatric hospitalizations. Preconception screening, together with genetic counseling of carriers, has resulted in remarkable declines in the incidence of several severe recessive diseases including Tay-Sachs disease and cystic fibrosis. However, extension of preconception screening to most severe disease genes has hitherto been impractical. Here, we report a preconception carrier screen for 448 severe recessive childhood diseases. Rather than costly, complete sequencing of the human genome, 7717 regions from 437 target genes were enriched by hybrid capture or microdroplet polymerase chain reaction, sequenced by next-generation sequencing (NGS) to a depth of up to 2.7 gigabases, and assessed with stringent bioinformatic filters. At a resultant 160× average target coverage, 93% of nucleotides had at least 20× coverage, and mutation detection/genotyping had ~95% sensitivity and ~100% specificity for substitution, insertion/deletion, splicing, and gross deletion mutations and single-nucleotide polymorphisms. In 104 unrelated DNA samples, the average genomic carrier burden for severe pediatric recessive mutations was 2.8 and ranged from 0 to 7. The distribution of mutations among sequenced samples appeared random. Twenty-seven percent of mutations cited in the literature were found to be common polymorphisms or misannotated, underscoring the need for better mutation databases as part of a comprehensive carrier testing strategy. Given the magnitude of carrier burden and the lower cost of testing compared to treating these conditions, carrier screening by NGS made available to the general population may be an economical way to reduce the incidence of and ameliorate suffering associated with severe recessive childhood disorders.

Development of Personalized Tumor Biomarkers Using Massively Parallel Sequencing

Rapid detection of specific aberrant rearrangements in tumors from individuals yields a well-poised technological advance toward personalized oncology. PARE Personalizes Cancer Genetics A diagnosis of cancer shatters the view of the world in an individual’s mind. A world that once moved as comfortably as the pace of one’s own life suddenly moves all too quickly. The individual starts asking questions and searching for possible remedies, and soon learns about the shockingly slow pace of successful cancer research. In the case of solid tumors, conventional surgical excision blanketed with “one size fits all” drug treatments simply fails to be universally effective in the long term. Cancer research has begun to shift to a more focused, personal approach that involves tailoring therapies directly to the complexity inherent in each individual—an area that holds considerable promise. But the differences among individuals are not the only layer of complexity hindering effective treatment. The intrinsic differences that accumulate over the course of tumor progression among similar tumor types hold the key to unlocking a truly personal remedy, a barcode, to cancer. Now, Leary et al. make use of a massively parallel sequencing technique—personalized analysis of rearranged ends (PARE)—to home in on the unique DNA rearrangements present in tumors that differ from the rearrangements present in nontumor DNA from a small subset of individuals. They provide evidence for a highly sensitive, reliable, and cost-effective method, a foundation from which the annotation of large numbers of such tumor signatures will yield a personal cancer code. In an arena that takes small steps, PARE offers a leap forward in the clinical management and treatment of solid tumors, revealing true biomarkers that enable monitoring of individual tumor progression, tailoring of response to therapeutic treatment, and identification of residual disease at a level previously undetectable by current methods. Clinical management of human cancer is dependent on the accurate monitoring of residual and recurrent tumors. The evaluation of patient-specific translocations in leukemias and lymphomas has revolutionized diagnostics for these diseases. We have developed a method, called personalized analysis of rearranged ends (PARE), which can identify translocations in solid tumors. Analysis of four colorectal and two breast cancers with massively parallel sequencing revealed an average of nine rearranged sequences (range, 4 to 15) per tumor. Polymerase chain reaction with primers spanning the breakpoints was able to detect mutant DNA molecules present at levels lower than 0.001% and readily identified mutated circulating DNA in patient plasma samples. This approach provides an exquisitely sensitive and broadly applicable approach for the development of personalized biomarkers to enhance the clinical management of cancer patients.

Rapid whole-genome mutational profiling using next-generation sequencing technologies.

Forward genetic mutational studies, adaptive evolution, and phenotypic screening are powerful tools for creating new variant organisms with desirable traits. However, mutations generated in the process cannot be easily identified with traditional genetic tools. We show that new high-throughput, massively parallel sequencing technologies can completely and accurately characterize a mutant genome relative to a previously sequenced parental (reference) strain. We studied a mutant strain of Pichia stipitis, a yeast capable of converting xylose to ethanol. This unusually efficient mutant strain was developed through repeated rounds of chemical mutagenesis, strain selection, transformation, and genetic manipulation over a period of seven years. We resequenced this strain on three different sequencing platforms. Surprisingly, we found fewer than a dozen mutations in open reading frames. All three sequencing technologies were able to identify each single nucleotide mutation given at least 10-15-fold nominal sequence coverage. Our results show that detecting mutations in evolved and engineered organisms is rapid and cost-effective at the whole-genome level using new sequencing technologies. Identification of specific mutations in strains with altered phenotypes will add insight into specific gene functions and guide further metabolic engineering efforts.

Genome Sequencing Highlights the Dynamic Early History of Dogs

To identify genetic changes underlying dog domestication and reconstruct their early evolutionary history, we generated high-quality genome sequences from three gray wolves, one from each of the three putative centers of dog domestication, two basal dog lineages (Basenji and Dingo) and a golden jackal as an outgroup. Analysis of these sequences supports a demographic model in which dogs and wolves diverged through a dynamic process involving population bottlenecks in both lineages and post-divergence gene flow. In dogs, the domestication bottleneck involved at least a 16-fold reduction in population size, a much more severe bottleneck than estimated previously. A sharp bottleneck in wolves occurred soon after their divergence from dogs, implying that the pool of diversity from which dogs arose was substantially larger than represented by modern wolf populations. We narrow the plausible range for the date of initial dog domestication to an interval spanning 11–16 thousand years ago, predating the rise of agriculture. In light of this finding, we expand upon previous work regarding the increase in copy number of the amylase gene (AMY2B) in dogs, which is believed to have aided digestion of starch in agricultural refuse. We find standing variation for amylase copy number variation in wolves and little or no copy number increase in the Dingo and Husky lineages. In conjunction with the estimated timing of dog origins, these results provide additional support to archaeological finds, suggesting the earliest dogs arose alongside hunter-gathers rather than agriculturists. Regarding the geographic origin of dogs, we find that, surprisingly, none of the extant wolf lineages from putative domestication centers is more closely related to dogs, and, instead, the sampled wolves form a sister monophyletic clade. This result, in combination with dog-wolf admixture during the process of domestication, suggests that a re-evaluation of past hypotheses regarding dog origins is necessary.

New insights into the Tyrolean Iceman's origin and phenotype as inferred by whole-genome sequencing

The Tyrolean Iceman, a 5,300-year-old Copper age individual, was discovered in 1991 on the Tisenjoch Pass in the Italian part of the Ötztal Alps. Here we report the complete genome sequence of the Iceman and show 100% concordance between the previously reported mitochondrial genome sequence and the consensus sequence generated from our genomic data. We present indications for recent common ancestry between the Iceman and present-day inhabitants of the Tyrrhenian Sea, that the Iceman probably had brown eyes, belonged to blood group O and was lactose intolerant. His genetic predisposition shows an increased risk for coronary heart disease and may have contributed to the development of previously reported vascular calcifications. Sequences corresponding to ~60% of the genome of Borrelia burgdorferi are indicative of the earliest human case of infection with the pathogen for Lyme borreliosis.