Zoya Kingsbury

Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA

Cancers acquire resistance to systemic treatment as a result of clonal evolution and selection. Repeat biopsies to study genomic evolution as a result of therapy are difficult, invasive and may be confounded by intra-tumour heterogeneity. Recent studies have shown that genomic alterations in solid cancers can be characterized by massively parallel sequencing of circulating cell-free tumour DNA released from cancer cells into plasma, representing a non-invasive liquid biopsy. Here we report sequencing of cancer exomes in serial plasma samples to track genomic evolution of metastatic cancers in response to therapy. Six patients with advanced breast, ovarian and lung cancers were followed over 1–2 years. For each case, exome sequencing was performed on 2–5 plasma samples (19 in total) spanning multiple courses of treatment, at selected time points when the allele fraction of tumour mutations in plasma was high, allowing improved sensitivity. For two cases, synchronous biopsies were also analysed, confirming genome-wide representation of the tumour genome in plasma. Quantification of allele fractions in plasma identified increased representation of mutant alleles in association with emergence of therapy resistance. These included an activating mutation in PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha) following treatment with paclitaxel; a truncating mutation in RB1 (retinoblastoma 1) following treatment with cisplatin; a truncating mutation in MED1 (mediator complex subunit 1) following treatment with tamoxifen and trastuzumab, and following subsequent treatment with lapatinib, a splicing mutation in GAS6 (growth arrest-specific 6) in the same patient; and a resistance-conferring mutation in EGFR (epidermal growth factor receptor; T790M) following treatment with gefitinib. These results establish proof of principle that exome-wide analysis of circulating tumour DNA could complement current invasive biopsy approaches to identify mutations associated with acquired drug resistance in advanced cancers. Serial analysis of cancer genomes in plasma constitutes a new paradigm for the study of clonal evolution in human cancers.

A comprehensive catalogue of somatic mutations from a human cancer genome

All cancers carry somatic mutations. A subset of these somatic alterations, termed driver mutations, confer selective growth advantage and are implicated in cancer development, whereas the remainder are passengers. Here we have sequenced the genomes of a malignant melanoma and a lymphoblastoid cell line from the same person, providing the first comprehensive catalogue of somatic mutations from an individual cancer. The catalogue provides remarkable insights into the forces that have shaped this cancer genome. The dominant mutational signature reflects DNA damage due to ultraviolet light exposure, a known risk factor for malignant melanoma, whereas the uneven distribution of mutations across the genome, with a lower prevalence in gene footprints, indicates that DNA repair has been preferentially deployed towards transcribed regions. The results illustrate the power of a cancer genome sequence to reveal traces of the DNA damage, repair, mutation and selection processes that were operative years before the cancer became symptomatic.

Rapid Whole-Genome Sequencing for Genetic Disease Diagnosis in Neonatal Intensive Care Units

Rapid whole-genome sequencing of neonates can shorten time to genetic disease diagnosis and thus genetic and prognostic counseling. Speed Heals The waiting might not be the hardest part for families receiving a diagnosis in neonatal intensive care units (NICUs), but it can be destructive nonetheless. While they wait on pins and needles for their newborn baby’s diagnosis, parents anguish, nurture false hope, wrestle with feelings of guilt—and all the while, treatment and counseling are delayed. Now, Saunders et al. describe a method that uses whole-genome sequencing (WGS) to achieve a differential diagnosis of genetic disorders in 50 hours rather than the 4 to 6 weeks. Many of the ~3,500 genetic diseases of known cause manifest symptoms during the first 28 days of life, but full clinical symptoms might not be evident in newborns. Genetic screens performed on newborns are rapid, but are designed to unearth only a few genetic disorders, and serial gene sequencing is too slow to be clinically useful. Together, these complicating factors lead to the administration of treatments based on nonspecific or obscure symptoms, which can be unhelpful or dangerous. Often, either death or release from the hospital occurs before the diagnosis is made. The new WGS protocol cuts analysis time by using automated bioinformatic analysis. Using their newly developed protocol, the authors performed retrospective 50-hour WGS to confirm, in two children, known molecular diagnoses that had been made using other methods. Next, prospective WGS revealed a molecular diagnosis of a BRAT1-related syndrome in one newborn; identified the causative mutation in a baby with epidermolysis bullosa; ruled out the presence of defects in candidate genes in a third infants; and, in a pedigree, pinpointed BCL9L as a new recessive gene (HTX6) that gives rise to visceral heterotaxy—the abnormal arrangement of organs in the chest and abdominal cavities. WGS of parents or affected siblings helped to speed up the identification of disease genes in the prospective cases. These findings strengthen the notion that WGS can shorten the differential diagnosis process and quicken to move toward targeted treatment and genetic and prognostic counseling. The authors note that the speed and cost of WGS continues to rise and fall, respectively. However, fast WGS is clinically useful when coupled with fast and affordable methods of analysis. Monogenic diseases are frequent causes of neonatal morbidity and mortality, and disease presentations are often undifferentiated at birth. More than 3500 monogenic diseases have been characterized, but clinical testing is available for only some of them and many feature clinical and genetic heterogeneity. Hence, an immense unmet need exists for improved molecular diagnosis in infants. Because disease progression is extremely rapid, albeit heterogeneous, in newborns, molecular diagnoses must occur quickly to be relevant for clinical decision-making. We describe 50-hour differential diagnosis of genetic disorders by whole-genome sequencing (WGS) that features automated bioinformatic analysis and is intended to be a prototype for use in neonatal intensive care units. Retrospective 50-hour WGS identified known molecular diagnoses in two children. Prospective WGS disclosed potential molecular diagnosis of a severe GJB2-related skin disease in one neonate; BRAT1-related lethal neonatal rigidity and multifocal seizure syndrome in another infant; identified BCL9L as a novel, recessive visceral heterotaxy gene (HTX6) in a pedigree; and ruled out known candidate genes in one infant. Sequencing of parents or affected siblings expedited the identification of disease genes in prospective cases. Thus, rapid WGS can potentially broaden and foreshorten differential diagnosis, resulting in fewer empirical treatments and faster progression to genetic and prognostic counseling.

Monitoring chronic lymphocytic leukemia progression by whole genome sequencing reveals heterogeneous clonal evolution patterns

Chronic lymphocytic leukemia is characterized by relapse after treatment and chemotherapy resistance. Similarly, in other malignancies leukemia cells accumulate mutations during growth, forming heterogeneous cell populations that are subject to Darwinian selection and may respond differentially to treatment. There is therefore a clinical need to monitor changes in the subclonal composition of cancers during disease progression. Here, we use whole-genome sequencing to track subclonal heterogeneity in 3 chronic lymphocytic leukemia patients subjected to repeated cycles of therapy. We reveal different somatic mutation profiles in each patient and use these to establish probable hierarchical patterns of subclonal evolution, to identify subclones that decline or expand over time, and to detect founder mutations. We show that clonal evolution patterns are heterogeneous in individual patients. We conclude that genome sequencing is a powerful and sensitive approach to monitor disease progression repeatedly at the molecular level. If applied to future clinical trials, this approach might eventually influence treatment strategies as a tool to individualize and direct cancer treatment.

Genome Sequencing and Analysis of the Tasmanian Devil and Its Transmissible Cancer

Summary The Tasmanian devil (Sarcophilus harrisii), the largest marsupial carnivore, is endangered due to a transmissible facial cancer spread by direct transfer of living cancer cells through biting. Here we describe the sequencing, assembly, and annotation of the Tasmanian devil genome and whole-genome sequences for two geographically distant subclones of the cancer. Genomic analysis suggests that the cancer first arose from a female Tasmanian devil and that the clone has subsequently genetically diverged during its spread across Tasmania. The devil cancer genome contains more than 17,000 somatic base substitution mutations and bears the imprint of a distinct mutational process. Genotyping of somatic mutations in 104 geographically and temporally distributed Tasmanian devil tumors reveals the pattern of evolution and spread of this parasitic clonal lineage, with evidence of a selective sweep in one geographical area and persistence of parallel lineages in other populations. PaperClip

Multifocal clonal evolution characterized using circulating tumour DNA in a case of metastatic breast cancer

Circulating tumour DNA analysis can be used to track tumour burden and analyse cancer genomes non-invasively but the extent to which it represents metastatic heterogeneity is unknown. Here we follow a patient with metastatic ER-positive and HER2-positive breast cancer receiving two lines of targeted therapy over 3 years. We characterize genomic architecture and infer clonal evolution in eight tumour biopsies and nine plasma samples collected over 1,193 days of clinical follow-up using exome and targeted amplicon sequencing. Mutation levels in the plasma samples reflect the clonal hierarchy inferred from sequencing of tumour biopsies. Serial changes in circulating levels of sub-clonal private mutations correlate with different treatment responses between metastatic sites. This comparison of biopsy and plasma samples in a single patient with metastatic breast cancer shows that circulating tumour DNA can allow real-time sampling of multifocal clonal evolution.

Whole-genome sequencing provides new insights into the clonal architecture of Barrett's esophagus and esophageal adenocarcinoma.

The molecular genetic relationship between esophageal adenocarcinoma (EAC) and its precursor lesion, Barretts esophagus, is poorly understood. Using whole-genome sequencing on 23 paired Barretts esophagus and EAC samples, together with one in-depth Barretts esophagus case study sampled over time and space, we have provided the following new insights: (i) Barretts esophagus is polyclonal and highly mutated even in the absence of dysplasia; (ii) when cancer develops, copy number increases and heterogeneity persists such that the spectrum of mutations often shows surprisingly little overlap between EAC and adjacent Barretts esophagus; and (iii) despite differences in specific coding mutations, the mutational context suggests a common causative insult underlying these two conditions. From a clinical perspective, the histopathological assessment of dysplasia appears to be a poor reflection of the molecular disarray within the Barretts epithelium, and a molecular Cytosponge technique overcomes sampling bias and has the capacity to reflect the entire clonal architecture.

Detection of long repeat expansions from PCR-free whole-genome sequence data

Identifying large expansions of short tandem repeats (STRs), such as those that cause amyotrophic lateral sclerosis (ALS) and fragile X syndrome, is challenging for short-read whole-genome sequencing (WGS) data. A solution to this problem is an important step toward integrating WGS into precision medicine. We developed a software tool called ExpansionHunter that, using PCR-free WGS short-read data, can genotype repeats at the locus of interest, even if the expanded repeat is larger than the read length. We applied our algorithm to WGS data from 3001 ALS patients who have been tested for the presence of the C9orf72 repeat expansion with repeat-primed PCR (RP-PCR). Compared against this truth data, ExpansionHunter correctly classified all (212/212, 95% CI [0.98, 1.00]) of the expanded samples as either expansions (208) or potential expansions (4). Additionally, 99.9% (2786/2789, 95% CI [0.997, 1.00]) of the wild-type samples were correctly classified as wild type by this method with the remaining three samples identified as possible expansions. We further applied our algorithm to a set of 152 samples in which every sample had one of eight different pathogenic repeat expansions, including those associated with fragile X syndrome, Friedreichs ataxia, and Huntingtons disease, and correctly flagged all but one of the known repeat expansions. Thus, ExpansionHunter can be used to accurately detect known pathogenic repeat expansions and provides researchers with a tool that can be used to identify new pathogenic repeat expansions.

Microbial Metabolism Shifts Towards an Adverse Profile with Supplementary Iron in the TIM-2 In vitro Model of the Human Colon.

Oral iron administration in African children can increase the risk for infections. However, it remains unclear to what extent supplementary iron affects the intestinal microbiome. We here explored the impact of iron preparations on microbial growth and metabolism in the well-controlled TNOs in vitro model of the large intestine (TIM-2). The model was inoculated with a human microbiota, without supplementary iron, or with 50 or 250 μmol/L ferrous sulfate, 50 or 250 μmol/L ferric citrate, or 50 μmol/L hemin. High resolution responses of the microbiota were examined by 16S rDNA pyrosequencing, microarray analysis, and metagenomic sequencing. The metabolome was assessed by fatty acid quantification, gas chromatography-mass spectrometry (GC-MS), and 1H-NMR spectroscopy. Cultured intestinal epithelial Caco-2 cells were used to assess fecal water toxicity. Microbiome analysis showed, among others, that supplementary iron induced decreased levels of Bifidobacteriaceae and Lactobacillaceae, while it caused higher levels of Roseburia and Prevotella. Metagenomic analyses showed an enrichment of microbial motility-chemotaxis systems, while the metabolome markedly changed from a saccharolytic to a proteolytic profile in response to iron. Branched chain fatty acids and ammonia levels increased significantly, in particular with ferrous sulfate. Importantly, the metabolite-containing effluent from iron-rich conditions showed increased cytotoxicity to Caco-2 cells. Our explorations indicate that in the absence of host influences, iron induces a more hostile environment characterized by a reduction of microbes that are generally beneficial, and increased levels of bacterial metabolites that can impair the barrier function of a cultured intestinal epithelial monolayer.

Clinical whole-genome sequencing from routine formalin-fixed, paraffin-embedded specimens: pilot study for the 100,000 Genomes Project

PurposeFresh-frozen (FF) tissue is the optimal source of DNA for whole-genome sequencing (WGS) of cancer patients. However, it is not always available, limiting the widespread application of WGS in clinical practice. We explored the viability of using formalin-fixed, paraffin-embedded (FFPE) tissues, available routinely for cancer patients, as a source of DNA for clinical WGS.MethodsWe conducted a prospective study using DNAs from matched FF, FFPE, and peripheral blood germ-line specimens collected from 52 cancer patients (156 samples) following routine diagnostic protocols. We compared somatic variants detected in FFPE and matching FF samples.ResultsWe found the single-nucleotide variant agreement reached 71% across the genome and somatic copy-number alterations (CNAs) detection from FFPE samples was suboptimal (0.44 median correlation with FF) due to nonuniform coverage. CNA detection was improved significantly with lower reverse crosslinking temperature in FFPE DNA extraction (80 °C or 65 °C depending on the methods). Our final data showed somatic variant detection from FFPE for clinical decision making is possible. We detected 98% of clinically actionable variants (including 30/31 CNAs).ConclusionWe present the first prospective WGS study of cancer patients using FFPE specimens collected in a routine clinical environment proving WGS can be applied in the clinic.