Iwanka Kozarewa

Target-enrichment strategies for next-generation sequencing

We have not yet reached a point at which routine sequencing of large numbers of whole eukaryotic genomes is feasible, and so it is often necessary to select genomic regions of interest and to enrich these regions before sequencing. There are several enrichment approaches, each with unique advantages and disadvantages. Here we describe our experiences with the leading target-enrichment technologies, the optimizations that we have performed and typical results that can be obtained using each. We also provide detailed protocols for each technology so that end users can find the best compromise between sensitivity, specificity and uniformity for their particular project.

Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G+C)-biased genomes

Amplification artifacts introduced during library preparation for the Illumina Genome Analyzer increase the likelihood that an appreciable proportion of these sequences will be duplicates and cause an uneven distribution of read coverage across the targeted sequencing regions. As a consequence, these unfavorable features result in difficulties in genome assembly and variation analysis from the short reads, particularly when the sequences are from genomes with base compositions at the extremes of high or low G+C content. Here we present an amplification-free method of library preparation, in which the cluster amplification step, rather than the PCR, enriches for fully ligated template strands, reducing the incidence of duplicate sequences, improving read mapping and single nucleotide polymorphism calling and aiding de novo assembly. We illustrate this by generating and analyzing DNA sequences from extremely (G+C)-poor (Plasmodium falciparum), (G+C)-neutral (Escherichia coli) and (G+C)-rich (Bordetella pertussis) genomes.

Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer.

Noninvasive mutation tracking in plasma can detect circulating tumor DNA arising from residual micrometastatic disease and thus identify patients at high risk of recurrence. Risk of recurrence Predicting whether a cancer patient will relapse remains a formidable challenge in modern medicine. Fortunately, circulating tumor DNA (ctDNA) present in the blood may give clues on residual disease—cancer cells left behind to seed new tumors even after treatment. Garcia-Murillas et al. developed a personalized ctDNA assay based on digital polymerase chain reaction to track mutations over time in patients with early-stage breast cancer who had received apparently curative treatments, surgery, and chemotherapy. Mutation tracking in serial samples accurately predicted metastatic relapse—in several instances, months before clinical relapse (median of ~8 months). Such unprecedented early prediction could allow for intervention before the reappearance of cancer in high-risk patients. In addition, the authors were able to shed light on the genetic events driving such metastases, by massively parallel sequencing of the ctDNA, which could inform new drug-based therapies on the basis of the patients’ individual mutations. The identification of early-stage breast cancer patients at high risk of relapse would allow tailoring of adjuvant therapy approaches. We assessed whether analysis of circulating tumor DNA (ctDNA) in plasma can be used to monitor for minimal residual disease (MRD) in breast cancer. In a prospective cohort of 55 early breast cancer patients receiving neoadjuvant chemotherapy, detection of ctDNA in plasma after completion of apparently curative treatment—either at a single postsurgical time point or with serial follow-up plasma samples—predicted metastatic relapse with high accuracy [hazard ratio, 25.1 (confidence interval, 4.08 to 130.5; log-rank P < 0.0001) or 12.0 (confidence interval, 3.36 to 43.07; log-rank P < 0.0001), respectively]. Mutation tracking in serial samples increased sensitivity for the prediction of relapse, with a median lead time of 7.9 months over clinical relapse. We further demonstrated that targeted capture sequencing analysis of ctDNA could define the genetic events of MRD, and that MRD sequencing predicted the genetic events of the subsequent metastatic relapse more accurately than sequencing of the primary cancer. Mutation tracking can therefore identify early breast cancer patients at high risk of relapse. Subsequent adjuvant therapeutic interventions could be tailored to the genetic events present in the MRD, a therapeutic approach that could in part combat the challenge posed by intratumor genetic heterogeneity.

Intraclonal heterogeneity and distinct molecular mechanisms characterize the development of t(4;14) and t(11;14) myeloma.

We have used whole exome sequencing to compare a group of presentation t(4;14) with t(11;14) cases of myeloma to define the mutational landscape. Each case was characterized by a median of 24.5 exonic nonsynonymous single-nucleotide variations, and there was a consistently higher number of mutations in the t(4;14) group, but this number did not reach statistical significance. We show that the transition and transversion rates in the 2 subgroups are similar, suggesting that there was no specific mechanism leading to mutation differentiating the 2 groups. Only 3% of mutations were seen in both groups, and recurrently mutated genes include NRAS, KRAS, BRAF, and DIS3 as well as DNAH5, a member of the axonemal dynein family. The pattern of mutation in each group was distinct, with the t(4;14) group being characterized by deregulation of chromatin organization, actin filament, and microfilament movement. Recurrent RAS pathway mutations identified subclonal heterogeneity at a mutational level in both groups, with mutations being present as either dominant or minor subclones. The presence of subclonal diversity was confirmed at a single-cell level using other tumor-acquired mutations. These results are consistent with a distinct molecular pathogenesis underlying each subgroup and have important impacts on targeted treatment strategies. The Medical Research Council Myeloma IX trial is registered under ISRCTN68454111.

Analysis of ESR1 mutation in circulating tumor DNA demonstrates evolution during therapy for metastatic breast cancer.

ESR1 mutations evolve during the treatment of metastatic breast cancer. An evolving problem A large number of breast cancers express the estrogen receptor, making them susceptible to hormonal treatments. Unfortunately, these tumors can develop mutations in the estrogen receptor gene (ESR1) and become resistant to hormonal therapies that were previously effective. Schiavon et al. used three independent cohorts of breast cancer patients to demonstrate that these mutations only evolved in cases where hormonal therapy was started late in the course of the disease, after development of metastasis, and not during the initial course of treatment. If these findings are confirmed in prospective clinical trials, then they will explain why starting hormonal treatment early decreases the risk of subsequent resistance to hormonal therapy. Acquired ESR1 mutations are a major mechanism of resistance to aromatase inhibitors (AIs). We developed ultra high–sensitivity multiplex digital polymerase chain reaction assays for ESR1 mutations in circulating tumor DNA (ctDNA) and investigated the clinical relevance and origin of ESR1 mutations in 171 women with advanced breast cancer. ESR1 mutation status in ctDNA showed high concordance with contemporaneous tumor biopsies and was accurately assessed in samples shipped at room temperature in preservative tubes. ESR1 mutations were found exclusively in estrogen receptor–positive breast cancer patients previously exposed to AI. Patients with ESR1 mutations had a substantially shorter progression-free survival on subsequent AI-based therapy [hazard ratio, 3.1; 95% confidence interval (CI), 1.9 to 23.1; P = 0.0041]. ESR1 mutation prevalence differed markedly between patients who were first exposed to AI during the adjuvant and metastatic settings [5.8% (3 of 52) versus 36.4% (16 of 44), respectively; P = 0.0002]. In an independent cohort, ESR1 mutations were identified in 0% (0 of 32; 95% CI, 0 to 10.9) tumor biopsies taken after progression on adjuvant AI. In a patient with serial sampling, ESR1 mutation was selected during metastatic AI therapy to become the dominant clone in the cancer. ESR1 mutations can be robustly identified with ctDNA analysis and predict for resistance to subsequent AI therapy. ESR1 mutations are rarely acquired during adjuvant AI but are commonly selected by therapy for metastatic disease, providing evidence that mechanisms of resistance to targeted therapy may be substantially different between the treatment of micrometastatic and overt metastatic cancer.

Whole genome sequencing of matched primary and metastatic acral melanomas

Next generation sequencing has enabled systematic discovery of mutational spectra in cancer samples. Here, we used whole genome sequencing to characterize somatic mutations and structural variation in a primary acral melanoma and its lymph node metastasis. Our data show that the somatic mutational rates in this acral melanoma sample pair were more comparable to the rates reported in cancer genomes not associated with mutagenic exposure than in the genome of a melanoma cell line or the transcriptome of melanoma short-term cultures. Despite the perception that acral skin is sun-protected, the dominant mutational signature in these samples is compatible with damage due to ultraviolet light exposure. A nonsense mutation in ERCC5 discovered in both the primary and metastatic tumors could also have contributed to the mutational signature through accumulation of unrepaired dipyrimidine lesions. However, evidence of transcription-coupled repair was suggested by the lower mutational rate in the transcribed regions and expressed genes. The primary and the metastasis are highly similar at the level of global gene copy number alterations, loss of heterozygosity and single nucleotide variation (SNV). Furthermore, the majority of the SNVs in the primary tumor were propagated in the metastasis and one nonsynonymous coding SNV and one splice site mutation appeared to arise de novo in the metastatic lesion.

Secondary mutations in BRCA2 associated with clinical resistance to a PARP inhibitor

PARP inhibitors (PARPi) for the treatment of BRCA1 or BRCA2 deficient tumours are currently the focus of seminal clinical trials exploiting the concept of synthetic lethality. Although clinical resistance to PARPi has been described, the mechanism underlying this has not been elucidated. Here, we investigate tumour material from patients who had developed resistance to the PARPi olaparib, subsequent to showing an initial clinical response. Massively parallel DNA sequencing of treatment‐naive and post‐olaparib treatment biopsies identified tumour‐specific BRCA2 secondary mutations in olaparib‐resistant metastases. These secondary mutations restored full‐length BRCA2 protein, and most likely cause olaparib resistance by re‐establishing BRCA2 function in the tumour cells. Copyright

Circadian Clock Components Regulate Entry and Affect Exit of Seasonal Dormancy as Well as Winter Hardiness in Populus Trees

This study addresses the role of the circadian clock in the seasonal growth cycle of trees: growth cessation, bud set, freezing tolerance, and bud burst. Populus tremula × Populus tremuloides (Ptt) LATE ELONGATED HYPOCOTYL1 (PttLHY1), PttLHY2, and TIMING OF CAB EXPRESSION1 constitute regulatory clock components because down-regulation by RNA interference of these genes leads to altered phase and period of clock-controlled gene expression as compared to the wild type. Also, both RNA interference lines show about 1-h-shorter critical daylength for growth cessation as compared to the wild type, extending their period of growth. During winter dormancy, when the diurnal variation in clock gene expression stops altogether, down-regulation of PttLHY1 and PttLHY2 expression compromises freezing tolerance and the expression of C-REPEAT BINDING FACTOR1, suggesting a role of these genes in cold hardiness. Moreover, down-regulation of PttLHY1 and PttLHY2 causes a delay in bud burst. This evidence shows that in addition to a role in daylength-controlled processes, PttLHY plays a role in the temperature-dependent processes of dormancy in Populus such as cold hardiness and bud burst.

Genome-wide Profiling of Genetic Synthetic Lethality Identifies CDK12 as a Novel Determinant of PARP1/2 Inhibitor Sensitivity

Small-molecule inhibitors of PARP1/2, such as olaparib, have been proposed to serve as a synthetic lethal therapy for cancers that harbor BRCA1 or BRCA2 mutations. Indeed, in clinical trials, PARP1/2 inhibitors elicit sustained antitumor responses in patients with germline BRCA gene mutations. In hypothesizing that additional genetic determinants might direct use of these drugs, we conducted a genome-wide synthetic lethal screen for candidate olaparib sensitivity genes. In support of this hypothesis, the set of identified genes included known determinants of olaparib sensitivity, such as BRCA1, RAD51, and Fanconis anemia susceptibility genes. In addition, the set included genes implicated in established networks of DNA repair, DNA cohesion, and chromatin remodeling, none of which were known previously to confer sensitivity to PARP1/2 inhibition. Notably, integration of the list of candidate sensitivity genes with data from tumor DNA sequencing studies identified CDK12 deficiency as a clinically relevant biomarker of PARP1/2 inhibitor sensitivity. In models of high-grade serous ovarian cancer (HGS-OVCa), CDK12 attenuation was sufficient to confer sensitivity to PARP1/2 inhibition, suppression of DNA repair via homologous recombination, and reduced expression of BRCA1. As one of only nine genes known to be significantly mutated in HGS-OVCa, CDK12 has properties that should confirm interest in its use as a biomarker, particularly in ongoing clinical trials of PARP1/2 inhibitors and other agents that trigger replication fork arrest.

Genome-wide functional screen identifies a compendium of genes affecting sensitivity to tamoxifen

Therapies that target estrogen signaling have made a very considerable contribution to reducing mortality from breast cancer. However, resistance to tamoxifen remains a major clinical problem. Here we have used a genome-wide functional profiling approach to identify multiple genes that confer resistance or sensitivity to tamoxifen. Combining whole-genome shRNA screening with massively parallel sequencing, we have profiled the impact of more than 56,670 RNA interference reagents targeting 16,487 genes on the cellular response to tamoxifen. This screen, along with subsequent validation experiments, identifies a compendium of genes whose silencing causes tamoxifen resistance (including BAP1, CLPP, GPRC5D, NAE1, NF1, NIPBL, NSD1, RAD21, RARG, SMC3, and UBA3) and also a set of genes whose silencing causes sensitivity to this endocrine agent (C10orf72, C15orf55/NUT, EDF1, ING5, KRAS, NOC3L, PPP1R15B, RRAS2, TMPRSS2, and TPM4). Multiple individual genes, including NF1, a regulator of RAS signaling, also correlate with clinical outcome after tamoxifen treatment.