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.
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.
Durvalumab is a programmed death-ligand 1 (PD-L1) inhibitor with clinical activity in advanced urothelial cancer (AUC)
. AUC is characterized by several recurrent targetable genomic alterations
. ...This study ( NCT02546661 , BISCAY) combined durvalumab with relevant targeted therapies in biomarker-selected chemotherapy-refractory AUC populations including: (1) fibroblast growth factor receptor (FGFR) inhibitors in tumors with FGFR DNA alterations (FGFRm); (2) pharmacological inhibitor of the enzyme poly-ADP ribose polymerase (PARP) in tumors with and without DNA homologous recombination repair deficiency (HRRm); and (3) TORC1/2 inhibitors in tumors with DNA alteration to the mTOR/PI3K pathway
.This trial adopted a new, biomarker-driven, multiarm adaptive design. Safety, efficacy and relevant biomarkers were evaluated. Overall, 391 patients were screened of whom 135 were allocated to one of six study arms. Response rates (RRs) ranged 9-36% across the study arms, which did not meet efficacy criteria for further development. Overall survival (OS) and progression-free survival (PFS) were similar in the combination arms and durvalumab monotherapy arm. Biomarker analysis showed a correlation between circulating plasma-based DNA (ctDNA) and tissue for FGFRm. Sequential circulating tumor DNA analysis showed that changes to FGFRm correlated with clinical outcome. Our data support the clinical activity of FGFR inhibition and durvalumab monotherapy but do not show increased activity for any of the combinations. These findings question the targeted/immune therapy approach in AUC.
Genome-wide association studies have identified more than 70 common variants that are associated with breast cancer risk. Most of these variants map to non-protein-coding regions and several map to ...gene deserts, regions of several hundred kilobases lacking protein-coding genes. We hypothesized that gene deserts harbor long-range regulatory elements that can physically interact with target genes to influence their expression. To test this, we developed Capture Hi-C (CHi-C), which, by incorporating a sequence capture step into a Hi-C protocol, allows high-resolution analysis of targeted regions of the genome. We used CHi-C to investigate long-range interactions at three breast cancer gene deserts mapping to 2q35, 8q24.21, and 9q31.2. We identified interaction peaks between putative regulatory elements ("bait fragments") within the captured regions and "targets" that included both protein-coding genes and long noncoding (lnc) RNAs over distances of 6.6 kb to 2.6 Mb. Target protein-coding genes were IGFBP5, KLF4, NSMCE2, and MYC; and target lncRNAs included DIRC3, PVT1, and CCDC26. For one gene desert, we were able to define two SNPs (rs12613955 and rs4442975) that were highly correlated with the published risk variant and that mapped within the bait end of an interaction peak. In vivo ChIP-qPCR data show that one of these, rs4442975, affects the binding of FOXA1 and implicate this SNP as a putative functional variant.
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 Fanconi's 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.
Genetic perturbation screens have the potential to dissect a wide range of cellular phenotypes. Such screens have historically been difficult in diploid mammalian cells. The recent derivation of ...haploid embryonic stem cells provides an opportunity to cause loss of function mutants with a random mutagen in a mammalian cell with a normal genetic background. We describe an approach to genetic screens that exploits the highly active piggyBac transposon in haploid mammalian cells. As an example of haploid transposon (HTP) screening, we apply this approach to identifying determinants of cancer drug toxicity and resistance. In a screen for 6-thioguanine resistance we recovered components of the DNA mismatch repair pathway, a known requirement for toxicity. In a further screen for resistance to the clinical poly(ADP-ribose) polymerase (PARP) inhibitor olaparib we recovered multiple Parp1 mutants. Our results show that olaparib toxicity to normal cells is mediated predominantly via Parp1, and suggest that the clinical side effects of olaparib may be on target. The transposon mutant libraries are stable and can be readily reused to screen other drugs. The screening protocol described has several advantages over other methods such as RNA interference: it is rapid and low cost, and mutations can be easily reverted to establish causality.
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 x 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.
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, INGS, 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.
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.
Trees are carbon dioxide sinks and major producers of terrestrial biomass with distinct seasonal growth patterns. Circadian clocks enable the coordination of physiological and biochemical temporal ...activities, optimally regulating multiple traits including growth. To dissect the clock's role in growth, we analysed Populus tremula × P. tremuloides trees with impaired clock function due to down‐regulation of central clock components. late elongated hypocotyl (lhy‐10) trees, in which expression of LHY1 and LHY2 is reduced by RNAi, have a short free‐running period and show disrupted temporal regulation of gene expression and reduced growth, producing 30–40% less biomass than wild‐type trees. Genes important in growth regulation were expressed with an earlier phase in lhy‐10, and CYCLIN D3 expression was misaligned and arrhythmic. Levels of cytokinins were lower in lhy‐10 trees, which also showed a change in the time of peak expression of genes associated with cell division and growth. However, auxin levels were not altered in lhy‐10 trees, and the size of the lignification zone in the stem showed a relative increase. The reduced growth rate and anatomical features of lhy‐10 trees were mainly caused by misregulation of cell division, which may have resulted from impaired clock function.
Trees undergo annual cycles of significant primary and secondary growths. They possess an endogenous clock that enables them to adjust growth to different seasons. This study demonstrates the importance of the circa 24‐hr (circadian) clock in sustaining growth of Populus trees under diel cycles and shows the clock times expression of CYCD3 and cell division. Notably, growth hormones are disrupted in trees with broken clocks. The clock, acting via the key clock components, LHY1 and LHY2 regulates gene expression and protein function. Reducing expression of LHY1 and LHY2 reduces levels of cytokinins, which regulate plant growth, and causes a misalignment of CYCD3 expression with cell division. Together, these changes alter the balance between growth and lignification while also reducing biomass. LHY1 and LHY2 may also promote expression of BBX19 and BBX32, whose proteins appear to be part of the mechanism regulating timing of growth. Understanding clock regulation may be valuable for breeding projects targeting these important traits.
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