Mutant Molecules Research Articles

Ultraspecific and Amplification-Free Quantification of Mutant DNA by Single-Molecule Kinetic Fingerprinting.

Conventional techniques for detecting rare DNA sequences require many cycles of PCR amplification for high sensitivity and specificity, potentially introducing significant biases and errors. While amplification-free methods exist, they rarely achieve the ability to detect single molecules, and their ability to discriminate between single-nucleotide variants is often dictated by the specificity limits of hybridization thermodynamics. Here we show that a direct detection approach using single-molecule kinetic fingerprinting can surpass the thermodynamic discrimination limit by 3 orders of magnitude, with a dynamic range of up to 5 orders of magnitude with optional super-resolution analysis. This approach detects mutations as subtle as the drug-resistance-conferring cancer mutation EGFR T790M (a single C → T substitution) with an estimated specificity of 99.99999%, surpassing even the leading PCR-based methods and enabling detection of 1 mutant molecule in a background of at least 1 million wild-type molecules. This level of specificity revealed rare, heat-induced cytosine deamination events that introduce false positives in PCR-based detection, but which can be overcome in our approach through milder thermal denaturation and enzymatic removal of damaged nucleobases.

Validation Strategy for Ultrasensitive Mutation Detection.

Ultrasensitive detection of low-abundance DNA point mutations is a challenging molecular biology problem, because nearly identical mutant and wild-type molecules exhibit crosstalk. Reliable ultrasensitive point mutation detection will facilitate early detection of cancer and therapeutic monitoring of cancer patients. The objective of this study was to develop a method to correct errors in low-level cell line mixes. We tested sample mixes with digital-droplet PCR (ddPCR) and next-generation sequencing. We introduced two corrections: baseline variant allele frequency (VAF) in the parental cell line was used to correct for copy number variation; and haplotype counting was used to correct errors in cell counting and pipetting. We found ddPCR to have better correlation for detecting low-level mutations without applying any correction (R2 = 0.80) and be more linear after introducing both corrections (R2 = 0.99). The VAF correction was found to be more significant than haplotype correction. It is imperative that various technologies be evaluated against each other and laboratories be provided with defined quality control samples for proficiency testing.

Anti-replicative recombinant 5S rRNA molecules can modulate the mtDNA heteroplasmy in a glucose-dependent manner.

Mutations in mitochondrial DNA are an important source of severe and incurable human diseases. The vast majority of these mutations are heteroplasmic, meaning that mutant and wild-type genomes are present simultaneously in the same cell. Only a very high proportion of mutant mitochondrial DNA (heteroplasmy level) leads to pathological consequences. We previously demonstrated that mitochondrial targeting of small RNAs designed to anneal with mutant mtDNA can decrease the heteroplasmy level by specific inhibition of mutant mtDNA replication, thus representing a potential therapy. We have also shown that 5S ribosomal RNA, partially imported into human mitochondria, can be used as a vector to deliver anti-replicative oligoribonucleotides into human mitochondria. So far, the efficiency of cellular expression of recombinant 5S rRNA molecules bearing therapeutic insertions remained very low. In the present study, we designed new versions of anti-replicative recombinant 5S rRNA targeting a large deletion in mitochondrial DNA which causes the KSS syndrome, analyzed their specific annealing to KSS mitochondrial DNA and demonstrated their import into mitochondria of cultured human cells. To obtain an increased level of the recombinant 5S rRNA stable expression, we created transmitochondrial cybrid cell line bearing a site for Flp-recombinase and used this system for the recombinase-mediated integration of genes coding for the anti-replicative recombinant 5S rRNAs into nuclear genome. We demonstrated that stable expression of anti-replicative 5S rRNA versions in human transmitochondrial cybrid cells can induce a shift in heteroplasmy level of KSS mutation in mtDNA. This shift was directly dependent on the level of the recombinant 5S rRNA expression and the sequence of the anti-replicative insertion. Quantification of mtDNA copy number in transfected cells revealed the absence of a non-specific effect on wild type mtDNA replication, indicating that the decreased proportion between mutant and wild type mtDNA molecules is not a consequence of a random repopulation of depleted pool of mtDNA genomes. The heteroplasmy change could be also modulated by cell growth conditions, namely increased by cells culturing in a carbohydrate-free medium, thus forcing them to use oxidative phosphorylation and providing a selective advantage for cells with improved respiration capacities. We discuss the advantages and limitations of this approach and propose further development of the anti-replicative strategy based on the RNA import into human mitochondria.

The Role of Proteasome Inhibitor MG132 in 2,4-Dinitrofluorobenzene-Induced Atopic Dermatitis in NC/Nga Mice

Background: Although immunosuppressants for therapy of atopic dermatitis (AD) are still being sought, proteasome inhibitors are also potential candidates for the treatment of AD. Proteasome inhibitors exert various effects by blocking proteasomal degradation and help regulate processes such as apoptosis induction via caspase-9, cell cycle progression via cyclins, NF-κB inactivation via IκB, and downregulation of antigen cross-presentation. The cells targeted by proteasome inhibitors are therefore activated cells undergoing proliferation or differentiation, and antigen-presenting cells carrying out protein degradation. Objectives: This study investigated the therapeutic effects and side effects of a proteasome inhibitor, MG132, on the treatment of AD. Methods: AD-like disease in NC/Nga mice housed under specific pathogen-free conditions was induced by repeated application of 2,4-dinitrofluorobenzene (DNFB). Disease progression was evaluated by inflammation score, histopathology, and serum IgE level, and the effects of systemic MG132 administration were investigated. The percentages and absolute numbers for each population of Th1, Th2, and Th17 cells in the axillary lymph nodes were analyzed by flow cytometry. Results: DNFB application increased the expression of a unique major histocompatibility complex class I mutant molecule D/L^dm7 in dendritic cells (DCs), and increased Th1 and Th17 cells in NC/Nga mice. In vivo MG132 administration to NC/Nga mice with DNFB-induced dermatitis reduced Th17 cells but maintained the level of Th1 cells, resulting in the alleviation of dermatitis lesions by decreasing both serum IgE hyperproduction and mast cell migration. To understand the mechanisms maintaining Th1 cell levels following in vivo MG132-administration, we focused on the role of proteasomes regulating D/L^dm7 expression. Interestingly, 20S proteasome activity was higher in NC/Nga DCs than in BALB/c DCs. In vitro MG132 administration partially increased D/L^dm7 expression in a dose-dependent manner during DC maturation, and induced IFN-γ production from autoreactive CD8+ T cells but not from CD4+ T cells following coculturing with D/L^dm7-upregulated DCs. Conclusion: Although MG132 administration temporarily alleviated AD pathogenesis in NC/Nga mice, prolonged MG132 treatment may result in immunopathogenesis leading to chronic AD due to its side effect of maintaining Th1 levels via autoreactive CD8+ T cells.

Epiphyseal growth plate architecture is unaffected by early postnatal activation of the expression of R992C collagen II mutant.

Spondyloepiphyseal dysplasia (SED) exemplifies a group of heritable diseases caused by mutations in collagenous proteins of the skeletal system. Its main feature is altered skeletal growth. Pathomechanisms of SED include: changes in the stability of collagen II molecules, inability to form proper collagen fibrils, excessive intracellular retention of mutant molecules, and endoplasmic reticulum stress. The complexity of this pathomechanism presents a challenge for designing therapies for SED. Our earlier research tested whether such therapies only succeed when applied during a limited window of development. Here, employing an inducible mouse model of SED caused by the R992C mutation in collagen II, we corroborate our earlier observations that a therapy must be applied at the prenatal or early postnatal stages of skeletal growth in order to be successful. Moreover, we demonstrate that blocking the expression of the R992C collagen II mutant at the early prenatal stages leads to long-term positive effects. Although, we could not precisely mark the start of the expression of the mutant, these effects are not significantly changed by switching on the mutant production at the early postnatal stages. By demonstrating the need for early therapeutic interventions, our study provides, for the first time, empirically-based directions for designing effective therapies for SED and, quite likely, for other skeletal dysplasias caused by mutations in key macromolecules of the skeletal system.

PD56-03 DETECTION OF PRIVATE CLONAL MUTATIONS IN LOCALISED PROSTATE CANCER FROM RARE MOLECULES OF CIRCULATING TUMOUR DNA

You have accessJournal of UrologyProstate Cancer: Markers II1 Apr 2018PD56-03 DETECTION OF PRIVATE CLONAL MUTATIONS IN LOCALISED PROSTATE CANCER FROM RARE MOLECULES OF CIRCULATING TUMOUR DNA Keval Patel, Charlie Massie, Francesco Marass, James Morris, Andy Lynch, Florent Mouliere, David Neal, Vincent Gnanapragasam, Tim Forshew, and Nitzan Rosenfeld Keval PatelKeval Patel More articles by this author , Charlie MassieCharlie Massie More articles by this author , Francesco MarassFrancesco Marass More articles by this author , James MorrisJames Morris More articles by this author , Andy LynchAndy Lynch More articles by this author , Florent MouliereFlorent Mouliere More articles by this author , David NealDavid Neal More articles by this author , Vincent GnanapragasamVincent Gnanapragasam More articles by this author , Tim ForshewTim Forshew More articles by this author , and Nitzan RosenfeldNitzan Rosenfeld More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2635AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate cancer (PCa) is the most common solid tumour in men. Current methods of distinguishing between indolent and aggressive PCa can be inaccurate due to PCa heterogeneity and multi-focality. To compensate, many centres now take upwards of 20 biopsies of the prostate in one sitting, and monitor men with further biopsies to ensure that the disease is not progressing. There is therefore a need for a non-invasive biomarker than can monitor tumour heterogeneity and aggression. Circulating-tumour DNA (ctDNA) represents an exciting opportunity to monitor cancer status non-invasively. We developed a method for analysis of multiple genomic regions whilst retaining sensitivity for detection of individual mutant molecules, using multiplex replicate dilution sequencing, or MRD-Seq. METHODS MRD-Seq distributes a DNA sample into multiple replicate PCR reactions, where each reaction contains a small number of initial template molecules. As a result, the detection of a single mutant molecule generates a signal that is clearly discernible above the background noise. Dilutions series determined the limit of detection (0.09% AF) and background noise (0.02%AF). To assess ctDNA detection in the context of intra-tumoural heterogeneity, prostatectomy specimens and pre-operative plasma was obtained from 2 men with localised but multifocal PCa, with appropriate ethical approval (MREC:01/4/061). Whole genome sequencing of spatially 5-7 distinct regions of prostate cancer for each man was used to design probes to interrogate private mutations. RESULTS Plasma MRD-Seq demonstrated a mutant AF of 0.9% and 0.4% in the 2 men from ~1,000 genomic equivalent copies input. MRD-Seq detected the presence of private mutations from multiple private clones in the plasma, with evidence that more aggressive tumour regions (according to Gleason Grade) were over-represented in the plasma. CONCLUSIONS This proof of principle study suggests that ctDNA is detectable in the plasma of localised PCa patients and could be used to monitor men on active surveillance and identify aggressive changes at an early stage. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e1062-e1063 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Keval Patel More articles by this author Charlie Massie More articles by this author Francesco Marass More articles by this author James Morris More articles by this author Andy Lynch More articles by this author Florent Mouliere More articles by this author David Neal More articles by this author Vincent Gnanapragasam More articles by this author Tim Forshew More articles by this author Nitzan Rosenfeld More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Biochemical Chgaracterization of Recombinant Baculovirus 373 K/E p53 Protein

In this study, we expressed and purified the recombinant baculovirus 373 K/E p53 protein in a baculovirus expression system to characterize this mutant and compare it with wild type p53. Gel- filtration chromatography and chemical cross-linking experiments indicated that purified recombinant baculovirus 373 K/E p53 protein assembles into multimeric forms ranging from tetramers to polymers. Gel-mobility shift assays and protein-DNA cross-linking studies demonstrated that the recombinant protein binds, to a consensus DNA target as a dimer but that additional p53 mutant molecules may then associate with the preformed p53-dimer-DNA complexes to form a larger p53_DNA complexes. These observations suggest that the p53 mutant tetramers and polymers that forms the minimal p53 mutant complex in solution dissociated upon DNA binding to form p53 mutant dimmer DNA complexes. The DNA binding activity of this mutant was then investigated using electrophoretic mobility shift assays as well as supershift assay with anti-p53 antibodies. Binding of the anti-p53 antibody PAb421to the oligomerization promoting domain on p53 stimulated the sequential formation of both the p53_dimer DNA and larger p53-DNA complexes

Is There Still Any Role for Oxidative Stress in Mitochondrial DNA-Dependent Aging?

Recent deep sequencing data has provided compelling evidence that the spectrum of somatic point mutations in mitochondrial DNA (mtDNA) in aging tissues lacks G > T transversion mutations. This fact cannot, however, be used as an argument for the missing contribution of reactive oxygen species (ROS) to mitochondria-related aging because it is probably caused by the nucleotide selectivity of mitochondrial DNA polymerase γ (POLG). In contrast to point mutations, the age-dependent accumulation of mitochondrial DNA deletions is, in light of recent experimental data, still explainable by the segregation of mutant molecules generated by the direct mutagenic effects of ROS (in particular, of HO· radicals formed from H2O2 by a Fenton reaction). The source of ROS remains controversial, because the mitochondrial contribution to tissue ROS production is probably lower than previously thought. Importantly, in the discussion about the potential role of oxidative stress in mitochondria-dependent aging, ROS generated by inflammation-linked processes and the distribution of free iron also require careful consideration.

In Vitro Mutagenesis Using Double-Stranded DNA Templates: Selection of Mutants with DpnI

In this protocol, two oligonucleotides are used to prime DNA synthesis by a high-fidelity polymerase on a denatured plasmid template. The two oligonucleotides both contain the desired mutation and have the same starting and ending positions on opposite strands of the plasmid DNA. The entire lengths of both strands of the plasmid DNA are amplified in a linear fashion during several rounds of thermal cycling, generating a mutated plasmid containing staggered nicks on opposite strands. Because of the amount of template DNA used in the amplification reaction, the background of transformed colonies containing wild-type plasmid DNA can be quite high unless steps are taken to enrich for mutant molecules. In this protocol, the products of the linear amplification reaction are treated with the restriction enzyme DpnI, which specifically cleaves fully methylated GMe6ATC sequences. DpnI will therefore digest the bacterially generated DNA used as template for amplification, but it will not digest DNA synthesized during the course of the reaction in vitro. DpnI-resistant molecules, which are rich in the desired mutants, are recovered by transforming E. coli cells to antibiotic resistance. Because the method works well with virtually any plasmid of moderate size (<7 kb), it can be used to introduce mutations directly into full-length cDNAs and eliminates the need for subcloning into specialized vectors.

Resolving the Enigma of the Clonal Expansion of mtDNA Deletions.

Mitochondria are cell organelles that are special since they contain their own genetic material in the form of mitochondrial DNA (mtDNA). Damage and mutations of mtDNA are not only involved in several inherited human diseases but are also widely thought to play an important role during aging. In both cases, point mutations or large deletions accumulate inside cells, leading to functional impairment once a certain threshold has been surpassed. In most cases, it is a single type of mutant that clonally expands and out-competes the wild type mtDNA, with different mutant molecules being amplified in different cells. The challenge is to explain where the selection advantage for the accumulation comes from, why such a large range of different deletions seem to possess this advantage, and how this process can scale to species with different lifespans such as those of rats and man. From this perspective, we provide an overview of current ideas, present an update of our own proposal, and discuss the wider relevance of the phenomenon for aging.

Abstract LB-B03: Exosome-based Detection of EGFR T790M in Plasma from Non-Small Cell Lung Cancer Patients

Abstract About 60% of non-small cell lung cancer (NSCLC) patients develop resistance to targeted epidermal growth factor receptor (EGFR) inhibitor therapy through the EGFR T790M mutation. Although these patients respond well to third generation tyrosine kinase inhibitors like osimertinib, obtaining tissue biopsies for mutation profiling poses risks and is often not feasible. Detecting the mutation in a liquid biopsy from plasma has emerged as a viable alternative to biopsy, however existing liquid biopsy tests using cell-free DNA (cfDNA) often suffer from low sensitivity due to the low copy number of mutant molecules in circulation. As an example, the Food and Drug Adminstration (FDA) approved companion diagnostic for T790M mutation status in plasma (cobas® EGFR mutation Test version 2) only has a 58% sensitivity at a specificity of 80%. In this study we aimed to achieve a higher performance by combining the mutated alleles on cfDNA as well as exosomal RNA and DNA (exoNA) . ExoNA and cfDNA were extracted from the plasma of NSCLC patients with biopsy-confirmed T790M-positive (N = 102) or T790M-negative (N = 108) samples. The T790M mutation status in plasma exoNA and cfDNA was determined using an analytically validated allele-specific qPCR assay in a CLIA laboratory. The training cohort consisted of 105 samples (51 positives, 54 negatives). The validation cohort consisted of 105 samples (51 positives, 54 negatives), where ~40% of the patients had intrathoracic disease(M0/M1a). Detection of the T790M mutation in exoNA and cfDNA achieved 92% sensitivity and 89% specificity when compared to tumor biopsy results in the validation cohort. We also observed a very high sensitivity (88%) in patients with intrathoracic disease (M0/M1a), patients for whom detection by liquid biopsy has been proven particularly challenging. The combination of exoNA and cfDNA for EGFR T790M detection has a higher sensitivity and specificity compared to historical cohorts using ctDNA alone. This is especially important as it could aid giving the right treatment to patients where biopsy is not available or avoid unnecessary tumor biopsies for T790M mutation testing. Citation Format: Elena Castellanos, Dominik G. Grimm, Vasisht Tadigotla, Stefan Bentink, James Hurley, John Healy, Patricia L. Neal, Mia Sher, Dan Baughman, Chris Karlovich, Mitch Raponi, Anne Krug, Mikkel Noerholm, Luis E. Raez, Johan Skog. Exosome-based Detection of EGFR T790M in Plasma from Non-Small Cell Lung Cancer Patients [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B03.

Saturation mutagenesis reveals manifold determinants of exon definition.

To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts, we conducted saturation mutagenesis of a 51-nt internal exon in a three-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high-throughput genetics, 5560 minigene molecules were assayed for splicing in human HEK293 cells. Up to 70% of mutations produced substantial (greater than twofold) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements, only a single 15-nt stem–loop showed a strong correlation with splicing, acting negatively. The in vitro formation of exon-protein complexes between the mutant molecules and proteins associated with spliceosome formation (U2AF35, U2AF65, U1A, and U1-70K) correlated with splicing efficiencies, suggesting exon definition as the step affected by most mutations. The measured relative binding affinities of dozens of human RNA binding protein domains as reported in the CISBP-RNA database were found to correlate either positively or negatively with splicing efficiency, more than could fit on the 51-nt test exon simultaneously. The large number of these functional protein binding correlations point to a dynamic and heterogeneous population of pre-mRNA molecules, each responding to a particular collection of binding proteins.

Biochemical and structural studies of mutants indicate concerted movement of the dimer interface and ligand-binding region of Mycobacterium tuberculosis pantothenate kinase.

Two point mutants and the corresponding double mutant of Mycobacterium tuberculosis pantothenate kinase have been prepared and biochemically and structurally characterized. The mutants were designed to weaken the affinity of the enzyme for the feedback inhibitor CoA. The mutants exhibit reduced activity, which can be explained in terms of their structures. The crystals of the mutants are not isomorphous to any of the previously analysed crystals of the wild-type enzyme or its complexes. The mycobacterial enzyme and its homologous Escherichia coli enzyme exhibit structural differences in their nucleotide complexes in the dimer interface and the ligand-binding region. In three of the four crystallographically independent mutant molecules the structure is similar to that in the E. coli enzyme. Although the mutants involve changes in the CoA-binding region, the dimer interface and the ligand-binding region move in a concerted manner, an observation which might be important in enzyme action. This work demonstrates that the structure of the mycobacterial enzyme can be transformed into a structure similar to that of the E. coli enzyme through minor perturbations without external influences such as those involving ligand binding.

Novel signal transducer and activator of transcription 1 mutation disrupts small ubiquitin-related modifier conjugation causing gain of function

Sumoylation is a posttranslational reversible modification of cellular proteins through the conjugation of small ubiquitin-related modifier (SUMO) and comprises an important regulator of protein function. We sought to characterize the molecular mechanism of a novel mutation at the SUMO motif on signal transducer and activator of transcription 1 (STAT1). STAT1 sequencing and functional characterization were performed in transfection experiments by using immunoblotting and immunoprecipitation in STAT1-deficient cell lines. Transcriptional response and target gene activation were also investigated in PBMCs. We identified a novel STAT1 mutation (c.2114A>T, p.E705V) within the SUMO motif (702IKTE705) in a patient with disseminated Rhodococcus species infection, Norwegian scabies, chronic mucocutaneous candidiasis, hypothyroidism, and esophageal squamous cell carcinoma. The mutation is located in the tail segment and is predicted to disrupt STAT1 sumoylation. Immunoprecipitation experiments performed in transfected cells confirmed absent STAT1 sumoylation for E705V, whereas it was present in wild-type (WT) STAT1 cells, as well as the loss-of-function mutants L706S and Y701C. Furthermore, stimulation with IFN-γ led to enhanced STAT1 phosphorylation, enhanced transcriptional activity, and target gene expression in the E705V-transfected compared with WT-transfected cells. Computer modeling of WT and mutant STAT1 molecules showed variations in the accessibility of the phosphorylation site Y701, which corresponded to the loss-of-function and gain-of-function variants. This is the first report of a mutation in the STAT1 sumoylation motif associated with clinical disease. These data reinforce sumoylation as a key posttranslational regulatory modification of STAT1 and identify a novel mechanism for gain-of-function STAT1 disease in human subjects.

Abstract 2715: Precision-designed, rapid and low-cost single molecule detection of mutations from circulating tumor DNA

Abstract Detecting and quantifying mutations from circulating tumor DNA (ctDNA) is a new approach for longitudinally monitoring cancer patients throughout the course of treatment. However, general limitations to the clinical utility of ctDNA include sample concentration, overall assay cost, and result turnaround time. We have developed a novel molecular assay that utilize single color digital droplet PCR (ddPCR) to both genotype and quantify the number of tumor derived DNA molecules in a given sample. Our assay routinely detects as few as three mutant DNA molecules per a reaction volume, can be tested efficiently for fewer than ten dollars per reaction, and generates useable mutation information within four hours. Additionally, the assay can be rapidly configured to detect different mutations specifically identified in any cancer patient. To demonstrate the single molecule sensitivity and specificity for clinically relevant hotspot mutations, we have validated the assay using multiple input sources including high quality cell line DNA, formalin-fixed paraffin embedded tissue (FFPET) DNA, and ctDNA. Our ddPCR assay utilizes a novel primer design that is not rely on fluorescent probes. The overall simplicity of assay design enables one to detect nearly any coding mutation; practically, this means that any cancer or DNA sample can be tested efficiently. We have created customized precision mutation assays for two individual cancer patients. After extracting ctDNA from 500µL of plasma we prepare a controlled mixed wild type and mutant standard curves which contain between 150-3 detectable mutant molecules of interest per reaction. We then assay the patient ctDNA sample in parallel to the controlled standard curve and generate clustering confidence intervals. We have designed precision mutation assays targeting KRAS A146V, AKT1 E17K, and TP53 R175H for Patient A as well as KRAS G12V and PIK3CA H1047R for Patient B. The results of our single-color precision mutation quantification assays support the original mutation findings from the primary tumors. In one case, from 1µL of un-amplified ctDNA equivalent to less than 0.5ng of DNA, we identified 4 mutant molecules per ddPCR reaction volume. We have verified the presence of hotspot mutations from un-amplified patient derived ctDNA and quantified the number of mutant molecules present in each ctDNA sample. This assay has many features that make it amenable to automation. We are currently developing a massively high throughput process to design assays for tens of thousands of mutations in a matter of weeks. Overall this is an extremely low cost, highly sensitive and scalable diagnostic technology. We anticipate that this technology will be a valuable tool for rapidly monitoring ctDNA longitudinally in cancer patients. Citation Format: Christina M. Wood-Bouwens, Christine Handy, Billy Lau, Hanlee Ji. Precision-designed, rapid and low-cost single molecule detection of mutations from circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2715. doi:10.1158/1538-7445.AM2017-2715

Abstract AP05: ULTRA–DEEP SEQUENCING DETECTS OVARIAN CANCER CELLS IN PERITONEAL FLUID AND REVEALS SOMATIC TP53 MUTATIONS IN NON–CANCEROUS TISSUES

Abstract High–grade serous ovarian carcinoma (HGSOC), the most common and most aggressive type of ovarian cancer, is characterized by near–universal prevalence of TP53 mutations, even at the early stages of the disease. Thus, the detection of tumor–specific TP53 mutations in clinical samples could potentially enable early cancer detection, monitoring, and screening. A main limitation, however, is the high error rate of conventional DNA sequencing technologies (estimated at 1 in 1000 nucleotides), which precludes the identification of low frequency tumor mutant molecules from technical error. Duplex Sequencing is a next–generation sequencing technology that uniquely tags each strand of a DNA molecule and quantifies mutations only when present in both strands of DNA. This reduces the error rate of sequencing to 1 in 10 million nucleotides, an unprecedented sensitivity that could enable the detection of extremely low frequency tumor mutant molecules by ultra–deep sequencing. The goal of this study was to provide proof–of–principle of the ability of Duplex Sequencing to detect extremely rare TP53 mutated cancer cells disseminated into the peritoneal cavity of women with HGSOC. We analyzed 17 peritoneal fluid samples from women with HGSOC and known TP53 tumor mutation (cases) and 20 peritoneal fluid samples from women without cancer (controls). The tumor–specific TP53 mutation was detected in matched peritoneal fluid from 94% of women with HGSOC (16/17), including 2 patients with occult tubal intraepithelial neoplasia, 7 patients with early stage HGSOC, and 8 with negative peritoneal fluid cytopathology. Tumor–specific alleles were detected as low as 1 mutant molecule per 24,737 normal genomes. Additionally, we detected extremely low–frequency TP53 mutations (median mutant fraction 1/13,139) in peritoneal fluid from nearly all patients with and without cancer (35/37). These mutations were mostly deleterious, clustered in hotspots, increased with age, and were more abundant in women with cancer than in controls. The total burden of TP53 mutations in peritoneal fluid distinguished cases from controls with 82% sensitivity (14/17) and 90% specificity (18/20). Age–associated, low frequency TP53 mutations were also found in 100% of matched peripheral blood samples from 15 women with and without ovarian cancer (none with hematologic cancer). Our results demonstrate the ability of Duplex Sequencing to detect very rare cancer cells and provide evidence of widespread, low frequency, age–associated somatic TP53 mutation in non–cancerous tissue. These mutations likely represent a premalignant mutational background that accumulates in cancer and aging. Citation Format: Krimmel JD, Schmitt MW, Kennedy SR, Harrell MI, Agnew KJ, Loeb LA, Swisher EM, Risques RA. ULTRA–DEEP SEQUENCING DETECTS OVARIAN CANCER CELLS IN PERITONEAL FLUID AND REVEALS SOMATIC TP53 MUTATIONS IN NON–CANCEROUS TISSUES [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP05.

Twoddpcr: an R/Bioconductor package and Shiny app for Droplet Digital PCR analysis.

SummaryDroplet Digital PCR (ddPCR) is a sensitive platform used to quantify specific nucleic acid molecules amplified by polymerase chain reactions. Its sensitivity makes it particularly useful for the detection of rare mutant molecules, such as those present in a sample of circulating free tumour DNA obtained from cancer patients. ddPCR works by partitioning a sample into individual droplets for which the majority contain only zero or one target molecule. Each droplet then becomes a reaction chamber for PCR, which through the use of fluorochrome labelled probes allows the target molecules to be detected by measuring the fluorescence intensity of each droplet. The technology supports two channels, allowing, for example, mutant and wild type molecules to be detected simultaneously in the same sample. As yet, no open source software is available for the automatic gating of two channel ddPCR experiments in the case where the droplets can be grouped into four clusters. Here, we present an open source R package ‘twoddpcr’, which uses Poisson statistics to estimate the number of molecules in such two channel ddPCR data. Using the Shiny framework, an accompanying graphical user interface (GUI) is also included for the package, allowing users to adjust parameters and see the results in real-time.Availability and implementationtwoddpcr is available from Bioconductor (3.5) at https://bioconductor.org/packages/twoddpcr/. A Shiny-based GUI suitable for non-R users is available as a standalone application from within the package and also as a web application at http://shiny.cruk.manchester.ac.uk/twoddpcr/.Package maintainer anthony.chiu@cruk.manchester.ac.uk

Prospects and limitations of improving skeletal growth in a mouse model of spondyloepiphyseal dysplasia caused by R992C (p.R1192C) substitution in collagen II.

Skeletal dysplasias form a group of skeletal disorders caused by mutations in macromolecules of cartilage and bone. The severity of skeletal dysplasias ranges from precocious arthropathy to perinatal lethality. Although the pathomechanisms of these disorders are generally well defined, the feasibility of repairing established aberrant skeletal tissues that developed in the presence of mutant molecules is currently unknown. Here, we employed a validated mouse model of spondyloepiphyseal dysplasia (SED) that enables temporal control of the production of the R992C (p.R1192C) collagen II mutant that causes this disease. Although in our earlier studies we determined that blocking the expression of this mutant at the early prenatal stages prevents a SED phenotype, the utility of blocking the R992C collagen II at the postnatal stages is not known. Here, by switching off the expression of R992C collagen II at various postnatal stages of skeletal development, we determined that significant improvements of cartilage and bone morphology were achieved only when blocking the production of the mutant molecules was initiated in newborn mice. Our study indicates that future therapies of skeletal dysplasias may require defining a specific time window when interventions should be applied to be successful.

In vivo severity ranking of Ras pathway mutations associated with developmental disorders

Germ-line mutations in components of the Ras/MAPK pathway result in developmental disorders called RASopathies, affecting about 1/1,000 human births. Rapid advances in genome sequencing make it possible to identify multiple disease-related mutations, but there is currently no systematic framework for translating this information into patient-specific predictions of disease progression. As a first step toward addressing this issue, we developed a quantitative, inexpensive, and rapid framework that relies on the early zebrafish embryo to assess mutational effects on a common scale. Using this assay, we assessed 16 mutations reported in MEK1, a MAPK kinase, and provide a robust ranking of these mutations. We find that mutations found in cancer are more severe than those found in both RASopathies and cancer, which, in turn, are generally more severe than those found only in RASopathies. Moreover, this rank is conserved in other zebrafish embryonic assays and Drosophila-specific embryonic and adult assays, suggesting that our ranking reflects the intrinsic property of the mutant molecule. Furthermore, this rank is predictive of the drug dose needed to correct the defects. This assay can be readily used to test the strengths of existing and newly found mutations in MEK1 and other pathway components, providing the first step in the development of rational guidelines for patient-specific diagnostics and treatment of RASopathies.

The Impact of Collisions on the Ability to Detect Rare Mutant Alleles Using Barcode-Type Next-Generation Sequencing Techniques

Barcoding techniques are used to reduce error from next-generation sequencing, with applications ranging from understanding tumor subclone populations to detecting circulating tumor DNA. Collisions occur when more than one sample molecule is tagged by the same unique identifier (UID) and can result in failure to detect very-low-frequency mutations and error in estimating mutation frequency. Here, we created computer models of barcoding technique, with and without amplification bias introduced by the UID, and analyzed the effect of collisions for a range of mutant allele frequencies (1e−6 to 0.2), number of sample molecules (10 000 to 1e7), and number of UIDs (410-414). Inability to detect rare mutant alleles occurred in 0% to 100% of simulations, depending on collisions and number of mutant molecules. Collisions also introduced error in estimating mutant allele frequency resulting in underestimation of minor allele frequency. Incorporating an understanding of the effect of collisions into experimental design can allow for optimization of the number of sample molecules and number of UIDs to minimize the negative impact on rare mutant detection and mutant frequency estimation.