Enrichment Of Mutants Research Articles

SLAM-MS: Mutation scanning of stem-loop amplicons with TaqMan probes by quantitative DNA melting analysis

DNA Melting Analysis (DMA) with a TaqMan probe covering the mutation “hot spot” is a simple, sensitive, and “closed tube” method of mutation detection. However, DMA requires asymmetric PCR to produce single-stranded amplicons capable of interacting with TaqMan probes. This makes quantitative analysis impossible owing to low amplification efficiency. Moreover, bi-strand mutation detection necessitates two independent PCRs. The SLAM-MS (Stem-Loop AMplicon Mutation Scanning) assay, in which symmetric PCR is performed using primers with 5'-universal primer sequence (UPS), has been developed to detect KRAS mutations. Some of the resulting amplicons, sense and antisense, adopt single-stranded stem-loop conformation and become unable to renature, but able to hybridize with TaqMan probes. Hybrids of stem-loops and complementary TaqMan probes are suitable for melting analysis and simultaneous bi-strand mutation scanning. In addition, the areas under the melting peaks are determined by the PeakFit software, a non-linear iterative curve fitting program, to evaluate the wild-type/mutant allele ratio. Thus, the SLAM-MS assay permits quantification of both the number of copies of the target sequence and the percentage of mutant alleles. For mutant enrichment, the SLAM-MS assay uses TaqMan probes as PCR blocking agents allowing an ~10 times higher mutation detection sensitivity than High Resolution Melting (HRM) assay.

Overexpression of a developing xylem cDNA library in transgenic poplar generates high mutation rate specific to wood formation.

SummaryWe investigated feasibility of the Full‐length complementary DNA OvereXpression (FOX) system as a mutagenesis approach in poplar, using developing xylem tissue. The main goal was to assess the overall mutation rate and if the system will increase instances of mutants affected in traits linked to the xylem tissue. Indeed, we found a high mutation rate of 17.7%, whereas 80% of all mutants were significantly affected in cellulose, lignin and/or hemicellulose. Cell wall biosynthesis is a major process occurring during xylem development. Enrichment of mutants affected in cell wall composition suggests that the tissue source for the FOX library influenced the occurrence of mutants affected in a trait linked to this tissue. Additionally, we found that FLcDNAs from mutants affected in cell wall composition were homologous to genes known to be involved in cell wall biosynthesis and most recovered FLcDNAs corresponded to genes whose native expression was highest in xylem. We characterized in detail a mutant line with increased diameter. The phenotype was caused by a poplar homolog of LONELY GUY 1 (LOG1), which encodes an enzyme in cytokinin biosynthesis and significantly increased xylem proliferation. The causative role of LOG1 in the observed phenotype was further reaffirmed by elevated cytokinin concentration in the mutant and recapitulation overexpression experiment wherein multiple independent lines phenocopied the original FOX mutant. Our experiments show that the FOX approach can be efficiently used for gene discovery and molecular interrogation of traits specific to woody perennial growth and development.

Growth-coupled evolution of phosphoketolase to improve L-glutamate production by Corynebacterium glutamicum.

The introduction of the key non-oxidative glycolytic (NOG) pathway enzyme, phosphoketolases (PKTs), into heterologous hosts can improve the yield of a variety of acetyl CoA-derived products of interest. However, the low specific activity of existing PKTs compared with that of 6-phosphofructokinase (PFK), the key EMP pathway enzyme, largely limits their potential applications. To improve PKT activity, previous attempts have focused on increasing intracellular PKT concentration via the use of strong promoters. Herein, we report the establishment of a growth-coupled evolution strategy for the enrichment and selection of PKT mutants with improved specific activity in Corynebacterium glutamicum hosts with defective PFK. Five mutants from 9 Bifidobacterium adolescentis-source PKT (BA-PKT) mutant libraries were obtained. Site-directed mutagenesis analysis revealed 11 mutant sites which contributed to improved BA-PKT specific activity. Further structural analysis revealed that the mutant sites were located far away from the enzyme active site, which makes them almost unpredictable using a rational design approach. Mutant site recombination led to the construction of a novel mutant, PKTT2A/I6T/H260Y, with Vmax 29.77 ± 1.58 U/mg and Kcat/Km 0.32 ± 0.01 s-1/mM, which corresponds to 73.27 ± 3.25% and 80.16 ± 3.38% improvements, respectively, compared with the wildtype (Vmax;17.17 ± 0.59 U/mg, Kcat/Km; 0.17 ± 0.01 s-1/mM). Expression of PKTT2A/I6T/H260 in C. glutamicum Z188 resulted in 16.67 ± 2.24% and 18.19 ± 0.53% improvement in L-glutamate titer and yield, respectively, compared with the wildtype BA-PKT. Our findings provide an efficient approach for improving the activity of PKTs. Furthermore, the novel mutants could serve as useful tools in improving the yield of L-glutamate and other acetyl CoA-associated products.

A novel parameter to predict the effects of antibiotic combinations on the development of Staphylococcus aureus resistance: in vitro model studies at subtherapeutic daptomycin and rifampicin exposures

The search for optimal predictors of anti-mutant effects remains a pressing problem in studies of antibiotic-associated bacterial resistance. To relate the emergence of bacterial resistance with the antibiotic mutant prevention concentration (MPC), a novel integral parameter – the area around the resistance threshold, i.e. MPC level (AAMPC) is proposed. The AAMPC is the algebraic sum of the area under the antibiotic concentration–time curve that is above the MPC (positive area) and the area above the concentration–time curve that is under the MPC (negative area). To assess the predictive performance of AAMPC, the enrichment of resistant Staphylococcus aureus was studied by simulating treatment with daptomycin and rifampicin alone and in combination in an in vitro dynamic model. The enhanced anti-mutant effects of the antibiotic combinations were due to lowering the negative 24-h AAMPCs. These findings suggest that a novel MPC-related parameter is a reliable predictor of mutant enrichment.

Next-generation sequencing with comprehensive bioinformatics analysis facilitates somatic mosaic APC gene mutation detection in patients with familial adenomatous polyposis

BackgroundFamilial adenomatous polyposis (FAP) is an autosomal dominant colorectal tumor characterized by numerous adenomatous colonic polyps that often lead to colon cancer. Although most patients with FAP harbored germline mutations in APC gene, it was recently recognized that patients with clinical FAP, but without detectable pathogenic mutations, could be associated with somatic mosaic APC mutation.MethodsWe reanalyzed the nest-generation sequencing (NGS) gene panel testing results of patients who were diagnosed with FAP, but did not have APC mutations, at Yonsei Cancer Prevention Center between July 2016 and March 2018. We tested several variant calling algorithms to identify low level mosaic variants. In one patient with a low frequency APC mutation, NGS analysis was performed together with endoscopic biopsy. Variant calling tools HaplotypeCaller, MuTect2, VarScan2, and Pindel were used. We also used 3′-Modified Oligonucleotides (MEMO)-PCR or conventional PCR for confirmation.ResultsAmong 28 patients with clinical suspicion of FAP but no detectable pathogenic variants of colonic polyposis associated genes, somatic mosaic pathogenic variants were identified in seven patients. The variant allele frequency ranged from 0.3 to 7.7%. These variants were mostly detected through variant caller MuTect2 and Pindel, and were further confirmed using mutant enrichment with MEMO-PCR.ConclusionsThe NGS with an adequate combination of bioinformatics tools is effective to detect low level somatic variants in a single assay. Because mosaic APC mutations are more frequent than previously thought, the presence of mosaic mutations must be considered when analyzing genetic tests of patients with FAP.

Evaluating the optimal time for amikacin administration with respect to haemodialysis using an in vitro pharmacodynamic simulation against epidemic nosocomial OXA-48 producing Klebsiella pneumoniae ST405 strains.

Bacterial viability and enrichment of resistance resulting from three different amikacin administration schedules with respect to haemodialysis (HD) were assessed against three OXA-48-producing Klebsiella pneumoniae isolated during an outbreak in a Spanish hospital. A previously described two-compartment dynamic system was used. Three possible amikacin administration schedules were simulated: (i) haemodialysis immediately after amikacin infusion (pre-HD); (ii) infusion immediately after haemodialysis (post-HD); and (iii) infusion at 50% interdialytic period. Amikacin standard dose (SD) and double dose (DD) were simulated for each schedule. Values of Cmax/MIC, Cmax/MPC (mutant prevention concentration), AUC0-48h/MIC, AUC0-48h/MPC and %TMSW (percentage of time that the concentration was inside the mutant selection window) were determined with experimental data and were correlated with the area under the bacterial killing curve of the total population and the resistant subpopulation. Both with SD and DD, the pre-HD schedule resulted in increases at 48h in bacterial counts of the total population at the expense of enrichment of pre-existing resistant subpopulations from 12h onwards for all strains. The estimated %TMSW that prevented enrichment of resistant mutants was <61.5%. The AUC0-48h/MPC (with values of ≈40 being associated with countering of increases in resistant subpopulations) was better than the %TMSW as a predictive parameter. This study showed that the longest times concentrations were above the MPC (i.e. highest AUC0-48h/MPC, lowest %TMSW), the lowest enrichment of resistant subpopulations. This implies use of the highest possible amikacin dose (limited by toxicity) and post-HD as the best administration schedule.

A novel mutant-allele capture method for enrichment of low abundant cancer mutants.

e14632 Background: High sensitivity detection of cancer mutants can facilitate cancer diagnosis, treatment selection, and disease monitoring. Improved methods to detect low abundance of cancer driver mutants are needed. Methods: We developed a novel method, termed PEAC, with a super high sensitivity to detect low abundant mutant from cancer driver genes through mutant capturing followed by Sanger sequencing. Circulating free DNA (cfDNA) standards containing 20 ng of 5%, 1%, 0% (wild-type), or 40 ng of 0.1% cancer driver mutants were used to establish the method. After pre-amplification of both wild-type and mutant alleles, mutant fragments were specifically bound by biotin-labelled, locked–nucleic acid modified probes, enriched by streptavidin beads, amplified through PCR and sequenced using Sanger for detection. Results: The wild-type standard did not show any mutation signal either with or without PEAC, nor did the standards with 5%, 1%, or 0.1% mutants prior to PEAC enrichment. However, after PEAC enrichment, 5%, 1%, and 0.1% of cancer driver mutants ( EGFR L858R, EGFR T790M, and BRAF V600E) predicting sensitivities to target therapies were enriched to ~90%, ~80% and 30-40% abundance, respectively. In addition to the application for point mutations, PEAC methodology was also used to enrich deletion mutant; 5%, 1%, and 0.1% of EGFR exon 19 deletion, E746_A750del, were all enriched to 90-100% frequency after PEAC. To address the limit of detection of PEAC technology, 100 ng of cfDNA standard containing 0.01% EGFR L858R and EGFR exon 19 deletion mutant were enriched to 15% and 50%, respectively, after PEAC, which indicates up to 5000-fold enrichment for EGFR exon 19 deletion by PEAC technology. Conclusions: PEAC technology can enrich very low abundance of key cancer driver mutants predicting sensitivities to tyrosine kinase inhibitors. PEAC enrichment in combination with Sanger sequencing may guide the application of cancer target therapy and represent a useful technology in the era of precision medicine.

Population divergence of Pseudomonas aeruginosa can lead to the coexistence with Escherichia coli in animal suppurative lesions

Purulent disease is the main factor that prevents the population increase of forest musk deer in artificial breeding, and especially the intracorporal suppurative lesions in late-stage with complex bacterial communities normally bring more difficulties for veterinary treatment. Although it is well-recognized that Pseudomonas aeruginosa and Escherichia coli are the two main bacterial pathogens which can be frequently co-isolated from the lung pus of forest musk deer, few studies have explored the interspecific relationship and coexistent mechanism of the two species. In this study, we identified a P. aeruginosa strain MYL-2, which harbored a loss-of-function mutation in the central regulator (LasR) of quorum-sensing (QS) system, from the lung pus of a dying forest musk deer with co-infecting E. coli strain MYL-58. Interestingly, P. aeruginosa MYL-2 could coexist with E. coli MYL-58 compared to the dominant role of lasR-intact P. aeruginosa strain MYL-1 in the competitive experiments. The results of in vitro coevolution assay further revealed that the QS-mediated competitive advantage of P. aeruginosa MYL-1 would be decreased along with the enrichment of lasR mutants in the communities, and P. aeruginosa could finally coexist with E. coli by forming a relatively stable equilibrium. Therefore, these findings provide an evolutionary explanation for the coexistence of P. aeruginosa and E. coli in the suppurative lesions of forest musk deer, and may also contribute to further understanding the pathology of animal purulent disease and the development of novel veterinary therapy.

Rational high-throughput system for screening of high sophorolipids-producing strains of Candida bombicola

A rational high-throughput screening can significantly improve the efficiency of strain screening with high performance. In this study, based on the addition reaction of unsaturated fatty acids in the sophorolipids (SLs) and I2 molecules, a simple and rapid high-throughput detection method for SLs was established which demonstrated a correlation coefficient (R2) of 0.9106 with high-performance liquid chromatography (HPLC) method. Moreover, chlorpromazine, as a rational selecting pressure for enrichment of mutants with high cytochrome P450 enzyme activity, which was a key enzyme in the synthesis of SLs, was introduced into the high-throughput screening model. Consequently, with the aid of this effective screening system, a high-yielding mutant designated as Candida bombicola F6.5 was successfully screened out from 1500 single colonies, which presented improvements of 40.3% and 11.4% on SLs titer and yield, respectively, compared to the parent strain in a 1L bioreactor.

Concentration-dependent enrichment of resistant Enterococcus faecium exposed to linezolid in an in vitro dynamic model

To explore the relationship between pharmacokinetic variables and enterococcal resistance to linezolid, a vancomycin-resistant strain whose mutant prevention concentration (MPC) exceeded the MIC by two fold was selected among six clinical isolates of Enterococcus faecium. The selected strain was exposed to simulated pharmacokinetics of twice-daily linezolid for five days. Mutants resistant to 2 × MIC of the antibiotic were enriched at ratios of the 24-h area under the concentration-time curve (AUC24) to the MIC of 15 and 30 h but not at 60 and 120 h. These observations could be explained by the different times when antibiotic concentrations exceed the MPC (T>MPC): 0 to 14, 63 and 100% of the dosing interval. Using the area under the bacterial mutant concentration-time curve (AUBCM) determined in this study and in previous work with other E. faecium strains (MPC/MIC 4), a strain-independent T>MPC relationship with mutant enrichment was established.

Mutational monitoring of EGFR T790M in cfDNA for clinical outcome prediction in EGFR-mutant lung adenocarcinoma.

Several ultra-sensitive methods for T790M in plasma cell-free DNA (cfDNA) have been developed for lung cancer. The correlation between mutation-allele frequency (MAF) cut-off, drug responsiveness, and outcome prediction is an unmet needs and not fully addressed. An innovative combination of peptide nucleic acid (PNA) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) was used to proof of concept for monitoring cfDNA T790M in EGFR-mutant patients. Mutant enrichment by PNA was optimized and the detection limit was evaluated through serial dilutions. The cut-off value was identified by receiver-operating-characteristic (ROC) curve analysis utilizing serial sampled plasmas of patients from EGFR-tyrosine kinase inhibitor (TKI) pretreatment to progressive-disease (PD). Results, comparisons, and objective response rate (ORR) were analyzed in 103 patients’ tumor and cfDNA T790M, with 20 of them receiving an additional COBAS test. The detection limit was 0.1% MAF. The cut-off for PD and imminent PD was 15% and 5% with an ROC area under the curve (AUC) of 0.96 and 0.82 in 2 ml plasma. Detection sensitivity of cfDNA T790M was 67.4% and overall concordance was 78.6%. ORR was similar in T790M-positive cfDNA (69.6%) and tumor samples (70.6%) treated with osimertinib. Among 65 T790M-positive tumors, 15 were negative in cfDNA (23.1%). Seven of 38 T790M-positive cfDNA samples were negative in the tumors (18.4%). PNA-MALDI-TOF MS had a higher detection rate than COBAS. In conclusion, identification of T790M cut-off value in cfDNA improves cancer managements. We provide a strategy for optimizing testing utility, flexibility, quality, and cost in the clinical practice.

Hypermutation-induced in vivo oxidative stress resistance enhances Vibrio cholerae host adaptation.

Bacterial pathogens are highly adaptable organisms, a quality that enables them to overcome changing hostile environments. For example, Vibrio cholerae, the causative agent of cholera, is able to colonize host small intestines and combat host-produced reactive oxygen species (ROS) during infection. To dissect the molecular mechanisms utilized by V. cholerae to overcome ROS in vivo, we performed a whole-genome transposon sequencing analysis (Tn-seq) by comparing gene requirements for colonization using adult mice with and without the treatment of the antioxidant, N-acetyl cysteine. We found that mutants of the methyl-directed mismatch repair (MMR) system, such as MutS, displayed significant colonization advantages in untreated, ROS-rich mice, but not in NAC-treated mice. Further analyses suggest that the accumulation of both catalase-overproducing mutants and rugose colony variants in NAC- mice was the leading cause of mutS mutant enrichment caused by oxidative stress during infection. We also found that rugose variants could revert back to smooth colonies upon aerobic, in vitro culture. Additionally, the mutation rate of wildtype colonized in NAC- mice was significantly higher than that in NAC+ mice. Taken together, these findings support a paradigm in which V. cholerae employs a temporal adaptive strategy to battle ROS during infection, resulting in enriched phenotypes. Moreover, ΔmutS passage and complementation can be used to model hypermuation in diverse pathogens to identify novel stress resistance mechanisms.

Efficient transposon mutagenesis mediated by an IPTG-controlled conditional suicide plasmid

BackgroundTransposon mutagenesis is highly valuable for bacterial genetic and genomic studies. The transposons are usually delivered into host cells through conjugation or electroporation of a suicide plasmid. However, many bacterial species cannot be efficiently conjugated or transformed for transposon saturation mutagenesis. For this reason, temperature-sensitive (ts) plasmids have also been developed for transposon mutagenesis, but prolonged incubation at high temperatures to induce ts plasmid loss can be harmful to the hosts and lead to enrichment of mutants with adaptive genetic changes. In addition, the ts phenotype of a plasmid is often strain- or species-specific, as it may become non-ts or suicidal in different bacterial species.ResultsWe have engineered several conditional suicide plasmids that have a broad host range and whose loss is IPTG-controlled. One construct, which has the highest stability in the absence of IPTG induction, was then used as a curable vector to deliver hyperactive miniTn5 transposons for insertional mutagenesis. Our analyses show that these new tools can be used for efficient and regulatable transposon mutagenesis in Escherichia coli, Acinetobacter baylyi and Pseudomonas aeruginosa. In P. aeruginosa PAO1, we have used this method to generate a Tn5 insertion library with an estimated diversity of ~ 108, which is ~ 2 logs larger than the best transposon insertional library of PAO1 and related Pseudomonas strains previously reported.ConclusionWe have developed a number of IPTG-controlled conditional suicide plasmids. By exploiting one of them for transposon delivery, a highly efficient and broadly useful mutagenesis system has been developed. As the assay condition is mild, we believe that our methodology will have broad applications in microbiology research.

Predicting antibiotic combination effects on the selection of resistant Staphylococcus aureus: In vitro model studies with linezolid and gentamicin

To explore whether combinations of linezolid with gentamicin restrict Staphylococcus aureus resistance, the enrichment of resistant mutants was studied in an in vitro dynamic model. A clinical isolate S. aureus 10 and its linezolid-resistant mutant selected by passaging on antibiotic-containing media were used in the study. The minimum inhibitory concentration (MIC) and the mutant prevention concentration (MPC) of antibiotics in combination were determined at a linezolid-to-gentamicin concentration ratio corresponding to the ratio of 24-h areas under the concentration-time curve (AUC24s) used in the pharmacokinetic simulations. Five-day treatments of S. aureus 10 supplemented with linezolid-resistant mutants (mutation frequency 10-8) with twice-daily linezolid and once-daily gentamicin, alone and in combination, were simulated at therapeutic and subtherapeutic AUC24s. Numbers of linezolid-resistant mutants increased at both therapeutic and subtherapeutic AUC24s, whereas gentamicin-resistant mutants were enriched only at the subtherapeutic AUC24 in single drug treatments. Linezolid-gentamicin combinations prevented the enrichment of linezolid-resistant S. aureus and restricted the enrichment of gentamicin-resistant mutants. The pronounced anti-mutant effect of the combinations was attributed to lengthening the time above MPC of both linezolid and gentamicin as their MPCs were lowered. Unlike resistant S. aureus, killing of the total bacterial burden exposed to linezolid-gentamicin combinations was less than in treatments with gentamicin alone, but greater than with linezolid alone. These findings indicate that (1) the anti-mutant effects of antibiotic combinations can be predicted by MPC determinations at pharmacokinetically-derived concentration ratios, and (2) a given antibiotic combination may be optimal against resistant but not susceptible subpopulations.

Abstract 4538: LiquidGxTMqPCR, an extremely sensitive qPCR-based liquid biopsy platform

Abstract In recent years, circulating cell-free tumor DNA has emerged as an excellent source of biomarkers for cancer detection, diagnosis and monitoring. One great challenge of detecting cell-free tumor DNA molecules is that they are usually found in a large background of wild-type DNA; therefore, assays with high sensitivity and low limit of detection are required. We have developed an extremely sensitive qPCR-based liquid biopsy platform, LiquidGxTM qPCR, to detect and track key driver mutations from patient blood. The platform takes advantage of our patent-pending technology BESTTM (Blocker-based Enrichment System of Tumor DNA) to specifically enrich mutant alleles from as low as 0.01% to more than 90%. With this platform, we currently have developed a CLIA-approved non-small cell lung cancer panel (EGFR T790M/L858R/C797S/Exon19Del, KRAS G12X/G13CD, BRAF V600E, ALK-EML4 fusion), and a colorectal cancer recurrence monitoring panel (KRAS G12X/G13CD, NRAS G12DV/Q61KR, PIK3CA E542K/E545K/H1047R, BRAF V600E). Our validation data show that the NSCLC panel has superior performance, with a LOD as low as 0.005%, analytical sensitivity more than 95% and analytical specificity of 100%. We have used this panel to test samples from nearly 200 NSCLC patients, and the results are highly concordant with those obtained by error-reducing NGS-based tests, despite some samples only detected via LiquidGxTM qPCR due to superior LOD. Other features of LiquidGxTM qPCR include (1) simultaneous enrichment of multiple mutants in the same reaction; (2) cost-effective and no special equipment required; and (3) fast turnaround time that takes as little as 0.5 days to get the results. In summary, we have developed a potent liquid biopsy platform for highly sensitive genomic biomarker test. With the technology, we have developed two cancer panels and this platform is also available as an assay development service. Citation Format: Yang Song, Danielle Quintanilha, Si Chen, Jun Huang. LiquidGxTMqPCR, an extremely sensitive qPCR-based liquid biopsy platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4538.

Genome-wide identification of tolerance mechanisms toward p-coumaric acid in Pseudomonas putida.

The soil bacterium Pseudomonas putida KT2440 has gained increasing biotechnological interest due to its ability to tolerate different types of stress. Here, the tolerance of P. putida KT2440 toward eleven toxic chemical compounds was investigated. P. putida was found to be significantly more tolerant toward three of the eleven compounds when compared to Escherichia coli. Increased tolerance was for example found toward p‐coumaric acid, an interesting precursor for polymerization with a significant industrial relevance. The tolerance mechanism was therefore investigated using the genome‐wide approach, Tn‐seq. Libraries containing a large number of miniTn5‐Km transposon insertion mutants were grown in the presence and absence of p‐coumaric acid, and the enrichment or depletion of mutants was quantified by high‐throughput sequencing. Several genes, including the ABC transporter Ttg2ABC and the cytochrome c maturation system (ccm), were identified to play an important role in the tolerance toward p‐coumaric acid of this bacterium. Most of the identified genes were involved in membrane stability, suggesting that tolerance toward p‐coumaric acid is related to transport and membrane integrity.

Testing the mutant selection window hypothesis with Staphylococcus aureus exposed to linezolid in an in vitro dynamic model.

To test the mutant selection window (MSW) hypothesis applied to linezolid-exposed Staphylococcus aureus and to delineate the concentration-resistance relationship, a mixed inoculum of linezolid-susceptible S. aureus cells and linezolid-resistant mutants (RMs) was exposed to linezolid multiple dosing. Three S. aureus strains (MIC of linezolid 2 mg/L), S. aureus 479, S. aureus 688 and S. aureus ATCC 700699, and their RMs (MIC 8 mg/L) selected by passaging on antibiotic-containing media were used in the study. RMs of S. aureus 479 and S. aureus ATCC 700699 contained a G2576T replacement (Escherichia coli numbering) in one of the copies of the 23S rRNA gene, which had been reported in clinically isolated mutants. Five-day treatments with twice-daily linezolid were simulated over a 32-fold range of the 24 h AUC (AUC24) to the MIC ratio. A pronounced enrichment of mutants resistant to 2×, 4× and 8× MIC was observed at AUC24/MIC ratios of 30 and 60 when linezolid concentrations were within the MSW for more than half of the dosing interval for each strain. The number of viable mutant cells decreased significantly at the simulated AUC24/MIC ratio of 120, while the AUC24/MIC ratio of 240 completely prevented mutant enrichment in vitro. Bell-shaped AUBCM-AUC24/MIC and AUBCM-AUC24/MPC relationships (r2 0.91 and 0.79, respectively) were strain independent. The bell-shaped pattern of AUC24/MIC and AUC24/MPC relationships with S. aureus resistance to linezolid is consistent with the MSW hypothesis. 'Antimutant' AUC24/MIC ratios were predicted based on the AUC24/MIC relationship with AUBCM.

Abstract LB-030: Specific missense mutations in TP53 elicit contrasting biochemical and biological outcomes affecting prostate cancer progression

Abstract Prostatic adenocarcinoma (PCa) remains the most frequently diagnosed cancer type and the third leading cause of cancer death in men in the United States. While organ-confined disease is manageable, treatment options for disseminated PCa remain limited. First-line therapy for metastatic PCa functions through targeting the androgen receptor (AR) signaling axis, as AR activity is required for tumor maintenance. However, while treatment is initially effective, these tumors invariably recur, displaying reactivation of AR signaling despite continued therapeutic targeting. Moreover, no durable treatment currently exists for this ultimately fatal disease stage, termed castrate-resistant PCa (CRPC). Significantly, recent high profile, genome-wide studies have revealed that missense mutations in the TP53 gene occur in nearly all cancer types, and these mutations frequently occur at specific TP53 “hotspots”. Interestingly, enrichment of certain hotspot p53 mutants specifically occurs in PCa. While the underlying causes of PCa-enriched mutations remain undefined, their presence elicits contrasting, pro-oncogenic biochemical and biological phenotypes. Unpublished data suggests that mutant p53 isoforms act in a mutation-specific fashion to drive PCa progression through alterations in the canonical activity of p53, identified through characterization of their related transcriptomes and cistromes. Biologically, these mutant p53 isoforms also confer differential proliferative and survival phenotypes. Moreover they act in a mutation-dependent fashion in response to castration therapy both in vitro and in vivo. Mechanistically, pro-oncogenic mutant p53 appears to manipulate both conventional and novel pathways leading to PCa progression. These findings both corroborate and expand the recent findings that mutations in p53 drive cancer progression, and further suggest a critical role for p53 mutations as regulators of disease progression in PCa. Citation Format: Jennifer J. McCann, Jeffry L. Dean, Renee De Leeuw, Matthew J. Schiewer, Christopher M. McNair, Karen E. Knudsen. Specific missense mutations in TP53 elicit contrasting biochemical and biological outcomes affecting prostate cancer progression [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 LB-030. doi:10.1158/1538-7445.AM2017-LB-030

Abstract 759: Highly-sensitive detection of EGFR mutation in non-small cell lung cancer using mutant-enrichment method

Abstract Detection of mutations in EGFR in non-small cell lung cancer is important for the clinical decision-making process of molecularly targeted therapies, including Iressa®, Tarceva®, and Tagrisso®. In this study we developed an assay allowing the detection of EGFR gene mutations being conducted in the routine diagnostic procedure and its analytic and clinical performance were evaluated using different types of specimens. The EGFR mutation assay in this study allows for detecting EGFR exon 19 deletion (Del), exon 20 insertion (Ins), and mutation in T790M, L858R, S768I and L861Q of rare amounts in a normal genetic background. Enhancement of detection sensitivity was achieved with the developed mutant enrichment method which combines mutation-specific primers and 3'-phosphorylation oligonucleotides as blockers for inhibiting wild-type gene from DNA amplification. Analytic performance for each mutant was assessed using the control plasmids containing the mutant genes of known amounts as well as the genomic DNA extracted from mutant cell lines and Horizon FFPE samples. The assay performance were also evaluated using clinical samples in parallel with the FDA-approval companion diagnostic product therascreen® EGFR RGQ PCR Kit . The sensitivity of the assay evaluated using control plasmids for Del, T790M , L858R, S768I, L861Q mutation and Ins was 5%, 0.5%, 1%, 0.5%, 0.1% and 0.5% respectively, and was 1%, 0.5%, 0.5%, 0.5%, 0.1% and 0.5% respectively when applying on HDx reference standards. Diagnostic results for the 116 clinical samples for identifying mutations of del, T790M, L858R, S768I, L861Q and Ins was 92.2%, 100%, 99.1%, 100%, 97.4% and 100% respectively concordance with the therascreen® EGFR RGQ PCR Kit, including 17 cases of multiple mutations. The overall diagnostic results for all mutations evaluated with PPA (positive percent agreement), NPA (negative percentage agreement) and OPA (overall percentage agreement) were 83.3%, 98.2% and 90.5%, respectively. The three samples firstly diagnostic as false positive were retested and confirmed to be Exon 19 del by DNA sequencing. The assay demonstrated good sensitivity when compared with commercial product. More clinical studies are being performed to establish relationship between drug efficacy and different mutation combinations. Citation Format: Hung-Chi Chien. Highly-sensitive detection of EGFR mutation in non-small cell lung cancer using mutant-enrichment method [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 759. doi:10.1158/1538-7445.AM2017-759

In Vitro Resistance Selection in Shigella flexneri by Azithromycin, Ceftriaxone, Ciprofloxacin, Levofloxacin, and Moxifloxacin.

Shigella flexneri continues to be a major cause of diarrhea-associated illness, and increasing resistance to first-line antimicrobials complicates the treatment of infections caused by this pathogen. We investigated the pharmacodynamics of current antimicrobial treatments for shigellosis to determine the likelihood of resistance promotion with continued global antimicrobial use. The mutant prevention concentration (MPC) and mutant selection window (MSW) were determined for azithromycin, ceftriaxone, ciprofloxacin, levofloxacin, and moxifloxacin against a wild-type strain of S. flexneri (ATCC 12022) and an isogenic gyrA mutant (m-12022). Time-kill assays were performed to determine antimicrobial killing. Concentrations of approved doses of ciprofloxacin, levofloxacin, and moxifloxacin are predicted to surpass the MPC for a majority of the dosage interval against ATCC 12022. However, against m-12022, concentrations of all fluoroquinolones are predicted to fall below the MPC and remain in the MSW for a majority of the dosage interval. Concentrations of ceftriaxone fall within the MSW for the majority of the dosage interval for both strains. All agents other than azithromycin displayed bactericidal activity in time-kill assays. Results of pharmacodynamic analyses suggest that all tested fluoroquinolones would achieve a favorable area under the concentration-time curve (AUC)/MPC ratio for ATCC 12022 and would restrict selective enrichment of mutants but that mutant selection in m-12022 would be likely if ciprofloxacin were used. Based on pharmacodynamic analyses, azithromycin and ceftriaxone are predicted to promote mutant selection in both strains. Confirmation of these findings and examination of novel treatment regimens using in vivo studies are warranted.