Mutant Fraction Research Articles

High-Accuracy Determination of Microsatellite Instability Compatible with Liquid Biopsies.

Microsatellite instability (MSI) has recently emerged as a predictive pan-tumor biomarker of immunotherapy efficacy, stimulating the development of diagnostic tools compatible with large-scale screening of patients. In this context, noninvasive detection of MSI from circulating tumor DNA stands as a promising diagnostic and posttreatment monitoring tool. We developed drop-off droplet-digital PCR (ddPCR) assays targeting BAT-26, activin A receptor type 2A (ACVR2A), and defensin beta 105A/B (DEFB105A/B) microsatellite markers. Performances of the assays were measured on reconstitution experiments of various mutant allelic fractions, on 185 tumor samples with known MSI status, and on 72 blood samples collected from 42 patients with advanced colorectal or endometrial cancers before and/or during therapy. The 3 ddPCR assays reached analytical sensitivity <0.1% variant allelic frequency and could reliably detect and quantify MSI in both tumor and body fluid samples. High concordance between MSI status determination by the three-marker ddPCR test and the reference pentaplex method were observed (100% for colorectal tumors and 93% for other tumor types). Moreover, the 3 assays showed correlations with r ≥ 0.99 with other circulating tumor DNA markers and their dynamic during treatment correlated well with clinical response. This innovative approach for MSI detection provides a noninvasive, cost-effective, and fast diagnostic tool, well suited for large-scale screening of patients that may benefit from immunotherapy agents, as well as for monitoring treatment responses.

Unequal Allelic Expression of Mutated Cardiac Troponin I from Cell-To-Cell May Induce Contractile Imbalance in Hypertrophic Cardiomyopathy

Hypertrophic Cardiomyopathy (HCM) is mostly caused by inherited, heterozygous mutations in sarcomeric proteins. A common mechanism why different functional effects of different mutations induce similar HCM features is unclear. In previous studies, unequal expression of mutated vs. wildtype mRNA and large variability in force generation between individual cardiomyocytes from HCM-patients with mutations in the myosin heavy chain gene (MYH7) was found. This contractile imbalance of adjacent cardiomyocytes could trigger development of common HCM features like cardiomyocyte disarray and fibrosis. For mutations in MYH7 we provided evidence that the unequal fractions of mutated mRNA from cell to cell most likely are due to burst-like, stochastic transcription, independent for the two alleles. Here we asked, if this hypothesis is unique for MYH7 or if it is a mechanism also in other sarcomeric proteins like cardiac troponin I (cTnI, TNNI3). We analyzed TNNI3 mRNA expression and force generation in a donor and HCM-patients with TNNI3-mutation (c.433C>T, R145W). RNA-fluorescence in situ hybridization experiments revealed that most nuclei contain no active transcription sites (aTS) (95% in donor and 81% in patients), while others show active transcription of one or both alleles. This indicates stochastic and independent burst-like transcription of TNNI3 rather than continuous transcription. Single cardiomyocytes of both, patients and donor, showed highly variable fractions of TNNI3 mutant and wildtype mRNA ranging from essentially only wildtype to essentially only mutant mRNA in allele-specific RT-PCR experiments. Calcium dependent force measurements on cardiomyocytes of the same patient presented substantially larger variability of force compared to donor cardiomyocytes. These results suggest that not only MYH7, but also TNNI3 is transcribed burst-like. This may induce cell-to-cell allelic imbalance and, as a result, contractile imbalance in the myocardium as a trigger for HCM.

Statistical Inference for the Evolutionary History of Cancer Genomes

Recent years have seen considerable work on inference about cancer evolution from mutations identified in cancer samples. Much of the modeling work has been based on classical models of population genetics, generalized to accommodate time-varying cell population size. Reverse-time, genealogical views of such models, commonly known as coalescents, have been used to infer aspects of the past of growing populations. Another approach is to use branching processes, the simplest scenario being the classical linear birth-death process. Inference from evolutionary models of DNA often exploits summary statistics of the sequence data, a common one being the so-called Site Frequency Spectrum (SFS). In a bulk tumor sequencing experiment, we can estimate for each site at which a novel somatic point mutation has arisen, the proportion of cells that carry that mutation. These numbers are then grouped into collections of sites which have similar mutant fractions. We examine how the SFS based on birth-death processes differs from those based on the coalescent model. This may stem from the different sampling mechanisms in the two approaches. However, we also show that despite this, they are quantitatively comparable for the range of parameters typical for tumor cell populations. We also present a model of tumor evolution with selective sweeps, and demonstrate how it may help in understanding the history of a tumor as well as the influence of data pre-processing. We illustrate the theory with applications to several examples from The Cancer Genome Atlas tumors.

ACB-PCR Quantification of Low-Frequency Hotspot Cancer-Driver Mutations.

Allele-specific competitive blocker PCR (ACB-PCR) is a sensitive and quantitative approach for the selective amplification of a specific base substitution. Using the ACB-PCR technique, hotspot cancer-driver mutations (tumor-relevant mutations in oncogenes and tumor suppressor genes, which confer a selective growth advantage) are being developed as quantitative biomarkers of cancer risk. ACB-PCR employs a mutant-specific primer (with a 3'-penultimate mismatch relative to the mutant DNA sequence, but a double 3'-terminal mismatch relative to the wild-type DNA sequence) to selectively amplify rare mutant DNA molecules. A blocker primer having a non-extendable 3'-end and a 3'-penultimate mismatch relative to the wild-type DNA sequence, but a double 3'-terminal mismatch relative to the mutant DNA sequence is included in ACB-PCR to selectively repress amplification from abundant wild-type molecules. Consequently, ACB-PCR can quantify the level of a single base pair substitution mutation in a DNA population when present at a mutant:wild-type ratio of 1×10-5 or greater. Quantification of rare mutant alleles is achieved by parallel analysis of unknown samples and mutant fraction (MF) standards (defined mixtures of mutant and wild-type DNA sequences). The ability to quantify specific mutations with known association to cancer has several important applications in evaluating the carcinogenic potential of chemical exposures in rodent models. Further, the measurement of cancer-driver mutant subpopulations is important for precision cancer treatment (selecting the most appropriate targeted therapy and predicting the development of therapeutic resistance). This chapter provides a step-by-step description of the ACB-PCR methodology as it has been used to measure human PIK3CA codon 1047, CAT→CGT (H1047R) mutation.

Manipulation of mitochondrial genes and mtDNA heteroplasmy.

Most patients with mitochondrial DNA (mtDNA) mutations have a mixture of mutant and wild-type mtDNA in their cells. This phenomenon, known as mtDNA heteroplasmy, provides an opportunity to develop therapies by selectively eliminating the mutant fraction. In the last decade, several enzyme-based gene editing platforms were developed to cleave specific DNA sequences. We have taken advantage of these enzymes to develop reagents to selectively eliminate mutant mtDNA. The replication of intact mitochondrial genomes normalizes mtDNA levels and consequently mitochondrial function. In this chapter, we describe the methodology used to design and express these nucleases in mammalian cells in culture and in vivo.

Rationale and Roadmap for Developing Panels of Hotspot Cancer Driver Gene Mutations as Biomarkers of Cancer Risk.

Cancer driver mutations (CDMs) are necessary and causal for carcinogenesis and have advantages as reporters of carcinogenic risk. However, little progress has been made toward developing measurements of CDMs as biomarkers for use in cancer risk assessment. Impediments for using a CDM‐based metric to inform cancer risk include the complexity and stochastic nature of carcinogenesis, technical difficulty in quantifying low‐frequency CDMs, and lack of established relationships between cancer driver mutant fractions and tumor incidence. Through literature review and database analyses, this review identifies the most promising targets to investigate as biomarkers of cancer risk. Mutational hotspots were discerned within the 20 most mutated genes across the 10 deadliest cancers. Forty genes were identified that encompass 108 mutational hotspot codons overrepresented in the COSMIC database; 424 different mutations within these hotspot codons account for approximately 63,000 tumors and their prevalence across tumor types is described. The review summarizes literature on the prevalence of CDMs in normal tissues and suggests such mutations are direct and indirect substrates for chemical carcinogenesis, which occurs in a spatially stochastic manner. Evidence that hotspot CDMs (hCDMs) frequently occur as tumor subpopulations is presented, indicating COSMIC data may underestimate mutation prevalence. Analyses of online databases show that genes containing hCDMs are enriched in functions related to intercellular communication. In its totality, the review provides a roadmap for the development of tissue‐specific, CDM‐based biomarkers of carcinogenic potential, comprised of batteries of hCDMs and can be measured by error‐correct next‐generation sequencing. Environ. Mol. Mutagen. 61:152–175, 2020. Published 2019. This article is a U.S. Government work and is in the public domain in the USA. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

P1617Contractile imbalance as trigger for HCM pathogenesis: evidence from mutations in different sarcomeric proteins

Abstract Hypertrophic cardiomyopathy (HCM) is mostly caused by mutations in sarcomeric proteins. About 90% of mutation-positive patients have mutations in one of four proteins: the β-myosin heavy chain (β-MyHC, MYH7), cardiac myosin binding protein C (cMyBP-C, MYBPC3), cardiac troponin I (cTnI, TNNI3), and cardiac troponin T. Almost all patients are heterozygous; they express the wildtype and the mutant protein isoform. For patients with β-MyHC missense mutations we have reported previously that individual cardiomyocytes show a significant variability in force generation and calcium-sensitivity, ranging from essentially donor–like to highly altered function. We provided evidence that the MYH7-alleles are switched on and off stochastically and independently from each other in each cell. This burst-like expression leads to highly variable fractions of mutant and wildtype mRNA between the cardiomyocytes, presumably causing variable fractions of mutant protein. We assume that this variability underlies the determined contractile imbalance leading to stronger cells that over-contract and over-stretch weaker cells. This could trigger development of HCM-hallmarks like myocyte disarray, fibrosis and hypertrophy. To test whether contractile imbalance may provide a common mechanism of HCM-development, we extended our analysis to additional sarcomeric proteins with HCM-mutations. Analysis of cardiomyocytes from a patient with missense mutation R145W in cTnI revealed highly variable calcium-sensitivity between individual cardiomyocytes, substantially higher than for donor cardiomyocytes. This functional heterogeneity was associated with highly variable fractions of mutant TNNI3-mRNA from cell-to-cell. This suggests that not only missense mutations in β-MyHC but also in cTnI induce hallmarks of HCM via the contractile imbalance mechanism. In contrast to missense mutations, truncation mutations in cMyBP-C presumably cause HCM via haploinsufficiency. Degradation of truncated proteins causes a lack of functional cMyBP-C and thereby alters function of the sarcomere. We hypothesized that different levels of haploinsufficiency from cell-to-cell may also cause contractile imbalance. Therefore we examined a patient with truncation mutation c.927–2A&gt;G in cMyBP-C. Western blot analysis revealed no truncated protein and reduced levels of wildtype-cMyBP-C, consistent with haploinsufficiency. We also observed a significantly higher variability in fluorescence intensity ratio (MyBPC/Alpha-Actinin) for cardiomyocytes of the HCM-patient than in donor cardiomyocytes. The patchy distribution of cMyBP-C in histological tissue section indicated variable levels of functional protein from cell-to-cell. Functional analysis revealed significantly more variable isometric force generation from cell to cell of patient cardiomyocytes compared to donor, suggesting contractile imbalance. We conclude that contractile imbalance may be a potential common mechanism of HCM pathogenesis. Acknowledgement/Funding German Research Foundation (DFG)

Abstract 2239: Analysis of nucleosomal DNA as an extraction control for plasma-based circulating tumor DNA assays

Abstract Purpose: Challenges to accurate quantitative measurements of circulating tumor DNA (ctDNA) include variable contamination with large DNA fragments released during mononuclear cell lysis, and potential variability in extraction efficiency. We investigated optimal procedures for plasma processing and the potential utility of a DNA spike-in as a process control for extraction efficiency. Methods: Initial experiments analyzed the potential benefits of different centrifugation protocols (1600 x g vs. 1600 x g followed by 16000 x g) for plasma separation by analyzing the size distribution of extracted DNA using a BioAnalyzer. In subsequent experiments we used droplet digital PCR (ddPCR) to compare differences in extraction efficiency between nucleosomal DNA (nDNA) and genomic DNA (gDNA) spiked into healthy donor plasma and extracted using the QIAamp DSP Circulating NA Kit (Qiagen). To evaluate the properties of the purification columns with respect to the yield of different DNA sizes, we compared the plasma results to experiments extracting purified nDNA and gDNA spiked into PBS instead of plasma. We investigated the potential competitive effect of large DNA on the extraction efficiency of small DNA by mixing nDNA with commercially available human gDNA in different concentrations. To assess if nDNA, when spiked into patient plasma, would track with endogenous ctDNA, we spiked a pool of plasma from patients with BRAFV600E mutations with known quantities of nDNA purified from NRASQ61K mutant cells (CHP212). The spiked plasma pool was divided and extracted using multiple separate columns. Copies/ml and mutant fraction were determined by ddPCR assays for BRAFV600E and NRASQ61K. Coefficients of variation were also calculated. Results: Comparison of different centrifugation speeds for plasma processing before and after freeze-thaw indicated that a 2nd high-speed centrifugation (16000 x g), either before or after a freeze-thaw cycle, minimizes large DNA in plasma samples prior to ctDNA extraction. In initial spike-in experiments extraction efficiency for nDNA was 87.7% compared to 42% for gDNA. We obtained similar results when purifying the nDNA and gDNA from PBS instead of plasma. We observed greater variability in nDNA extraction efficiency when increasing amounts of gDNA were added as a contaminant. Finally, we found that the slight, paralell variations in extraction yields of externally spiked NRASQ61K mutant nDNA and endogenous BRAFV600E ctDNA in 7/9 replicate extractions. The overall coefficients of variation (total copies/mL) for 9 extractions for BRAFV600E and NRASQ61K were 3.9 and 4.2 respectively. Conclusion: A second centrifugation step helps reduce potential contamination of ctDNA with large DNA, and may improve the recovery of ctDNA from plasma samples. Nucleosomal DNA can potentially be used as a process control for extracting ctDNA from plasma samples. Citation Format: Mahrukh M. Syeda, George Karlin-Neumann, Iman Osman, David Polsky. Analysis of nucleosomal DNA as an extraction control for plasma-based circulating tumor DNA assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2239.

Abstract 2286: Liquid biopsy for early cancer detection

Abstract Introduction: Early cancer detection remains as a critical challenge to improve patient’s survival and clinical outcome. A non-invasive liquid biopsy permits the analysis of multiple circulating biomarkers including cell free nucleic acids and extracellular vesicles that facilitate the discovery of disease. We hypothesize that cancer-derived circulating biomarkers contain heterogeneous surface membrane proteins and/or nucleic acids representing the original tumor, requiring tools which can identify multiple biomarkers to be detected simultaneously. However, challenges including a lack of established isolation standards and determining sufficiently sensitive detection platforms remain for clinical implementation. Our goal is to develop a multiplexed biomarker based assay which can be leveraged to capture the molecular heterogeneity of distinct subpopulations of tumor derived circulating biomarkers. Materials and Methods: Materials and methods vary greatly by project, each project design will be briefly summarized: 1) Single molecule imaging of ctDNA was conducted using PEG-coated surface whereby biotin streptavidin linked DNA reference strands were exposed to fluorophore conjugated mutant DNA strands for FRET imaging of mutant specific sequences. 2) Extracellular vesicle imaging and profiling was conducted using antibody based surface capture and high-resolution flow cytometry for high throughput exosome characterization. 3) RNA-biomarkers were identified by converting RNA into cDNA using target gene panels selected from data bases at each cycle during a PCR. Results and Discussion: RNA profiling of initial sample cohort distinguished cancer from healthy, while detecting early cancer patients with high sensitivity. Differentiating genes with biological relevance were identified which can classify pancreatic cancer patients from healthy donor. cfDNA imaging method had nonspecific signal &amp;lt; 0.1-1%, wherein 1-10% mutant fraction can be genotyped. High resolution flow cytometry identified distinct subpopulations of plasma exosomes and revealed their molecular heterogeneity and cancer specific marker candidates can be screened against purified exosomes. Conclusions: Extracellular vesicle imaging and profiling co-validated by high resolution flow cytometry, exemplifying the utility of a multi-platform detection scheme to characterize plasma-derived extracellular vesicle populations. Cell free RNA biomarkers of cancer by cell-free RNA sequencing were analyzed that can improve early cancer detection outcome, this proof of concept was done on pancreatic patient plasma and is being expanded into other cancer cohorts. Citation Format: Hyunji Kim, Fehmi Civitci, Josiah Wagner, Pavana Anur, Matthew Rames, Xiaolin Nan, Terry Morgan, Thuy Ngo. Liquid biopsy for early cancer detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2286.

Abstract 4461: Analysis of circulating tumor DNA (ctDNA) identifies on-treatment genomic alterations in a phase 2 study of veliparib plus FOLFIRI ± bevacizumab vs. placebo plus FOLFIRI ± bevacizumab in metastatic colorectal cancer (mCRC)

Abstract Changes in plasma ctDNA are used as a surrogate molecular marker of response to determine clinical efficacy and provide insights into potential resistance mechanisms to targeted therapeutics. Detection of genomic alterations in circulating tumor DNA (ctDNA) in colorectal cancer has been widely employed as an approach to measure real-time changes in the tumor genome. In order to understand mechanisms of resistance to the PARP inhibitor, Veliparib (ABT-888), pathogenic alterations in oncogenes and DNA Damage Response (DDR) genes were profiled longitudinally in plasma ctDNA and associated to clinical response to Veliparib concurrent to FOLFIRI +/- Bevacizumab. Methods: 215 serial plasma samples collected at Baseline, Cycle 2 (28 days post-treatment) and at disease progression (Final Visit) from 78 patients (pts) with mCRC in the NCT02305758 clinical trial were selected for the current study, consisting of ~50% partial responders (PR), ~30% stable disease (SD) pts and 5% Non-responders (PD). Detection of ctDNA mutations was accomplished using a targeted, error-correctible NGS assay encompassing 63 cancer driver genes, tumor suppressors &amp; DDR genes. Paired pre- and on-treatment plasma samples were evaluable from 81% pts selected. Results: Veliparib (VPRB) pts with longest PFS experienced the largest fold change reductions in total cfDNA concentration by end of treatment (EOT). Interestingly, in both treatment arms, the ctDNA mutant fraction per pt decreased by EOT (median = -41%). In support of this correlation, the ctDNA mutational burden also decreased by 28% in VPRB pts and 39% in PLAC pts, largely driven by PR pts with longer PFS. Overall, tumor-specific mutations detected in ctDNA had 88% concordance with tissue, particularly in common mCRC driver mutations. Baseline KRAS mutations were detected in 46% pts (mostly PR &amp; PD) and are eliminated or diminished in 28% pts (14% VPRB, 14% Placebo) by EOT. 30 DDR &amp; MMR genes were found to be frequently altered in all pts, notably APC &amp; MSH3, at both baseline and EOT. However, by EOT, RAD1, XRCC3, BRCA1 and CHEK1 mutations were acquired or increased, while TP53BP1, POLD1 and ERCC5 mutations were eliminated or reduced, prominently in VPRB PR pts. Conclusions: Broadly, a reduction in ctDNA fraction and pathogenic mutation burden, notably in KRAS, is evidenced in both treatment arms and appears to trend with longer PFS at least in Veliparib-treated pts. Despite these prognostic ctDNA changes, VPRB-specific DDR mutation changes may provide insight into VPRB mechanism of action and resistance in mCRC and is under further exploration at earlier post-treatment timepoints and in SD pts. These data could add insights into alterations acquired earlier in the treatment regimen and the relationship of these alterations with resistance to PARPi in mCRC. Citation Format: Cyril Y. Ramathal, Erin Murphy, Elizabeth Asque, Arne Jason Grundstad, Peter Ansell, Lei He, Yan Luo, Jordan Berlin, Jeff Waring. Analysis of circulating tumor DNA (ctDNA) identifies on-treatment genomic alterations in a phase 2 study of veliparib plus FOLFIRI ± bevacizumab vs. placebo plus FOLFIRI ± bevacizumab in metastatic colorectal cancer (mCRC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4461.

Comparison of the Clinical Sensitivity of the Idylla Platform and the OncoBEAM RAS CRC Assay for KRAS Mutation Detection in Liquid Biopsy Samples

KRAS mutations are common in colorectal cancer (CRC). In this setting, mutation status determination in circulating-free DNA from blood samples (liquid biopsy) has been shown to be a viable alternative to tissue testing. The objective of this study was to compare the sensitivity of two liquid biopsy methods for detecting KRAS mutations in plasma samples from metastatic CRC patients. Samples with a positive (KRAS-MUT+) result and a mutant allelic fraction (MAF) < 5% according to the OncoBEAM RAS CRC assay were pairly analyzed by the Idylla ctKRAS Mutation Test (n = 116). In a cohort of 71 patients with at least 1 year of follow-up, the progression-free survival (PFS) was determined according to MAF values. Idylla detected KRAS mutations in 81/116 OncoBEAM KRAS-MUT+ samples with MAF < 5% and in 48/79 samples with MAF < 1%. Concordance between OncoBEAM and Idylla significantly improved at higher MAF values. PFS rates at 6 and 12 months tended to be lower in patients with MAF levels between 1% and 5% than in those with levels <1%. OncoBEAM demonstrated greater sensitivity for plasma detection of KRAS mutations than Idylla. Importantly, our data identified a “gray zone” below 1% MAF where Idylla showed reduced KRAS mutation detection, highlighting the importance of an accurate method to provide the mutational status of CRC patients.

CtDNA and copy-number variant quantification in mCRPC at the time of progression.

265 Background: Tumor burden is a prognostic biomarker in cancer; however, in metastatic castration resistant prostate cancer (mCRPC), quantification of tumor burden markers is difficult. Circulating ctDNA is an accessible biomarker which can be used to characterize somatic DNA. The goal of this study was to evaluate the utility of ctDNA quantification and characterization at time of disease progression in mCRPC. Methods: From 2015 to 2018, 221 mCRPC patients underwent ctDNA screening with Guardant 360 (Guardant Health, Inc. Redwood City, CA) at times of disease progression. This analysis consisted of exonic coverage of 70 genes as well as amplifications in 18 genes, with pathogenic mutations categorized by effect and mutant fraction ctDNA. Clinical annotation including lines of life-extending therapies (LET) before and after testing were collected. Statistical analyses included repeated measure ANOVA, logistic regression, and parameter estimations. Results: An increasing number of LETs resulted in a clinically significant increase in mutant ctDNA fraction (P &lt; 0.001). Subjects that received ≥ 5 or 3-4 LETs had significantly higher total mutant ctDNA fraction compared to subjects exposed with either 0 (p = 0.0007 and 0.0196, respectively) or 1-2 LETs (p = 0.0013 and 0.0432, respectively). 1-2 LETs did not demonstrate a significantly different total mutant ctDNA compared to subjects with 0 LETs (p = 0.4578). The estimated mean total mutant ctDNA fraction demonstrated a positive trend with an increasing number of LETs, including 2.08% (95% CI 1.24-3.49) without a LET, 2.62% (95% CI 1.71–4.01%) with 1-2 LETs, 4.58% (95% CI 2.92–7.17) with 3–4 LETs, and 7.79% (95% CI 4.51-13.48) for subjects with ≥5 LETs. The presence of copy number gain variations (CNVs) demonstrated an increased frequency with exposure to more LETs (p = 0.0005). Subjects with ≥5 or 3-4 LETs were 5.192 (p = 0. 0.0011, 95% CI 1.934-13.942) and 4.808 (p = 0.0019, 1.780-12.983) times more likely to have CNVs present compared to those without LETs. Conclusions: As mCRPC patients progress through lines of therapy, both the mutant ctDNA fraction and frequency of copy number gain variations increase.

Low-Frequency Mutational Heterogeneity of Invasive Ductal Carcinoma Subtypes: Information to Direct Precision Oncology.

Information regarding the role of low-frequency hotspot cancer-driver mutations (CDMs) in breast carcinogenesis and therapeutic response is limited. Using the sensitive and quantitative Allele-specific Competitor Blocker PCR (ACB-PCR) approach, mutant fractions (MFs) of six CDMs (PIK3CA H1047R and E545K, KRAS G12D and G12V, HRAS G12D, and BRAF V600E) were quantified in invasive ductal carcinomas (IDCs; including ~20 samples per subtype). Measurable levels (i.e., ≥ 1 × 10−5, the lowest ACB-PCR standard employed) of the PIK3CA H1047R, PIK3CA E545K, KRAS G12D, KRAS G12V, HRAS G12D, and BRAF V600E mutations were observed in 34/81 (42%), 29/81 (36%), 51/81 (63%), 9/81 (11%), 70/81 (86%), and 48/81 (59%) of IDCs, respectively. Correlation analysis using available clinicopathological information revealed that PIK3CA H1047R and BRAF V600E MFs correlate positively with maximum tumor dimension. Analysis of IDC subtypes revealed minor mutant subpopulations of critical genes in the MAP kinase pathway (KRAS, HRAS, and BRAF) were prevalent across IDC subtypes. Few triple-negative breast cancers (TNBCs) had appreciable levels of PIK3CA mutation, suggesting that individuals with TNBC may be less responsive to inhibitors of the PI3K/AKT/mTOR pathway. These results suggest that low-frequency hotspot CDMs contribute significantly to the intertumoral and intratumoral genetic heterogeneity of IDCs, which has the potential to impact precision oncology approaches.

Rezidüel Tümör Tespiti İçin Bir Sıvı Biyopsi Yöntemi Olarak Alelspesifik Emülsiyon PCR (asePCR)

AimThis study aimed to develop an allele-specific emulsion polymerase chain reaction (asePCR)technique for detection of cell-free circulating tumor DNA (ctDNA) in the blood plasma of cancer patients. Materials and MethodsGenomic DNA extracted from normal (wild-type) and thyroid cancer (mutant) cell lines (MCF10A and B-CPAP) were used to obtain specific dilution series (spike-in) of mutant DNA with wild-type DNA. Two sequential PCR steps (regular and asePCR) were performed with specifically designed primers for each step to detect the spiked-in mutant DNA fraction within the wild-type normal DNA. The DNA products amplified by regular PCR were used as templates in the asePCR where specific reverse primers were designed to amplify either the mutant or wild-type allele while a common forward primer was covalently bound to the beads. The asePCR reaction mix was encapsulated in water-in-oil emulsion compartments and incubated in a thermocycler with specific PCR program settings. At the end of asePCR reaction, oil compartments were broken and DNA-coated beads were evaluated by FACS to determine the mutant and wild-type fractions for each sample.ResultsThe asePCR technique successfully detected the mutant DNA in the background of wildtype DNA (0% mutant) as a proof-of-principle. A strong correlation (r2&amp;gt;0.99) was found between the expected mutant fraction and the fraction measured by asePCR. asePCR was sensitive and specific enough to consistently detect various low mutant DNA fractions in the background of wild-type DNA with no false positives in the wild-type sample.Discussion and Conclusion Our asePCR can be used to detect mutant ctDNA in the plasma of cancer patients as a liquid biopsy method for tumor response evaluation and early detection of possible relapses.

EGFR C797S, EGFR T790M and EGFR sensitizing mutations in non-small cell lung cancer revealed by six-color crystal digital PCR.

BackgroundDetection of EGFR sensitizing and p.T790M and p.C797S resistance mutations is particularly important for non-small cell lung cancer (NSCLC) patient therapy management. Non-invasive blood-based monitoring of these mutations may pave the way to a fine-tuned personalized treatment. Digital PCR has emerged as an extremely sensitive method to detect rare mutations, however its major limitation is the number of hotspots that can be simultaneously differentiated.MethodsWe developed a 6-color digital PCR assay for the detection and quantification of 19 most prevalent EGFR sensitizing and resistance mutations and evaluated this assay on 82 tumor and plasma samples from NSLC patients.ResultsLimits of detection (LOD) for the 6-color digital PCR assay were assessed on serial dilutions of DNA standards. We found that the 6-color assay enabled detection of mutant fractions as low as 1 mutant in 1025 wild-type molecules, depending on the mutation targeted, when assayed in a background of 10 000 wild-type DNA copies. EGFR mutant allelic fraction was also measured on tumor and plasma samples by 6-color digital PCR, and displayed a highly significant correlation with next generation sequencing and 3-color digital PCR. Lastly, the 6-color digital PCR assay was performed on several longitudinal plasma samples from four patients and revealed levels of sensitizing and resistance EGFR mutations that reflected well the course of the disease.ConclusionThis 6-color Crystal digital PCR assay could represent a robust solution using digital PCR for the monitoring of EGFR mutations.

Bordetella bronchiseptica Bcr4 antagonizes the negative regulatory function of BspR via its role in type III secretion.

Bordetella species, including B. pertussis, have a type III secretion system that is highly conserved among gram-negative pathogenic bacteria. Genes encoding the component proteins of the type III secretion system are localized at the bsc locus in the Bordetella genome. Here, the function of a hypothetical protein Bcr4 encoded at the bsc locus in the B. bronchiseptica genome was investigated. A Bcr4-deficient mutant was created and the amounts of type III secreted proteins (e.g., BopB, BopN and Bsp22) in both the supernatant fraction and whole-cell lysates of the Bcr4-deficient mutant were determined. It was found that the amounts of these proteins were significantly lower than in the wild-type strain. The amounts of type III secreted proteins in the supernatant fraction and whole-cell lysates were much greater in a Bcr4-overproducing strain than in the wild-type strain. The type III secreted protein BspR reportedly negatively regulates the type III secretion system. Here, it was observed that a Bcr4 + BspR double-knockout mutant did not secrete type III secreted proteins, whereas the amounts of these proteins in whole-cell lysates of this mutant were nearly equal to those in whole-cell lysates of the BspR-deficient mutant. Bcr4 thus appears to play an essential role in the extracellular secretion of type III secreted proteins. Our data also suggest that Bcr4 antagonizes the negative regulatory function of BspR.

Sensitive and rapid detection of TERT promoter and IDH mutations in diffuse gliomas.

Mutations in telomerase reverse transcriptase promoter (TERTp) and isocitrate dehydrogenase 1 and 2 (IDH) offer objective markers to assist in classifying diffuse gliomas into genetic subgroups. However, traditional mutation detection techniques lack sensitivity or have long turnaround times or high costs. We developed GliomaDx, an allele-specific, locked nucleic acid-based quantitative PCR assay to overcome these limitations and sensitively detect TERTp and IDH mutations. We evaluated the performance of GliomaDx on cell line DNA and frozen tissue diffuse glioma samples with variable tumor percentage to mimic use in clinical settings and validated low percentage variants using sensitive techniques including droplet digital PCR (ddPCR) and next-generation sequencing. We also developed GliomaDx Nest, which incorporates a high-fidelity multiplex pre-amplification step prior to allele-specific PCR for low-input formalin-fixed paraffin embedded (FFPE) samples. GliomaDx detects the TERTp and IDH1 alterations at an analytical sensitivity of 0.1% mutant allele fraction, corresponding to 0.2% tumor cellularity. GliomaDx identified TERTp/IDH1 alterations in a cohort of frozen tissue samples with variable tumor percentage of all major diffuse glioma histologic types. GliomaDx Nest is able to detect these hotspot mutations with similar sensitivity from pre-amplified samples and was successfully tested on a cohort of clinical FFPE samples. Testing of a cohort of previously identified TERTpWT-IDHWT gliomas (by Sanger sequencing) revealed that 26.3% harbored low-percentage mutations. Analysis by ddPCR and whole exome sequencing of these tumors confirmed the low mutant fraction of these alterations and overall mutation-based tumor purity. Our results show that GliomaDx can rapidly detect TERTp/IDH mutations with high sensitivity, identifying cases that might be missed due to the lack of sensitivity of other techniques. This approach may facilitate more objective classification of diffuse glioma samples in clinical settings such as intraoperative diagnosis or in testing cases with low tumor purity.

Lifespan Kras mutation levels in lung and liver of B6C3F1 mice.

Somatic mutations accumulate in the human genome and are correlated with increased cancer incidence as humans age. The standard model for studying the carcinogenic effects of exposures for human risk assessment is the rodent 2-year carcinogenicity assay. However, there is little information regarding the effect of age on cancer-driver gene mutations in these models. The mutant fraction (MF) of Kras codon 12 GGT to GAT and GGT to GTT mutations, oncogenic mutations orthologous between humans and rodents, was quantified over the lifespan of B6C3F1 mice. MFs were measured in lung and liver tissue, organs that frequently develop tumors following carcinogenic exposures. The MFs were evaluated at 4, 6, 8, 12, 21, and 85 weeks, with the 12-week and 21-week time points being coincident with the conclusion of 28-day and 90-day exposure durations used in short-term toxicity testing. The highly sensitive and quantitative Allele-specific Competitive Blocker PCR (ACB-PCR) assay was used to quantify the number of mutant Kras codon 12 alleles. The mouse lung showed a slight, but significant trend increase in the Kras codon 12 GAT mutation over the 85-week period. The trend with age can be equally well-fit by several non-linear functions, but not by a linear function. In contrast, the liver GAT mutation did not increase, and the GTT mutation did not increase for either organ. Even with the slight increase in the lung GAT MFs, our results indicate that the future use of Kras mutation as a biomarker of carcinogenic effect will not be confounded by animal age. Environ. Mol. Mutagen. 59:715-721, 2018. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

METABOLIC ACTIVATION OF 2,6-DIMETHYLANILINE: MUTATIONAL SPECIFICITY IN THE GPT GENE OF AS52 CELLS

Objective: The purpose of the current work was to characterize the mechanisms of cytotoxicity and mutagenesis of a potential human bladder carcinogen 2,6-dimethylaniline (2,6-DMA).Methods: Chinese hamster ovary (CHO) AS52 cells were exposed to either human S9 activated 2,6-DMA for 6 h or its N-hydroxylamine and aminophenol metabolites for 1 h in serum-free medium. Cell survival was determined by trypan blue exclusion 24 h after treatment, and 6-thioguanine-resistant mutants at the xanthine-guanine phosphoribosyl transferase (gpt) gene locus were assessed with doses, of which relative survival is 30% or more. Nested polymerase chain reaction-based deletion analysis was also performed.Results: AS52 cells exhibited a dose-dependent increase in cytotoxicity and mutant fraction on treatment of 2,6-DMA and its metabolites but show a considerable variation in potency with aminophenol metabolites having the highest potency and parent compound at least; at the highest 2,6-dimethylaminophenol dose (10 μM), the mutant fraction in AS52 cells was 8-fold (13.2×10−5) greater than the spontaneous fraction of 1.62×10−5. Total deletion of the gpt gene sequences was found in 1 (4%) spontaneous and 2 (6%) the 6-thioguanine mutants generated by N-hydroxy-2,6-DMA.Conclusions: These findings indicate the mutagenicity of 2,6-DMA at the gpt gene, which is mediated through hydroxylamine and aminophenol metabolites, and contribute to the elucidation of mechanisms through which 2,6-DMA may exert its effects in vivo.

GENOTOXICITY OF N-HYDROXY AND AMINOPHENOL METABOLITES OF 2,6- AND 3,5-DIMETHYLANILINE AT THE HYPOXANTHINEGUANINE PHOSPHORIBOSYLTRANSFERASE LOCUS IN TK6 CELLS

Objective: The objective of this study as to characterize the genotoxicity of reactive metabolites of 2,6-dimethylaniline (2,6-DMA) and 3,5-DMA in the hypoxanthineguanine phosphoribosyltransferase (HPRT) gene of human lymphoblastoid TK6 cells.Methods: Cultures were exposed to N-hydroxylamine and aminophenol metabolites of 2,6- and 3,5-DMA for 1 h in serum-free medium. Cell survival 24 h after exposure was determined by trypan blue exclusion. Cells were then subcultured for 7–10 days to allow to phenotypic expression of HPRT mutants. After the expression period, cells were plated in the presence of 2 μg/ml 6-thioguanine for the selection of HPRT mutants. Plating efficiency was determined and mutant fraction calculated. Electron paramagnetic resonance (EPR) was also used to determine whether 3,5- dimethylaminophenol (DMAP) produced reactive oxygen species (ROS).Results: All of the metabolites tested were cytotoxic to these cells but exhibited a considerable variation in potency. The aminophenol metabolites of 2,6- and 3,5-DMA were considerably more toxic than the corresponding N-hydroxylamines. Furthermore, each metabolite of 3,5-DMA was more toxic than its 2,6-DMA counterpart; N-OH-3,5-DMA and 3,5-DMAP were clearly mutagenic at a level of 50 μM. EPR studies showed intracellular oxidative stress induced under 3,5-DMAP treatment.Conclusions: Our findings suggest that genotoxic responses of 2,6- and 3,5-DMA are mediated through the generation of ROS by hydroxylamine and/ or aminophenol metabolites.