Low-level Mutations Research Articles

53. Clinical utility of single-gene ultra-deep coverage NGS assay for TP53 mutations for detection of low-level mutations

TP53 is a tumor suppressor gene most frequently mutated in malignancies. Mutations in TP53 span the entire coding region without any hot-spots. In serial follow-up samples, identification of previously known TP53 mutation serves as a marker of minimal residual disease and can potentially influence treatment decisions. Conventional standard for single gene TP53 mutation testing is by Sanger sequencing. However, Sanger sequencing is laborious and is limited by a low-detection sensitivity of ∼20%, feasibility to test only limited number of exons and limited ability for multiplexing. To overcome these problems, in our laboratory, we validated and recently implemented a single-gene NGS-based mutation assay for TP53 gene. The assay (Multiplicom, Belgium) is composed of 17 amplicons in 2 pools covering the entire coding region of TP53 gene (1.8 kb).). Using 20 ng of genomic DNA from either fresh peripheral blood/ bone marrow or formalin fixed paraffin embedded samples, library amplification followed by sequencing on Miseq flow cell (24 samples/ run) resulted in a median depth of coverage of ∼7000x per sample. The limit of detection of this assay was determined at 1%. In the meeting, we will present and illustrate clinical scenarios where the (1) high sensitivity of this assay and (2) comprehensive coverage of the entire coding region (compared to Sanger sequencing) added clinical value for minimal residual disease monitoring including post-transplant setting and early detection of sub-clonal mutations.

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.

Abstract 4522: An optimized NGS workflow for detection of FLT3 internal tandem duplication (ITD) in AML samples

Abstract FLT3 ITD, a prognostic marker and drug target, is present in ~19% of AML patients. FLT3 ITD is a tandem repeat of the entire or partial exon 13-15 region. FLT3 ITDs range in size from 3bp to above 300bp and impact RTK signaling. Recent publications have shown that FLT3 ITD can be detected at single nucleotide resolution by NGS panels. However, detection of large FLT3 ITD and accurate reporting of ITD frequency remain challenging for NGS methods. False negative ITD results or inaccurate mutant/wild type allele ratio measurements could negatively alter treatment decisions for AML patients. The Illumina TruSight Myeloid panel (TMP) is a commercial NGS assay that covers 54 genes involved in myeloid malignancies. Although the TMP assay can detect SBS and small indel, medium to large size FLT3 ITD cannot be detected. Here we present an enhanced workflow to overcome these challenges. Positive controls, dynamic range, accuracy, and inter-run reproducibility were validated for the detection of SBS, indel, and FLT3 ITD using reference standards and DNA extracted from cell lines and AML patient EDTA blood and bone marrow aspirate samples. NGS libraries were prepared following Illumina's standard protocol. 275bp X2 sequencing instead of the standard 150bp X2 was run on MiSeq flowcell v3. Data analysis was performed using a MolecularMD custom pipeline. The analysis of FLT3 ITD was conducted using three methods including two off-the-shelf programs, Pindel and ITDseek, as well as Cigar, a MolecularMD developed ITD detection algorithm. FLT3 ITDs at 126 bp and 21 bp in PL-21 and MOLM-13 cell lines, respectively, were detected by TMP assay using the three algorithms. In contrast to ITDseek which only reports ITD sequence without frequency information, Pindel and Cigar report both ITD sequence and frequency. Compared to Pindel, ITD frequency reported by the custom algorithm is closer to that determined by the fragment analysis. Serial dilution of the 126bp ITD into wild type DNA showed the 0.55% ITD (from fragment analysis) can be reproducibly detected at ~0.6% by the Cigar algorithm. FLT3 ITDs ranging in size from 6bp to 300bp were detected in clinical AML samples using the enhanced TMP workflow and confirmed by a fragment analysis method. Limit of detection, reproducibility and accuracy of SBS and indel detection in other key genes by the TMP assay have also been validated. Low-level mutations, i.e. NRAS G12D at 1.7%, NRAS G13D at 2%, and IDH2 R140Q at 1.4%, were detected by TMP panel from AML samples and further confirmed with an independent assay. A long deletion (52bp) in exon 9 of CALR was also detected by the custom pipeline. In summary, the enhanced TMP workflow allows for accurate detection and reporting of SBS, indel and FLT3 ITDs including large duplications. Optimized detection of FLT3 ITDs will improve AML patient selection for targeted therapy. Citation Format: Xiaodong Wang, Zhenyu Yan, Peng Fang, Weihua Liu, Scott Glynn, Jennifer Biroschak, Chad Galderisi, Cindy Spittle, Jin Li. An optimized NGS workflow for detection of FLT3 internal tandem duplication (ITD) in AML samples [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 4522.

PO-317 Improved performance and workflow using sanger sequencing for low-level somatic mutation detection from low amount of formalin-fixed paraffin-embedded (FFPE) DNA

IntroductionSanger sequencing is often used in oncology research applications for molecular profiling of cancers. Applied Biosystems Minor Variant Finder (MVF) Software now enables low-level variant detection in Sanger sequencing traces.RAS mutational testing is frequently performed by clinical researchers due to the strong correlation between RAS mutational profiles of colorectal cancers and their anti–epidermal growth factor receptor (EGFR) response.Material and methodsWe have improved and simplified the workflow for the extended RAS research assay. The ready-to-use plates are preloaded with primers for all the eight frequently studied hotspot regions of KRAS and NRAS genes thus only the PCR mix and templates need to be added. Minimised hands-on time, extra convenience and flexibility gained by using the SeqStudio Genetic Analyzer which utilises an all-in-one cartridge and provides simple plate setup with easy-to-use touch-screen interface and real-time/remote data monitoring.Results and discussionsThe performance of the assay on the SeqStudio instrument was tested in the laboratory of Dr. Quagliata (University of Basel, Switzerland) on twenty-two FFPE DNA samples derived from colon cancer biopsies. Variants (ranging from 5.25% to 91.1%) were successfully detected even from research samples where less than 1 ng of input DNA per reaction was used. Only limited amount of microdissected FFPE sections were available for 6 research samples where DNA concentrations were below 1 ng (down to 0.094 ng).ConclusionRAS mutations down to 5% level can be detected even from less than 1 ng FFPE DNA with simplified workflow, fast turnaround time and low cost per sample.

Abstract 941: Method for sensitive detection of tumor fingerprints in plasma

Abstract Presence of excess unaltered, wild-type DNA providing no information of biologic or clinical value may often mask rare alterations containing diagnostic or therapeutic clues for cancer. There is a growing demand for removing unaltered DNA over large pools-of-sequences, especially for applications in liquid biopsies with circulating DNA (cfDNA). We recently developed nuclease-assisted minor-allele enrichment with probe-overlap (NaME-PrO), a single-step approach that removes WT-DNA prior to DNA amplification, following which current genomic analysis processes remain unchanged. NaME-PrO employs a double-strand DNA-specific nuclease (DSN) and overlapping oligonucleotide-probes interrogating multiple DNA targets. Following genomic-DNA denaturation, the temperature is lowered and the probes form transient double-stranded regions with their targets, and DSN is added to provide nuclease digestion to the selected sites. Mutations create mismatches that inhibit DSN digestion; thus, subsequent amplification yields DNA with alterations enhanced at multiple targets. In this manner, WT DNA at hundreds or thousands of DNA regions can be digested simultaneously. We also show that addition of organic solvents like DMSO in the NaME-PrO reaction enables DNA denaturation at lower temperatures that do not inactivate the enzyme (DSN). This enables DSN addition from the beginning of the reaction, enabling homogeneous, closed-tube application of the approach and avoids contamination-prone sample manipulations due to open-tube addition of DSN enzyme after denaturation. Using NaME-PrO ± DMSO we demonstrate several-hundred-fold multiplexed mutation enrichment in diverse human samples on multiple clinically relevant targets in 50-plex reactions. Application with targeted resequencing of tumor-circulating DNA demonstrated detection of mutations at the 0.01-0.1% levels with few sequence reads. In ongoing studies aiming to detect minimal residual disease using cfDNA in late-stage breast cancer, we used tumor exome sequencing to identify multiple clonal mutations for individual patients. We then performed multiplexed NaME-PrO to trace the "tumor fingerprint" in plasma. In ~50-plex reactions we showed that the majority (>60%) of the tumor-specific mutations were also detected in plasma. To evaluate the ultimate sensitivity of the method to identify such "tumor fingerprints," we applied NaME-PrO in serial dilutions of cfDNA from breast cancer patients into cfDNA from cancer-free, normal patients. We demonstrated that dilutions down to 1,000-10,000-fold, corresponding to mutations at <0.01% allelic frequency, lead to identification of at least one tumor-specific mutation in plasma. NaME-PrO can be combined with platforms that utilize molecular barcodes to provide "maximum efficiency sequencing," enabling sensitive detection of low-level mutations without requiring "deep sequencing" and excessive sequence reads. Citation Format: Fangyan Yu, Mariana Fitarelli-Kiehl, Ka Wai Leong, Viktor Adalsteinsson, Heather Parsons, Mike G. Makrigiorgos. Method for sensitive detection of tumor fingerprints in plasma [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 941.

Health Profiles of Mosaic Versus Non-mosaic FMR1 Premutation Carrier Mothers of Children With Fragile X Syndrome

The FMR1 premutation is of increasing interest to the FXS community, as questions about a primary premutation phenotype warrant research attention. 100 FMR1 premutation carrier mothers (mean age = 58; 67–138 CGG repeats) of adults with fragile X syndrome were studied with respect to their physical and mental health, motor, and neurocognitive characteristics. We explored the correlates of CGG repeat mosaicism in women with expanded alleles. Mothers provided buccal swabs from which DNA was extracted and the FMR1 CGG genotyping was performed (Amplidex Kit, Asuragen). Mothers were categorized into three groups: Group 1: premutation non-mosaic (n = 45); Group 2: premutation mosaic (n = 41), and Group 3: premutation/full mutation mosaic (n = 14). Group 2 mothers had at least two populations of cells with different allele sizes in the premutation range besides their major expanded allele. Group 3 mothers had a very small population of cells in the full mutation range (>200 CGGs) in addition to one or multiple populations of cells with different allele sizes in the premutation range. Machine learning (random forest) was used to identify symptoms and conditions that correctly classified mothers with respect to mosaicism; follow-up comparisons were made to characterize the three groups. In categorizing mosaicism, the random forest yielded significantly better classification than random classification, with overall area under the receiver operating characteristic curve (AUROC) of 0.737. Among the most important symptoms and conditions that contributed to the classification were anxiety, menopause symptoms, executive functioning limitations, and difficulty walking several blocks, with the women who had full mutation mosaicism (Group 3) unexpectedly having better health. Although only 14 premutation carrier mothers in the present sample also had a small population of full mutation cells, their profile of comparatively better health, mental health, and executive functioning was unexpected. This preliminary finding should prompt additional research on larger numbers of participants with more extensive phenotyping to confirm the clinical correlates of low-level full mutation mosaicism in premutation carriers and to probe possible mechanisms.

Genetic and epigenetic alterations in normal tissues have differential impacts on cancer risk among tissues

Genetic and epigenetic alterations are both involved in carcinogenesis, and their low-level accumulation in normal tissues constitutes cancer risk. However, their relative importance has never been examined, as measurement of low-level mutations has been difficult. Here, we measured low-level accumulations of genetic and epigenetic alterations in normal tissues with low, intermediate, and high cancer risk and analyzed their relative effects on cancer risk in the esophagus and stomach. Accumulation of genetic alterations, estimated as a frequency of rare base substitution mutations, significantly increased according to cancer risk in esophageal mucosae, but not in gastric mucosae. The mutation patterns reflected the exposure to lifestyle risk factors. In contrast, the accumulation of epigenetic alterations, measured as DNA methylation levels of marker genes, significantly increased according to cancer risk in both tissues. Patients with cancer (high-risk individuals) were precisely discriminated from healthy individuals with exposure to risk factors (intermediate-risk individuals) by a combination of alterations in the esophagus (odds ratio, 18.2; 95% confidence interval, 3.69-89.9) and by only epigenetic alterations in the stomach (odds ratio, 7.67; 95% confidence interval, 2.52-23.3). The relative importance of epigenetic alterations upon genetic alterations was 1.04 in the esophagus and 2.31 in the stomach. The differential impacts among tissues will be critically important for effective cancer prevention and precision cancer risk diagnosis.

An optimized targeted Next-Generation Sequencing approach for sensitive detection of single nucleotide variants

Monitoring of minimal residual disease (MRD) has become an important clinical aspect for early relapse detection during follow-up care after cancer treatment. Still, the sensitive detection of single base pair point mutations via Next-Generation Sequencing (NGS) is hampered mainly due to high substitution error rates. We evaluated the use of NGS for the detection of low-level variants on an Ion Torrent PGM system. As a model case we used the c.1849G > T (p.Val617Phe) mutation of the JAK2-gene. Several reaction parameters (e.g. choice of DNA-polymerase) were evaluated and a comprehensive analysis of substitution errors was performed. Using optimized conditions, we reliably detected JAK2 c.1849G > T VAFs in the range of 0.01–0.0015% which, in combination with results obtained from clinical data, validated the feasibility of NGS-based MRD detection. Particularly, PCR-induced transitions (mainly G > A and C > T) were the major source of error, which could be significantly reduced by the application of proofreading enzymes. The integration of NGS results for several common point mutations in various oncogenes (i.e. IDH1 and 2, c-KIT, DNMT3A, NRAS, KRAS, BRAF) revealed that the prevalent transition vs. transversion bias (3.57:1) has an impact on site-specific detection limits of low-level mutations. These results may help to select suitable markers for MRD detection and to identify individual cut-offs for detection and quantification.

Abstract A032: Circulating tumor (ct)DNA mutations in EGFR pathway genes and clinical outcomes for patients with metastatic colorectal cancer (mCRC) treated with panitumumab from the ASPECCT study

Abstract Background: Mutations in RAS family genes are a negative predictor for response to anti-EGFR therapy. ASPECCT was the first prospective study to show that panitumumab was noninferior to cetuximab for overall survival (OS) in chemorefractory wild-type (WT) KRAS exon 2 mCRC. This analysis used next-generation sequencing of ctDNA before and after panitumumab therapy to explore mutations in EGFR pathway genes and their association with outcomes. Methods: Plasma samples collected at baseline (BL) and safety follow-up (SFU) were analyzed for ctDNA mutations using the PlasmaSelect-R™ 63-gene panel. Mutations for six major genes within the EGFR pathway (BRAF, KRAS, MAP2K1, NRAS, PIK3CA, and PTEN) were analyzed as categorical and continuous variables, and were evaluated for association with OS using univariate Cox proportional hazards (PH) model. Results: Of the 499 patients randomized to panitumumab, 208 had paired plasma samples at BL and SFU. Of the 113 (54.3%) patients who were WT for all genes at BL, 59 (52%) remained WT at SFU. At BL, 65 patients had single gene mutations in either BRAF (7.7%), KRAS (6.7%), PIK3CA (6.3%), NRAS (5.8%), PTEN (3.8%), or MAP2K1 (1.0%), and 30 patients had mutations in multiple genes (at <2% prevalence). The majority of mutations at BL were in BRAF (16.3%), PIK3CA (14.4%), and KRAS (13.5%). At SFU, 62 patients gained single gene mutations in either KRAS (9.1%), BRAF (7.7%), PIK3CA (4.8%), MAP2K1 (3.8%), NRAS (3.4%), or PTEN (1.0%). The majority of mutations gained at SFU were in KRAS (22.6%) and BRAF (21.2%). Categorical analysis of mutations at BL found that patients with WT status for all six genes survived longer, and that KRAS and PTEN WT status had the most significant association to OS. Furthermore, continuous analysis indicated that patients with low mutation levels survived longer (table). Conclusions: Patients were more likely to have a mutation in a single gene at BL and in multiple genes at SFU. Based on the Cox PH model, KRAS had the smallest P value for correlation with OS at BL. Continuous analysis of ctDNA fraction suggests that patients with low-level KRAS mutations may still benefit from anti-EGFR therapy; however, a threshold for treatment benefit cannot be determined from a single-arm trial. These findings suggest that the tumor genetic landscape can become increasingly complex after treatment despite a single selection pressure. Categorical and Continuous Analysis for EGFR Pathway Genes and Median Overall Survival (mOS)*Categorical (Y/N)ContinuousGeneWild-typeMutantLow Risk†High Risk†Any/Sumn (%)113 (54.3)95 (45.7)153 (73.6)55 (26.4)mOS, months (95% CI)38.17 (31.58-48.08)21.92 (19.25-25.67)34.92 (29.25-38.67)18.42 (14.50-21.25)Cox PH P value2.803E-074.185E-09BRAFn (%)174 (83.7)34 (16.3)199 (95.7)9 (4.3)mOS, months (95% CI)31.58 (26.25-36.17)21.25 (15.67-28.67)29.25 (25.25-34.75)18.42 (6.08-21.92)Cox PH P value0.0030.014KRASn (%)180 (86.5)28 (13.5)192 (92.3)16 (7.7)mOS, months (95% CI)31.58 (27.33-36.17)19.92 (12.42-21.83)30.33 (26.67-35.33)15.67 (11.08-21.25)Cox PH P value1.597E-052.953E-06MAP2K1‡n (%)201 (96.6)7 (3.4)N/AN/AmOS, months (95% CI)29.17 (24.67-34.58)14.50 (0.00-43.42)Cox PH P value0.025NRASn (%)188 (90.4)20 (9.6)201 (96.6)7 (3.4)mOS, months (95% CI)29.17 (24.67-34.83)24.25 (16.33-33.67)29.17 (24.75-34.58)15.25 (0.00-45.58)Cox PH P value0.0450.002PIK3CAn (%)178 (85.6)30 (14.4)200 (96.2)8 (3.8)mOS, months (95% CI)29.50 (25.67-35.33)21.83 (15.67-29.42)29.17 (24.75-34.75)21.83 (6.08-33.17)Cox PH P value0.0090.021PTENn (%)190 (91.3)18 (8.7)197 (94.7)11 (5.3)mOS, months (95% CI)30.33 (26.25-35.50)19.75 (6.42-23.92)29.25 (25.25-34.83)15.25 (6.25-21.83)Cox PH P value6.907E-065.715E-05*A limitation of this analysis is the low number of mutants in some cases.†The cutoff was defined as the mutation level where the predicted hazard equals the baseline hazard.‡Insufficient numbers above cutoff to perform continuous analysis.CI, confidence interval; PH, proportional hazards; Y/N, yes/no Citation Format: Michael Boedigheimer, Agnes Lee Ang, Tae Won Kim, Anne Thomas, Peter Gibbs, Paul Ruff, Kristina Hool, Timothy Price. Circulating tumor (ct)DNA mutations in EGFR pathway genes and clinical outcomes for patients with metastatic colorectal cancer (mCRC) treated with panitumumab from the ASPECCT study [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 A032.

Detection of low-abundance point mutations by competitive strand assisted endonuclease IV signal amplification system.

Genetic mutations are important molecular biomarkers for cancer diagnosis and surveillance. Therefore, the development of methods for mutation detection characterized with straightforward, highly specific and sensitive to low-level mutations within various sequence contexts is extremely needed. Although some of the currently available methods have shown very encouraging results, their discrimination efficiency is still very low. Herein, we demonstrate a fluorescent probe coupled with blocker and property of melting temperature discrimination, which is able to identify the presence of known or unknown single-base variations at abundances down to 0.1% within 20 min. The discrimination factors between the perfect-match target and single-base mismatched target are determined to be 10.15-38.48. The method is sequence independent, which assures a wide range of application. The new method would be an ideal choice for high-throughput in vitro diagnosis and precise clinical treatment.

Evaluation of digital PCR for detecting low-level EGFR mutations in advanced lung adenocarcinoma patients: a cross-platform comparison study

Emerging evidence has indicated that circulating tumor DNA (ctDNA) from plasma could be used to analyze EGFR mutation status for NSCLC patients; however, due to the low level of ctDNA in plasma, highly sensitive approaches are required to detect low frequency mutations. In addition, the cutoff for the mutation abundance that can be detected in tumor tissue but cannot be detected in matched ctDNA is still unknown. To assess a highly sensitive method, we evaluated the use of digital PCR in the detection of EGFR mutations in tumor tissue from 47 advanced lung adenocarcinoma patients through comparison with NGS and ARMS. We determined the degree of concordance between tumor tissue DNA and paired ctDNA and analyzed the mutation abundance relationship between them. Digital PCR and Proton had a high sensitivity (96.00% vs. 100%) compared with that of ARMS in the detection of mutations in tumor tissue. Digital PCR outperformed Proton in identifying more low abundance mutations. The ctDNA detection rate of digital PCR was 87.50% in paired tumor tissue with a mutation abundance above 5% and 7.59% in paired tumor tissue with a mutation abundance below 5%. When the DNA mutation abundance of tumor tissue was above 3.81%, it could identify mutations in paired ctDNA with a high sensitivity. Digital PCR will help identify alternative methods for detecting low abundance mutations in tumor tissue DNA and plasma ctDNA.

Validation of the denaturing capillary electrophoresis (DCE) assay for non-invasive liquid biopsy in lung carcinoma: A study of concordance with the cobas EGFR mutation test v2.

e23040 Background: The detection of circulating tumor DNA (ctDNA), referred to as “liquid biopsy” is becoming a preferred option for some patients with lung adenocarcinomas. The key for a suitable technology is high sensitivity enabling to detect small fractions of mutated alleles, the method has to be solid against false-positives. The aim of this work was to compare our method based on capillary electrophoresis under denaturing conditions (DCE) to FDA and CE IVD approved commercial kit - cobas EGFR Mutation Test v2. Methods: A total of 44 plasma samples were acquired from patients with clinicaly confirmed stage IV lung adenocarcinoma. There were samples from patients prior to receiving therapy (with a known tissue EGFR genotype) as well as several ones showing progression on a previously administered antiEGFR-therapy (with no tissue info available at the time of plasma sampling). DNA was isolated from multiple 2 mL aliquots of each plasma. The first aliquot was run on cobas z480 real-time instrument using unmodified manufacturer's protocol. Another 2ml aliquot was run by DCE assay (accredited method under ISO 15189:2012), which is based on a PCR followed by a fragment analysis on an ABI capillary analyzer (Sanger sequencer). Results: The mutual concordance was 84.1% (37/44). cobas assay identified 11 EGFR-positive plasma samples (6 x Ex19del, 3 x L858R, 1x T790M combined with Ex19del and 1x T790M combined with L858R). DCE identified 8 EGFR-positive samples (6 x Ex19del, 1 x L858R, 1xT790M combined with L858R). DCE missed 5 of cobas-positive samples (2x Ex19del, 2x L858R and 1x T790M) while in turn identifying 2x Ex19del plasma samples that were concordant with tissue, but missed by cobas. Overall concordance with tissue was 73.3% (22/30) for cobas, 70.0% (21/30) for DCE assay and 80.0% (24/30) for both combined. Conclusions: DCE approach is reliable for low levels of mutations in plasma of lung cancer patients and comparable (and complementary) to cobas. Its advantage is simplicity, low cost and a universal, straighforward adaptability to virtually any mutation at any site allowing to detect oncogenic as well as tumor supressor mutations in ctDNA. Supported by grant 17-30748A.

Double mutation in DNA gyrase confers moxifloxacin resistance and decreased fitness of Mycobacterium smegmatis.

Ofloxacin and moxifloxacin are the most commonly used fluoroquinolones (FQs) for the treatment of tuberculosis. As a new generation FQ, moxifloxacin has been recommended for the treatment of ofloxacin-resistant TB. However, the mechanism by which ofloxacin-resistant Mycobacterium tuberculosis further gains resistance to moxifloxacin remains unclear. We used Mycobacterium smegmatis as a model for studying FQ resistance in M. tuberculosis . Moxifloxacin-resistant M. smegmatis was selected in vitro based on strains with primary ofloxacin resistance. The gyrA and gyrB genes of the resistant strains were sequenced to identify resistance-associated mutations. An in vitro competition assay was applied to explore the influence of gyrA / gyrB mutations on bacterial fitness. Finally, we evaluated the clinical relevance of our findings by analysing the WGS data of 1984 globally collected M. tuberculosis strains. A total of 57 moxifloxacin-resistant M. smegmatis strains based on five ofloxacin-resistant strains were obtained. Sequencing results revealed that all moxifloxacin-resistant strains harboured second-step mutations in gyrA or gyrB . The relative fitnesses of the double-mutation strains varied from 0.65 to 0.93 and were mostly lower than those of their mono-mutation parents. From the genomic data, we identified 37 clinical M. tuberculosis strains harbouring double mutations in gyrA and/or gyrB and 36 of them carried at least one low-level FQ-resistance mutation. Double mutation in DNA gyrase leads to moxifloxacin resistance and decreased fitness in M. smegmatis . Under current dosing of moxifloxacin, double mutations mainly happened in M. tuberculosis strains with primary low-level resistance mutations.

Frequent low-level mutations of protein kinase D2 in angiolipoma.

Tumours displaying differentiation towards normal fat constitute the most common subgroup of soft tissue neoplasms. A series of such tumours was investigated by whole-exome sequencing followed by targeted ultra-deep sequencing. Eighty per cent of angiolipomas, but not any other tumour type, displayed mutations in the protein kinase D2 (PRKD2) gene, typically in the part encoding the catalytic domain. The absence of other aberrations at the chromosome or RNA level suggests that PRKD2 mutations are critical for angiolipoma development. Consistently, the mutated PRKD2 alleles were present at low (3-15%) frequencies, indicating that only a subset of the tumour cells is affected. Indeed, by sequencing mature fat cells and other cells separately, the former typically showed the highest mutation frequencies. Thus, we hypothesize that altered PRKD2 signalling in the adipocytic cells drives tumourigenesis and, in agreement with its pivotal role in angiogenesis, induces the vessel formation that is characteristic for angiolipoma. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A branch-migration based fluorescent probe for straightforward, sensitive and specific discrimination of DNA mutations

Genetic mutations are important biomarkers for cancer diagnostics and surveillance. Preferably, the methods for mutation detection should be straightforward, highly specific and sensitive to low-level mutations within various sequence contexts, fast and applicable at room-temperature. Though some of the currently available methods have shown very encouraging results, their discrimination efficiency is still very low. Herein, we demonstrate a branch-migration based fluorescent probe (BM probe) which is able to identify the presence of known or unknown single-base variations at abundances down to 0.3%-1% within 5 min, even in highly GC-rich sequence regions. The discrimination factors between the perfect-match target and single-base mismatched target are determined to be 89–311 by measurement of their respective branch-migration products via polymerase elongation reactions. The BM probe not only enabled sensitive detection of two types of EGFR-associated point mutations located in GC-rich regions, but also successfully identified the BRAF V600E mutation in the serum from a thyroid cancer patient which could not be detected by the conventional sequencing method. The new method would be an ideal choice for high-throughput in vitro diagnostics and precise clinical treatment.

Abstract P3-09-03: Low-level gonosomal mosaicism of a de novo BRCA1 gene mutation – The origin of a constitutional mutation in a breast cancer family

Abstract P3-09-03: Low-level gonosomal mosaicism of a de novo <i>BRCA1</i> gene mutation – The origin of a constitutional mutation in a breast cancer family

Accurate clinical genetic testing for autoinflammatory diseases using the next-generation sequencing platform MiSeq

Autoinflammatory diseases occupy one of a group of primary immunodeficiency diseases that are generally thought to be caused by mutation of genes responsible for innate immunity, rather than by acquired immunity. Mutations related to autoinflammatory diseases occur in 12 genes. For example, low-level somatic mosaic NLRP3 mutations underlie chronic infantile neurologic, cutaneous, articular syndrome (CINCA), also known as neonatal-onset multisystem inflammatory disease (NOMID). In current clinical practice, clinical genetic testing plays an important role in providing patients with quick, definite diagnoses. To increase the availability of such testing, low-cost high-throughput gene-analysis systems are required, ones that not only have the sensitivity to detect even low-level somatic mosaic mutations, but also can operate simply in a clinical setting. To this end, we developed a simple method that employs two-step tailed PCR and an NGS system, MiSeq platform, to detect mutations in all coding exons of the 12 genes responsible for autoinflammatory diseases. Using this amplicon sequencing system, we amplified a total of 234 amplicons derived from the 12 genes with multiplex PCR. This was done simultaneously and in one test tube. Each sample was distinguished by an index sequence of second PCR primers following PCR amplification. With our procedure and tips for reducing PCR amplification bias, we were able to analyze 12 genes from 25 clinical samples in one MiSeq run. Moreover, with the certified primers designed by our short program—which detects and avoids common SNPs in gene-specific PCR primers—we used this system for routine genetic testing. Our optimized procedure uses a simple protocol, which can easily be followed by virtually any office medical staff. Because of the small PCR amplification bias, we can analyze simultaneously several clinical DNA samples with low cost and can obtain sufficient read numbers to detect a low level of somatic mosaic mutations.

Detecting Accumulated Somatic Mutations in Healthy Aged Female Myeloid Cells

Background: Acute Myeloid Leukemia and Myelodysplasia are diseases associated with age and with genetic mutations driving evolution of clonal dominance. The earliest steps leading to genetic variation occur in a milieu of normal hematopoiesis by as a matter of course because genomic divergence is a fundamental element of biology. Organism speciation implicates genetic variation is the first step of evolution, followed by resource restriction pressure (a bottleneck). Such conditions create an environment where competitive advantages arise for genetically unique sub-species. Our work is based on the hypothesis that these rules also apply to the rise of pathologic clonal dominance. Consequentially, a low level of mutations in normal hematopoietic stem cells should be detectable and increase with age. Mutations can serve as lineage markers to allow the definition of the kinetics of competing myeloid sub-clones. Also, individual clones with higher rates of mutation might serve as a pre-condition for pathological clonal dominance and disease initiation.Methods: Healthy 78 year old female myeloid progenitors were single cell sorted and cultured for 6 weeks in expansion media. Clones were harvested and typed apropos X-Chromosome Inactivation state (XCI state) as either "Xa" or "Xb" using a methylation specific polymerase chain reaction strategy after DNA was bisulfite modified. Remaining unmodified DNA was whole genome amplified and placed on an Affymetrix 6.0 Single Nucleotide Polymorphism (SNP) array (2.5 million SNP calls/array). Partek Genome Suite software interpreted raw SNP intensity signals as copy number variants (CNV) or loss of heterozygosity (LOH) calls using a stringent search strategy of a > 30 SNP markers, and an average variance of 0.5 (p<0.001). Mutations were compared between XCI states and within XCI states (i.e: individual Xa or Xb clones).Results: SNP comparison between XCI defined phylogenetic lineages showed significant somatic LOH and CNV variance, as did comparison of clones within specific XCI states (e.g: between Xa clones). Mutations represent accumulated genetic drift over the 78.75 years since subject conception and are summarized in Table 1. Regarding call parameters, LOH analysis was unpaired, using Partek internal references. For CNV calls, subtractive comparison was made between related data sets. For example, raw Xa data from clones A1 and A2 were averaged, CNV calls were generated and compared to Xb averaged clone data (B1 and B2) to reveal specific XCI state variance (i.e.: A-Ave vs B-Ave). Individual clones were compared within the XCI state (i.e.: clone A1 compared directly to A2 to reveal unique A1 calls). An example of our data interpretation: 8 LOH and 558 CNV are specific to and shared among XCI state Xa clones (i.e.: A-both). Clone A1 had 35 specific, unique LOH and 1402 CNV calls, while clone A2 had 15 unique, specific LOH and 650 CNV calls. Xb clone data (B1, B2, B-both) are displayed in Table 1.Conclusions: Recent studies reveal frequent mutations in the blood of healthy individuals; however within a person there a no model to identify normal myeloid sub-clones. Therefore we have no accurate data on stem cell kinetics or clonal evolution in healthy marrow. Herein we describe the feasibility of using SNP calls variation in un-diseased aged human myeloid cells to define stem cell specific progeny. To our knowledge, this is the first use of both inclusive (somatic mutations) and exclusive (XCI state) markers to develop a phylogenetic lineage map of normal aged marrow. Importantly, all clones had progenitors with an identical genetic background (the fertilized egg) at a single time point (conception). Therefore, this innovative approach allows for the somatic variation rate to be monitored in primary cells occupying the same organism environment as a function of age and stress events (i.e.: chemo, radiation, etc). [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Molecular monitoring in chronic myeloid leukemia-how low can you go?

Molecular monitoring of BCR-ABL1 transcripts for patients with chronic myeloid leukemia (CML) is now used to assess response to tyrosine kinase inhibitors (TKIs), including treatment failure that mandates a change of therapy. Therefore, many centers have adopted the molecular technique for measuring BCR-ABL1 and rely on conversion of values to the international reporting scale for appropriate clinical interpretation. However, the technique has a degree of inherent variability despite standardized procedures, which means care should be taken by the clinician when assessing response based on BCR-ABL1 cutoff limits. The last few years have witnessed the emergence of a new molecular response target, which is the achievement and maintenance of a deep molecular response. The ability to achieve treatment-free remission for some patients has shifted the relevant boundary for molecular response. However, the definitive safe BCR-ABL1 transcript level and length of the maintenance phase after which treatment cessation can be attempted has not yet been determined. For patients with TKI resistance, BCR-ABL1 kinase domain mutation analysis remains an essential assessment to guide therapy. Furthermore, low-level mutation detection is clinically relevant for response prediction to subsequent TKI therapy for some patients. Multiple low-level mutations may be a biomarker of a clonally diverse disease with the propensity for resistance evolution. Overall, molecular monitoring, including low-level monitoring is a fundamental component of management for patients with CML.

Prevalence and Dynamics of BCR-ABL Independent Gene Mutations in Patients with Chronic Myeloid Leukemia

Living without treatment has become a realistic target for patients with chronic myeloid leukemia (CML) who achieved durable deep molecular remission under treatment with tyrosine kinase inhibitors (TKI). Recently, we have identified novel BCR-ABL-independent gene mutations in newly diagnosed CML patients that may function as important cofactors in the evolution of CML and persistence of the disease (Schmidt M, Rinke J, et al. Leukemia 2014; 28(12): 2292-9). Here, we sought to investigate the dynamics of such mutations during a 2-year treatment with the second generation TKI nilotinib. To check if BCR-ABL independent mutations are an age associated phenomenon we also screened a cohort of children and adolescents with CML. A total of 51 chronic phase CML patients were analyzed including 31 adults (male, n=23; median age 57 years, range 42 to 73 years) and 20 children/adolescents (male, n= 14; median age 14 years, range 10 months to 27 years). For each adult patient, samples were analyzed at diagnosis, in subsequent complete cytogenetic remission (CCyR) after 3 months and in deep molecular remission (≤ MMR) after 24 months of nilotinib therapy. Children/adolescents samples were investigated at diagnosis only. Targeted deep next-generation sequencing (NGS) was used to analyze a panel of 30 commonly mutated genes in myeloid disorders. Constitutional DNA obtained from buccal swabs at diagnosis was investigated to check whether mutations are of somatic origin. BCR-ABL independent gene mutations were found in 8/31 (26%) adult CML patients at diagnosis affecting the genes ASXL1 (n=3), DNMT3A (n=3), RUNX1 (n=1) and RUNX1 plus TET2 (n=1). No mutation was recognized in corresponding constitutional DNA specimens indicating that all mutations were somatically acquired. Analysis of individual hematopoietic colonies from three adult patients revealed that most mutations were part of the BCR-ABL-positive clone. NGS of subsequent samples obtained after three months of TKI therapy identified two DNMT3A mutations in BCR-ABL-negative cells that were also present in BCR-ABL-positive cells at diagnosis, implying that this mutation preceded the BCR-ABL rearrangement. After 24 months of TKI treatment, NGS revealed these two DNMT3A mutations again as well as a novel low-level EZH2 mutation in a patient with deep molecular remission (MR4.5) that was neither detectable at diagnosis nor after 3 months of treatment indicating the emergence of a BCR-ABL-independent subclone in BCR-ABL-negative cells during long-term nilotinib treatment. Within the children/adolescents cohort BCR-ABL independent gene mutations were identified in 5/20 (25%) subjects all affecting the ASXL1 gene. All mutations were stop codon or frameshift mutations implying loss of ASXL1 gene function. We conclude that BCR-ABL independent gene mutations can be found frequently in CML patients and affect predominately epigenetic modifier genes. The finding of frequent mutations also in children/adolescents implicates that BCR-ABL independent gene mutations are not just age-related events. Such molecular aberrations may play an important role in the evolution and persistence of the disease and may affect therapy in both children and adults with CML. These findings may provide important novel genetic information regarding CML biology and for the design and performance of discontinuation trials. DisclosuresHochhaus:Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; BMS: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding.