Cell Line DNA Research Articles

Validation and implementation of a modular targeted capture assay for the detection of clinically significant molecular oncology alterations

ObjectivesThe rapid discovery of clinically significant genetic variants has translated to next-generation sequencing assays becoming out-of-date by the time they are designed, validated, and implemented. UW-OncoPlex addresses this through the adoption of a modular panel capable of redesign as significant alterations are identified. We describe the validation of OncoPlex version 6 (OPXv6) for the detection of single nucleotide variants (SNVs), insertions and deletions (indels), copy number variants (CNVs), structural variants (SVs), microsatellite instability (MSI), and tumor mutational burden (TMB) in a panel of 340 genes. DesignOne hundred twelve samples with diverse diagnoses were comprised of formalin-fixed-paraffin-embedded tissue, fresh-frozen tissue, plasma, peripheral blood, bone marrow, saliva, and cell-line DNA. Libraries were prepared from genomic and cell-free DNA, hybridized to a custom panel of xGen Lockdown probes, and sequenced on Illumina platforms. Sequences were processed through a custom bioinformatics pipeline, and variant calls were compared to prior orthogonal clinical results. ResultsAccuracy was 99% for SNVs ≥5% allele frequency, 98% for indels, 97% for SVs, 99% for CNVs, 100% for MSI, and 100% for TMB (compared to previous OncoPlex versions). Library preparation turnaround time decreased by 40%, and sequencing quality improved with a 2.5-fold increase in average sequencing coverage and 4-fold increase in percent on-target. ConclusionsOPXv6 demonstrates improvements over prior UW-OncoPlex versions including reduced capture cost, improved sequencing quality, and decreased time to results. The modular capture probe design also provides a nimble laboratory response in addressing the expansions necessary to meet the needs of the continuously evolving field of molecular oncology.

Absence of bovine leukemia virus proviral DNA in Japanese human blood cell lines and human cancer cell lines.

Bovine leukemia virus (BLV) infects cattle worldwide and causes B-cell lymphoma in cattle. BLV has been identified in human breast and lung cancer and in blood, but the association of BLV and human cancer is controversial. In this study, we investigated the existence of BLV in 145 Japanese human blood cell lines and 54 human cancer cell lines, using a new highly sensitive PCR assay that can amplify even one copy of BLV using LTR primers different from those in previous studies on BLV provirus in breast cancer. All samples were found negative for BLV provirus, suggesting that BLV is unlikely to infect humans.

Detection of Low Frequency T-Cell Clones Via a Next Generation Sequencing (NGS) Based Immune-Oncology Research Assay

Introduction: The ability to detect and quantitate low frequency T-cell clones enables numerous hematology/oncology research applications, including identification and assessment of biomarkers associated minimal residual disease (MRD). Rare clone detection via NGS requires highly efficient library preparation and accurate sequencing methodologies, because single nucleotide substitution sequencing errors mimic the natural variation in the T-cell repertoire, resulting in detection of artifactual low frequency clones. Here we present an experimental framework and corresponding performance of rare clone detection utilizing the OncomineTM TCR Beta short read (TCRb-SR) assay, using Ampliseq-based library preparation targeting the highly variable CDR3 region of TCRb using either DNA or RNA as input, with sample-to-result in 2 days. Methods: To evaluate detection sensitivity of the TCRB-SR assay, we utilized Jurkat cell line DNA and RNA because the presence of a single T cell clone enables precise control of dilution studies. Commercially procured Jurkat gDNA or RNA was spiked into peripheral blood leukocyte gDNA or RNA from 10-1 to 10-6 absolute clone frequency to create specimens with a known T-cell clone at frequencies commonly observed in MRD research applications. Peripheral blood leukocyte gDNA or RNA was used as the background for spike in studies due to its high T-Cell diversity. Six to twenty technical replicates were analyzed per dilution point, with DNA inputs ranging from 100ng to 1ug and RNA inputs ranging from 25ng to 100ng to evaluate the minimum detectable clone frequency as a function of nucleic acid input. Libraries were prepared following the TCRB-SR manufacturer's instructions for both DNA and RNA, followed by templating and sequencing using Ion Chef and S5 systems. Data processing was performed in Torrent Suite software (v5.10) followed by read alignment to the IMGT database of variable, diversity, and joining genes using Ion Reporter software (v5.10). For the 10-6 target frequency with gDNA as the input, four 1ug libraries were combined for analysis in Ion Reporter. Analytical sensitivity was calculated at each target clone frequency by detection of the Jurkat clone as defined by V-gene, Joining gene, and CDR3 nucleotide sequence. Results: Detection sensitivity was dependent on the amount gDNA or RNA input. For gDNA inputs, we observed 100% sensitivity at 10-3 with 100ng input, 100% sensitivity at 10-4 with 250ng input, 95% sensitivity at 10-5 with 1ug input, and 100% sensitivity at 10-6 with 4ug input. For RNA inputs, we observed 100% sensitivity at 10-5 with 25ng input and 100% sensitivity at 10-6 with 100ng input. In addition, we observe a strong linearity of observed clone frequencies at each dilution level, with an r-squared of 0.97. Conclusions: Here we demonstrate the ability to detect T-cell clones down to 10-6 from gDNA or RNA inputs with high sensitivity and linearity utilizing the OncomineTM TCR Beta short read assay. We present data demonstrating detection of clones with absolute frequencies of 10-6 utilizing 4ug gDNA input or 100ng RNA input, highlighting strong performance at nucleic acid input levels typically seen in clinical research samples. Taken together, we show feasibility for rare clone detection in either gDNA or RNA enabling research and development for T-cell minimal residual disease applications. For research use only, not for use in diagnostic procedures. Disclosures Williamson: Thermo Fisher Scientific: Employment. Looney:Thermo Fisher Scientific: Employment. Lowman:Thermo Fisher Scientific: Employment. Sidhu:Thermo Fisher Scientific: Employment. Solano:Thermo Fisher Scientific: Employment. Salazar:Thermo Fisher Scientific: Employment. Tian:Thermo Fisher Scientific: Employment. Ramsamooj:Thermo Fisher Scientific: Consultancy.

Redefining “Gold Standard”: Ultra-Sensitive Characterization of Commercial DNA Standards with Duplex Sequencing

DNA standards are a critical resource in diagnostic and research labs. Positive and negative controls are essential for validating the sensitivity and specificity of next generation sequencing (NGS) and other genetic assays. Several commercial DNA standards have been widely used to validate clinical oncology assays. However, as genomic technologies have become more sensitive, the metrics defining what a "gold standard" entails have not been carefully re-evaluated. Cell culture and synthetic chemical means of generating DNA standards have the potential to artificially introduce mutations. Cell lines are often exposed to supraphysiologic levels of reactive oxygen. Oligonucleotide synthesis has an error rate orders of magnitude above biological levels. This background may not interfere with detection of clonal or sub clonal variants with moderately sensitive assays, but it will obscure the presence of very rare mutations when assessing with extremely sensitive ones and it will decrease apparent assay performance. In this study, we applied Duplex Sequencing (DS), an extremely sensitive NGS technology with an error rate below one-in-ten-million, to characterize widely used commercial myeloid DNA standards. Duplex sequencing is an error correction method that independently sequences and compares the two strands of original DNA molecules to enable error correction. The cross-strand comparison eliminates errors caused by DNA damage, early cycle PCR errors and various technical errors that escape standard NGS and other error-corrected NGS methods. We performed DS on two commercial myeloid leukemia DNA standards and an in-house control DNA. Commercial standard A (CS-A) is constructed of a mix of DNA from multiple mutation-containing cell lines. Commercial standard B (CS-B) is constructed of synthetic mutant DNA molecules mixed into DNA from a single well-characterized cell line. The control DNA was obtained from the donated apheresis product of a healthy 25-year old never-smoker. We used a hybrid capture panel covering 15 variants with a 5%-40% expected variant allele frequency (VAF) in CS-A, and 12 variants with a 5%-15% expected VAF in CS-B. CS-A, CS-B and control DNA were sequenced to maximum Duplex depths of 12,256x, 15,844x and 38,535x, respectively. In the two commercial DNA standards, all targeted variants were detected by DS. The majority of variants were within +/- 1.5-fold from the expected VAF. For CS-A, the correlation of DS vs. vendor target VAF was strong (r2 = 0.96). For CS-B, the correlation between DS and vendor target VAF was very weak (r2 = 0.07), although was better correlated to vendor-reported ddPCR and conventional NGS (r2 = 0.49 and 0.61). We also performed DS on a specially designed in-house standard, comprised of apheresis-derived control DNA with a series of mutant cell lines spiked at low frequencies from 1/100 to 1/100,000, that we sequenced to more than 1 million-fold Duplex depth. 9/9 mutations were detected down to a target VAF of 10-5 with r2 = 0.96 for DS vs. target values. We performed DS on a second in-house mutation dilution series which included the CS-A standard and other samples diluted into apheresis control DNA, and detected 9/9 mutations down to a target VAF of 4x10-5 with r2 = 0.93. The overall mutation frequencies (number of unexpected mutations / total number of base-pairs sequenced) of CS-A, CS-B and the control were 1.6x10-6, 2.1x10-6 and 4.7x10-7, respectively. The in-house mutation mixes were both 5.4x10-7. CS-A carried 96 low-frequency clonal variants (≥2 counts, <1% VAF) and CS-B had 102 low-frequency clonal variants. The apheresis control, despite being sequenced to nearly 3 times greater Duplex depth, had only 6 low-frequency clonal variants. The variants in the control sample are in genes associated with Clonal Hematopoiesis of Indeterminate Potential (CHIP) and reflect true biological background rather than technical noise. As genomic technologies increase in accuracy, so too does the need for ultra-pure DNA standards. As shown in this study the use of cell line DNA as the primary substrate for mutation standards, whether spiking in synthetic mutant molecules or simply mixing DNA from multiple cell lines, is not sufficient for highly sensitive assays due to accumulation of mutations in culture. Improved standards are necessary for high-precision technologies, especially for detection of residual disease or early cancer screening. Disclosures Higgins: TwinStrand Biosciences: Employment. Pratt:TwinStrand Biosciences: Employment. Valentine:TwinStrand Biosciences: Employment. Williams:TwinStrand Biosciences: Employment. Salk:TwinStrand Biosciences: Employment.

The application of whole exome sequencing in different metastatic potential of heptaocellular carcinoma

Objective Genome-wide exome sequencing was used to analyze DNA of hepatocellular carcinoma (HCC) tissues and cell lines with different metastatic potentials to explore the mutation genes and sites related to the HCC metastasis. Methods Genomic DNA extracted from twelve HCC tumors and its adjacent tissues and six HCC cells (HCC-LM3, MHCC-97L, MHCC-97H, HepG2, SMCC-7721 and PLC-RFP-5) with different metastatic potentials were analyzed by whole exome sequencing. Bioinformatics analysis and screening of mutations was used to investigated mutation genes and sites potentially associated with HCC metastasis. Results Comparing high and low metastatic potential HCC, high metastatic potential HCC group had more than three gene mutations including UIMC1 (rs3733876 C/T), SEMA3C (rs1527482 G/A), SAMD9 (rs10279499 G/T), RECK (rs16932912 G/A), PDLIM4 (rs4877 G/T, LIMK2 (rs3747154 A/G), CRIM1 (rs3821169 G/T) which was associated with HCC metastasis. Mutate genes and its sites such as CAPZB (rs79308175C/T), CSF1R (rs10079250A/G), HSPA5 (rs143920039A/T), SORBS3 (rs2449331T/C, rs3758036C/T) only occured in high metastatic potential HCC cells (HCC-LM3, MHCC-97L and MHCC-97H), while mutation sites RASAL3 (rs146624357G/A, rs142945276 C/T) only appeared in three low metastatic potential cells (HepG2, SMCC-7721 and PLC-RFP-5). The DNA comparative analysis of three homologous high metastatic potential HCC cell lines (HCC-LM3, MHCC-97Land MHCC-97H) showed that only EHBP1 (rs77403174T/C) was presented in HCC-LM3, while FAM189B (rs150296362C/T), SERPINB13 (rs144903442G/A) and ARHGEF1 (rs2303797C/T) only presented in MHCC-97L and MHCC-97H. Conclusion Mutations and its sites such as UIMC1 (rs3733876C/T), SEMA3C (rs1527482G/A), SAMD9 (rs10279499G/T) was detected to potentially related to the HCC metastasis by genome-wide exome sequencing. Key words: Whole exome sequencing; Hepatocellular carcinoma; Mutations; Metastasis

Analytical validation of the Target Selector ctDNA platform featuring single copy detection sensitivity for clinically actionable EGFR, BRAF, and KRAS mutations.

BackgroundPersonalized medicine requires accurate molecular profiling for targeted therapy decisions. Insufficient tissue yield or tumor heterogeneity frequently limits the correct tissue biomarker determination. As a noninvasive complement to traditional tissue biopsies, liquid biopsies detect and track cancer driver mutations from biofluids (e.g., blood, urine). Here we present the analytical validation of Target Selector™ ctDNA assays capable of single mutant DNA copy detection.MethodsThe Target Selector ctDNA assay applies a patented Switch-Blocker technology to suppress amplification of background (wild-type) WT alleles, while allowing specific amplification of very low frequency mutant alleles. In contrast to allele specific enrichment technologies like ddPCR, one Switch-Blocker inhibits amplification of a DNA target up to 15 bp in length (e.g., one Switch-Blocker covers all KRAS exon 2, codon 12 and 13 variants). Target enrichment is achieved through a quantitative PCR reaction; subsequent DNA sequencing confirms mutation identity. Analytical validation with cancer cell line DNA was conducted by three independent operators using five instruments across five days.ResultsA total of 3086 samples were tested on EGFR, BRAF and KRAS Target Selector ctDNA assays, with EGFR WT as a reference. All assays showed >99% analytical sensitivity and specificity. Single mutant copy detection is confirmed by experimental data and theoretical estimates. In the presence of 14000 WT DNA copies, limits of detection were: EGFR Del19, 0.01%; EGFR L858R, 0.02%; EGFR T790M, 0.01%; BRAF V600E, 0.01%; KRAS G12C, 0.02%. Inter- and intra-assay analyses showed r2>0.94, suggesting consistent performance among operational variables. Healthy donor samples (100 tests) showed clinical specificity at >99%. Finally, Target Selector clinical experience data of >2200 patient samples is consistent with published tissue mutation prevalence.ConclusionsHighly sensitive Target Selector ctDNA assays with single mutant copy detection and limit of detection at 0.02% or better enable accurate molecular profiling vital for disease management.

1416P - Enhanced access to EGFR molecular testing in NSCLC using a cell-free DNA tube for liquid biopsy

BackgroundThe clinical utility of non-invasive liquid biopsy and ability to accurately detect EGFR mutations in circulating-tumor DNA of patients with NSCLC has been well demonstrated. This expands the pool of patients eligible for molecular testing. The cobas® EGFR Mutation Test v2 (cobas test) is a real-time PCR test for qualitative and semi-quantitative detection of 42 EGFR mutations in DNA from tissue and circulating free DNA (cfDNA) from K2 EDTA plasma. Blood collected in K2 EDTA requires plasma be separated within 8hours, which can be a barrier to molecular testing and thus impact treatment decisions. The Roche Cell-Free DNA Collection Tube (Roche cfDNA) stabilizes blood up to 8 days, allowing greater flexibility in transportation and time to plasma separation. Here the suitability of plasma from Roche cfDNA tubes for use with the cobas test is demonstrated. MethodsTest performance with Roche cfDNA plasma was verified with NSCLC patient specimens or surrogate samples (sheared cell line DNA in healthy donor plasma). Correlation was tested using paired draws in K2 EDTA and Roche cfDNA tubes for 51 NSCLC patients and 20 healthy donor surrogate samples. Limit of detection (LoD) and linearity established with K2 EDTA plasma were verified with Roche cfDNA plasma. Reproducibility was assessed using surrogate samples at two levels with multiple operators, Roche cfDNA tube lots, instruments, days and sites. Blood storage conditions were established at an external laboratory with 6 NSCLC patient specimens. ResultsCompared to K2 EDTA plasma, Roche cfDNA plasma had a PPA, NPA and OPA of 100.0% with the cobas test. LoD for EGFR mutation detection in Roche cfDNA plasma was verified as≤100 cp/mL. Linearity for EGFR mutations in Roche cfDNA plasma was verified. The reproducibility study had a call agreement of≥98.6%. Blood in Roche cfDNA tubes was stable for up to 8 days at 18-25ºC with one excursion of up to 24hours at 15-30ºC prior to separation. ConclusionsUse of the Roche cfDNA tube with the cobas® EGFR Mutation Test v2 provides the flexibility to store blood for up to 8 days prior to separation, with equivalent performance to K2 EDTA plasma, and facilitates use of liquid biopsy for NSCLC patients needing molecular testing. Legal entity responsible for the studyRoche Molecular Systems Inc. FundingAstraZeneca PLC. DisclosureT.E. May: Full / Part-time employment: Roche Molecular Solutions. S.A. Scudder: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche Molecular Solutions. S.J. Joshi: Full / Part-time employment: Roche Molecular Solutions. M. Kohlmann: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca, PLC. N. Shrestha: Full / Part-time employment: Roche Molecular Solutions. N. Lee: Full / Part-time employment: Roche Molecular Solutions. J. Lai: Full / Part-time employment: Roche Molecular Systems Inc. M. Tsourounis: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca PLC. A. Kohlmann: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca PLC. P. O’Donnell: Full / Part-time employment, Spouse / Financial dependant: Roche Molecular Systems. H. Halait: Shareholder / Stockholder / Stock options, Full / Part-time employment: Roche Molecular Systems. All other authors have declared no conflicts of interest.

Development of conventional and real time PCR assays for rapid species authentication of mammalian cell lines commonly used in veterinary diagnostic laboratories

Mammalian cell lines are valuable tools in biomedical fields, with applications ranging from disease diagnosis to the production of biological reagents and vaccines. Here we report the development of new conventional (cPCR) and real time PCR (qPCR) assays for species identification of several mammalian kidney cell lines originated from swine, green monkey, hamster and bovine tissues that are extensively used in veterinary diagnostic laboratories. The PCR primers and probes were selected from highly conserved mitochondrial genes and analyzed in silico by nucleotide BLAST in the National Center for Biotechnology Information (NCBI) website to ensure target specificity. The assays were highly species-specific and had no cross-reactivity against other tested cell lines originated from different mammalian species. Assay sensitivity (limit of detection; LOD) was determined using serial dilutions of cell line DNA as template. The estimated LODs were between 2.95 and 48 pg (picogram) DNA/assay for cPCR, and between 1.5 × 10−3 and 4.8 × 10−2 pg DNA/assay for qPCR. Multiplex qPCR assays were developed for simultaneous detection of up to three species in a single assay. The multiplex qPCR assays exhibited the same sensitivity as the corresponding singleplex assays with the exception of the green monkey species that demonstrated a 10–100 fold decline in the sensitivity. Contamination of swine cells was detected in one of the rabbit cell lines. The contamination was further confirmed by Sanger and Next-Generation sequencing.

Role of miR-182-5p overexpression in trichloroethylene-induced abnormal cell cycle functions in human HepG2 cells

ABSTRACTTrichloroethylene (TCE), a widely used industrial solvent, occurs frequently in the global environment. TCE was found to induce hepatocarcinogenesis in mice and one of the underlying mechanisms was reported to involve miR-182-5p overexpression. Subsequently, miR-182-5p overexpression was shown to contribute to chemical-induced enhanced cell proliferation in mouse liver cells by targeting the gene Cited2. The aim of this study was to compare our findings in mice with those in a human hepatoma cell line HepG2. Data demonstrated that TCE at 0.1mM exerted no marked effect on human hepatoma cell line HepG2 cell migration, cell cycle, apoptosis, and DNA damage, but significantly stimulated cell proliferation rate and increased mRNA expression levels of proliferating cell nuclear antigen (PCNA), a cell proliferation biomarker. In addition, TCE enhanced miR-182-5p expression levels but lowered Cited2 mRNA expression. In summary, data showed that similar to mouse liver cells, TCE exposure also upregulated cells miR-182-5p expression and inhibited Cited2 expression in human hepatoma cell line HepG2. Our results suggest that the TCE-mediated alterations in the observed cellular functions involve interaction with miR-182-5p. It is of interest that utilization of liver cancer tissues from the Cancer Genome Atlas (TCGA) database also demonstrated that upregulated miR-182-5p expression and reduced Cited2 mRNA expression was detected suggesting that TCE-induced hepatocarcinogenesis involved processes similar to those in humans.

Establishment and verification of immortalized lymphoblastic cell lines derived from platelets CD36 deficient individuals

Objective To investigate the establishment and verification of immortal lymphoblastic cell lines from platelets CD36 deficient individuals. Methods From January 2012 to January 2018, eight individuals with platelet CD36 deficiency were selected as the study subjects. Among them, six cases were blood donors from Nanning Blood Center, one case was patient with platelet transfusion refractoriness (PTR) hospitalized in Guangxi 923 Hospital, and 1 case was mother of a child with fetal/neonatal alloimmune thrombocytopenia (FNAIT) hospitalized in Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region. All the subjects′ ages ranged from 15 to 66 years. The subjects were identified as platelet CD36 deficiency by flow cytometry, monoclonal antibody immobilization of platelet (MAIPA) and CD36 genotyping. Among them, one case was a CD36 mutant heterozygote of 538T>C, three cases were mutant heterozygote of 380C>T, one case was mutant homozygous of 329-330del, and 3 cases were mutant heterozygote of 329-330del. Volume of 5 mL heparin anticoagulant blood was collected from participants and their lymphocytes were isolated. Epstein-Barr virus (EBV) and cyclosporine were used to treat peripheral blood lymphocytes of participants with platelet CD36 deficiency to obtain immortal lymphoblast cell lines, which were frozen after stable passages. Resuscitation activity and mycoplasma detection of these cell lines were performed, and the CD36 gene was sequenced for verification. The procedure of this study is accordance with the requirement of the revised World Medical Association Declaration of Helsinki in 2013. Informed consent was obtained from each participant. Results ① After EBV and cyclosporine treatment, the peripheral blood lymphocytes of the participants were cultured for 7 d, and blastoformation of the cells with prickly edges were observed, and some of the cells were agglutinated in clusters. After continuous replacement of the complete culture medium for about 1 month, a large number of cells proliferated and grew vigorously, and more clonal spheres could be seen to form by naked eyes, and immortalized lymphoblastic cell line was successfully obtained. ② CD36 deficient immortalized lymphoblastic cell lines were subcultured for 20 generations, then frozen in liquid nitrogen. All of them were successfully resuscitated, and the cell lines were observed growing well under inverted phase contrast microscope. ③ Mycoplasma test results of the CD36 deficient immortalized lymphoblastic cell lines showed negative. ④ DNA of CD36 deficient immortalized lymphoblastic cell lines were amplified and sequenced by PCR, and comparison results showed that the DNA of immortalized lymphoblastic cell lines were identical to those of individuals with CD36 deficiency, and no gene mutation occurred. The genotypes of CD36 deficient immortalized lymphoblastic cell lines established in this study included 1 case of CD36 gene 538T>C mutant heterozygote, three cases of 380C>T mutant heterozygotes, one case of 329-330del mutant homozygote and 3 cases of 329-330del mutant heterozygotes. Conclusions CD36 deficient immortalized lymphoblastic cell lines passaged stably, the genotypes of these platelets CD36 deficient individuals are permanently preserved and can be used as long-term experimental reliable materials for the study of CD36 related platelet immunology. Key words: Antigens, CD36; Platelets; Herpesvirus 4, human; Cell line; Lymphoblast cell lines

Abstract 422: Duplex sequencing for MRD detection in acute myeloid leukemia

Abstract The purpose of this study is to develop a universally applicable Duplex Sequencing (DS) assay for extremely sensitive detection of minimal residual disease (MRD) in acute myeloid leukemia (AML). The presence of MRD plays a critical role in AML recurrence and mortality, and is an important prognostic marker to guide clinical care and clinical trial risk stratification. Unfortunately, current MRD detection methods, including light microscopy, cytogenetics, qPCR, and flow cytometry have shortcomings in terms of sensitivity, general applicability, and/or inter-laboratory reproducibility. Duplex Sequencing eliminates technical artefacts observed with other sequencing technologies and delivers an unprecedented level of accuracy and sensitivity, on widely available NGS platforms. Here, we report validation of our DS MRD mutation detection assay, with a background mutation level more than 100-fold below single-stranded tag-based error correction (SSCS). Our complete DS MRD probe panel targets mutation hotspots or full coding sequences in 29 genes recurrently mutated in AML, representing mutations found in approximately 90% of patients. Supplementing with a targeted RNA-seq panel for recurrent gene fusions would approach 100%. To simulate low frequency mutations in MRD, we generated 2 DNA dilution series: either AML patient DNA or cell line DNA with known AML mutations that were serially diluted into normal human DNA. Replicate libraries were prepared with custom targeted probe panels and sequenced to over 50,000x or over 1,000,000x total Duplex depth (original Duplex DNA molecules sequenced per site), respectively. Expected mutations were detected down to MAFs of 1.9x10-5 and 6.0x10-6. Background mutation levels of 5.7x10-7 and 5.8x10-7, were respectively seen in each series, and likely reflect the true age-associated biological background, rather than technical noise. SSCS artefacts in these samples were approximately 100x higher than the frequency of DS mutations, and obscured expected mutations in the lower dilutions. We are further validating our method in both retrospective and prospective studies. In retrospective samples from a completed clinical trial, we are comparing the sensitivity and accuracy of DS vs. multicolor flow cytometry for MRD with known clinical outcomes. The relative prognostic value of DNA from blood vs. bone marrow will be quantified with DS as well. We will also perform DS on prospective samples to examine the prognostic value of specific genes and MAFs to eliminate uninformative variants, which will also allow accurate quantification of low level mutations in non-leukemic clones that accumulate with aging and can confound detection of MRD. Elimination of sequencing artefacts is critical for the application of NGS assays to MRD detection, especially when expected mutations are unknown, and DS is an important new tool in high sensitivity cancer diagnostics. Citation Format: Jacob E. Higgins, Lindsey N. Williams, Christopher S. Hourigan, Jerald P. Radich, Jesse J. Salk. Duplex sequencing for MRD detection in acute myeloid leukemia [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 422.

Abstract 2299: Development of a novel next-generation sequencing (NGS)-based assay for measurable residual disease (MRD) in FLT3-ITD acute myeloid leukemia (AML) and its potential clinical application in patients

Abstract Introduction The presence of MRD in patients with AML who are in morphologic remission has been shown to be a powerful predictor of eventual relapse. FMS-like tyrosine kinase 3 (FLT3) internal tandem duplications (ITD) confer a negative prognostic impact by increasing risk of relapse. The ability to detect FLT3-ITD mutations in remission bone marrow specimens is hampered by the limited sensitivity at 1% of PCR-based assays. To address such limitations, we developed a novel NGS-based MRD assay for the detection of FLT3-ITD mutations. Method Genomic DNA was isolated from bone marrow (BM) aspirates or peripheral blood (PB) samples. PCR was performed to amplify exons 13 to 15 of the FLT3 gene. Highly diverse NGS libraries were then generated and sequenced using Illumina’s sequencer. Using a custom bioinformatics approach, unique FLT3-ITD mutations of varying lengths were identified and mutant allelic frequency calculated. The assay was validated using clinical samples and contrived samples. For accuracy assessment, 30 samples (including PB or BM remnant patient DNA and cell line DNA, above DNA diluted in normal DNA) were included. Data were compared with a Fragment Size Analysis by Capillary Electrophoresis assay. To assess precision, the assay was validated at multiple levels evaluating intra-assay, inter-assay, inter-operator, inter-instrument and inter-reagent lot precision. DNA samples from selected mutant cell lines representing different FLT3-ITD lengths were spiked into normal DNA to evaluate assay sensitivity and linearity. Summary The ideal DNA input range was established as 300 ng to 500 ng. In all FLT3-ITD-positive cell line samples covering diverse FLT3-ITD lengths (6 bp to 156 bp), the FLT3-ITD MRD NGS assay showed 100% concordance with the reference assay. The assay is also capable of detecting ITDs as expected after the samples were diluted in normal DNA to mimic samples from patients in remission. All acceptance criteria for the different precision parameters were met. The lower limit of detection of 0.013% regardless of ITD length was established but the assay was capable of detecting FLT3-ITD mutations at a level as low as 0.003% without false-positive results. The assay was linear (R2 = 0.958) down to FLT3-ITD allele frequency levels of 0.035% or the lower limit of quantitation. Conclusion Analytical validation results established the role of this NGS-based MRD assay for the clinical management of FLT3-ITD AML. The FLT3-ITD MRD NGS assay demonstrated high sensitivity and high specificity by detecting the unique length of each patient’s mutation with no false-positive results in expected negative samples. This assay might be helpful in defining the depth of remission, identifying persistent disease, and helping to guide decision making in the use of FLT3 inhibitors as continuation therapy. Citation Format: Wenge Shi, Christian Laing, Wei Ding, Marc Mycoco, Jelveh Lameh, Reinhold Pollner. Development of a novel next-generation sequencing (NGS)-based assay for measurable residual disease (MRD) in FLT3-ITD acute myeloid leukemia (AML) and its potential clinical application in patients [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 2299.

Abstract 817: Assessment and manipulation of DNA methylation in cancer cells using terahertz radiation

Abstract Carcinogenesis is closely associated with abnormal regulation of gene expression, among which DNA methylation in one of the key epigenetic modifications. Therefore, several studies have been directed towards the evaluation of DNA methylation. Here, we aimed to experimentally observe the specific signal of methylated DNA in various blood cancer cell lines using terahertz (THz) radiation, which is presented as a resonance in the spectrum. THz radiations are electromagnetic waves having frequencies of 0.1−10 THz, which falls between those of infrared radiation and microwaves. Molecular low-energy motions in THz region, such as collective vibrations, torsion of molecular structures, and hydrogen bonds, is correlated with biological characteristics, i.e., vibrational modes of DNA, structural modes in proteins, cell membrane vibrations, or enzyme activity. These features are usually presented as a characteristic resonance of the modes in the THz spectrum. In our study, the degree of global DNA methylation was obtained by the quantification of the resonance around 1.7 THz, and the results were in good agreement with those obtained by enzyme-linked immunosorbent assay (ELISA). Further, DNA demethylation was performed using high-power THz radiation around the resonance frequency of 1.7 THz, which lead to a 10% to 72% decrease in the degree of DNA methylation as shown in Table 1. Thus, our data shows the feasibility of using THz radiation for the assessment and manipulation of DNA methylation. This is the first study wherein the sensing and manipulation of DNA methylation could be achieved using a non-contact and non-invasive optical technique. Use of THz radiation for the assessment of DNA methylation might be helpful for its rapid processing, while THz manipulation of DNA methylation can avoid the toxicity of chemical demethylating agents. We propose that THz radiation may be useful for the diagnosis of cancer and we expect that THz demethylation might find potential application in cancer therapy. Change of methylation status of blood cancer DNA using high-power THz radiation.SamplesNo ExposureHigh-power THz exxpsoureChange (%) THzChange (%) ELISATHzELISARatio1THzELISARatio2119.621.8510.6111.661.0611.0040.5742.70211.341.388.2210.201.198.5710.0513.77315.151.4310.596.240.738.5558.8148.95417.731.4712.063.510.447.9880.2070.07511.271.437.8810.001.367.3511.274.90STDEV1.58STDEV1.24THz, the amplitude of terahertz resonance; ELISA, optical density of Enzyme linked immunosorbent assay; STDEV, standard deviation1 THz/ELISA of no exposure2 THz/ELISA of high-power THz exposure Citation Format: Hee-Jin Yang, Hwayeong Cheon, Jin Ho Paik, Joo-Hiuk Son. Assessment and manipulation of DNA methylation in cancer cells using terahertz radiation [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 817.

Abstract 3526: Incorporation of unique molecular identifiers (UMIs) into unique dual sample indexing (UDI) improves the accuracy of quantitative next generation sequencing

Abstract Accurate analysis of quantitative NGS data is critical for low frequency variant detection, identification of differentially expressed transcripts, and correct diagnosis and patient care in a clinical NGS setting. Two major factors affecting sequencing accuracy are 1) PCR duplication arising from amplification of library molecules; and 2) errors introduced during library preparation and actual sequencing on the flow cell. Standard practice for identification and removal of PCR duplicates relies on aligning reads to the same genomic coordinates. However, this approach can not differentiate between PCR duplicates and reads originating from unique molecules with identical ends. The most effective method for error correction is Duplex Sequencing, which utilizes UMIs to tag both strands of each individual DNA duplex followed by building consensus sequences. Unfortunately, this approach is labor intensive and cost-prohibitive for complex genomes and large target panels. Therefore, a simple and reliable approach for duplicate removal and error correction would greatly facilitate the wider adoption of NGS technology for diagnostics and clinical applications. In this study, we incorporate UMIs into UDI systems and assess the effect on the accuracy of quantitative sequencing assays. We first study the effectiveness of various computational methods to account for UMIs and remove base-calling errors introduced during sequencing. We then analyze the utility of UMIs for 1) unique and low abundance transcript identification and accurate transcript quantification; and 2) error correction in low frequency variant detection in genomic sequencing from both high quality cell line DNA and low quality FFPE DNA. In addition, we demonstrate that combining unique dual sample indexing with UMI molecular barcoding further improves data analysis accuracy, especially on patterned flow cells. Our approach involves a simple new UMI-containing UDI adaptor design that can also be applied to other sequencing methods and platforms. Citation Format: Pingfang Liu, Keerthana Krishnan, Camille X. Devoe, Bradley W. Langhorst, Eileen Dimalanta, Theodore B. Davis. Incorporation of unique molecular identifiers (UMIs) into unique dual sample indexing (UDI) improves the accuracy of quantitative next generation sequencing [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 3526.

Synthesis of polymeric chitosan derivative Nanoparticles and their MTT and flow cytometry evaluation against breast carcinoma cell

Chitosan extracted from shrimp cortex was grafted with some different anhydride compounds like; acetic anhydride, propionic anhydride, succinic anhydride, and phthalic anhydride. The chitosan-grafted-anhydrides were copolymerized with L-lactide in ring opening polymerization. All grafted chitosan derivatives were obtained in very good yield, and they were characterized by FTIR and the resulted spectra confirmed the right structures of chitosan and its different synthesized derivatives, and then they were converted to nanoparticles in size by subjecting them to sonication method.In vitro cytotoxicity detection of different chitosan anhydride derivative nanoparticles and their grafted-polylactide were concerning three different types of human breast cancer cell lines as MTT assay, the results exhibit the highly significant cell growth inhibition of these tumor cells compared with a positive control; furthermore, the chitosan anhydride derivatives grafted polylactide demonstrated increasing in reducing cell viability in comparison with their non-grafted form. The DNA fragmentation index percentage was evaluated for some of the studied polymer nanoparticles, using acridine orange dye and the results were shown no or less effective against BT breast carcinoma cell lines DNA

Design and synthesis of phenylpiperazine derivatives as potent anticancer agents for prostate cancer.

Novel thiourea (5a, 5b) and thiazolidinone derivatives (6a, 6b) were synthesized by hybridizing molecules starting from the compound 6-(4-phenylpiperazin-1-yl)pyridin-3-amine (4) which is known to show anticancer activity. The synthesis of the leading compound was carried out by using 1-(5-nitropyridin-2-yl)-4-phenylpiperazine (3) which was obtained by a novel method of the reaction of 2-chloro-5-nitropyridine (1) and N-phenylpiperazine (2). The structures of the compounds were confirmed using FTIR, 1 H NMR, 13 C NMR, HRMS spectroscopic methods and elemental analysis. The organic molecules were tested for their anticancer activities against prostate cancer (PC) cell lines: DU 145, PC-3 and LNCaP. As the compound 5a exerted the highest cytotoxic activity, IC50 concentrations of compound 5a were further investigated in terms of morphology, colony-forming ability, RNA expression, fragmented DNA and cell cycle distributions of PC cell lines. Overall data revealed that compound 5a treatment induces apoptosis and DNA fragmentation in PC cell lines and inhibits cell cycle progression resulting in the accumulation of cells in either the G1 or the S phases.

Low-pass Whole-genome Sequencing of Circulating Cell-free DNA Demonstrates Dynamic Changes in Genomic Copy Number in a Squamous Lung Cancer Clinical Cohort.

We developed a method to monitor copy number variations (CNV) in plasma cell-free DNA (cfDNA) from patients with metastatic squamous non-small cell lung cancer (NSCLC). We aimed to explore the association between tumor-derived cfDNA and clinical outcomes, and sought CNVs that may suggest potential resistance mechanisms. Sensitivity and specificity of low-pass whole-genome sequencing (LP-WGS) were first determined using cell line DNA and cfDNA. LP-WGS was performed on baseline and longitudinal cfDNA of 152 patients with squamous NSCLC treated with chemotherapy, or in combination with pictilisib, a pan-PI3K inhibitor. cfDNA tumor fraction and detected CNVs were analyzed in association with clinical outcomes. LP-WGS successfully detected CNVs in cfDNA with tumor fraction ≥10%, which represented approximately 30% of the first-line NSCLC patients in this study. The most frequent CNVs were gains in chromosome 3q, which harbors the PIK3CA and SOX2 oncogenes. The CNV landscape in cfDNA with a high tumor fraction generally matched that of corresponding tumor tissue. Tumor fraction in cfDNA was dynamic during treatment, and increases in tumor fraction and corresponding CNVs could be detected before radiographic progression in 7 of 12 patients. Recurrent CNVs, such as MYC amplification, were enriched in cfDNA from posttreatment samples compared with the baseline, suggesting a potential resistance mechanism to pictilisib. LP-WGS offers an unbiased and high-throughput way to investigate CNVs and tumor fraction in cfDNA of patients with cancer. It may also be valuable for monitoring treatment response, detecting disease progression early, and identifying emergent clones associated with therapeutic resistance.

A Simple and Highly Specific MassARRAY-Based Stool DNA Assay to Prioritize Follow-up Decisions in Fecal Immunochemical Test-Positive Individuals.

Background: Seventy-five percent of fecal immunochemical test (FIT)-positive individuals are false positives and undergo unnecessary colonoscopies. Here, we established a stool DNA (sDNA) test that uses the Single Allele Base Extension Reaction (SABER) MassARRAY platform to improve the accuracy of FIT-based CRC detection. Methods: Twenty-one variants in five CRC-associated genes were selected for the sDNA panel. Cell line DNA and matched mutation-confirmed tissue and stool samples from 34 patients were used for accuracy assessment (cohort 1). The clinical performance of the sDNA assay was further evaluated in 101 independent FIT-positive stool samples (cohort 2). Results: In cohort 1, we obtained a 62% mutation concordance rate in paired tissue and stool samples of the CRC group, regardless of the FIT status. In cohort 2, 100% specificity in normal controls with positive FIT results was observed. By weighting the FIT value and the presence of a given variant type in stool and then summing the two scores, we found that a one-increment increase in the score was associated with a 4.538-fold risk (95% CI = 2.121–9.309) for malignancy in the FIT-positive setting. Conclusions: Our highly specific sDNA assay can help prioritize the most at-risk FIT-positive persons to receive prompt colonoscopic confirmation of CRC.

Hybrid Cell-Penetrating Foldamer with Superior Intracellular Delivery Properties and Serum Stability.

Sequence specific molecules with high folding ability (i.e., foldamers) can be used to precisely control the distribution and projection of side chains in space and have recently been introduced as tailored systems for delivering nucleic acids into cells. Designed oligourea sequences with an amphipathic distribution of Arg- and His-type residues were shown to form tight complexes with plasmid DNA, and to effectively promote the release of DNA from the endosomes. Herein, we report the synthesis of new cell-penetrating foldamer sequences in which the foldamer segment is conjugated via a reducible disulfide bond to a ligand that binds cell-surface expressed nucleoproteins with the idea that this system could facilitate both assemblies with nucleic acids and cell entry. This new system was evaluated for delivery of DNA in several cell lines and was found to compare favorably with all comparators tested (DOTAP and b-PEI as well as a number of known cell penetrating peptides) in various cell lines and particularly in culture medium containing up to 50% of serum. These results suggest that this dual molecular platform which is long lasting and noncytotoxic could be of practical use for in vivo applications.

Diversity and characterisation of polymorphic 3' untranslated region haplotypes of MICA and MICB genes.

MICA and MICB genes encode ligands that interact with the natural killer (NK) cell activating receptor, NKG2D. These ligands display a highly polymorphic allelic repertoire, although the true functional significance of this polymorphism remains elusive. We previously reported additional polymorphism in the 5' untranslated region (UTR) proximal promoter region of these genes by sequencing international histocompatibility workshop (IHW) cell line DNA promoter and coding regions. The present study extends this analysis by further characterising the 3'UTR region of the same IHW reference panel to achieve a more complete understanding of MICA and MICB haplotype diversity and possible functional relevance. We found 17 extended MICA haplotypes encompassing the coding region and 3'UTR, including four novel haplotypes identified in IHW cell line DNA. This increased to 21 when also considering the 5'UTR proximal promoter region. Analysis of the MICB 3'UTR revealed two novel sequences in cell lines KLO and WIN designated MICB-UTR8 and UTR9, respectively. A total of 11 MICB haplotypes were identified in this study and five were unique. The present study, characterising MICA/B 3'UTR polymorphism utilising IHW reference cell lines, could be useful for future studies investigating the role of microRNA in post-transcriptional repression of gene expression and for immunotherapy strategies to combat cancer progression.