Cell Line DNA Research Articles

Monitoring Minimal Residual Disease in Acute Myeloid Leukemia Using Genomic or cfDNA with MyMRD®, a Targeted NGS Panel

Acute myeloid leukemia (AML) is a genetically and phenotypically heterogeneous disorder. Precision therapies for AML have been developed that target specific driver mutations. The efficacies of these therapies are variable, making it critical to determine successful therapies prior to patient relapse. For patients achieving a first complete remission, minimum residual disease (MRD) is an important prognostic factor, as MRD may provide a powerful and timely tool to evaluate therapeutic efficacy. There is a growing demand that new and promising drugs are approved as quickly as possible and accelerated approvals will require biomarker surrogate endpoints, such as MRD, rather than long-term survival endpoints.We have developed a sensitive NGS gene panel (MyMRD®), which identifies pathogenic variants in AML. The MyMRD panel targets single nucleotide variants (SNVs), insertions and deletions (indels) in coding exons of hotspots of 21 genes (ASXL1 BRAF CALR CEBPA CSF3R DNMT3A FLT3 IDH1 IDH2 JAK2 KIT KRAS MPL NPM1 NRAS PTPN11 RUNX1 SF3B1 SRSF2 TP53 ZRSR2), and structural variants of potential genomic breakpoint hotspots within 3 somatic gene fusion partners (CBFB-MYH11 KMT2A RUNX1-RUNX1T1). This 23 gene targeted panel can identify driver mutations that cause relapse in >90% of all AML patients, as well as common drivers in other myeloid neoplasms and myelodysplasic syndromes.MyMRD panel validation included determining the limit of blank (LoB), limit of detection (LoD), and linearity. Validation samples were generated using known variant containing DNA from cell lines and clinical samples diluted into NA12878 (“Genome in a Bottle”) DNA. The average background, crossover, and carryover rates were determined to be 0.017%, 0.023%, and 0.016%, respectively. The LoB was determined to be 0.13% from calculating each of expected variants using the 95th percentile of all negative samples. Overall, we established an LoD of 0.5% for >95% of the targeted SNV and indel sites in the assay with lower LoDs for specific mutations of interest, such as 0.17% for FLT3 TKD and 0.34% for NPM1. The LoD for structure variants was determined to be 1.8%. The assay shows strong linearity with R2 = 0.969 - 0.994 of 11 selected targets in the entire range (0.17% - 50%) of variant allele frequencies (VAFs) tested. Linearity and LoD determined from clinical samples containing SNVs and indels were consistent with conclusions obtained from contrived cell line samples.Cell-free DNA (cfDNA) isolated from plasma is becoming a readily available source ofcirculating tumor DNA for testing. A similar molecular profile has been observed from cfDNA to that of bone-marrow tumor cells from multiple myeloma patients. Therefore, we investigated whether the MyMRD assay, developed for genomic DNA analysis, could be applied to cfDNA to assess variants at a level comparable to testing of genomic DNA. To overcome the limitation of cfDNA availability, DNA fragments with size similar to cfDNA (140-170bp), were generated from contrived cell line DNA to assess the LoD and linearity of assay. The cfDNA assay also showed strong linearity (R2=0.975 - 0.998) in the range of VAFs (0.1- 20%) tested. With cfDNA input of 25ng, the LoD was established to be 0.5% for targeted SNV sites, and 1% for targeted indel sites. The MyMRD cfDNA assay detected variants at the same LoD for SNVs and a slightly higher LoD for indels.Additional clinical samples were tested with the MyMRD assay. Variants detected were verified with capillary electrophoresis and/or amplicon-based NGS assays. The results showed 100% concordance with the MyMRD panel assay.The MyMRD gene panel is a sensitive and reliable assay that provides monitoring of MRD using genomic DNA and cfDNA. The assay is shown to detect clinically important driver variants and to have excellent linearity and LoD for targeted sites. The assay provides an accurate method for detecting mutations in multiple targets in patients and can be used to stratify patients for therapy and clinical trials, and may provide the sensitivity necessary to serve as a surrogate endpoint assay for targeted therapies in AML. DisclosuresXie:Invivoscribe Inc.: Employment. Chamberlain:Invivoscribe Inc.: Employment. Carson:Invivoscribe Inc.: Employment. Atkinson:Invivoscribe Inc.: Employment. McClain:Invivoscribe Inc.: Employment. Sprague:Invivoscribe, Inc.: Employment. Kiya:Invivoscribe Inc.: Employment. Xia:Invivoscribe Inc.: Employment. Huang:Invivoscribe Inc.: Employment. Patay:Invivoscribe Inc.: Consultancy, Equity Ownership, Patents & Royalties. Blankfard:Invivoscribe Inc.: Employment. Miller:Invivoscribe, Inc.: Employment, Equity Ownership.

Induction of apoptosis in ovarian cancer cells by miR-493-3p directly targeting AKT2, STK38L, HMGA2, ETS1 and E2F5.

Apoptosis is a form of directed programmed cell death with a tightly regulated signalling cascade for the destruction of single cells. MicroRNAs (miRNAs) play an important role as fine tuners in the regulation of apoptotic processes. MiR-493-3p mimic transfection leads to the induction of apoptosis causing the breakdown of mitochondrial membrane potential and the activation of Caspases resulting in the fragmentation of DNA in several ovarian carcinoma cell lines. Ovarian cancer shows with its pronounced heterogeneity a very high death-to-incidence ratio. A target gene analysis for miR-493-3p was performed for the investigation of underlying molecular mechanisms involved in apoptosis signalling pathways. Elevated miR-493-3p levels downregulated the mRNA and protein expression levels of Serine/Threonine Kinase 38 Like (STK38L), High Mobility Group AT-Hook 2 (HMGA2) and AKT Serine/Threonine Kinase 2 (AKT2) by direct binding as demonstrated by luciferase reporter assays. Notably, the protein expression of RAF1 Proto-Oncogene, Serine/Threonine Kinase (RAF1) was almost completely downregulated by miR-493-3p. This interaction, however, was indirect and regulated by STK38L phosphorylation. In addition, RAF1 transcription was diminished as a result of reduced transcription of ETS proto-oncogene 1 (ETS1), another direct target of miR-493-3p. Taken together, our observations have uncovered the apoptosis inducing potential of miR-493-3p through its regulation of multiple target genes participating in the extrinsic and intrinsic apoptosis pathway.

220 - Pharmacologic ascorbate induces DNA damage, alters the bioenergetic profile, but does not influence cellular respiration or glycolysis in human pancreatic cancer cells

We have investigated the effects of high dose ascorbate, (pharmacological ascorbate, P-AscH-) as an adjuvant to common cancer treatment modalities. We have proposed that P-AscH- acts as a pro-drug for delivery of extracellular H2O2 to tumors. This H2O2 enhances or exacerbates DNA damage. Many cancer treatment modalities, whether biophysical (ionizing radiation) or chemical (drug), are believed to involve direct damage to DNA or inhibit DNA repair processes. We have observed that DNA damage occurs in both nuclear as well as mitochondrial DNA in human pancreatic cancer cell lines upon exposure to P-AscH- with concomitant decreases in NAD+, NADH, and ATP. After removal of ascorbate, all the above resolve with time suggesting a link to cellular bioenergetics. To address steady-state changes in these redox-related species, we used Seahorse XF analysis. In media as ascorbate oxidizes it consumes oxygen in a concentration-dependent manner; this could lead to misinterpretation of data on cellular bioenergetics. To avoid this we incubated cells (0.5 to 1 h) with lethal doses of P-AscH- and then rapidly removed it before initiating bioenergetic experiments. We detected no significant changes in cellular bioenergetics following lethal doses (clonogenic) of P-AscH- at 0.5 h and up to 24 h after exposure. These results indicate that the decreased levels of NAD+, NADH, and ATP observed following short-term exposure to P-AscH- is not due to decreased production but rather due solely to increased demand. In additional experiments we observed no changes in metabolism upon exposure to dehydroascorbate (DHA) or ascorbate 2-phosphate, forms that load cells with ascorbate, but produce no extracellular H2O2. These data indicate that P-AscH- generates extracellular H2O2 leading to DNA damage resulting in depletion (demand) of cellular NAD+, NADH, and ATP. There appears to be no bioenergetic crisis due to a loss (production) in bioenergetic capacity.

Development of Novel Mutation-Specific Droplet Digital PCR Assays Detecting TERT Promoter Mutations in Tumor and Plasma Samples

Detecting mutations in the plasma of patients with solid tumors is becoming a valuable method of diagnosing and monitoring cancer. The TERT promoter is mutated at high frequencies in multiple cancer types, most commonly at positions -124 and -146 (designated C228T and C250T, respectively). Detection of these mutations has been challenging because of the high GC content of this region (approximately 80%). We describe development of novel probe-based droplet digital PCR assays that specifically detect and quantify these two mutations, along with the less common 242-243 CC>TT mutation, and demonstrate their application using human tumor and plasma samples from melanoma patients. Assay designs and running conditions were optimized using cancer cell line genomic DNAs with the C228T or C250T mutations. The limits of detection were 0.062% and 0.051% mutant allele fraction for the C228T and C250T assays, respectively. Concordance of 100% was observed between droplet digital PCR and sequencing-based orthogonal methods in the detection of TERT mutant DNA in 32 formalin-fixed, paraffin-embedded melanoma tumors. TERTmutant DNA was also identified in 21 of 27 plasma samples (78%) from patients with TERTmutant tumors, with plasma mutant allele fractions ranging from 0.06% to 15.3%. There were no false positives in plasma. These data demonstrate the potential of these assays to specifically detect and quantify TERTmutant DNA in tumors and plasma of cancer patients.

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.

The biological performance of purified supercoiled p53 plasmid DNA in different cancer cell lines

Abstract Tumor suppressor p53 remains one of the most interesting therapeutic targets in cancer gene therapy due to its consistent mutation in numerous cancers. Thus, the reinstatement of the p53 expression and function can be seen as an effective alternative for cancer treatment, motivating research in this field. In this study, l -methionine matrix was used to purify the supercoiled topoisoform of a plasmid DNA encoding the p53 protein. This pure biopharmaceutical was conjugated with liposomes to comprehensively analyze its in vitro performance and therapeutic potential in different cancer cell lines, including the lung and cervix models. A different profile of cellular responses was attained after the transfection of these cancer cell lines with the p53-pDNA. Actually, the in vitro transfection with pure sc p53-pDNA resulted in a higher expression of the tumor suppressor protein in cancer cells when compared with the native pDNA samples (oc + sc topoisoforms). Also, wild-type p53 expression following transfection was significantly higher in HeLa cervix cancer cells compared to that obtained in A549 lung cancer cells. Overall, our findings emphasize the potential of sc pDNA gene-based therapy, also raising awareness of the need to adjust the therapeutics, considering the feature of high heterogeneity of cancer cells.

Targeted next-generation sequencing as a comprehensive test for Mendelian diseases: a cohort diagnostic study

With the development of next generation sequencing, more and more common inherited diseases have been reported. However, accurate and convenient molecular diagnosis cannot be achieved easily because of the enormous size of disease causing mutations. In this study, we introduced a new single-step method for the genetic analysis of patients and carriers in real clinical settings. All kinds of disease causing mutations can be detected at the same time in patients with Mendelian diseases or carriers. First, we evaluated this technology using YH cell line DNA and 9 samples with known mutations. Accuracy and stability of 99.80% and 99.58% were achieved respectively. Then, a total of 303 patients were tested using our targeted NGS approaches, 50.17% of which were found to have deleterious mutations and molecular confirmation of the clinical diagnosis. We identified 219 disease causing mutations, 43.84% (96/219) of which has never been reported before. Additionally, we developed a new deleteriousness prediction method for nonsynonymous SNVs, and an automating annotation and diagnosis system for Mendelian diseases, thus greatly assisting and enhancing Mendelian diseases diagnosis and helping to make a precise diagnosis for patients with Mendelian diseases.

DNA Repair Deficient Chinese Hamster Ovary Cells Exhibiting Differential Sensitivity to Charged Particle Radiation under Aerobic and Hypoxic Conditions

It has been well established that hypoxia significantly increases both cellular and tumor resistance to ionizing radiation. Hypoxia associated radiation resistance has been known for some time but there has been limited success in sensitizing cells to radiation under hypoxic conditions. These studies show that, when irradiated with low linear energy transfer (LET) gamma-rays, poly (ADP-ribose), polymerase (PARP), Fanconi Anemia (FANC), and mutant Chinese Hamster Ovary (CHO) cells respond similarly to the non-homologous end joining (NHEJ) and the homologous recombination (HR) repair mutant CHO cells. Comparable results were observed in cells exposed to 13 keV/μm carbon ions. However, when irradiated with higher LET spread out Bragg peak (SOBP) carbon ions, we observed a decrease in the oxygen enhancement ratio (OER) in all the DNA of repair mutant cell lines. Interestingly, PARP mutant cells were observed as having the largest decrease in OER. Finally, these studies show a significant increase in the relative biological effectiveness (RBE) of high LET SOBP carbon and iron ions in HR and PARP mutants. There was also an increase in the RBE of NHEJ mutants when irradiated to SOBP carbon and iron ions. However, this increase was lower than in other mutant cell lines. These findings indicate that high LET radiation produces unique types of DNA damage under hypoxic conditions and PARP and HR repair pathways play a role in repairing this damage.

Stimulation of exosome release by extracellular DNA is conserved across multiple cell types.

Exosomes are distinguished from other types of extracellular vesicles by their small and relatively uniform size (30-100 nm) and their composition which reflects their endo-lysosomal origin. Involvement of these extracellular organelles in intercellular communication and their implication in pathological conditions has fuelled intensive research on mammalian exosomes; however, currently, very little is known about exosomes in lower vertebrates. Here we show that, in primary cultures of head kidney leukocytes from Atlantic salmon (Salmo salar), phosphorothioate CpG oligodeoxynucleotides induce secretion of vesicles with characteristics very similar to these of mammalian exosomes. Further experiments revealed that the oligonucleotide-induced exosome secretion did not depend on the CpG motifs but it relied on the phosphorothioate modification of the internucleotide linkage. Exosome secretion was also induced by genomic bacterial and eukaryotic DNA in toll-like receptor 9-negative piscine and human cell lines demonstrating that this is a phylogenetically conserved phenomenon which does not depend on activation of immune signaling pathways. In addition to exosomes, stimulation with phosphorothioate oligonucleotides and genomic DNA induced secretion of LC3B-II, an autophagosome marker, which was associated with vesicles of diverse size and morphology, possibly derived from autophagosome-related intracellular compartments. Overall, this work reveals a previously unrecognized biological activity of phosphorothioate ODNs and genomic DNA - their capacity to induce secretion of exosomes and other types of extracellular vesicles. This finding might help shed light on the side effects of therapeutic phosphorothioate oligodeoxynucleotides and the biological activity of extracellular genomic DNA which is often upregulated in pathological conditions.

Abstract 5335: A highly efficient targeted bisulfite sequencing method for accurate DNA methylation profiling

Abstract Background DNA methylation plays an important role in gene regulation. Bisulfite sequencing (BS), in which unmethylated cytosines are converted to uracil while methylated cytosines remain intact, is considered as the gold standard for DNA methylation detection with single nucleotide resolution. Due to the high cost of whole genome BS and the low throughput of amplicon BS, there remains the need for new targeted bisulfite sequencing methods with high efficiency and flexibility. Results We developed a targeted bisulfite sequencing method, by incorporating single primer enrichment (SPE) and molecular barcoding (UMI) technology. This enables flexible and scalable targeted DNA methylation analysis with high efficiency. We first studied the ability of our assay to detect CpG methylation at various levels in reference materials. We also evaluated its utility in profiling CpG methylation in different cell line DNA, and compared the results with published data from alternative technologies (WBS, RRBS and microarray). Conclusions We demonstrated the successful implementation of SPE and UMI technology on bisulfite converted DNA with high enrichment specificity and low bias. We showed that our method accurately detected CpG methylation at different levels, in concordance with expected titration mixture or published datasets. Comparing to reported amplicon BS, our technology enables much higher multiplexing scale and much simpler workflow. Comparing to probe capture based enrichment, our technology enables much lower DNA input, much higher sequencing efficiency (on-target) and shorter turnaround time. Citation Format: Quan Peng, Daniel Kim, Mohammad Nezami Ranjbar, Sascha Strauss, Scott Winter, Yexun Wang. A highly efficient targeted bisulfite sequencing method for accurate DNA methylation profiling [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 5335.

Abstract 430: Detection of low-frequency variants from highly degraded DNA samples using a novel library preparation method

Abstract Diagnostic tools based on next generation sequencing are fundamentally transforming clinical oncology. However, there is a lack of adequate library preparation strategies for highly degraded, clinically relevant samples, such as cell-free DNA (cfDNA) and FFPE DNA. Furthermore, clinical samples are often available in limited quantity, making the detection of pathologic variants particularly challenging. Due to the extreme heterogeneity of these sample types, targeted sequencing is often used to achieve deep coverage of genomic loci and enable detection of low-frequency variants. Commercially available protocols for library preparation require stringent size-selection to remove adapter-dimers, which reduces library complexity. Achieving high specificity can be challenging because low-frequency artifacts arise from a variety of sources, including DNA extraction, library construction, PCR, hybrid selection, and sequencing. These artifacts can be identified by “duplex sequencing”, where strand-specific unique molecular identifiers (UMIs) are used to confirm the presence of an alteration on both strands of an input molecule. However, duplex sequencing typically delivers low conversion rates with degraded samples due to poor ligation efficiency and template loss during size-selection. We present a novel library construction chemistry that eliminates adapter dimers and utilizes duplexed UMIs to increase sensitivity. The method employs a two-step ligation procedure that does not require size-selection or adjustment of AMPure bead ratios during cleanup. We achieve maximal library conversion using a unique, mutant DNA ligase and proprietary sequencing adapters that increase ligation efficiency and suppress chimera formation. We demonstrate performance using three sample types: sheared genomic DNA, cfDNA, and FFPE DNA, across a wide input range (1-1000 ng). To mimic performance with degraded DNA, we created libraries from mixtures of NA12878 and NA24385 cell line DNA sheared to 150 bp with inputs of 1-25 ng and mutant allele fractions (MAFs) down to 0.25%. Libraries were enriched using a 75 kb panel of xGen Lockdown Probes, followed by ultra-deep sequencing and variant calling. When compared to commercially available methods, our approach yielded a 50-100% increase in library complexity with significantly improved sensitivity to <1% variants. We also obtained 100% specificity using duplexed UMI correction, which removed all false-positive calls. To highlight clinical utility, we extended our study to cfDNA samples with inputs of 5-25 ng and MAFs down to 1%, and FFPE DNA samples with inputs of 25-100 ng and MAFs down to 1%. Citation Format: Ushati Das Chakravarty, Zac Zwirko, Yu Zheng, Madelyn Light, Kevin Lai, Keith Bryan, Scott Rose, Yun Bao, Mirna Jarosz, Caifu Chen. Detection of low-frequency variants from highly degraded DNA samples using a novel library preparation method [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 430.

Abstract 2888: DNA Repair Enzyme Signature as a biomarker of MAPK pathway inhibition by targeted therapies in melanoma cell lines

Abstract About 50% of melanomas carry activating mutation in BRAF or NRAS genes. BRAF/MEK inhibitors elicit a transient effective response but resistance rapidly develops through various MAPK/PI3K/AKT pathway activating mechanisms. DNA Repair mechanisms are regulated by these signaling pathways. We hypothesized that effective inhibition of the MAPK pathway should translate into decrease of DNA Repair capacities. To gain insights into this hypothesis we measured the DNA Repair capacities of 12 melanoma cell lines treated or not by BRAF and MEK inhibitors (respectively Vemurafenib V and Cobimetinib C, Roche laboratories), alone and in combination. We evaluated various excision synthesis repair mechanisms using a multiplexed functional DNA Repair assay using cell extracts. We thus obtained a comprehensive overview of the cell lines DNA Repair capacities. Significant qualitative and quantitative differences were observed between the DNA Repair profiles of the 3 mutation groups in non-treated cells. Globally DNA Repair capacities of BRAFm cells significantly decreased following treatment by V and V+C confirming that excision synthesis repair mechanisms are under the control of the MAPK pathway. When the cell lines were examined individually, interesting features were observed. 3/7 showed drastic decrease of DNA repair with V and V+C treatment. On the contrary, C alone activated repair in 3/7, possibly reflecting a paradoxical activation of some pathways. In addition, among the 3 NRASm cell lines, only 1 exhibited a marked decreased of DNA Repair after C treatment. Surprisingly V and V+C activated some repair activities in one WT cell line, possibly reflecting some paradoxical activation of the complex kinase network. Our results suggest that mutations in signaling kinase pathways impact the so-called DNA Damage Response and translate into specific DNA Repair Enzyme Signatures. Crosstalk of tyrosine kinases with the DNA damage signaling pathway is well known. It is possible to take advantage of their intricate regulation through characterization of DNA Repair Enzymatic Signature to gain information on the inhibition efficacy of targeted drugs. Interestingly this approach could be conducted on clinical samples and we propose to use the DNA Repair Enzyme Signature as a new tool to investigate effectiveness of targeted therapies in metastatic melanoma. This study was sponsored by Institut Roche. Citation Format: Sylvie Sauvaigo, Manel Benkhiat, Florian Braisaz, Florence de Fraipont, Caroline Aspord, Stéphane Mouret, Joël Plumas, Fanny Bouquet, Marie-Thérèse Leccia. DNA Repair Enzyme Signature as a biomarker of MAPK pathway inhibition by targeted therapies in melanoma cell lines [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 2888.

Abstract 4534: Validation of highly sensitive TargetSelectorTM ctDNA assays for EGFR, BRAF, and KRAS mutations

Abstract Background: Accurate detection of driver mutations in cancer patients is vital for targeted therapy. Compared to tissue biopsy, “liquid biopsy” offers a non-invasive and more systemic approach to identify tumor mutations by assessing circulating tumor DNA (ctDNA) released from tumor cells into peripheral blood. We have developed TargetSelectorTM Real-Time PCR based assays to detect low frequency mutant alleles in ctDNA. The TargetSelectorTM assay uses a proprietary blocker to suppress amplification of excess WT alleles released from normal cells, while allowing specific amplification of mutants. Here we focus on five important targets: EGFR (Del19, L858, and T790), BRAF (V600), and KRAS (G12/G13), which are relevant to lung cancer, melanoma, and colorectal cancer. Methods: The TargetSelectorTM assay applies a specific blocker to cover variants on a short stretch of target DNA (up to 10 bp for nucleotide variants). For example, one KRAS exon 2 blocker covers all variants on both G12 and G13 positions. The TargetSelectorTM assays were first validated with cancer cell line DNA carrying mutation targets on QuantStudio 5 Real-Time PCR instruments (QS5). Sanger Sequencing was subsequently performed to confirm the mutation. Analytical validation was conducted by 3 independent operators using 5 instruments across 5 days in our CAP/CLIA certified laboratory. For ctDNA testing, whole blood samples were collected in CEE-SureTM Blood Collection tubes and DNA extraction from plasma was performed on the QIAsymphony. Results: In total, we tested 3086 samples for EGFR, BRAF and KRAS TargetSelectorTM ctDNA assays, with EGFR WT assay as the background reference. All five ctDNA assays showed >99% analytical sensitivity and >99% analytical specificity. Based on practical and theoretical estimates, each ctDNA assay demonstrated single mutant copy detection sensitivity. In the presence of 14,000 copies of WT background, the sensitivity of our ctDNA assays are: EGFR Del19, 0.01%; EGFR L858R, 0.02%; EGFR T790M, 0.01%; BRAF V600E, 0.01%; KRAS G12C, 0.02%. The inter-assay and intra-assay analyses showed r2 >0.94, suggesting a consistent performance among operational variables. Samples tested from 20 healthy donors (100 tests in total) showed clinical specificity >99%. In the concordance study of 13 clinical samples (31 tests in total) between QS5 and ABI 7900HT platforms, TargetSelectorTM ctDNA assays with the QS5 identified the same plus additional mutations compared to the 7900HT. Conclusions: TargetSelectorTM ctDNA assays were validated both analytically and clinically, showing single mutant copy detection and sensitivity at 0.02% or better in a background of excess WT DNA. Implementation of the QS5 qPCR platform into our TargetSelectorTM ctDNA assays leads to a higher sensitivity and faster turnaround time. These factors enable sensitive and efficient testing crucial for guiding treatment decisions and patient care. Citation Format: Shan-Fu Wu, Timothy T. Lu, Anh Pham, Jeffrey Chen, Tony Daher, Errin Samuelsz, Manisha Patel, Veena M. Singh, Lyle J. Arnold, Jason C. Poole. Validation of highly sensitive TargetSelectorTM ctDNA assays for EGFR, BRAF, and KRAS mutations [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 4534.

The experimental study of genomic alterations of hepatocellular carcinoma by panel-based sequencing

Objective To explore the value of high-throughput capture sequencing technology of tumor gene in detection of somatic mutation and mutation patterns of hepatocellular carcinoma cells. Methods Based on the target gene sequencing technology of the second generation sequencing technology, a custom sequence capture system containing 325 tumor related genes panel, NimbleGen, was used, and the target region of 7 hepatoma cell lines (HuH-7, Hep3B, SK-HEP-1, MHCC97H, MHCC97L, HepG2, HepG2.2.15) DNA gene sequencing were captured and sequenced. The tumor-related mutant gene and mutation load rate, as well as the difference between the gene mutation trend were analysed. Results The results showed that the average coverage rate of the target area was 99.7%, and the average sequencing depth was about 1 000×, and 100 genes(mutation frequency≥5%)were detected, including 344 non synonymous mutations. There are 38 gene mutations closely related to cancer enriching in 5 oncogenic signaling pathways: chromatin remodeling, Notch, mitogen-activated protein kinase (MAPK), p53, and Wnt/beta-catenin pathways. Compared with the previous reports of somatic mutations in hepatocellular carcinoma (HCC) patients, the chromatin remodeling and gene mutations in the Notch signaling pathway were higher in HCC cell lines. Three mutant genes not reported in HCC patients ever were identified, including two tumor suppressor genes [MSH6, sex determining region Y box 9 (SOX9)] and one oncogene (ALK).44 mutant genes were identified in MHCC97 cell lines (MHCC97H and MHCC97L), of which 30 (68%) were co-mutations. Mutation genes are enriched in oxidative stress pathways (KEAP1), chromatin remodeling (NCOR1, KMT2D, ARID1A), MAPK (MAP3K1, MAP2K3) and Wnt/beta-catenin (AXIN1, SOX9) pathways. ALK and ERBB2 mutations were detected only in MHCC97L, APOB mutations only in MHCC97H. Notch pathway (Notch1, Notch2) had higher mutation frequency in MHCC97L, TERT in MHCC97H.37 mutant genes were identified in HepG2 and HepG2.2.15, of which 19 (51%) were co-mutations. HepG2 and HepG2.2.15 had the same mutation sites and similar mutation frequency in Notch pathway (Notch1, Notch2) and chromatin remodeling pathway (KMT2D, ZFHX4). Tp53 mutations were detected only in HepG2, adenomatous polyposis coli (APC) and ataxia-telangiectasia mutated (ATM) mutations only in HepG2.2.15. The four HCC cell lines (HuH-7, Hep3B, SK-HEP-1, HepG2) from different HCC patients had different mutation modes, but the same clone derived HCC cell lines showed a large similarity. Conclusion High throughput sequencing technology of tumor gene has high application value in the detection of cell lines and mutation patterns in hepatocellular carcinoma cell lines. Key words: Hepatocellular carcinoma; Gene mutation; Panel-based Sequencing

Identification of strong intron enhancer in the heparanase gene: effect of functional rs4693608 variant on HPSE enhancer activity in hematological and solid malignancies

Heparanase is an endo-β-glucuronidase that specifically cleaves the saccharide chains of heparan sulfate (HS) proteoglycans and releases HS-bound cytokines, chemokines, and bioactive growth-promoting factors. Heparanase plays an important role in the nucleus as part of an active chromatin complex. Our previous studies revealed that rs4693608 correlates with heparanase levels and increased risk of acute and extensive chronic graft vs. host disease (GVHD). Discrepancy between recipient and donor in this SNP significantly affected the risk of acute GVHD. In the present study, we analyzed the HPSE gene region, including rs4693608, and demonstrated that this region exhibits SNPs-dependent enhancer activity. Analysis of nuclear proteins from normal leukocytes revealed their binding to DNA probe of both alleles with higher affinity to allele G. All malignant cell lines and leukemia samples disclosed a shift of the main bands in comparison to normal leukocytes. At least five additional shifted bands were bound to allele A while allele G probe was bound to only one main DNA/protein complex. Additional SNPs rs4693083, rs4693084, and rs4693609 were found in strong linkage disequilibrium (LD) with rs11099592 (exon 7). Only rs4693084 affected protein binding to DNA in cell lines and leukemia samples. As a result of the short distance between rs4693608 and rs4693084, both SNPs may be included in a common DNA/protein complex. DNA pull-down assay revealed that heparanase is involved in self-regulation by negative feedback in rs4693608-dependent manner. During carcinogenesis, heparanase self-regulation is discontinued and the helicase-like transcription factor begins to regulate this enhancer region. Altogether, our study elucidates conceivable mechanism(s) by which rs4693608 SNP regulates HPSE gene expression and the associated disease outcome.

PO-025 Antiproliferative effects of rosemary (rosmarinus officinalis, L.) extract in colon cancer cells lines

IntroductionInfections by oncoviruses are an important risk factor for the development of neoplasms. Particularly, anogenital cancers are strongly associated with persistent infections by high-risk human papillomaviruses (HPV). In the case of cervical cancer the physical state of the virus seems to be an important prognostic marker. Especially the integration of viral DNA into the host cell genome bears the risk of following degenerations. Therefore, the early and sensitive detection is of high interest. Besides Pap-test and PCR, fluorescence in situ hybridization (FISH) is used for a quantitative and qualitative detection of HPV infections and risk assessment.Material and methodsFor analysis high-risk HPV FISH probes should be developed for standardised, type-specific and quantitative detection of HPV nucleic acid in host cells. Due to the short viral DNA sequence, present with just few copies, conventional FISH probes have little sensitivity. Therefore, we tested specific FISH probes targeting different regions of the HPV genome in combination with signal amplification. PCR-produced FISH probes were biotin labelled by direct incorporation of biotinylated nucleotides or nick translation. Our biotinylated HPV probes were tested on HPV-positive and –negative cells and detected using fluorescence labelled streptavidin or subsequent tyramide signal amplification. In addition to FISH, probes were used for chromogenic in situ hybridization. All analysis were performed with our fully automatized multicolor fluorescence imaging platform VideoScan.Results and discussionsThe presence of HPV DNA in cell lines was confirmed by (digital) PCR. The protocol for synthesis of biotinylated probes using HPV sequence specific primers and genomic DNA as template was successful. Biotin incorporation was checked with agarose gel electrophoresis and modified dot blot hybridization. In in situ hybridization experiments, our probes showed high signals in HPV high-copy number CaSki cells and a tissue model, compared to HPV negative samples.ConclusionHighly sensitive and specific FISH probes offer a great potential for the detection of HPV DNA in patient samples and thereby for the assessment of tumour risks. Using our probes, we got positive results in hybridization experiments using chromogenic and fluorescent detection methods with or without further amplification steps. Next, comparative FISH experiments will be performed to check probe sensitivity in cells containing few HPV copies and their practicability in biopsy derived tissue samples.

Murine Endogenous Retroviruses Are Detectable in Patient-Derived Xenografts but Not in Patient-Individual Cell Lines of Human Colorectal Cancer.

Endogenous retroviruses are remnants of retroviral infections. In contrast to their human counterparts, murine endogenous retroviruses (mERV) still can synthesize infectious particles and retrotranspose. Xenotransplanted human cells have occasionally been described to be mERV infected. With genetic engineered mice and patient-derived xenografts (PDXs) on the rise as eminent research tools, we here systematically investigated, if different tumor models harbor mERV infections. Relevant mERV candidates were first preselected by next generation sequencing (NGS) analysis of spontaneous lymphomas triggered by colorectal cancer (CRC) PDX tissue. Two primer systems were designed for each of these candidates (AblMLV, EcoMLV, EndoPP, MLV, and preXMRV) and implemented in an quantitative real-time (RT-qPCR) screen using murine tissues (n = 11), PDX-tissues (n = 22), PDX-derived cell lines (n = 13), and patient-derived tumor cell lines (n = 14). The expression levels of mERV varied largely both in the PDX samples and in the mouse tissues. No mERV signal was, however, obtained from cDNA or genomic DNA of CRC cell lines. Expression of EcoMLV was higher in PDX than in murine tissues; for EndoPP it was the opposite. These two were thus further investigated in 40 additional PDX. In addition, four patient-derived cell lines free of any mERV expression were subcutaneously injected into immunodeficient mice. Outgrowing cell-derived xenografts barely expressed EndoPP. In contrast, the expression of EcoMLV was even higher than in surrounding mouse tissues. This expression gradually vanished within few passages of re-cultivated cells. In summary, these results strongly imply that: (i) PDX and murine tissues in general are likely to be contaminated by mERV, (ii) mERV are expressed transiently and at low level in fresh PDX-derived cell cultures, and (iii) mERV integration into the genome of human cells is unlikely or at least a very rare event. Thus, mERVs are stowaways present in murine cells, in PDX tissues and early thereof-derived cell cultures. We conclude that further analysis is needed concerning their impact on results obtained from studies performed with PDX but also with murine tumor models.

Abstract P5-06-03: Generation and characterization of a novel invasive lobular breast carcinoma cell line WCRC-25

Abstract Background: Invasive Lobular Breast Carcinoma (ILC) is the second most common histologic subtype of breast cancer, comprising 10-15% of all cases. ILC is clinically and molecularly distinguished from the major subtype–Invasive Ductal Carcinoma (IDC)–by loss of E-cadherin (CDH1). Studies on ILC remain sparse, in part due to limited cell culture models. The Women's Cancer Research Center (WCRC) has therefore set up a combined effort of breast surgical oncologists, medical oncologists, pathologists, and cancer biologists to collect fresh tissues and establish additional ILC cell lines. Methods: Tumor cells were grown using irradiated fibroblast conditioned media (CM) in hypoxic (5% O2) conditions. Sanger sequencing and Droplet Digital PCR (ddPCR) were utilized to test for mutation in CDH1 in tumor and circulating DNA (cfDNA), using germline DNA as control. DNA copy number status in the tumor was detected using nanoString approach. Expression of E-cadherin and a series of lineage markers was elucidated using Immunoblotting (IB) and Immunofluorescence (IF). A panel of ILC (MDA-MB-134, Sum44PE, IPH-926) and IDC (MCF-7, MDA-MB-231) cell lines was included for comparison. After establishment, WCRC-25 population doubling was compared between growth in CM or Dulbecco's Modified Eagle's Medium (DMEM), and hypoxic (5% O2) or normoxic (21% O2) conditions. Growth phenotypes were characterized in 2D, Ultra Low Attachment (ULA), and soft agar. Results: WCRC-25 was successfully established from the pleural effusion of a 77-year old patient with metastatic ILC. The patient had stage IV (T3N3M1) ER+/PR-/HER2- ILC; was treated with bilateral mastectomy, radiation therapy, and multiple lines of chemotherapy (FOLFOX due to initial misdiagnosis; 2 cycles carboplatin/paclitaxel; anastrazole, fulvestrant, pegylated liposomal doxorubicin, gemcitabine, exemastane, and eribulin (with denosumab for bone lesions); had metastatic lesions in stomach, bone, pleura, and pericardium; and ultimately passed away from progressive pleural effusions roughly 3 years after diagnosis. A novel nonsense mutation of CDH1 was observed in exon 13 (Q705*) in cell line DNA, resulting in a premature stop codon. The same mutation was confirmed in cfDNA obtained from longitudinal blood samples from the patient. Copy number analysis revealed deletion of the second CDH1 copy in tumor, and E-cadherin protein loss was confirmed by IB and IF. WCRC-25 cells expressed epithelial cell markers CK8/18 and EpCAM, and as expected did not express stromal marker αSMA. ERα expression was very low, and not sufficient for measurable hormone response. Population doubling was significantly faster in hypoxic compared to normoxic conditions, regardless of media type, but minimal in 3D. Conclusions: WCRC-25 is a novel ILC cell line, defined by CDH1 nonsense truncating mutation and LOH, resulting in loss of E-cadherin protein expression. Cells show favorable growth characteristics in hypoxic conditions and maintain epithelial-dominated protein expression. We are currently performing RNA seq analysis of the matched primary tumor and metastatic samples from the stomach, peritoneum/falciform ligament, pleural effusion and skin, which we will compare and contrast with that of the established WCRC-25 cell line. Citation Format: Kota K, Bossart E, Basudan A, Minteer T, Meier C, Brown D, Gurda GT, Miller L, Dabbs DJ, Lee A, Puhalla S, Jankowitz R, McAuliffe P, Lucas P, Oesterreich S. Generation and characterization of a novel invasive lobular breast carcinoma cell line WCRC-25 [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-06-03.

Oxidative stress increases M1dG, a major peroxidation-derived DNA adduct, in mitochondrial DNA.

Reactive oxygen species (ROS) are formed in mitochondria during electron transport and energy generation. Elevated levels of ROS lead to increased amounts of mitochondrial DNA (mtDNA) damage. We report that levels of M1dG, a major endogenous peroxidation-derived DNA adduct, are 50–100-fold higher in mtDNA than in nuclear DNA in several different human cell lines. Treatment of cells with agents that either increase or decrease mitochondrial superoxide levels leads to increased or decreased levels of M1dG in mtDNA, respectively. Sequence analysis of adducted mtDNA suggests that M1dG residues are randomly distributed throughout the mitochondrial genome. Basal levels of M1dG in mtDNA from pulmonary microvascular endothelial cells (PMVECs) from transgenic bone morphogenetic protein receptor 2 mutant mice (BMPR2R899X) (four adducts per 106 dG) are twice as high as adduct levels in wild-type cells. A similar increase was observed in mtDNA from heterozygous null (BMPR2+/−) compared to wild-type PMVECs. Pulmonary arterial hypertension is observed in the presence of BMPR2 signaling disruptions, which are also associated with mitochondrial dysfunction and oxidant injury to endothelial tissue. Persistence of M1dG adducts in mtDNA could have implications for mutagenesis and mitochondrial gene expression, thereby contributing to the role of mitochondrial dysfunction in diseases.

Multicenter validation of cancer gene panel-based next-generation sequencing for translational research and molecular diagnostics

The simultaneous detection of multiple somatic mutations in the context of molecular diagnostics of cancer is frequently performed by means of amplicon-based targeted next-generation sequencing (NGS). However, only few studies are available comparing multicenter testing of different NGS platforms and gene panels. Therefore, seven partner sites of the German Cancer Consortium (DKTK) performed a multicenter interlaboratory trial for targeted NGS using the same formalin-fixed, paraffin-embedded (FFPE) specimen of molecularly pre-characterized tumors (n = 15; each n = 5 cases of Breast, Lung, and Colon carcinoma) and a colorectal cancer cell line DNA dilution series. Detailed information regarding pre-characterized mutations was not disclosed to the partners. Commercially available and custom-designed cancer gene panels were used for library preparation and subsequent sequencing on several devices of two NGS different platforms. For every case, centrally extracted DNA and FFPE tissue sections for local processing were delivered to each partner site to be sequenced with the commercial gene panel and local bioinformatics. For cancer-specific panel-based sequencing, only centrally extracted DNA was analyzed at seven sequencing sites. Subsequently, local data were compiled and bioinformatics was performed centrally. We were able to demonstrate that all pre-characterized mutations were re-identified correctly, irrespective of NGS platform or gene panel used. However, locally processed FFPE tissue sections disclosed that the DNA extraction method can affect the detection of mutations with a trend in favor of magnetic bead-based DNA extraction methods. In conclusion, targeted NGS is a very robust method for simultaneous detection of various mutations in FFPE tissue specimens if certain pre-analytical conditions are carefully considered.