Detection Of DNA Mutations Research Articles

(Invited) Chemical Sensors Based on Thermal Resistance Analysis at Solid-Liquid Interfaces – Applications and Challenges

Introduction Over the past few decades, researchers have devoted considerable effort to the integration of synthetic receptors into chemical sensing platforms. In general, molecularly imprinted polymers (MIPs) have been developed for targets ranging from small molecules to proteins while surface imprinted polymers (SIPs) are generally used to detect larger entities such as microorganisms or whole cells [1]. These synthetic receptors have been combined with traditional electrochemical, optical or microgravimetric readout approaches to create sensors for various applications including medical diagnostics and environmental screening [2]. In 2012, a surprisingly versatile readout tool for label-free sensing was developed; the so-called heat-transfer method (HTM) [3]. The technique was originally developed for the detection of point mutations in DNA but was soon extended towards chemical sensing by coating the chips with synthetic receptors. In this way, it can be used for a wide variety of applications ranging from e.g. bacteria detection in urine samples for UTI diagnosis to cardiac biomarker testing in serum [4-5].The benefit of these thermal chemical sensing platforms over existing state-of-the-art technology lies in the unique combination of highly selective synthetic receptors and a low-cost, user-friendly readout platform that requires minimal instrumentation. The challenges towards commercial application mainly involve standardizing and upscaling the MIP/SIP synthesis procedure as well as the limited sensitivity of the readout approach. However, progress is being made in both aspects in recent years. Method Bacteria imprints were made by spin coating aluminum chips with semi-cured polyurethane layers. Template bacteria are immobilized onto a PDMS stamp and pressed into the polyurethane layer that was cured over-night. Removal of the stamp and template leads to a SIP containing microcavities that are complementary to the template. Thermal resistance over the solid-liquid interface was monitored by sending a thermal wave through the chip with an average temperature of 37.0°C and an amplitude of 0.1 °C. The phase shift of the transmitted thermal wave at 0.03 Hz was analyzed in function of the bacteria concentration in spiked urine samples [4].MIPs can be made in a more reproducible manner than SIPs, through the so-called solid phase synthesis approach. MIP nanoparticles were obtained by immobilizing selected cardiovascular biomarkers onto glass beads that were packed in a column. Radical polymerization was performed at room temperature and the colum was eluded at different temperatures. The particles that elute at elevated temperatures were retained, as their affinity for the target is the highest. The MIP particles were directly immobilized onto the surface of the thermocouples for thermal resistance measurements. Results and Conclusions The results shown in Figure 1a illustrate that the phase shift observed in the transmitted thermal wave will increase when exposing the SIP-coated aluminum chip to the target bacterium, in this case E. coli. These findings can be explained by the fact that bacteria binding to the cavities in the SIP layer will displace the urine (water-based so a good thermal conductor) that was previously present, leading to an increase of the thermal-resistance at the solid-liquid interface and a tempered propagation of the thermal wave that was send through the chip.The experiments using the nano-sized MIP particles for biomarker illustrate that it is possible to multiplex the thermal analysis for the detection of cardiac biomarkers in serum. The data in figure 1b show that the thermal resistance of the MIP particles at the interface only change when exposed to their target (red, ST2). Exposing the MIPs to a similar cardiac biomarker (blue, H-FABP) results in a response that is similar to the response of the non-imprinted reference channel (black) with no significant change in the phase shift observed.

Highly sensitive and specific detection of hepatitis B virus DNA and drug resistance mutations utilizing the PCR-based CRISPR-Cas13a system

ObjectivesUndetectable or low-level hepatitis B virus (HBV) DNA and drug resistance mutations in patients may increase the risk of HBV transmission or cause active viral replication and other clinical problems. Here, we established a highly sensitive and practical method for HBV and drug resistance detection using a polymerase chain reaction (PCR) -based CRISPR-Cas13a detection system (referred to as PCR-CRISPR) and evaluated its detection capability using clinical samples. MethodsSpecific CRISPR RNAs (crRNAs) are designed for HBV DNA detection and YMDD (tyrosine-methionine-aspartate-aspartate) variant identification. The HBV DNA was detected in 312 serum samples for HBV diagnosis using quantification PCR (qPCR) and PCR-CRISPR. Additionally, 424 serum samples for YMDD testing were detected by qPCR, direct sequencing, and our assay. ResultsUsing PCR-CRISPR, one copy per test of HBV DNA was detected with HBV-1 crRNA in 15 min after PCR amplification. Consistent results with qPCR were observed for 302 samples, while the remaining 10 samples with low-level HBV DNA were detectable by PCR-CRISPR and droplet digital PCR but not by qPCR. PCR-CRISPR diagnosed all 412 drug-resistant samples detected by the YMDD detection qPCR kit and direct sequencing, as well as the other 12 drug-resistant samples with low-level HBV DNA undetectable by qPCR and direct sequencing. ConclusionsWe developed a novel PCR-CRISPR method for highly sensitive and specific detection of HBV DNA and drug resistance mutations. One copy per test for HBV DNA and YMDD drug resistance mutations could be detected. This method has wide application prospects for the early detection of HBV infection, drug resistance monitoring and treatment guidance.

Unique dual indexing PCR reduces chimeric contamination and improves mutation detection in cell-free DNA of pregnant women

Allele fraction measurement is an essential component in nucleic acid analysis. The formation of chimeric amplicons during multiplex PCR amplification, however, greatly affects the allele fraction even before downstream analysis. Previous error correction strategy with unique molecular indexing (UMI) targets mainly points mutations rather than chimeras. Since the mutant allele detection in pregnant women cell-free DNA (cfDNA) is limited by chimeric amplicon contamination, a more direct error correction solution is demanded. Here we demonstrate effective reduction of chimeric amplicon contamination by unique dual indexing. With error corrected deep sequencing analysis, we achieved 100% accuracy in 16 tests of the parental mutation inheritance and de novo mutations in cfDNA of pregnant women, whose fetuses were at risk of tuberous sclerosis complex or Marfan syndrome. Our error correction strategy could offer a versatile solution for accurate multiplex PCR amplification.

Sensitive and Direct DNA Mutation Detection by Surface-Enhanced Raman Spectroscopy Using Rational Designed and Tunable Plasmonic Nanostructures.

Efficient DNA mutation detection methods are required for diagnosis, personalized therapy development, and prognosis assessment for diseases such as cancer. To address this issue, we proposed a straightforward approach by combining active plasmonic nanostructures, surface-enhanced Raman spectroscopy (SERS), and polymerase chain reaction (PCR) with a statistical tool to identify and classify BRAF wild type (WT) and V600E mutant genes. The nanostructures provide enhanced sensitivity, while PCR offers high specificity toward target DNA. A series of positively charged plasmonic nanostructures including gold/silver nanospheres, nanoshells, nanoflowers, and nanostars were synthesized with a one-pot strategy and characterized. By changing the shape of nanostructures, we are able to vary the surface plasmon resonance from 551 to 693 nm. The gold/silver nanostar showed the highest SERS activity, which was employed for DNA mutation detection. We reproducibly analyzed as few as 100 copies of target DNA sequences using gold/silver nanostars, thus demonstrating the high sensitivity of the direct SERS detection. By means of statistical analysis (principal component analysis-linear discriminant analysis), this method was successfully applied to differentiate the WT and V600E mutant both from whole genome DNA lysed from cell line and from cell-free DNA collected from cell culture media. We further proved that this assay is capable of specifically amplifying and accurately classifying a real plasma sample. Thus, this direct SERS strategy combined with the active plasmonic nanostructures has the potential for wide applications as an alternative tool for sensitively monitoring and evaluating important clinical nucleotide biomarkers.

Single-molecule detection of cancer mutations using a novel PCR-LDR-qPCR assay.

Detection of low-abundance mutations in cell-free DNA is being used to identify early cancer and early cancer recurrence. Here, we report a new PCR-LDR-qPCR assay capable of detecting point mutations at a single-molecule resolution in the presence of an excess of wild-type DNA. Major features of the assay include selective amplification and detection of mutant DNA employing multiple nested primer-binding regions as well as wild-type sequence blocking oligonucleotides, prevention of carryover contamination, spatial sample dilution, and detection of multiple mutations in the same position. Our method was tested to interrogate the following common cancer somatic mutations: BRAF:c.1799T>A (p.Val600Glu), TP53:c.743G>A (p.Arg248Gln), KRAS:c.35G>C (p.Gly12Ala), KRAS:c.35G>T (p.Gly12Val), KRAS:c.35G>A (p.Gly12Asp), KRAS:c.34G>T (p.Gly12Cys), and KRAS:c.34G>A (p.Gly12Ser). The single-well version of the assay detected 2-5 copies of these mutations, when diluted with 10,000 genome equivalents (GE) of wild-type human genomic DNA (hgDNA) from buffy coat. A 12-well (pixel) version of the assay was capable of single-molecule detection of the aforementioned mutations at TP53, BRAF, and KRAS (specifically p.Gly12Val and p.Gly12Cys), mixed with 1,000-2,250 GE of wild-type hgDNA from plasma or buffy coat. The assay described herein is highly sensitive, specific, and robust, and potentially useful in liquid biopsies.

Novel Mutation Hotspots within Non-Coding Regulatory Regions of the Chronic Lymphocytic Leukemia Genome

Mutations in non-coding DNA regions are increasingly recognized as cancer drivers. These mutations can modify gene expression in cis or by inducing high-order chormatin structure modifications with long-range effects. Previous analysis reported the detection of recurrent and functional non-coding DNA mutations in the chronic lymphocytic leukemia (CLL) genome, such as those in the 3′ untranslated region of NOTCH1 and in the PAX5 super-enhancer. In this report, we used whole genome sequencing data produced by the International Cancer Genome Consortium in order to analyze regions with previously reported regulatory activity. This approach enabled the identification of numerous recurrently mutated regions that were frequently positioned in the proximity of genes involved in immune and oncogenic pathways. By correlating these mutations with expression of their nearest genes, we detected significant transcriptional changes in genes such as PHF2 and S1PR2. More research is needed to clarify the function of these mutations in CLL, particularly those found in intergenic regions.

An ultrasensitive colorimetric test for the detection of somatic rare mutations in DNA.

Targeted therapies for cutaneous melanoma, such as those based on specific BRAF inhibitors, have improved the treatment and enhanced the survival rate of patients who harbor the V600E point mutation in the BRAF gene. However, tissue biopsies to characterize BRAF mutation status are prone to sampling bias, due to the intrinsic heterogeneity of a tumor mass. In contrast, blood biopsies, which analyze circulating tumor DNA (ctDNA), offer the most complete and sensitive characterization of the mutation status of a tumor, provide early and more accurate diagnosis, but they require instrumental and costly molecular tests. Therefore, the development of low-cost but highly sensitive tests for the non-invasive identification of BRAFV600E mutation in ctDNA would be of great clinical utility as a routine screening for the early identification of responsive patients and the follow-up of targeted therapy's response. The present work developed a naked-eye, inexpensive, yet very specific colorimetric assay, whose sensitivity is suitable for the detection of BRAFV600E rare mutation in ctDNA. Such test potentially may detect at an early stage the mutation in the tumor mass, when the first mutated cells appear in the blood, by using minimal instrumentation and thus enabling its widespread implementation in the clinics, even in local, minimally equipped laboratories. Indeed, the test detects 0.5% of BRAFV600E in an excess of BRAFWT DNA, which matches the sensitivity of some commercial instrumental assays. Such sensitivity is thus clinically relevant for diagnostic purposes, allowing the early identification of drug-sensitive patients.

Development and Analytical Validation of an Expanded Mutation Detection Panel for Next-Generation Sequencing of Thyroid Nodule Aspirates

Molecular analysis is used to evaluate the risk of malignancy for thyroid fine-needle aspirates, identified as indeterminate by microscopic cytology, on the basis of the detection of various oncogenic DNA mutations and fusion transcripts or on the use of various mRNAs or miRNA-based classifier algorithms. Our approach has been to use a combination test using the detection of oncogenic mutations/fusion transcripts and an miRNA expression-based classifier algorithm. To improve the performance of the combination test, the next-generation sequencing (NGS)-based mutational panel was expanded from the detection of 5 oncogenes to 10 oncogenes and tumor suppressor genes and the detection of fusion transcripts was increased from 6 to 38. Herein, we describe the assay development of the expanded panel NGS test and optimization of various steps for the library preparation of multiplexed target genes to maintain quality parameters for sequencing and to improve the robustness of the test for use in clinical testing in a College of American Pathologists/Clinical Laboratory Improvements Amendments-certified laboratory. Technical hurdles in NGS library preparation for the sequencing of both normal and high guanine-cytosine-rich regions, and balanced amplification of various amplicons in highly multiplexed PCRs, were successfully overcome. Analytical validation as a laboratory-developed test (ThyGeNEXT) included the demonstration of assay reproducibility, lower limit of detection, as well as other fundamental quality parameters.

Detection and discrimination of single nucleotide polymorphisms by exploiting metal ion-mediated DNA duplex

Detection of mutant DNA is necessary for early diagnosis of disease. In particular, detection of single nucleotide polymorphisms (SNPs) has become an important method for monitoring cancer and obtaining theragnostic information about it. SNPs are very difficult to detect selectively because of their similarity with wildtype DNA. In this paper, the specific binding between metal ion and DNA mismatch was used to achieve highly sensitive and selective SNP detection. Cytosine-cytosine (C-C) mismatched nucleobases capture silver (Ag) ions, and thymine-thymine (T-T) mismatched nucleobases capture mercury (Hg) ions. Metal ions bound between DNA could be reduced using a reducing agent (hydroquinone). In addition, gold (Au) amalgam could be formed around the DNA mismatch by the reduction of Au ion and mercury ion bound between T-T mismatches. Differential pulse voltammetry (DPV) was used to measure signals which are mediated by silver and Au amalgam. The detection limit of point mutation DNA obtained by the proposed detection method is 20 pM. In addition, by comparing the values of the DPV peaks by silver and Au amalgam, the types of DNA point mutations including cytosine or thymine (CT, CC, TT, AC, and TG) could be distinguished. The results obtained from our proposed method suggest its potential for diagnosing cancer patients.

Detection of KRAS mutations in plasma cell-free DNA of colorectal cancer patients and comparison with cancer panel data for tissue samples of the same cancers.

Robust identification of genetic alterations is important for the diagnosis and subsequent treatment of tumors. Screening for genetic alterations using tumor tissue samples may lead to biased interpretations because of the heterogeneous nature of the tumor mass. Liquid biopsy has been suggested as an attractive tool for the non-invasive follow-up of cancer treatment outcomes. In this study, we aimed to verify whether the mutations identified in primary tumor tissue samples could be consistently detected in plasma cell–free DNA (cfDNA) by digital polymerase chain reaction (dPCR). We first examined the genetic alteration profiles of three colorectal cancer (CRC) tissue samples by targeted next-generation sequencing (NGS) and identified 11 non-silent amino acid changes across six cancer-related genes (APC, KRAS, TP53, TERT, ARIDIA, and BRCA1). All three samples had KRAS mutations (G12V, G12C, and G13D), which were well-known driver events. Therefore, we examined the KRAS mutations by dPCR. When we examined the three KRAS mutations by dPCR using tumor tissue samples, all of them were consistently detected and the variant allele frequencies (VAFs) of the mutations were almost identical between targeted NGS and dPCR. When we examined the KRAS mutations using the plasma cfDNA of the three CRC patients by dPCR, all three mutations were consistently identified. However, the VAFs were lower (range, 0.166% to 2.638%) than those obtained using the CRC tissue samples. In conclusion, we confirmed that the KRAS mutations identified from CRC tumor tissue samples were consistently detected in the plasma cfDNA of the three CRC patients by dPCR.

PATH-06. A TARGETED NGS-BASED OVERALL SOLUTION TO SUPPORT DIAGNOSTICS AND THERAPY PREDICTION IN NEUROONCOLOGY: CONSIDERATIONS FOR A FLEXIBLE AND COST-EFFICIENT PLATFORM CHOICE AND PANEL DESIGN

Abstract Continuous updates add novel molecular markers to the WHO classification of CNS tumors. Additionally, molecularly informed trials may provide the basis for the introduction of additional predictive biomarkers. All these developments will prospectively require molecular testing to a yet unknown extent and still pose challenges to many neuropathology labs. Some of the relevant alterations (e.g. C11orf95-RELA) are hard to assess or complex subclassifications (e.g. for medulloblastomas) are required. Some labs rely on a two-tiered approach: i) methylation arrays for tumor classification and ii) targeted NGS panel sequencing for identifying actionable mutations. However, high initial investment and annual costs for running two platforms in parallel hinder a fast expansion of the new techniques in the breadth of neuropathology. We decided to focus on the sole use of the relatively cost-efficient Mini-Seq NGS platform (Illumina). First, we designed a DNA panel (FFPE, HaloPlex, Agilent; no Covaris required) that is suited for detection of DNA mutations, copy number alterations and chromosomal alterations (459 kbp, 58 genes, 4082 SNPs, 98.83% coverage). We successfully used this platform to support tumor classification in astrocytomas, oligodendrogliomas, meningiomas and medulloblastomas and identified actionable targets of clinical use. We then designed a RNA panel (FFPE, Sure Select, Agilent) that detects gene fusions and covers a broad range of alterations particularly relevant to pediatric tumors (e.g. pediatric glioblastomas, ependymomas, CNS PNETs) (148 kbp, coding sequence of 31 genes, 99.88% coverage). With these two panels we cover the most relevant molecular alterations in the field of neurooncology. The cost-efficient and flexible single platform approach enables state-of-the-art molecular diagnostics in virtually every neuropathology lab in a quality-controlled manner. Functionality is guaranteed for future revisions of the WHO classification as novel biomarkers can be easily included in an adapted panel design.

P2.03-42 Highly Sensitive Sel-CapTM Lung Cancer Panel NGS for Detection of Tumor-Derived EGFR Mutations in Plasma Cell-Free DNA in Lung Cancer

Next-generation sequencing is a genetic analysis platform for multiple analysis and large-scale analysis. Sensitivity is limited by methods for detecting cancer mutations that are contained at low rates in blood. With two highly sensitive detection techniques for genetic alterations, we evaluated their diagnostic value for tumor-derived EGFR mutations in plasma cell-free DNA (cfDNA) and measured concordance rate of activating EGFR mutations between cfDNA and tumor tissue. We conducted this study using FFPE tissue and plasma samples which were donated from NSCLC patients (N=316) between 2011 and 2013 and stored in the Human-derived Biomaterials Bank in Seoul St. Mary’s Hospital. Meanwhile, we prospectively collected plasma samples simultaneously when tumor tissue was taken out by surgery. We also collected peripheral blood just before administration of EGFR TKIs and each time imaging study was done during the administration of EGFR TKIs. We analyzed activating EGFR mutations in both cfDNA and tumor tissues using PNAclamping, Cancer panel and Sel-CapTM Lung Cancer Panel. The sensitivity and specificity of three detection techniques, PNAclamp, cancer panel and Sel-CapTM Lung Cancer Panel NGS for EGFR mutations in tumor tissues were more than 90% in all. In particular, EGFR T790M was found not only in the patients acquiring drug resistance approximately 10-12 months following the administration of 1st or 2nd generation EGFR TKIs, but also in the patients in whom EGFR TKI was continuously maintained over 30 months. The sensitivity of Sel-CapTM Lung Cancer Panel NGS for EGFR exon19 deletion and exon21 L858R in cfDNA was 76.2% and 61.1%, respectively. In addition, the sensitivity for double mutation of 19del and T790M was about 30% higher compared to that of cancer panel sold domestically (2/20, 10.0% vs. 9/20, 45.0%), suggesting that Sel-CapTM Lung Cancer Panel NGS enables to detect EGFR mutation up to 0.1% in plasma cfDNA. As a result of tracking of outcomes of EGFR TKIs treatment over time for patients with low frequency of EGFR mutations, we found that 1st or 2nd generation EGFR TKIs were switched into 3rd generation EGFR TKIs on the basis of disease progression on radiologic imaging, when an average of 6.8 months (0.2-25.4 months) elapsed from the first detection time of T790M in cfDNA. The presence of T790M in peripheral blood harvested periodically from patients receiving EGFR TKIs was initially identified about 3-5 months ahead of disease progress assessed by imaging study. With regard to the detection of EGFR mutations in lung cancer patients, Sel-CapTM Lung Cancer Panel NGS platform showed superior sensitivity to conventional NGS-based cancer panels as well as higher concordance rate between tumor tissues and plasma cfDNA. In longitudinal plasma cfDNA samples isolated from periodic liquid biopsies, quantitative changes of EGFR mutation were found to be significantly correlated to tumor response by EGFR TKIs. Our data suggest that Sel-CapTM Lung Cancer Panel NGS is a highly sensitive diagnostic platform for detection of EGFR T790M induced by long-term administration of EGFR TKIs and for determining when 3rd generation EGFR TKI be begun.

Point mutation in the TGFBI gene: surface-enhanced infrared absorption spectroscopy (SEIRAS) as an analytical method

A novel method to detect DNA mutations based on gold nanoparticles is described. This bioconjugate was prepared by conjugation of the TGFBI gene on the surface of gold nanoparticles. The surface plasmon resonance band observed in the ultraviolet–visible spectrum of the gold nanoparticles showed a shift to a lower energy after the surface was covered with the TGFBI gene. Surface-enhanced infrared spectroscopy (SEIRAS) showed the absorption bands of the bioconjugate due to their proximity to the nanoparticles. The SEIRAS effect was able to detect small and specific changes in the sequence of the TGFBI gene using interactions of the bioconjugates. The study was performed on patients clinically diagnosed with lattice corneal dystrophy (LCD). To our knowledge, this is the first report of FTIR spectroscopy employed to analyse this gene. We used genotyping results previously obtained by next-generation DNA sequencing technology to ensure that the analysed samples had the mutation. Our results show highly reproducible signals, which could be used for analytical studies of mutations in the genome.

Highly sensitive and selective detection of single-nucleotide polymorphisms using gold nanoparticle MutS enzymes and a micro cantilever resonator

Mutant DNAs are important markers useful for the diagnosis of human disease. Single-nucleotide polymorphisms (SNPs) represent the most common types of DNA mutations. As there is only a one base pair change in a single nucleotide between the SNP and the wild-type DNA, it is difficult to distinguish the SNPs. In this report, a highly sensitive and selective detection and discrimination of SNPs is performed using MutS, gold nanoparticles (AuNP) and a resonator. A single mismatched base exists between the SNP mutation and the probe DNA on the resonator, and MutS binds to the DNA at the location of the mismatch. As MutS is attached to AuNP (MutS-AuNP), both MutS and AuNP are adsorbed onto the resonator. The detection is based on the resonance frequency shift of the resonator following the adsorption of MutS-AuNP on the resonator. Highly sensitive detection of DNA mutations was achieved using AuNPs that act as mass amplifiers, and the obtained limit of detected was 100 fM. Additionally, our proposed method detected mutations in the presence of as little as 0.1% wild-type, and discrimination of specific mutations was also achieved. The results obtained from our proposed method suggest its potential for diagnosing cancer patients.

Abstract 2437: Microfluidic device integrated with lipid nanoprobe for extracellular vesicle isolation toward non-invasive cancer diagnosis

Abstract Extracellular vesicles (EVs) can mediate intercellular communication by transferring cargo proteins and nucleic acids between cells. Recently, they have been demonstrated for potential value in cancer diagnostics and treatment monitoring. However, relevant clinical translation is mainly limited by technical challenges in EV isolation. We previously reported lipid nanoprobes, which is a nanomaterial system of labeling probe and capture probe. The labeling probe is constructed with a lipid molecule as the active component to label EVs rapidly and spontaneously with high efficiency, while the capture probe can isolate labeled EVs magnetically. Herein, we report on the development of a microfluidic device with integrated lipid nanoprobe for on-chip EV isolation towards instrument development for clinical translation. The nanostructured substrate of the device is integrated with a micromixer for one-step isolation of EVs from 1-ml plasma. We validated the device with plasma collected from pancreatic cancer patients, followed by DNA mutation detection of EV cargo. Size-tunable nanostructures are fabricated by patterning high-resolution iron thin films on fused silica substrates. By judicious thermal annealing, homogeneously dispersed iron nanoparticles are formed by fission and fusion on the substrate, which are served as a mask for silica dry-etching. Subsequently, selected lipid nanoprobes, which can instantaneously insert into lipid bilayers of EVs, are grafted onto the nanostructured substrate in a micromixer. This platform is verified first to isolate cultured cancer cell-derived EVs spiked-in plasma samples that emulated clinical samples. This platform is further applied to isolate EVs from pancreatic cancer patients’ plasma and identified KRASmutations in EV DNA with highly sensitive droplet digital PCR. Together, this platform enables rapid and efficient isolation of EVs from plasma specimens, and thus, holds great potential in clinical translation. Citation Format: Siyang Zheng, Yuan Wan, Mackenzie Maurer, Yi-Qiu Xia, Si-Jie Hao, Wen-Long Zhang, Nelson S. Yee. Microfluidic device integrated with lipid nanoprobe for extracellular vesicle isolation toward non-invasive cancer diagnosis [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 2437.

Electrochemical detection of DNA mismatches using a branch-shaped hierarchical SWNT-DNA nano-hybrid bioelectrode.

Common approaches for DNA mutation detection are high cost and have difficult or complex procedure. We propose a fast quantitative method for recognition of DNA mutation based on SWNT/DNA self-assembled nanostructure. Covalent SWNT/DNA hybrid nanostructures are widely used in the fabrication of electrochemical biosensors. Interfacing carbon nanotubes with DNA in particular, is used as a detection method for the analysis of genetic disorders or the detection of mismatches in DNA hybridisation. We have designed a self-assembled, branch-shaped hybrid nanostructure by hybridisation of two sticky oligos that are attached to the ends of SWNTs via a linker oligo. These hybrid nanostructures showed a good conductivity that was greater than free SWNTs. Impedance spectroscopy studies illustrated that the conductivity of these hybrid nanostructures depended on the conformation and structure of the hybridised DNA. We demonstrated that the strategy of using SWNT/DNA self-assembled hybrid nanostructure fabrication yields sensitive and selective tools to discriminate mismatches in DNA. Cyclic voltammetry (CV) and impedance spectroscopy clearly revealed that the conductivity of the branch-shaped and hierarchical hybridised SWNT/DNA nanostructure is higher when matched, than when mismatched in a 1 and 1' hybridised SWNT/DNA nanostructure. Rapid biosensing of match and mismatch nanostructure based on carbon printed electrode showed similar results which can be used for rapid and fast detection of DNA mismatch.

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available inpatient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Analysis of mutation detection in cell-free DNA in ascites using comprehensive NGS panel.

e13029 Background: Ascites is one of complications for patients with malignancies in advanced stage. Detection and characterization of cell-free DNA (cfDNA) from ascites effusions have not yet been studied. Herein, we analysis genomic alterations present in advanced cancer with ascites. Methods: We implemented targeted capture NGS with a panel of 1021 cancer-relevant genes on 85 ascites ctDNA, also on 46 matched plasma and 9 tissue from 85 patients. Results: Among 85 patients with advanced cancer, 42 digest system cancer (DC), 16 gynecologic cancer (GC), 14 non-small cell lung cancer(NSCLC) and 13 other solid tumors included. Overall, 90%(77/85) patients detected somatic mutations, which were identified predominantly in TP53 and KRAS for DC and TP53 for GC. Among NSCLC patients with ascites, nine were treated with EGFR TKI-targeted therapy previously. Mutations lead to the resistance of EGFR-TKI were identified in 55.6% (5/9) cases, including acquired EGFR T790M/C797S and primary BRAF V600E. These indicate ctDNA in ascites can used to guide tumor treatment and monitor of resistance. For the 46 pairs of ascites and plasma samples, actionable variations with on label, off-label, or experimental drugs available, were identified in 61% (n = 28) of ascites effusion samples, whereas plasma was 46% (n = 21). Median number of ascites somatic mutations was 4, while the median number was 3 in plasma samples. Of that, 85% and 61% of plasma ctDNA and ascites cfDNA samples were observed with the highest MAF above 1%. In addition, 63% and 35% of plasma ctDNA and pleural cfDNA samples were observed with the highest MAF above 10%. For the 9 groups of plasma, ascites and tumor tissue samples, at least 1 mutations within tissue was detected in 8 ascites samples and 6 in plasma samples. Conclusions: Our results suggest that cfDNA from ascites is more sensitive than plasma and had higher similarity with the tumor tissues for advanced cancer patients. With higher MAF and detection rate of actionable variations, cfDNA from ascites might provide additional information not detected with plasma cell-free DNA guide more precise cancer treatment.

Tumor volumetric correlation with plasma cell free DNA (cfDNA) mutation detection in metastatic lung cancers.

e14610 Background: In patients with metastatic lung cancers, analysis of cfDNA is increasingly used to detect oncogenes, frequently allowing matched targeted therapy. Mutation detection in cfDNA may be influenced by several factors, such as disease burden. We hypothesized that volumetrics, as routinely analyzed during radiation simulation, correlates with mutation detection in cfDNA. Methods: Patients underwent cfDNA analysis between 10/2016 and 1/2018. Those with metastatic NSCLC, adequate imaging for volumetric analysis (PET/CT and MRI brain), and who were treatment naïve at plasma draw were included. Plasma underwent a 21-gene next generation sequencing assay (Resolution Bioscience). A single observer segmented tumor volumes. Volume of disease was correlated with mutation detection in cfDNA for all 21 genes including the 8 NCCN oncogenes (EGFR, ALK, ROS1, BRAF, KRAS, HER2, RET, MET) with univariate Mann-Whitney. Results: Of the 210 pts with plasma, 52 met inclusion criteria. There were 22 males (42%), median age 67yo (IQR 59-74), 24 former smokers (median pack-years: 25), 44 adenocarcinomas (85%), 21 matched to targeted therapy based on cfDNA results (40%), and median cfDNA extracted concentration was 1.56 ng/mL (IQR 1.1 – 2.6 ng/mL). Involved sites included lymph nodes (n = 43), bone (n = 25), brain (n = 15), liver (n = 13), and adrenals (n = 12). Median total tumor volume was 67.0 mL (IQR 36.6-184.6 mL). When evaluating all 21 genes, cfDNA was mutation positive in 40 pts and negative in 12 pts. Median tumor volume was 77.6 mL and 40.4 mL for positive and negative, respectively (p = 0.12). For the 8 NCCN oncogenes, cfDNA was positive in 18 pts and negative in 34 pts. Median tumor volume was 105.3 mL and 67.0 mL for positive and negative, respectively (p = 0.40). Conclusions: Volumetric analysis showed there was a non-significant trend toward larger tumor volume among pts with positive cfDNA mutation detection. As the use of cfDNA continues to expand into earlier disease stage settings, volumetrics at diagnosis may become an increasingly important tool to help predict cfDNA mutation detection to guide targeted therapy. Further studies of volumetrics and cfDNA analysis are warranted.

Detection of KRAS mutation via ligation-initiated LAMP reaction

KRAS mutations are abnormalities widely found in genomic DNA and circulating tumor DNA (ctDNA) of various types of cancers. Thus, highly sensitive detection of KRAS mutations in genomic DNA is of great significance in disease diagnosis and personalized medicine. Here, we developed a ligation-initiated loop-mediated isothermal amplification (LAMP) assaying method for ultrasensitive detection of KRAS mutation. In the presence of mutant KRAS DNA (mutDNA), the dumbbell-shaped structure (DSS) is formed by the specific ligation of two substrates (SLS1 and SLS2), which act as a template to initiate the following LAMP amplification. Making use of the outstanding specificity of ligation reaction and superior amplification of LAMP, 10 aM mutDNA can be accurately determined. In addition, as low as 0.1% mutDNA can be detected in the presence of a large excess of wild-type KRAS DNA (wtDNA), indicating the high sensitivity and specificity of the method. Furthermore, this strategy has been successfully applied for detection of a KRAS mutation from tissue samples of colorectal cancer patients. Thus, the developed ligation-initiated LAMP fluorescence assaying strategy presents a promising prospect for ultrasensitive detection of mutations.