Matthew Wiggin

Noise analysis and reduction in solid-state nanopores

The electrical noise characteristics of ionic current through organic and synthetic nanopores have been investigated. Comparison to proteinaceous alpha-Hemolysin pores reveals two dominant noise sources in silicon nitride nanometre-scale pores: a high-frequency noise associated with the capacitance of the silicon support chip (dielectric noise), and a low-frequency current fluctuation with 1/ f α characteristics (flicker noise). We present a technique for reducing the dielectric noise by curing polydimethylsiloxane (PDMS) on the nanopore support chip. This greatly improves the performance of solid-state nanopore devices, yielding an unprecedented signal-to-noise ratio when observing dsDNA translocation events and ssDNA probe capture for force spectroscopy applications. (Some figures in this article are in colour only in the electronic version)

Single-molecule bonds characterized by solid-state nanopore force spectroscopy.

Weak molecular interactions drive processes at the core of living systems, such as enzyme-substrate interactions, receptor-ligand binding, and nucleic acid replication. Single-molecule force spectroscopy is a remarkable tool for revealing molecular scale energy landscapes of noncovalent bonds, by exerting a mechanical force directly on an individual molecular complex and tracking its survival as a function of time and applied force. In principle, force spectroscopy methods can also be used for highly specific molecular recognition assays, by directly characterizing the strength of bonds between probe and target molecules. However, complexity and low throughput of conventional force spectroscopy techniques render such biosensing applications impractical. Here we demonstrate a straightforward single-molecule approach, suitable for both biophysical studies and molecular recognition assays, in which a approximately 3 nm silicon nitride nanopore is used to determine the bond lifetime spectrum of the biotin-neutravidin complex. Thousands of individual molecular complexes are captured and dissociated in the solid-state nanopore under constant applied forces, ranging from 400 to 900 mV, allowing us to extract the location of the energy barrier that governs the interaction, mapped at Deltax approximately 0.5 nm. These results highlight the capacity of a solid-state nanopore to detect and characterize intermolecular interactions and demonstrate how this could be applied to rapid, highly specific molecular detection assays.

Nonexponential Kinetics of DNA Escape from α-Hemolysin Nanopores ☆

Throughput and resolution of DNA sequence detection technologies employing nanometer scale pores hinge on accurate kinetic descriptions of DNA motion in nanopores. We present the first detailed experimental study of DNA escape kinetics from alpha-hemolysin nanopores and show that anomalously long escape times for some events result in nonexponential kinetics. From the distribution of first-passage times, we determine that the energy barrier to escape follows a Poisson-like distribution, most likely due to stochastic weak binding events between the DNA and amino acid residues in the pore.

Enumeration and targeted analysis of KRAS , BRAF and PIK3CA mutations in CTCs captured by a label-free platform: Comparison to ctDNA and tissue in metastatic colorectal cancer

Treatment of advanced colorectal cancer (CRC) requires multimodal therapeutic approaches and need for monitoring tumor plasticity. Liquid biopsy biomarkers, including CTCs and ctDNA, hold promise for evaluating treatment response in real-time and guiding therapeutic modifications. From 15 patients with advanced CRC undergoing liver metastasectomy with curative intent, we collected 41 blood samples at different time points before and after surgery for CTC isolation and quantification using label-free Vortex technology. For mutational profiling, KRAS, BRAF, and PIK3CA hotspot mutations were analyzed in CTCs and ctDNA from 23 samples, nine matched liver metastases and three primary tumor samples. Mutational patterns were compared. 80% of patient blood samples were positive for CTCs, using a healthy baseline value as threshold (0.4 CTCs/mL), and 81.4% of captured cells were EpCAM+ CTCs. At least one mutation was detected in 78% of our blood samples. Among 23 matched CTC and ctDNA samples, we found a concordance of 78.2% for KRAS, 73.9% for BRAF and 91.3% for PIK3CA mutations. In several cases, CTCs exhibited a mutation that was not detected in ctDNA, and vice versa. Complementary assessment of both CTCs and ctDNA appears advantageous to assess dynamic tumor profiles.

Forming an α-Hemolysin Nanopore for Single-Molecule Analysis

Nanopore analysis of single molecules can be performed by measuring the modulation in ionic current passing through the nanopore while an individual biomolecule such as DNA or RNA is resident in, translocating through, or otherwise interacting with the pore. The corresponding current signature has been shown to reveal properties of the biomolecule and information on its interactions with the pore. The alpha-hemolysin nanopore remains the pore of choice, particularly for single-molecule analysis of nucleic acids, because of its internal dimensions, hydrophilicity, and low-noise characteristics. In this chapter we present a detailed protocol for forming a robust alpha-hemolysin nanopore (or multiple nanopores) for single-molecule analysis.

Somatic mutation detection using various targeted detection assays in paired samples of circulating tumor DNA, primary tumor and metastases from patients undergoing resection of colorectal liver metastases

Assessing circulating tumor DNA (ctDNA) is a promising method to evaluate somatic mutations from solid tumors in a minimally-invasive way. In a group of twelve metastatic colorectal cancer (mCRC) patients undergoing liver metastasectomy, from each patient DNA from cell-free DNA (cfDNA), the primary tumor, metastatic liver tissue, normal tumor-adjacent colon or liver tissue, and whole blood were obtained. Investigated was the feasibility of a targeted NGS approach to identify somatic mutations in ctDNA. This targeted NGS approach was also compared with NGS preceded by mutant allele enrichment using synchronous coefficient of drag alteration technology embodied in the OnTarget assay, and for selected mutations with digital PCR (dPCR). All tissue and cfDNA samples underwent IonPGM sequencing for a CRC-specific 21-gene panel, which was analyzed using a standard and a modified calling pipeline. In addition, cfDNA, whole blood and normal tissue DNA were analyzed with the OnTarget assay and with dPCR for specific mutations in cfDNA as detected in the corresponding primary and/or metastatic tumor tissue. NGS with modified calling was superior to standard calling and detected ctDNA in the cfDNA of 10 patients harboring mutations in APC, ATM, CREBBP, FBXW7, KRAS, KMT2D, PIK3CA and TP53. Using this approach, variant allele frequencies in plasma ranged predominantly from 1 to 10%, resulting in limited concordance between ctDNA and the primary tumor (39%) and the metastases (55%). Concordance between ctDNA and tissue markedly improved when ctDNA was evaluated for KRAS, PIK3CA and TP53 mutations by the OnTarget assay (80%) and digital PCR (93%). Additionally, using these techniques mutations were observed in tumor-adjacent tissue with normal morphology in the majority of patients, which were not observed in whole blood. In conclusion, in these mCRC patients with oligometastatic disease NGS on cfDNA was feasible, but had limited sensitivity to detect all somatic mutations present in tissue. Digital PCR and mutant allele enrichment before NGS appeared to be more sensitive to detect somatic mutations.Assessing circulating tumor DNA (ctDNA) is a promising method to evaluate somatic mutations from solid tumors in a minimally‐invasive way. In a group of twelve metastatic colorectal cancer (mCRC) patients undergoing liver metastasectomy, from each patient DNA from cell‐free DNA (cfDNA), the primary tumor, metastatic liver tissue, normal tumor‐adjacent colon or liver tissue, and whole blood were obtained. Investigated was the feasibility of a targeted NGS approach to identify somatic mutations in ctDNA. This targeted NGS approach was also compared with NGS preceded by mutant allele enrichment using synchronous coefficient of drag alteration technology embodied in the OnTarget assay, and for selected mutations with digital PCR (dPCR). All tissue and cfDNA samples underwent IonPGM sequencing for a CRC‐specific 21‐gene panel, which was analyzed using a standard and a modified calling pipeline. In addition, cfDNA, whole blood and normal tissue DNA were analyzed with the OnTarget assay and with dPCR for specific mutations in cfDNA as detected in the corresponding primary and/or metastatic tumor tissue. NGS with modified calling was superior to standard calling and detected ctDNA in the cfDNA of 10 patients harboring mutations in APC, ATM, CREBBP, FBXW7, KRAS, KMT2D, PIK3CA and TP53. Using this approach, variant allele frequencies in plasma ranged predominantly from 1 to 10%, resulting in limited concordance between ctDNA and the primary tumor (39%) and the metastases (55%). Concordance between ctDNA and tissue markedly improved when ctDNA was evaluated for KRAS, PIK3CA and TP53 mutations by the OnTarget assay (80%) and digital PCR (93%). Additionally, using these techniques mutations were observed in tumor‐adjacent tissue with normal morphology in the majority of patients, which were not observed in whole blood. In conclusion, in these mCRC patients with oligometastatic disease NGS on cfDNA was feasible, but had limited sensitivity to detect all somatic mutations present in tissue. Digital PCR and mutant allele enrichment before NGS appeared to be more sensitive to detect somatic mutations.

Nanopore Force Spectroscopy on DNA Duplexes

Force spectroscopy can be applied using nanopores to study charged molecules such as nucleic acids. This technique can be used to study the binding energy of a DNA duplex by threading an anchored single-stranded DNA (ssDNA) probe molecule through a nanopore (having a diameter large enough to accommodate only a single strand) and allowing target DNA on the backside of the pore to hybridize to the probe. Electric potential can be used to apply a force to the charged ssDNA in a direction tending to translocate the duplex through the pore. If the pore is only large enough to accept ssDNA, the duplex must dissociate for the probe to escape the pore. The dissociation time of the duplex can therefore be measured under applied force, and (provided that enough dissociation events have been recorded) a characteristic time scale for dissociation can be determined. In this chapter, we present a detailed protocol for performing nanopore force spectroscopy on DNA duplexes using one or more alpha-hemolysin nanopores. We present the details of the measurement of the duplex survival probability under force, and show that dissociation time scales for duplexes that are perfectly complimentary differ by greater than approximately two orders of magnitude from those containing a single sequence mismatch, offering opportunities for sequence detection.

Abstract 3149: Enumeration and mutational profiling of CTCs and comparison to ctDNA and colorectal cancer liver metastases

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Background Colorectal cancer (CRC) is the 3rd most common cancer diagnosed worldwide in both men and women. Only 39% of cancers are diagnosed at a localized stage, and 5-year survival rates decrease rapidly for patients with advanced and metastasized disease (stage III 61%, stage IV 8%). Better markers for detection of disease progression, therapeutic resistance and minimal residual disease are still needed. Liquid biopsies, such as CTCs and ctDNA, are emerging biomarkers shed by the tumor into the blood stream. Both markers currently are attracting growing interest for their use in disease prognosis, early detection of recurrence and are promising candidates for guiding cancer therapy in real-time. Method For rapid label-free isolation of CTCs from peripheral blood we used the Vortex technology, a microfluidic device using inertia and laminar microvortices. From 15 patients with metastatic CRC to the liver that underwent liver metastatectomy with curative intent, we collected CTCs preoperatively, at the 5th postoperative day and during follow-up visits. Cells collected were immunostained for EpCAM, CD45 and DAPI, enumerated using standardized classification criteria, and subjected to Sanger sequencing. CTC enumeration and mutational patterns were compared to the primary tumor, liver metastases and ctDNA (detected by a multiplexed PCR and enrichment technology; Kidess E et al., 2015) as well as CEA levels when available. Results 41 blood samples from 15 patients were collected at different time points prior to and after surgical resection of liver metastases. More CTCs were found in preoperatively collected CRC patient samples (2.4 CTCs/mL, 0.1 - 5.5/mL) than in age-matched healthy controls (0.1 CTCs/mL, 0 - 0.4/mL). 80% of all CRC samples were identified as positive for CTCs (based on a calculated threshold from healthy controls), with varying levels of EpCAM expression (81.4% of CTCs being EpCAM+). The number of CTCs for each patient, showed a close correlation to clinical parameters and ctDNA levels: detection of CTCs, CTC mutational profiles as well as ctDNA revealed minimal residual disease and anticipated tumor recurrence earlier than carcinoembryonic antigen (CEA) value or imaging. For example, for P006, postoperative imaging surveillance revealed progressive disease, which was accompanied by rising levels of CTCs (up to 29 CTCs/mL at the last time point) and PIK3CA mutant DNA in both plasma ctDNA and CTC DNA, while CEA remained in the normal range. Conclusion Our data illustrate that CTCs as well as ctDNA can efficiently reveal disease recurrence as well as disease progression earlier than imaging and far more reliable compared to CEA, the currently standard biomarker for CRC. Beyond enumeration, CTC molecular analysis gives additional information and will potentially help to promote the development of tailored therapies for every individual patient. Citation Format: Evelyn Kidess-Sigal, Haiyan E. Liu, Melanie Triboulet, James Che, Georges A. Poultsides, Brendan C. Visser, Andre Marziali, Marc Lee, Valentina Vysotskaia, Matthew Wiggin, Vishnu C. Ramani, Ulrich Keilholz, Ingeborg Tinhofer, Amin Zia, John Coller, Jeffrey A. Norton, Elodie Sollier, Stefanie S. Jeffrey. Enumeration and mutational profiling of CTCs and comparison to ctDNA and colorectal cancer liver metastases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3149.