Liquid biopsy for colorectal cancer – from cfDNA extraction to the quantification of point mutations… - SLUB Dresden

Media type: E-Book

Title: Liquid biopsy for colorectal cancer – from cfDNA extraction to the quantification of point mutations by multiplexed ddPCR

Contributor: Schlenker, Franziska [Author]; Zengerle, Roland [Degree supervisor]; Zengerle, Roland [Other]; Eckert, Cornelia [Other]

Corporation: Hahn-Schickard-Institut für Mikroanalysesysteme ; Albert-Ludwigs-Universität Freiburg, Professur für Anwendungsentwicklung ; Albert-Ludwigs-Universität Freiburg, Fakultät für Angewandte Wissenschaften

Published: Freiburg: Universität, 2022

Extent: Online-Ressource

Language: English

DOI: 10.6094/UNIFR/228995

Identifier:

Keywords: Biopsy ; (local)doctoralThesis

Origination:

University thesis: Dissertation, Universität Freiburg, 2022

Footnote:

Description: Abstract: Novel, easily accessible biomarkers in body fluids with the so-called liquid biopsy have come into focus in cancer research. This is especially the case for circulating tumor DNA in patients’ blood streams. The simplicity of a blood sampling makes continuous monitoring of tumor status possible. The surveillance of the patient’s mutation pattern during treatment is essential to select the most effective therapy. This work aims to demonstrate the use of centrifugal microfluidics for colorectal cancer monitoring by liquid biopsy. For this, the complete workflow from circulating cell-free DNA (cfDNA) extraction to the quantification of point mutations by multiplexed digital droplet PCR (ddPCR) is demonstrated. Today’s standard methods for cfDNA extraction in a clinical environment are automated pipetting robots. However, the high purchasing costs, the precious laboratory space and lack of experts are a major problem, especially in smaller hospitals. This work shows a concept for an automated cfDNA extraction workflow in a monolithically integrated cartridge with pre-stored reagents in a small benchtop device. For the integration of the current gold standard assays for cfDNA extraction, it had to be transferred from a spin-column based workflow to a bead-based approach. Here, the most crucial influencing factors such as bead type and its volume, binding time, ethanol concentration during washing, drying after washing to remove remaining ethanol and cooling before DNA binding were all optimized in a way, that the bead-based approach yields the same results as the workflow with the spin-column. This means, that the bead-based approach in the manual workflow shows extraction recoveries close to the one from the spin-columns with approx. 100% recovery efficiency compared to the control. Furthermore, the integration of the manual bead-based cfDNA extraction assay into the LabDisk was successfully shown. Independent of the DNA concentration (104-102 DNA copies/ml plasma) spiked with synthetic mutant DNA into the plasma sample the achievable recovery for the LabDisk had an average of 95% (range 83% - 108%). The LabDisk showed the advantage of the automation with a higher reproducibility in DNA recovery rates shown in a lower standard deviation (±13%) compared to the manual approaches with ±17% (spin-column) and ±26% (beads). Comparison of automated cfDNA extraction on the LabDisk with the QIAsymphony shows very comparable results for the patient plasma samples while at the same time reducing technical complexity. Consequently it is shown that the LabDisk is a highly attractive alternative to huge automation robots such as the QIAsymphony or to a tedious manual workflow in clinical routine workflow for liquid biopsy. cfDNA quantification in a liquid biopsy context is most commonly performed by assessing comprehensive target panels to monitor the tumor burden of a cancer patient. In this work, optimization-free multiplex assays for ddPCR, with the use of the mediator probe (MP) technology are established for the seven most frequent point mutations in colorectal carcinoma (KRAS and BRAF). The limit of detection for the developed 2-plex and 4-plex MP ddPCR was measured with spiking experiments of synthetic mutant DNA in human genomic DNA. During therapy monitoring, the allele frequency which is the parameter to be determined. The MP ddPCR assays showed a limit of detection of 0.004% to 0.38% for the multiplex MP ddPCR assay with a R² of 0.98%. The limit of blank was determined to be 0 DNA copies/ml plasma for the 2-plex MP ddPCR assays whereas it was increased for KRAS G13D to be 9.82 DNA copies/ml plasma and for BRAF V600E to be 16.29 DNA copies/ml plasma in the 4-plex MP ddPCR assay. However, by the usage of patient plasma eluates, both MP ddPCR assay concepts (2-plex and 4-plex) were able to assign 100% of the tested patient samples correct positive or negative. Furthermore, they showed comparable performance to the reference locked nucleic acid assay from the university hospital Freiburg. The presented MP ddPCR assay needs no time consuming assay optimization as all presented assays uses the same concentrations for the PCR reagents and the same cycling conditions independent of the target to be analyzed. However, a high degree of multiplexing is essential in cancer diagnostics to monitor for example clonal evolution during treatment. Therefore, this work shows an increased degree of multiplexing by the usage of virtual fluorescence color channels by introducing a photobleaching-resistant and photobleaching-sensitive assay concept for the quantification of point mutations. This combination leads to the doubling of the multiplexing degree for each channel. The photobleaching effect, requires an additional readout step and a low-cost light-emitting diode (LED). This concept can increase the degree of multiplexing of the used ddPCR reader from three standard channels to six channels by the introduction of three additional virtual channels by photobleaching of the fluorophores. The usability of this concept for liquid biopsy was demonstrated using the two cancer-associated point mutations KRAS G12D and G12V. With a serial dilution experiment in the red channel, the required sensitivity of assays used for liquid biopsy the limit of detection for KRAS G12D was determined to 16 DNA copies/reaction (standard channel) and for KRAS G12V to nine DNA copies/reaction (virtual channel). As a final topic, the developed 4-plex MP ddPCR assay is integrated into a centrifugal microfluidic ddPCR cartridge. The latter is able to generate droplets with a diameter of approx. 82.7 µm in each of the 12 identical ddPCR unit. The oil-surfactant concentration and the oil-combination were determined to be the most crucial influencing parameter for a robust droplet generation and thermostable droplets during PCR. This ddPCR disk featured the usage of a standard fluorescence microscope for the evaluation of the fluorescence data from the ddPCR units. The integration of one of three 4-plex panels was demonstrated with KRAS wild type and the cancer-associated point mutations KRAS G12A, G12D and G12V. The limit of detection experiment showed the diagnostically relevant sensitivity with 3.5 mutant DNA copies in a background of 15,000 DNA copies from the wild type with a R² of 0.99. With both the aforementioned cfDNA extraction the ddPCR assay for cfDNA quantification integrated into the LabDisk in this work, the entire liquid biopsy workflow could be automated for the first time using centrifugal microfluidics. Comparative experiments showed excellent performance of the disks and the assays compared to gold standard methods demonstrating the feasibility and great potential of this technology for future introduction into routine clinical practice

Access State: Open Access

Rights information: In Copyright

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