Liquid biopsy

A liquid biopsy, also known as fluid biopsy or fluid-phase biopsy, is the sampling and analysis of non-solid biological tissue, primarily blood. It is mainly used as a diagnostic and monitoring tool for diseases such as cancer. Liquid biopsy is less invasive for the donor compared to tissue biopsy. When tumor cells die, they release ctDNA into the blood. Cancer mutations in ctDNA mirror those found in traditional tumor biopsies, allowing them to be used as molecular biomarkers to track the disease. The challenge is the low concentration of ctDNA from the tumor cells in the blood. The gold standard has been to use NGS, pyrosequencing or real-time qPCR, but the drawback of these methods has been their limitations in LOD. Pyrosequencing for tumor tissue is about 10%, NGS is between 1–5%, and qPCR can detect down to 1%. This creates an issue for relapse during residual disease monitoring of the donor because of the limitation in detection levels.

Benefits of using nanoplate dPCR for liquid biopsy analysis

Load up to 28 µl sample to increase the LOD and minimize subsampling error with the QIAcuity Nanoplate 26K – allows you to generate more data points to secure small changes in expression or for residual disease monitoring
Detect ultra-rare mutations down to 0.01% variant allele frequency
Handle more crude samples like whole blood and urine, as dPCR measurement is unaffected by amplification efficiency

Liquid biopsy-based detection of PIK3CA mutations from cfDNA using dPCR

In this application note, we demonstrate the utility of the QIAcuity Digital PCR System to confidently detect ultra-rare PIK3CA variants in cfDNA. The manual QIAamp workflows and the automated EZ2 and QIAsymphony workflows delivered cfDNA with high yield and purity from large plasma volumes of up to 10 mL. The automated QIAGEN workflows also eliminated the need for manual pre-enrichment or preparation of plates. Additionally, we demonstrated the comparability of dPCR and Qubit quantifications and the ability of dPCR to quantify PIK3CA mutation frequencies in cfDNA with high precision, cost and time-efficiency.

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Related publications

Cardoso GC, Ganzella FAO, Miniskiskosky G, Cunha RS, Ramos EAS. Digital methylation-specific PCR: New applications for liquid biopsy. Biomol Concepts. 2024;15(1).

Crucitta S, et al. Comparison of digital PCR systems for the analysis of liquid biopsy samples of patients affected by lung and colorectal cancer. Clin Chim Acta. 2023;541:117239.

Ruas JS, et al. Somatic copy number alteration in circulating tumor DNA for monitoring of pediatric patients with cancer. Biomedicines. 2023;11(4):1082.

Csoma SL, et al. Correlation analyses between histological staging and molecular alterations in tumor-derived and cell-free DNA of early-stage primary cutaneous melanoma. Cancers. 2023;15(21):5141.

Rosing F et al. Quantification of Human Papillomavirus cell-free DNA from low volume blood plasma samples by digital PCR. medRxiv. 2023.DOI:10.1101/2023.09.28.23296267.

Kilian AL, Lollies A, Weniger MA, Küppers R. P035: CRISPR/Cas9-mediated knockout reveals an important role of CD30 in Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma. Hemasphere. 2022;6:17.

Further resources

Kappmeier C et al. Advancing cancer research: A novel digital PCR tool for simultaneous detection of multiple hallmark mutations in BRAF and EGFR. QIAGEN, 2024.