Droplet digital PCR-based EGFR mutation detection with an internal quality control index to determine the quality of DNA

Abstract

In clinical translational research and molecular in vitro diagnostics, a major challenge in the detection of genetic mutations is overcoming artefactual results caused by the low-quality of formalin-fixed paraffin-embedded tissue (FFPET)-derived DNA (FFPET-DNA). Here, we propose the use of an ‘internal quality control (iQC) index’ as a criterion for judging the minimum quality of DNA for PCR-based analyses. In a pre-clinical study comparing the results from droplet digital PCR-based EGFR mutation test (ddEGFR test) and qPCR-based EGFR mutation test (cobas EGFR test), iQC index ≥ 0.5 (iQC copies ≥ 500, using 3.3 ng of FFPET-DNA [1,000 genome equivalents]) was established, indicating that more than half of the input DNA was amplifiable. Using this criterion, we conducted a retrospective comparative clinical study of the ddEGFR and cobas EGFR tests for the detection of EGFR mutations in non-small cell lung cancer (NSCLC) FFPET-DNA samples. Compared with the cobas EGFR test, the ddEGFR test exhibited superior analytical performance and equivalent or higher clinical performance. Furthermore, iQC index is a reliable indicator of the quality of FFPET-DNA and could be used to prevent incorrect diagnoses arising from low-quality samples.

Detection of the EGFR G719S Mutation in Non-small Cell Lung Cancer Using Droplet Digital PCR

“…In the present work, digital and non-digital platforms have been compared to evaluate G719S mutation detection when using tissue and liquid biopsy. Similar results were published by Kim et al, when comparing ddPCR and COBAS platforms for EGFR mutation detection using formalin-fixed paraffin embedded (FFPE) biospecimens to address clinical and quality control issues (28). In the current scenario and due to the wide range of analytical techniques, laboratories will be able to select the optimal platform for their needs.…”

Ultra-deep massively parallel sequencing with unique molecular identifier tagging achieves comparable performance to droplet digital PCR for detection and quantification of circulating tumor DNA from lung cancer patients

“…Tumor tissue biopsy is regarded as the gold standard for tumor genetic profiling in current clinical practice [22]. However, since this is an invasive procedure, it is not always feasible to carry out the biopsy to assess patients’ responses following initial treatment, particularly in those who are in advanced stages or do not have sufficient tumor tissues [23]. Liquid biopsy has recently been shown to better reflect the whole genetic complexity of tumor tissues and enables real-time monitoring of treatment-associated resistance [24, 25].…”

An on-chip wound healing assay fabricated by xurography for evaluation of dermal fibroblast cell migration and wound closure

“…In a final set of experiments, the role of activation of ERK signaling in cell migration was investigated. ERK phosphorylation can be inhibited using specific MEK inhibitors such as U0126 22 . ERK activation after wounding promotes keratinocyte and epithelial cell migration 9 , 23 , however, the ERK pathway is involved in the wound healing process through promotion of cell proliferation but not cell migration 24 .…”