Droplet-based digital PCR and next generation sequencing for monitoring circulating tumor DNA: a cancer diagnostic perspective

Abstract

Early detection of cancers through the analysis of ctDNA could have a significant impact on morbidity and mortality of cancer patients. However, using ctDNA for early cancer diagnosis is challenging partly due to the low amount of tumor DNA released in the circulation and its dilution within DNA originating from non-tumor cells. Development of new technologies such as droplet-based digital PCR (ddPCR) or optimized next generation sequencing (NGS) has greatly improved the sensitivity, specificity and precision for the detection of rare sequences. Areas covered: This paper will focus on the potential application of ddPCR and optimized NGS to detect ctDNA for detection of cancer recurrence and minimal residual disease as well as early diagnosis of cancer patients. Expert commentary: Compared to tumor tissue biopsies, blood-based ctDNA analyses are minimally invasive and accessible for regular follow-up of cancer patients. They are also described as a better picture of patients' pathology allowing to highlight both tumor heterogeneity and multiple tumor sites. After a brief introduction on the application of the follow-up of ctDNA using genetic or epigenetic biomarkers for prognosis and surveillance of cancer patients, potential perspectives of using ctDNA for early diagnosis of cancers will be presented.

TP53 Mutations in Serum Circulating Cell-Free Tumor DNA As Longitudinal Biomarker for High-Grade Serous Ovarian Cancer

“…Sensitivity and specificity are the main challenges for detecting cancer-specific alterations in cfDNA. Recent advances in NGS [41][42][43] and PCR protocols have allowed the quantitative detection of mutations with a sensitivity below 0.001% [44]. Compared to conventional PCR, dPCR is a reliable method and easy to set up.…”

Droplet digital polymerase chain reaction for rapid broad‐spectrum detection of bloodstream infections

“…The droplet digital polymerase chain reaction (ddPCR) is a novel molecular technique that has been developed to improve the sensitivity and quantification of rare target DNA sequences, for example in liquid biopsies for cancer monitoring, non‐invasive prenatal testing for genetic abnormalities and detection of DNA contaminants in bioprocessing (Hussain et al , ; Oellerich et al , ; Postel et al , ; Zhang et al , ; Wang et al , ; Tan et al , ; Galimberti et al , ). After DNA isolation, ddPCR generates approximately 20 000 miniscule droplets, which enriches target DNA sequences by reducing the competition with high‐copy templates (Fig.…”

Precision Medicine in Lymphoma by Innovative Instrumental Platforms

“…On the contrary, some situations may severely limit the availability of DNA or RNA, as well as in the presence of a small disease burden or for particular sites (i.e., the central nervous system or the eye), which make the biopsy impossible or too risky for the patient. Therefore, the collection of cell-free, circulating tumor DNA (ctDNA) in plasma, the socalled liquid biopsy, represents an optimal and easily accessible source of genetic material, which can be analyzed by techniques with the highest sensitivity (15)(16)(17). Moreover, pharmacogenetic analyses can exploit the very limited quantities of nucleic acids released by neoplastic cells in blood, cerebrospinal fluid (CSF) and aqueous humor.…”