Using circulating cell-free DNA to monitor personalized cancer therapy

Abstract

High-quality genomic analysis is critical for personalized pharmacotherapy in patients with cancer. Tumor-specific genomic alterations can be identified in cell-free DNA (cfDNA) from patient blood samples and can complement biopsies for real-time molecular monitoring of treatment, detection of recurrence, and tracking resistance. cfDNA can be especially useful when tumor tissue is unavailable or insufficient for testing. For blood-based genomic profiling, next-generation sequencing (NGS) and droplet digital PCR (ddPCR) have been successfully applied. The US Food and Drug Administration (FDA) recently approved the first such "liquid biopsy" test for EGFR mutations in patients with non-small cell lung cancer (NSCLC). Such non-invasive methods allow for the identification of specific resistance mutations selected by treatment, such as EGFR T790M, in patients with NSCLC treated with gefitinib. Chromosomal aberration pattern analysis by low coverage whole genome sequencing is a more universal approach based on genomic instability. Gains and losses of chromosomal regions have been detected in plasma tumor-specific cfDNA as copy number aberrations and can be used to compute a genomic copy number instability (CNI) score of cfDNA. A specific CNI index obtained by massive parallel sequencing discriminated those patients with prostate cancer from both healthy controls and men with benign prostatic disease. Furthermore, androgen receptor gene aberrations in cfDNA were associated with therapeutic resistance in metastatic castration resistant prostate cancer. Change in CNI score has been shown to serve as an early predictor of response to standard chemotherapy for various other cancer types (e.g. NSCLC, colorectal cancer, pancreatic ductal adenocarcinomas). CNI scores have also been shown to predict therapeutic responses to immunotherapy. Serial genomic profiling can detect resistance mutations up to 16 weeks before radiographic progression. There is a potential for cost savings when ineffective use of expensive new anticancer drugs is avoided or halted. Challenges for routine implementation of liquid biopsy tests include the necessity of specialized personnel, instrumentation, and software, as well as further development of quality management (e.g. external quality control). Validation of blood-based tumor genomic profiling in additional multicenter outcome studies is necessary; however, cfDNA monitoring can provide clinically important actionable information for precision oncology approaches.

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.…”

The evolution of molecular diagnosis using digital polymerase chain reaction to detect cancer via cell‐free DNA and circulating tumor cells

“…Hence, a blood sample is undoubtedly the more desirable alternative (Francis and Stein, 2015;Lowes et al, 2016). Furthermore, using this kind of methodology was successful in revealing pancreatic, lung, breast, and prostate cancer onset in patients but also melanomas in early-stage development through the cfDNA (Haber and Velculescu, 2014;Earl et al, 2015;Iwama et al, 2017;Oellerich et al, 2017;Takai and Yachida, 2016;Vendrell et al, 2017). Iwama and colleagues demonstrated that the use of dPCR in patients' liquid biopsy of lung adenocarcinoma was able to detect the EGFR "activating mutation" in cfDNA at a high rate of 81.3% and 100%, compared with Next Generation Sequencing (NGS) with 71.7 and 60.0%).…”

Quantification of HIV DNA Using Droplet Digital PCR Techniques

“…Second, the sensitivity of ddPCR makes it an ideal platform for studying rare‐event molecules in the presence of a large background of unaffected molecules. This level of sensitivity has made ddPCR an attractive modality in detecting rare mutations associated with cancer phenotypes in liquid biopsies (Oellerich et al., ), as well as in low‐level pathogen detection (Mu, Yan, Tang, & Liao, ; Roberts et al., ; Sedlak, Cook, Cheng, Magaret, & Jerome, ; Srisutham et al., ; Strain et al., ; Wilson et al., ). Indeed, ddPCR has been used to detect HIV DNA, with single targets of 60 to 80 nucleotides [reviewed in Rutsaert, Bosman, Trypsteen, Nijhuis, and Vandekerckhove (); also see Trypsteen, Kiselinova, Vandekerckhove, and De Spiegelaere ()].…”