Digital PCR has the characteristics of high sensitivity, absolute quantification, high specificity, and low amount of specimens used, which can provide more objective and automated quantitative detection results for clinical purposes, and better overcome the problems of poor quality of DNA in tumor tissue-embedded specimens and limited amount of specimens etc,.
With these advantages, digital PCR is widely used in several different fields, especially in biomedicine, including rare mutation detection, gene copy number variation detection, gene expression studies, methylation level detection, tumor liquid biopsy, noninvasive prenatal testing, microbial (viral, bacterial, etc.) detection, transplant rejection monitoring, and second-generation sequencing-assisted library building and is expected to become a reliable tool for diagnosis and monitoring of malignant tumors.
EGFR mutation is now a routine concomitant diagnosis for patients with Non-Small-Cell Lung Cancer (NSCLC) and is an important guide for the use of EGFR tyrosine kinase inhibitors (TKI). However, because most NSCLC is at an advanced stage, it is difficult to obtain enough tumor tissue to complete this test, which does not facilitate dynamic monitoring of patient efficacy and drug resistance. In contrast, body fluid specimens such as blood, pleural fluid, and cerebrospinal fluid, which will contain tumor-derived DNA (circulating tumor DNA (ctDNA)), are more readily available and are considered to be a valid alternative to tissue specimens. Since the amount of ctDNA in body fluid specimens is very low, it places a high demand on the assay. Compared with traditional assays, dPCR can significantly improve the detection rate of EGFR mutations in plasma. In addition, taking advantage of the absolute quantification of digital PCR, the abundance of EGFR mutations in plasma can be monitored dynamically, and this dynamic change is closely related to the efficacy of patients with TKI, which can better guide the treatment of patients
ALK fusion gene is another routine concomitant diagnosis for NSCLC patients and can guide the use of ALK-TKI drugs. Currently, immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and RT-PCR are the three methods routinely used in clinical practice. These three methods have their own advantages and disadvantages: IHC is relatively simple and inexpensive, but is susceptible to subjective judgment; FISH can detect various fusion types, but is cumbersome and expensive; RT-PCR method has better specificity and objective results, but can only be used for known types.
Including mutation detection, there exist more applications in oncology biomarker detection area. For instance, HER2 Copy number variation in breast cancer, the digital PCR has the advantages of standardization of operation and interpretation, good reproducibility of results, short test cycle time, and low specimen usage compared to the IHC method.
DNA methylation is a common epigenetic modification of mammalian DNA, which plays an important role in development and disease processes by regulating gene expression in cells. Aberrant DNA methylation involves overall hypomethylation of the entire genome and local hypermethylation of CpG islands located in gene promoter regions and intergenic DNA, a common epigenetic alteration in human malignancies. PCR-based methods have been widely used for the assessment of DNA methylation, based on the principle of first treating DNA with bisulfite, which will convert unmethylated cytosines to uracil by deamination, and then designing primers and probes that bind specifically to this DNA for amplification detection. However, conventional PCR assays are susceptible to PCR inhibitors and have limited sensitivity for detection of rare methylated alleles in the context of non-methylated alleles. Digital PCR, on the other hand, performs reactions in numerous independent droplets and is therefore less susceptible to inhibitors among each other, enabling accurate detection of rare methylation alleles that are not detected by conventional methods, and promising the future use of DNA methylation as a predictor of regional carcinogenesis or a cancer risk biomarker.
With the improvement of treatment modalities such as chemotherapy, targeted therapy, and hematopoietic stem cell transplantation, the treatment of hematologic tumors is increasingly improving. Recurrence due to microscopic residual disease (MRD) is currently a major challenge in the treatment of hematologic tumors. Dynamic monitoring of MRD is an important guideline for evaluating treatment efficacy, predicting disease recurrence, and implementing individualized treatment. Digital PCR technology with limited dilution reduces complex background interference and concentrates low-abundance target gene signals, thus improving the sensitivity of MRD assays.
Benefits and applications of mutation detection by dPCR
1. Absolute quantification of both mutant and wild-type sequence，allowing dynamic monitoring of disease biomarkers
2. Superior precision allows discriminating smaller change
3. Achieve detection sensitivity of 0.1%~0.001% mutant in background of abundant homogenous sequences
4. Less susceptible to PCR efficiency variation, superior tolerance to PCR inhibitors, offering reliable quantification
