Digital PCR application in non-invasive cancer treatment tracking

Article by Dr. Le Thi Thanh Huong – Share99 Institute of Stem Cell Research and Gene Technology.

In this article, we will learn the application of Digital PCR (Droplet Digital TM PCR, ddPCRTM) for liquid biopies in cancer diagnosis, monitoring and evaluating treatments as well as detecting recurrence after remission.

Liquid biopsy is a non-invasive method used to diagnose and monitor the condition, including cancer. Instead of taking a biopedet at the affected tissue, people collect "cell-free DNA" (cfDNA), circulating tumor DNA (ctDNA; a set of cfDNA and derived from the tumor), or suspension cancer cells (also known as circulating tumor cells). , CTCs) from blood or other body services. Digital PCR (Droplet Digital TM PCR, ddPCRTM) is applied for a liquid biosy procedure in cancer diagnosis, monitoring and evaluation of treatments as well as detecting recurrence after remission.

Liquid bios are non-invasive tests that detect fragments of DNA or cells in the blood or other body fluids. The term "free DNA" (cfDNA) refers to dna fragments mainly derived from cyclically dying cells (apoptotic) and necrosis. CfDNA circulating in the blood can be used to diagnose a wide range of diseases including cancer, diabetes, and even myocardial infarction, as well as monitoring transplanted organ in the recipient. In case of cancer, cfDNA is often referred to as 3gory tumor DNA, or ctDNA.

Today, cancer research and diagnosis is one of the important applications of liquid bios. This technical team helps to solve some specific work later.

• Monitoring and identifying treatment regimens – Liquid biopsies can help doctors monitor the effectiveness of a treatment regimen, optimize the regimen, determine if changes in the treatment regimen are beneficial.
• Help monitor changes in tumors – There are many tumors that change continuously over time; monitoring these changes can help doctors make changes in treatment.
• Monitor recurrence – Help screen patients in remission to detect newly developed tumors before physical signs appear clearly, whether the tumor appears in the same location or migrates to another location.
• Regular screening of high-risk patients – For some people with cancer-related genotypes, a liquid bios bios can help detect cancer early before clinical symptoms appear or are detected using routine imaging techniques.

The goal of liquid biopy is to detect and quand quantities of cfDNA, ctDNA, and CTC that carry mutations circulating in the blood. However, these target sequences are often present at low levels and around them there are other complex components such as white blood cells, normal DNA from the body's frail old cells, etc. In addition, resory DNA is often highly fragmented, so the intact target sequence content will not be high. These challenges require mutation screening techniques that require greater sensitivity to be able to detect and quant quantise low-content targeted protocols in the short term. Digital PCR or Digital Droplet PCR (ddPCRTM) is currently a suitable tool for liquid biopsies. This is a new technology, which allows the absolute dosing of targeted order with high sensitivity and accuracy.

A digital micro-droplet PCR process consists of 5 basic steps.

1. PCR response preparation: The preparation of the ddPCR reaction is similar to the usual PCR reaction. The reaction also includes basic components such as the enzyme Taq polymerase, bait, dNTP, magnesium, etc. and additional EvaGreen photonics or TaqMan fluorescent detectors. DNA samples will be added to these reactions. Just like when running PCR, in addition to reactions to samples, technicians often run additional positive test sample reactions (containing target DNA) and negative anti-negative reactions (not containing target DNA).
2. Micro-droplet creation: With a special device called droplet generator, a reaction will be randomly divided into micro-droplets with a volume of about 1 nL. Each of these micro-droplets is full of components of a Real-time PCR reaction. DNA molecules will be randomly distributed into these micro-droplets.
3. DNA replication in micro-droplets: All micro-droplets generated from an initial reaction will be transferred to a well in a 96-well PCR disk and placed into a regular PCR machine. As the heat rotation program takes place, the DNA present in each micro-droplet will be humane as a regular PCR reaction. Fluorescent signals generated by detectors or light-ray substances will accumulate in each micro-droplet,
4. Read the signal in micro-droplets: After the PCR reaction ends, the micro-droplets will be analyzed by a fluorescent reading device. Each micro-droplet in each tube contained in step 3 will be sucked into the pipe of the device and in turn pass through the fluorescent signal readers. The signal strength of each fluorescent color in each micro-droplet will be recorded by the reader.
5. Data analysis: The reading results of all micro-droplets generated from each initial reaction will be transferred to computer-based analysis software. For each initial 20 μL reaction, the results analyzed include:

• The total number of droplets generated from the initial reaction, including positive micro-droplets and negative micro-droplets.
• The total number of positive micro-droplets (containing target DNA and having a higher fluorescent signal strength than micro-droplets in a negative reaction).
• Total number of negative micro-droplets (does not contain target DNA and has fluorescent signal strength on par with micro-droplets in a negative reaction)

Based on the number of positive micro-droplets and the total number of micro-droplets generated from each initial reaction, the software will apply the Poisson distribution model to calculate the target DNA concentration in the initial reaction volume.

Today, there are many buildings in the world that have been applying digital PCR in detecting and tracking a variety of cancers. Through the examples below, readers will visualize the digital PCR applications as well as the excellent sensitivity of this technique.

Cell lung cancer is not small

Non-small cell lung cancer (NSCLC) is the most common form of lung cancer. NSCLC carries epidermis growth factor receptor activation mutations (EGFR) and responds to tyrosine kinase inhibitors. There are various EGFR activation mutations, and after a period of treatment, the majority of NSCLC becomes resistant to kinase inhibitors. This resistance is caused mainly by the EGFRT790M second-class mutation. Therefore, in addition to identifying EGFR mutations, doctors now believe that it is necessary to check the appearance of second-line mutations during treatment. In his 2015 work, Zhu and his associates applied digital PCR to ctDNA analysis and helped increase clinical sensitivity for EGFR activation mutations to 80%. In addition, digital PCR enables the detection of low-rate EGFRT790M second mutations (Oxnard and associates, 2014, Watanabe and associates, 2015, Zhang and associates 2015) before the tumor grows again due to resistance to tyrosine kinase inhibitors.

Malignant skin cancer

About 50% of malignant skin cancers carry mutations that convert valine into glutamic acid at codon 600 of serine/threonine kinase B-Raf (a pre-carcinogenic gene). Patients carrying this mutation are treated with BRAF inhibitors. The level of cfDNA circulating in the blood containing this mutation is very low (<0,01% của cfDNA). However, recently there has been a digital PCR application to detect this type of BRAFV600E mutation and achieve sensitivity to 0.005%. This work also applies digital PCR to demonstrate correlation between BRAFV600E mutation percentages in tumors and in cfDNA (Sanmamed and associates, 2015). In addition, in patients treated with BRAF inhibitors, the degree of reduction of BRAFV600E ctDNA will correspond to the degree of destruction of tumors and vice versa the increase in the percentage of CTDNA BRAFV600E will be a sign of resistance to treatments. Research by Sanmamed and associates has demonstrated that it is entirely possible to apply digital PCR to identify patients who are likely to respond to BRAF inhibitor treatments as well as monitor their course of treatment afterwards.

Breast cancer

In breast cancer, because the percentage of patients with postoperative relapses is significant, doctors may prescribe additional radiotherapy and/or chemotherapy to destroy the remaining tumor cells. Beaver and her partner applied digital PCR to analyze ctDNA samples before and after breast cancer surgery. The team was found to have ctDNA in several post-surgical samples (Beaver and associates, 2014). This suggests that micro-microeables are still present in these samples, and we can apply digital PCR to detect leftover solid tumors (Siravegna and Bardelli, 2014). From here, we can calculate the feasibility of applying digital PCR to detect low levels of ctDNA in order to predict the risk of recurrence after breast cancer surgery and make decisions about the appropriate form of postoperative care.

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References

• Beaver JA et al. (2014). Detection of cancer DNA in plasma of patients with early-stage breast cancer. Clin Cancer Res 20, 2643–2650. PMID: 24504125
• Oxnard GR et al. (2014). Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA. Clin Cancer Res 20, 1698–1705. PMID: 24429876
• Sanmamed MF et al. (2015). Quantitative cell-free circulating BRAFV600E mutation analysis by using of droplet digital PCR in the follow-up of patients with melanoma being treated with BRAF inhibitors. Clin Chem 61, 297–304. PMID: 25411185
• Siravegna G and Bardelli A (2014). Minimal residual disease in breast cancer: in blood veritas. Clin Cancer Res 20, 2505–2507. PMID: 24658155
• Watanabe M et al. (2015). Ultra-sensitive detection of the pretreatment EGFR T790M mutation in non-small cell lung cancer patients with an EGFR-activating mutation using droplet digital PCR. Clin Cancer Repub ahead of print. PMID: 25882755
• Zhang B et al. (2015). Comparison of droplet digital PCR and conventional quantitative PCR for measuring EGFR gene mutation. Exp Ther Med 9, 1383–1388. PMID: 25780439
• Zhu G et al. (2015). Highly sensitive Droplet Digital PCR method for detection of EGFR-activating mutations in plasma cell-free DNA from patients with advanced non-small cell lung cancer. J Mol Diagn 17, 265–272. PMID: 25769900

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