Microdroplet Digital Polymerase Chain Reaction for Precision Detection of Non-Small Cell Lung Cancer Mutations

Summary

Here, we present a protocol to sensitively and specifically detect EGFR T790M mutations using droplet digital polymerase chain reaction (ddPCR), which can also be extended to rapidly identify other tumor mutations such as G719S, L858R, L861Q, and S768I.

Abstract

On a global scale, lung cancer continues to be the primary contributor to cancer-related deaths, with its prevalence ranking second only to that of kidney cancer. Non-small cell lung cancer (NSCLC) constitutes approximately 80-85% of all reported lung cancer cases. The T790M mutation in the EGFR gene is a widely recognized primary determinant of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). The presence of this condition has been observed in approximately 50-60% of those experiencing progressive illness. Therefore, the implementation of an expeditious and highly responsive approach for detecting T790M mutations in the EGFR gene is essential for the expeditious identification of non-small cell lung cancer. In the current study, ddPCR was used to detect the T790M mutation with a detection limit of 10 copies/µL, which is 10-fold higher than the sensitivity of qPCR (1 × 10² copies/µL). For the 15 simulated samples, ddPCR was capable of detecting the presence of T790M mutations in the EGFR gene, which were observed to be at reduced levels when analyzed using qPCR. The detection of T790M mutations inside the EGFR gene by a fast detection method is being investigated to determine its suitability.

Introduction

Lung cancer is the leading cause of cancer-related mortality globally, and it stands as the second most prevalent malignancy overall. Non-small cell lung cancer (NSCLC) accounts for a significant majority, approximately 80% to 85%, of all reported lung cancer cases^1,2,3,4. For patients diagnosed with advanced NSCLC harboring EGFR mutations, the established therapeutic approach involves the application of first-, second-, or third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs)^5,6,7. However, most patients treated with first- or second-generation EGFR-TKIs eventually develop acquired resistance to the medication. This resistance is frequently attributed to the presence of the T790M mutation in the EGFR gene, which is found in roughly 50-60% of patients experiencing disease progression^8,9,10,11,12.

Traditionally, genetic abnormalities in tumor DNA from tissue samples are identified using techniques like fluorescence in situ hybridization (FISH) or next-generation sequencing (NGS)^13,14. Yet, tissue biopsies can be challenging due to tumor inaccessibility, poor patient health, or insufficient tumor cell content^15,16,17. As an alternative, liquid biopsies, particularly from plasma, offer a less invasive way to identify driver mutations by detecting circulating tumor DNA (ctDNA). Tumor cells shed DNA into the bloodstream, pleural fluid, saliva, or feces, providing a valuable source for genetic analysis^18,19. The genetic profile of ctDNA often mirrors the tumor's genetic makeup, offering a comprehensive view of tumor heterogeneity-a crucial factor for accurate diagnosis and personalized cancer treatment^20,21.

Among the various techniques for identifying EGFR mutations in peripheral blood, including sequencing, real-time polymerase chain reaction (RT-PCR), digital PCR, denaturing high-performance liquid chromatography (DHPLC), amplification refractory mutation system (ARMS), and mutant-enriched PCR (ME-PCR), microdroplet digital PCR (ddPCR) has emerged as a highly effective tool for ctDNA mutation detection^22,23. Its superior sensitivity, precision, and ability to detect low-abundance mutations with high accuracy set it apart. Unlike traditional PCR, ddPCR partitions samples into thousands of droplets, enabling the absolute quantification of target DNA sequences without standard curves. This makes it exceptionally suitable for detecting rare mutations like the T790M mutation in EGFR. Despite ddPCR's advantages, its widespread clinical application has been limited by challenges in sample preparation, system optimization, and comparative performance analysis with established techniques like qPCR. A critical gap exists in optimizing ddPCR protocols for ctDNA mutation detection, particularly regarding the sensitivity, specificity, and reproducibility essential for clinical adoption.

In this study, we hypothesize that ddPCR offers superior sensitivity and reliability for detecting the EGFR T790M mutation in ctDNA compared to traditional qPCR methods. We focused on optimizing the nucleic acid extraction process to enhance efficiency, followed by ddPCR optimization for mutation detection. Additionally, we compared ddPCR and qPCR performance in terms of sensitivity, specificity, and reproducibility using simulated samples. By systematically evaluating ddPCR's potential, we aim to establish it as a viable and superior alternative to qPCR for EGFR mutation detection in liquid biopsies.

Protocol

This study did not involve human participants, human tissues, or live animal experiments; thus, no formal ethical approval was required. All procedures were conducted in accordance with institutional and national guidelines. The T790M mutant plasmid was commercially synthesized. Pig blood used to simulate clinical samples was obtained from a certified supplier following animal welfare regulations, with no live animal involvement. All other reagents, including magnetic beads, primers, probes, and PCR enzymes, were commercially sourced and used as instructed by the manufacturers.

Nucleic acid extraction
Nucleic acid extraction was performed using a magnetic bead extraction kit and a fully automatic nucleic acid extractor instrument. The standard procedure involved adding 200 µL of sample and 20 µL of Proteinase K to column 1 or 7 of the pre-packed deep-well plate. Then, 15 µL of magnetic beads supplied with the kit were added to column 2 or 8. The extraction program was initiated on the instrument, with the magnetic sleeve placed in its designated location. After extraction, the liquid from column 6 or 12 was transferred to a new 1.5 mL nuclease-free tube and stored at -20 °C.

To optimize the extraction of low-concentration target nucleic acids, optimization experiments were conducted. The recommended extraction parameters were: lysis temperature 70 °C, lysis time 10 min, 1 min for each of the three wash solutions, 2 min drying, and 3 min elution at 56 °C. The most influential parameters were systematically optimized: lysis temperature (62 °C, 66 °C, 70 °C, 74 °C, and 78 °C), lysis time (1, 3, 5, 7, and 9 min), and magnetic bead size (100 nm, 200 nm, 300 nm, 400 nm, 500 nm, and 1000 nm). These optimizations were performed using the T790M mutant plasmid at 1 × 10⁵ copies/µL. The 100 nm magnetic beads were supplied with the extraction kit, while other particle sizes were obtained from a biotechnology company. The efficiency of each extraction condition was assessed by analyzing the qPCR amplification curves and Ct values of the extracted samples. Statistical analysis of these results was performed using statistical software.

Standard plasmid DNA
The T790M mutant plasmid of the EGFR gene was custom-synthesized by a biotechnology company. Four micrograms of the dry plasmid powder were resuspended in 100 µL of 1x TE Buffer. The plasmid concentration was determined using a spectrophotometer. Copy number per microliter was calculated using the formula: Copy number/µL = [(6.02 × 10²³) × (DNA concentration (ng/µL) × 10^-9)] / (DNA length in base pairs × 660). The concentration of the synthesized fragment was 1.26 × 10⁹ copies/µL. This stock plasmid was then diluted 126-fold to obtain a working concentration of 1 × 10⁸ copies/µL.

Primer design and synthesis
Primers and a TaqMan probe for the EGFR T790M mutation were designed using Primer 6 based on standard primer design principles. Upstream primer: 5′-CCTCACCTCCACCGTGC-3′; Downstream primer²⁴: 5′-AGGCAGCCGAAGGGCA-3′; Probe: 5′-FAM-AGCTCATCACGCAGCTCA-BHQ1-3′. All primers and probes were synthesized by a commercial oligonucleotide synthesis service.

Establishment and optimization of ddPCR and TaqMan fluorescence quantitative detection system
A T790M plasmid stock of 1 × 10⁸ copies/µL was diluted to 1 × 10⁶ copies/µL using 1× TE Buffer for use in ddPCR and qPCR system optimization experiments.

Optimization of ddPCR system: The ddPCR system was optimized based on the enzyme manufacturer's recommendations. The initial recommended system included: 7.5 µL of 4x PCR mix, 1.2-3 µL of upstream primer (10 µM), 1.2-3 µL of downstream primer (10 µM), 0.3-1.8 µL of probe (10 µM), 1-15 µL of DNA template, and nuclease-free water to a final volume of 30 µL. The recommended amplification cycle involved an initial 10 min at 95 °C, followed by 40 cycles of 30 s at 94 °C and 60 s at 55-65 °C. The annealing temperature (55-61.8 °C), primer concentration (1-3 µL), probe concentration (0.75-1.75 µL), and template concentration (3-15 µL) were systematically optimized. Optimization results were statistically analyzed using statistical software.

Optimization of qPCR system: The qPCR system was optimized according to the enzyme manufacturer's recommendations. The initial suggested system included: 5 µL of Taq polymerase, 0.45 µL of 250 mM MgCl2, 0.5-2.5 µL of forward primer (10 µM), 0.5-2.5 µL of reverse primer (10 µM), 0.5-2.5 µL of probe (10 µM), 5 µL of DNA sample, and nuclease-free water to a final volume of 25 µL. The recommended amplification program was 5 min at 95°C; followed by 45 cycles of 15 s at 95 °C and 30 s at 56-64 °C, with fluorescence collection at 56-64 °C. The annealing temperature (56-64 °C), primer concentration (1-3 µL), probe concentration (0.5-2.5 µL), and template concentration (1-5 µL) were optimized. Statistical analysis of these results was performed using statistical software.

ddPCR and Taqman fluorescence quantitative PCR sensitivity test
To evaluate the sensitivity of ddPCR versus qPCR, the 1 × 10⁸ copies/µL T790M plasmid was serially diluted 10-fold using 1x TE Buffer to create a concentration gradient ranging from 10⁶ to 10¹ copies/µL. For ddPCR, seven reaction solutions were prepared according to the optimized ddPCR system, one for each concentration, including a negative control. Samples were loaded onto a microdroplet generator chip and processed with a droplet generation instrument to generate microdroplets. These droplets were then amplified using a PCR thermal cycler and subsequently analyzed by a digital PCR detection instrument. Concurrently, seven reaction solutions were prepared based on the optimized qPCR system, with identical templates. These were run on a real-time PCR instrument to assess the respective sensitivities of the two approaches (n = 3).

ddPCR repeatability tests
To assess repeatability, standard T790M plasmid DNA at 1 × 10⁸ copies/µL was diluted to generate high (1 × 10⁶ copies/µL), medium (1 × 10⁴ copies/µL), and low (1 × 10² copies/µL) concentration standards. Six reaction solutions were prepared according to the optimized ddPCR system. Samples were loaded onto a microdroplet generator chip and processed with a droplet generation instrument to form microdroplets. The microdroplets were then amplified using a thermal cycler and analyzed by a digital PCR detection instrument. A negative control was included. The experiment was performed in triplicate. Reproducibility was evaluated by comparing amplification profiles and calculating the coefficient of variation (CV) values from the three experimental replicates.

Analog sample testing
In order to test the reliability of the T790M mutation ddPCR and qPCR systems, we used pig blood randomly mixed with high and low concentrations of T790M mutation standard plasmids respectively, and pig blood without mixed standard plasmids was used as negative samples, and 15 simulated samples were prepared randomly, and nucleic acid was extracted from the simulated samples, and detected by using the ddPCR and qPCR methods, respectively.

Statistical analysis
All experiments were performed in at least three technical replicates. Data are presented as mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 10 software. Differences between groups were analyzed using Student's t-test or one-way analysis of variance (ANOVA) as appropriate. A p-value of less than 0.05 was considered statistically significant.

Results

Optimization of nucleic acid extraction
The ddPCR-based workflow for detecting the EGFR T790M mutation is summarized schematically in Figure 1. First, we determined the optimal lysis temperature. As shown in Figure 2A, a cleavage temperature of 66 °C yielded the smallest average Ct value, indicating the most efficient cleavage. Therefore, 66 °C was selected for subsequent experiments. Next, we optimized the lysis time at the determined optim...

Discussion

Lung cancer, as the leading cause of cancer-related deaths worldwide, has always been a focal point in medical research. In this study, we explore multiple critical aspects of lung cancer, aiming to provide deeper insights into its diagnosis, treatment, and prognosis. The cornerstone of precise diagnosis and treatment for lung cancer is accurate genetic testing. As demonstrated by Zhao et al.²⁵, traditional genetic testing heavily relies on high-quality tissue samples, and is costly and time-consu...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1× TE Buffer	Sangon Biotech	B548144	Tris-EDTA buffer used for DNA dilution
4× Unimix-U enzyme mix	TargetingOne Bio	UNIMIXU-4X	ddPCR enzyme mix
A300 Fast Thermal Cycler	LongGene	A300	PCR amplification
Biochip analyzer	SEED Bio	DigitalPCR-Biochip	Detection system for digital PCR
Drop Marker Sample Preparation Instrument	Xinyi Biotechnology	Digital PCR Platform TD-2	Preparation of microdroplets
Magnetic Bead Nucleic Acid Extraction Kit	LemnisCare Technology	MXP-1005	Nucleic Acid Extraction
Microdroplet generator chip	TargetingOne Biotech	MG-Chip-01	Used to generate microdroplets for ddPCR
NanoDrop	Allsheng	NC-500	Nucleic acid concentration measurement after sample extraction
NanoDrop ND-500 spectrophotometer	Allsheng Instruments	ND-500	Spectrophotometer for DNA quantification
PCR instrument A300 Fast Thermal Cycler	LongGene	A300	Thermal cycler for PCR amplification
Primers and TaqMan probe	Sangon Biotech	Custom-synthesized	Primers and TaqMan probe for T790M detection
qPCR instrument Gentier mini	Tianlong Technology	Gentier mini	Real-time PCR instrument
T790M mutation plasmid	Tsingke biology	T790M	Standard Curve and Detection Limit Test