Human EGFR Mutation Gene Detection Kit (Multiplex Fluorescent PCR Method)_NMPA Approval in January 2018
Product Name: Human EGFR Mutation Gene Detection Kit (Multiplex Fluorescent PCR Method)
Category: Domestic Class III
Registration Pathway: Innovation
NMPA Approval Time: January 2018
Product Structure and Composition: This kit contains P-EGFR mixed enzyme, P-EGFR positive control, P-EGFR 8-plex reaction strip, and P-EGFR reaction solution.
Scope of Application: This kit is used for qualitative detection of human EGFR mutation genes in plasma DNA samples from patients with advanced non-small cell lung cancer (NSCLC). Specifically, the 19 exon deletion and L858R mutation are used for companion diagnostic testing of erlotinib hydrochloride tablets, and the T790M mutation is used for companion diagnostic testing of osimertinib mesylate tablets.
Working Principle: This kit detects EGFR mutation genes in plasma DNA samples based on the Amplification Refractory Mutation System (ARMS) and fluorescence PCR technology. Specific mutation detection primers are designed for EGFR mutation gene sites. During PCR amplification, the primer extends and amplifies the mutant template because the base at the 3' end of the primer perfectly matches the mutant template. In contrast, primer extension is blocked for the wild-type template, inhibiting amplification of the wild-type template, thereby detecting EGFR mutation genes. The kit combines fluorescence PCR technology using fluorescent probes labeled with FAM/ROX/CY5 for real-time detection of EGFR mutation genes. Additionally, the reaction system includes primers for detecting the conserved region of the human EGFR gene and fluorescent probes labeled with HEX for internal control used in result interpretation and sample quality monitoring. The PCR amplification reaction system also contains UNG enzyme, which selectively cleaves the uracil glycosidic bond in PCR fragments containing dU, effectively reducing false positives due to PCR product contamination.
Analytical Performance Research: The product's analytical performance includes the lowest detection limit, precision, concordance rate of negative/positive reference materials, specificity (cross-reactivity, interference tests), nucleic acid extraction and purification performance, etc.
In the evaluation of the lowest detection limit, using 3 batches of finished kits (P214072505Z, P214080103Z, P214080504Z), specific background DNA concentrations (3ng/reaction, 7.5ng/reaction, and 15ng/reaction) were evaluated with 3 mutation types set at different mutation ratios, repeated 20 times. The lowest detection limit with a detection rate of not less than 95% was determined to be a DNA concentration of 15ng/reaction with a mutation ratio of 0.2%-0.8%.
In the precision study, the applicant evaluated the intra-batch, inter-batch, intra-day, inter-day, and inter-operator precision of the product using negative precision reference materials, weak positive precision reference materials (1% mutation ratio), and strong positive precision reference materials (50% mutation ratio) on 3 batches of finished kits (P214072505Z, P214080103Z, P214080504Z). The results showed that the Ct value CV values were all within 5%, indicating good intra-batch, inter-batch, intra-day, inter-day, and inter-operator precision of the kit.
The kit was evaluated for specificity and accuracy using 91 positive reference materials and 12 negative reference materials on 3 batches of finished kits (P214072505Z, P214080103Z, P214080504Z), resulting in a concordance rate of 100% for both positive and negative reference materials.
In the cross-reactivity evaluation, the applicant assessed cross-reactivity with other mutation gene types that are homologous to the EGFR gene, wild-type likely to cause cross-reactivity, non-human genes, and lung-related infectious microorganisms. Samples included HER2 gene plasmid DNA (103 copies/µL), plasma sample DNA (1ng/µL), and DNA from bacteria/yeast/mycobacterium/tetanus (1ng/µL). It was verified that the above samples did not produce cross-reactivity with this product.
Interference test results showed that the kit's detection results were not interfered with by endogenous interferents in plasma samples (hemoglobin, triglycerides, ferritin, etc., at concentrations of 2g/L, 37mmol/L, and 200ng/mL, respectively) or exogenous interferents (paclitaxel, cisplatin, trastuzumab, sodium citrate, etc., at concentrations of 90µg/mL, 90µg/mL, 90µg/mL, and 0.645mol/L, respectively).
Regarding nucleic acid extraction and purification steps, the applicant compared the efficiency of Qiagen's QIAamp Circulating nucleic acid Kit and Xiamen Ed's nucleic acid extraction reagent for extracting DNA from 81 plasma samples. The results showed that the nucleic acid extraction efficiency of the two methods was comparable, and the extracted samples met the requirements for detection with this kit.
Based on the evaluations, Qiagen's QIAamp Circulating nucleic acid Kit and Xiamen Ed's nucleic acid extraction reagent were selected as the nucleic acid extraction methods for this product. The current product submission includes only one packaging specification (12 tests/box), and the applicant provided performance evaluation data for three batches of products (P214072505Z, P214080103Z, P214080504Z) on all applicable models.
Positive Judgment Value Research: The plasma samples used for the positive judgment value study of this product are sourced from Xiamen Ed BioTech Research Center Ltd., and the EGFR mutation status of these samples is verified using digital PCR methods. For the common clinical mutation types 19-del, L858R, and T790M, the applicant used 94 clinical samples for testing. Using 3 batches of commercial reagent kits (P214072505Z, P214080103Z, P214080504Z) on three instrument models - Stratagene Mx3000P™, ABI7500, and MacroSLAN systems, the extracted DNA was tested. By analyzing ROC curves using SPSS software, the positive judgment value was determined based on the maximum Youden index. For rare mutation types like 20-ins, S768I, G719X, L861Q, the applicant used 3 batches of reagent kits on the same instrument models to determine the positive judgment value through parallel comparison with digital PCR.
Stability Research: The applicant conducted real-time stability, accelerated stability, freeze-thaw stability, and bottle opening stability studies on this product to determine its effective storage time under various conditions. The real-time stability study showed that the product can be stored stably for 8 months at -20±5℃. The accelerated stability study demonstrated that the product can be stored stably for 7 days at 37℃. The freeze-thaw stability study indicated that the product maintained performance requirements even after 10 freeze-thaw cycles. The bottle opening stability study revealed that the opened kit stored at 2-8℃ remained stable for 7 days and at -20±5℃ for 8 months, meeting all performance criteria.
Thermal Accelerated Stability Research: Three batches of finished reagent kits (lot numbers: P214111401Z, P214112001Z, P214112602Z) were thawed and stored at 37°C. On days 0, 3, 5, and 7, the physical properties, accuracy, specificity, detection limit, and precision were assessed. The results showed that the product remained stable for 7 days at 37°C, and all performance criteria were met.
Freeze-Thaw Stability Research: Three batches of finished reagent kits (lot numbers: P214111401Z, P214112001Z, P214112602Z) were stored at -20±5°C until completely frozen, then thawed at room temperature, and subjected to multiple freeze-thaw cycles. After the 0th, 2nd, 4th, 6th, 8th, and 10th freeze-thaw cycles, the physical properties, accuracy, specificity, detection limit, and precision were evaluated. The results indicated that the product remained stable after 10 freeze-thaw cycles, meeting all performance requirements.
Bottle Opening Stability Research: Three batches of finished reagent kits (lot numbers: P214111401Z, P214112001Z, P214112602Z) were thawed, opened, and the P-EGFR 8-plex PCR reaction strip, P-EGFR reaction solution, P-EGFR mixed enzyme, and P-EGFR positive control were exposed to air before being tightly closed. The kits were stored at 2-8°C for 0, 3, 5, and 7 days, and at -20±5°C for 0, 4, 6, and 8 months. Physical properties, accuracy, specificity, detection limit, and precision were assessed. The results showed that the opened reagent kits remained stable for 7 days at 2-8°C and 8 months at -20±5°C, meeting all performance criteria.
Clinical Evaluation:
Consistency Clinical Study:
The applicant conducted a clinical trial at six institutions, including Fudan University Affiliated Zhongshan Hospital, Yunnan Cancer Hospital (Third Affiliated Hospital of Kunming Medical University), Wenzhou Medical University Affiliated First Hospital, Shanghai Chest Hospital, Peking Union Medical College Hospital, and Capital Medical University Affiliated Beijing Chest Hospital. The study aimed to evaluate the clinical performance of this product by comparing it with a marketed product on clinical samples. The enrolled samples included 1069 cases of advanced (IIIB-IV stage) lung adenocarcinoma and a small number of non-lung adenocarcinoma, as well as lung adenocarcinoma IA-IIIA stage plasma samples. The comparison reagent selected was a marketed human EGFR gene mutation detection kit (fluorescence PCR method) (registration number: NMPA 20143402001). For samples with inconsistent results between the test reagent and the comparison reagent or samples that were positive for both tests but could not be specifically typed, high-throughput sequencing (NGS) was used for sequence determination.
This clinical trial detected a total of 373 mutation-positive samples, with a positivity rate of 34.89%, including 168 cases of 19del mutation-positive, 168 cases of L858R mutation-positive, 69 cases of T790M mutation-positive, 11 cases of 20-ins mutation-positive, 14 cases of G719X mutation-positive, 13 cases of S768I mutation-positive, and 8 cases of L861Q mutation-positive. The comparison study results with the comparison reagent showed a positive agreement rate of 97.35%, a negative agreement rate of 89.7%, and an overall agreement rate of 91.86%. Statistical analysis using kappa test showed Kappa=0.81, indicating good detection consistency between the two.
Out of the 1069 samples, there were a total of 122 samples where the results of the two tests were inconsistent or partially consistent. Among these, 68 samples had NGS method verification results consistent with the test reagent results, 42 samples had NGS method verification results consistent with the comparison reagent results, and 12 samples had inconsistent results with both methods. The applicant classified 1041 cases of lung adenocarcinoma samples into different treatment regimens: initial treatment, recurrence, and TKI-resistant recurrence groups, and compared the differences in various mutations. The 19-del mutation had the highest detection rate in the tyrosine kinase inhibitor (TKI)-resistant recurrence group, significantly different from the other two groups; the L858R mutation had the highest detection rate in the TKI-resistant recurrence group, followed by the initial treatment group, with significant differences among the three groups; the T790M mutation had a significantly higher detection rate in the TKI-resistant recurrence group than in other groups; the detection rates of other mutations showed no significant differences among the groups.
Paired Study of Tissue and Blood Samples:
A total of 229 tissue EGFR test results from Wenzhou Medical University Affiliated First Hospital were analyzed, with 203 tissue samples collected at the same time as plasma samples. The test reagent was used to detect plasma samples, and the results were compared with the previous tissue test results of the same subjects (human EGFR gene mutation detection kit (fluorescence PCR method) NMPA 20143402001). The results showed a positive agreement rate of 65.55% between the test reagent plasma detection results and the tissue sample detection results, consistent with previous literature.
Clinical Study Related to the Efficacy of EGFR-TKI Drugs:
The AURA17 study evaluated the safety and efficacy of osimertinib tablets (Tagrisso) in patients with locally advanced or metastatic NSCLC (stage IIIB-IV), who had disease progression after previous approved EGFR-TKI drug treatment and were positive for EGFR T790M mutation in the Asia-Pacific region. A total of 306 subjects (255 from China) preliminarily met the inclusion criteria and were screened. Among them, 277 subjects provided sufficient tissue samples, and after central laboratory testing, 183 subjects were confirmed to be positive for EGFR T790M mutation in tissue samples. These 183 subjects were subsequently treated with osimertinib tablets (Tagrisso). Among these 183 subjects, 168 could provide concurrent plasma samples, and a total of 82 T790M mutation-positive cases were detected in these 168 plasma samples. Among the 77 subjects who took osimertinib tablets (Tagrisso) and were evaluable for efficacy, 49 showed disease improvement, with an objective response rate of 63.6% (95% CI: 52, 74).
Additionally, a retrospective clinical trial study was conducted on patients receiving treatment with osimertinib tablets (Tagrisso) at Beijing Chest Hospital of Capital Medical University, Affiliated First Hospital of Wenzhou Medical University, and Yunnan Cancer Hospital, where 7 subjects were found to be T790M mutation-positive in plasma samples after recurrence and progression of advanced non-small cell lung cancer following approved EGFR-TKI drug treatment. After taking osimertinib tablets (Tagrisso), 5 cases showed disease improvement, 2 cases remained stable, with an objective response rate of 71.43% and a disease control rate of 100%. These studies indicate that this product can help NSCLC patients choose osimertinib tablets (Tagrisso) for targeted tumor treatment.
Clinical Study Related to the Efficacy of Erlotinib Hydrochloride Tablets (Iressa):
A retrospective clinical trial study was conducted on patients receiving treatment with erlotinib hydrochloride tablets (Iressa) at Beijing Chest Hospital of Capital Medical University, Affiliated First Hospital of Wenzhou Medical University, and Yunnan Cancer Hospital, where 48 subjects were found to be EGFR sensitive mutation-positive in plasma samples of advanced non-small cell lung cancer. After taking erlotinib hydrochloride tablets (Iressa), 29 cases showed disease improvement, 18 cases remained stable, and 1 case showed disease progression, with an objective response rate of 60.42% and a disease control rate of 97.92%. This study suggests that this product can be used to help NSCLC patients choose erlotinib hydrochloride tablets (Iressa) for targeted tumor treatment.