Correlation of Circulating Tumor DNA EGFR Mutation Levels with Clinical Outcomes in Patients with Advanced Lung Adenocarcinoma
Introduction
Lung cancer remains a leading cause of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) accounting for the majority of cases. Among NSCLC subtypes, lung adenocarcinoma is the most prevalent. The discovery of epidermal growth factor receptor (EGFR) mutations as a driver of lung adenocarcinoma has revolutionized treatment strategies, with EGFR tyrosine kinase inhibitors (TKIs) becoming a cornerstone of targeted therapy. However, despite the efficacy of TKIs, a significant proportion of patients develop resistance within 9 to 12 months, underscoring the need for precise detection and monitoring of EGFR mutations.
Traditional methods of EGFR mutation detection rely on tissue biopsies, which have several limitations, including tumor heterogeneity, difficulty in obtaining samples after recurrence or metastasis, and potential complications from invasive procedures. In recent years, liquid biopsy, particularly the analysis of circulating tumor DNA (ctDNA), has emerged as a promising non-invasive alternative. ctDNA provides real-time information on tumor genetics, offering insights into treatment response and resistance mechanisms earlier than imaging or tissue biopsies.
Several molecular detection methods have been developed for ctDNA analysis, including the amplification-refractory mutation system (ARMS), droplet digital polymerase chain reaction (ddPCR), and next-generation sequencing (NGS). While these methods have shown utility, they each have limitations in terms of sensitivity, availability, and practicality for clinical use. The super-amplification-refractory mutation system (superARMS) is a newer technique approved for detecting EGFR mutations in plasma ctDNA. However, it remains a qualitative method, limiting its application in clinical research.
In this study, we introduce a reformed-superARMS (R-superARMS) method, which leverages the DCt value (mutant cycle threshold [Ct] value–internal control Ct value) generated during the PCR assay to transform superARMS into a semi-quantitative detection method. We evaluate the performance of R-superARMS in detecting EGFR mutations in plasma ctDNA and explore its correlation with clinical outcomes in patients with advanced lung adenocarcinoma.
Methods
This study was conducted at the Cancer Center of the First Hospital of Jilin University in Changchun, China. A total of 41 patients with advanced lung adenocarcinoma (stage IIIB or IV) were enrolled between January 2017 and June 2018. Inclusion criteria included pathologically confirmed lung adenocarcinoma, advanced clinical stage, newly diagnosed or recurrent disease, no prior treatment at the time of sample collection, and availability of both tumor tissue and blood samples collected within 14 days of each other.
All patients received first-generation TKIs (erlotinib, gefitinib, or icotinib) either as monotherapy or in combination with antiangiogenic drugs (bevacizumab or apatinib) as first-line treatment. Tumor response was assessed using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Plasma ctDNA was extracted from blood samples using the AmoyDx circulating DNA kit, and EGFR mutations were detected using the superARMS EGFR mutation detection kit. The DCt value was calculated to identify the presence of EGFR mutations, with a cut-off value defined as 11 for 19del/L858R/20ins, 8 for T790M, and 12 for G719X/L861Q/S768I.
The R-superARMS method was developed by utilizing the DCt value to convert superARMS from a qualitative to a semi-quantitative detection method. Patients were categorized based on baseline EGFR mutation levels and changes in DCt values after one month of treatment. The primary endpoint was progression-free survival (PFS), and the secondary endpoint was overall survival (OS). Statistical analyses included Kaplan-Meier survival analysis, receiver operating characteristic (ROC) curve analysis, and Fisher’s exact test for categorical variables.
Results
The study cohort comprised 41 patients with a mean age of 63 years. The majority were female (68.3%) and non-smokers. Approximately 51.2% of patients had extrathoracic metastatic disease, primarily involving bone and brain metastases. The concordance rate of EGFR mutations between tumor tissue and matched plasma samples was 68.3% (28/41). The most common EGFR mutations were 19del (31.7%) and L858R (29.3%), with other rare mutations including G719X and T790M.
Baseline levels of ctDNA EGFR mutations were found to significantly impact patient outcomes. Using ROC curve analysis, the cut-off DCt value for baseline EGFR mutations was set at 8.11. Patients with a DCt value ≤8.11 had a significantly longer median OS (not reached vs. 11.0 months; log-rank P = 0.024) compared to those with a DCt value >8.11. Although no significant difference in PFS was observed between the two groups, the survival curves were separated, with a trend toward longer PFS in the DCt ≤8.11 group.
Changes in ctDNA EGFR mutation levels during treatment also correlated with clinical outcomes. Patients were divided into mutation clearance (MC) and mutation incomplete clearance (MIC) groups based on whether the DCt value turned negative after one month of treatment. The MC group had a significantly longer median OS (not reached vs. 10.4 months; log-rank P = 0.021) and a trend toward longer PFS (not reached vs. 27.5 months; log-rank P = 0.088) compared to the MIC group.
Quantitative analysis of changes in DCt values after one month of treatment revealed that a cut-off value of 4.89 was predictive of patient outcomes. Patients with a change in DCt value >4.89 had a significantly longer median OS (not reached vs. 11.0 months; log-rank P = 0.014) compared to those with a change ≤4.89. Although no significant difference in PFS was observed, the results suggested that changes in EGFR mutation levels could serve as an early indicator of treatment response.
Discussion
The findings of this study highlight the potential of R-superARMS as a semi-quantitative method for detecting EGFR mutations in plasma ctDNA. By leveraging the DCt value, R-superARMS provides a simple, fast, and cost-effective approach to monitoring EGFR mutation levels, offering advantages over traditional qualitative methods. The ability to quantify EGFR mutations allows for a more precise assessment of treatment response and prognosis, addressing a critical unmet need in the management of advanced lung adenocarcinoma.
The study demonstrated that baseline levels of ctDNA EGFR mutations and changes in mutation levels during treatment are strongly correlated with patient outcomes. Patients with lower baseline DCt values (≤8.11) and those who achieved mutation clearance after one month of treatment had significantly longer OS, suggesting that these parameters could be used to identify patients who are more likely to benefit from targeted therapy. Additionally, the quantitative analysis of changes in DCt values provided early insights into treatment efficacy, potentially enabling clinicians to adjust therapeutic strategies before disease progression becomes evident on imaging.
The concept of molecular response, as measured by changes in ctDNA EGFR mutation levels, offers a complementary approach to traditional imaging-based assessments. The study found that patients in the MC group and those with a change in DCt value >4.89 had higher proportions of stable disease (SD) and partial response (PR) upon RECIST evaluation, further supporting the utility of ctDNA analysis in predicting treatment outcomes. This approach aligns with the emerging trend of incorporating liquid biopsy into cancer staging and treatment planning, as proposed by the TNMB (TNM staging by liquid biopsy) classification system.
While the study provides valuable insights, it is not without limitations. The small cohort size and potential bias from the time interval between tissue and blood sample collection may have influenced the results. Additionally, the impact of subsequent treatments on OS should be considered when interpreting the findings. Nevertheless, the study lays the groundwork for future large-scale clinical trials to validate the use of R-superARMS in routine clinical practice.
In conclusion, R-superARMS represents a significant advancement in the detection and monitoring of EGFR mutations in plasma ctDNA. By providing a semi-quantitative measure of mutation levels, this method enhances our ability to predict treatment response and prognosis in patients with advanced lung adenocarcinoma. The integration of ctDNA analysis into clinical practice has the potential to improve patient outcomes through more personalized and timely therapeutic interventions.
doi.org/10.1097/CM9.0000000000001760
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