EGFR T790M detection and osimertinib treatment response evaluation by liquid biopsy in lung adenocarcinoma patients with acquired resistance to first generation EGFR tyrosine kinase inhibitors

Plasma and tumor tissue samples were collected from patients screened or enrolled in the ongoing ASTRIS study. ASTRIS (D5160C00022) is open label, multinational, multicenter, real world treatment study of single agent osimertinib for patients with advanced/metastatic EGFR T790M mutation positive NSCLC who have received prior therapy with an first generation EGFR-TKI. Patients were required to have the diagnosis of EGFR activating mutation positive NSCLC. Drug resistance was defined as disease progression after first generation EGFR-TKI (gefetinib, elortinib or ecotinib) treatment, which was evaluated by RECIST 1.1 criteria. EGFR T790M positive patients received osimertinib 80 mg Qd until disease progression evaluated by investigators.

Tumor tissue or plasma samples were collected from patients screened or enrolled in ASTRIS study, following progression on a previous EGFR-TKI, but prior to dosing with osimertinib. CT-guided needle lung biopsy or Supraclavicular lymph node biopsy samples were snap frozen and stored at − 80 °C. A total of 8 ml of Plasma sample from each patient were assigned for cobas and ddPCR testing, and 4 ml of plasma from the time point of first evaluation (6 weeks) after osimertinib treatment were evaluated by ddPCR. ctDNA isolation followed standard protocols. Cobas® Mutation Test v2 kit was used to detect both EGFR activating mutation and T790M in FFPE or plasma samples. The Cobas is an allele-specific polymerase chain reaction assay to detect known common EGFR mutations. Analysis was confirmed by negative and positive controls. The PCR reactions were run on the cobas®z 480 analyzer with EGFR Blood Analysis Package Software. A semiquantitative index (SQI) was generated to reflect the proportion of mutated and wild-type copies of the EGFR gene. The SQI was derived from a dilution series with known copy numbers of mutated EGFR and a fixed amount of wild-type EGFR, with the wildtype DNA copy as internal control. Bio-rad PrimePCR™ ddPCR™ Mutation Assay kit were used to detect T790M in plsma ctDNA. The RNase P Copy Number Reference Assay (Life Technologies, Carlsbad, California) was used to determine the total amount of DNA in plasma samples. A total of 3000–3500 genome equivalents were analyzed per reaction with a sensitivity between 0.1–0.5%.

Associations between mutations and clinical and biological characteristics were analyzed by χ² or Fisher’s exact test. All data were analyzed using the Statistical Package for the Social Sciences Version 16.0 Software (SPSS Inc., Chicago, IL). The two-sided significance level was set at p < 0.05.

The flowchart of study design was shown in Fig. 1. Among the 69 screened NSCLC patients, there were 23 men and 46 women. There were 22 smokers and 47 non-smokers, accounting for 31.9 and 68.1%, respectively. There were 67 adenocarcinoma and 2 Adenosquamous carcinoma. The number of patients in stages IIIB, IVA and IVB was 20, 21, and 28, respectively. The numbers of M1a, M1b and M1c patients were 19, 4 and 26. The detailed information is listed in Table 1.

The T790M mutation rate of FFPE tissue cobas, plasma cobas and plasma ddPCR were 54.5, 21.3 and 30.4% respectively. The T790M positive rate was 52.2% considering all testing methods. T790M mutation was detected by ddPCR in all of the plasma cobas positive cases. However, in 10 tumor tissue biopsy cobas negative cases, 3 were positive by plasma ctDNA ddPCR. The proportion of T790M positive cases detected by ddPCR in stage IIIB, IVA and IVB patients were 30, 47.6 and 57.1%, respectively (P = 0.176) (Fig. 2a). However, T790M positive defined by cobas plasma ctDNA test were significantly different in stage IIIB, IVA and IVB (Fig. 2b) or M1a, M2b and M1c patients (P = 0.003, 0.004), suggesting the advantages of ddPCR test in any stages of disease. The T790M quantification by ddPCR significantly rises in stage IIIB, IVA and IVB cases (P = 0.033), while no such trend was observed in different M1a, M2b and M1c cases (P = 0.178). No association was observed between T790M status and duration of first generation EGFR TKI treatment (Table 2).

In 23 patients received osimertinib treatment, the OOR was 60.9%. There were 14 patients evaluated as partial response (PR) and 8 were stable disease (SD), 1 patient experienced PR of liver metastasis tumor but progression of primary lung lesion. Quantification of T790M after 6 weeks of treatment decreased to very low level, while no association was observed between response status and T790M mutation level decrease (Fig. 3).