Detection of mutations in EGFR in circulating lung-cancer cells

Abstract

Background: The use of tyrosine kinase inhibitors to target the epidermal growth factor receptor gene (EGFR) in patients with non-small-cell lung cancer is effective but limited by the emergence of drug-resistance mutations. Molecular characterization of circulating tumor cells may provide a strategy for noninvasive serial monitoring of tumor genotypes during treatment.

Methods: We captured highly purified circulating tumor cells from the blood of patients with non-small-cell lung cancer using a microfluidic device containing microposts coated with antibodies against epithelial cells. We performed EGFR mutational analysis on DNA recovered from circulating tumor cells using allele-specific polymerase-chain-reaction amplification and compared the results with those from concurrently isolated free plasma DNA and from the original tumor-biopsy specimens.

Results: We isolated circulating tumor cells from 27 patients with metastatic non-small-cell lung cancer (median number, 74 cells per milliliter). We identified the expected EGFR activating mutation in circulating tumor cells from 11 of 12 patients (92%) and in matched free plasma DNA from 4 of 12 patients (33%) (P=0.009). We detected the T790M mutation, which confers drug resistance, in circulating tumor cells collected from patients with EGFR mutations who had received tyrosine kinase inhibitors. When T790M was detectable in pretreatment tumor-biopsy specimens, the presence of the mutation correlated with reduced progression-free survival (7.7 months vs. 16.5 months, P<0.001). Serial analysis of circulating tumor cells showed that a reduction in the number of captured cells was associated with a radiographic tumor response; an increase in the number of cells was associated with tumor progression, with the emergence of additional EGFR mutations in some cases.

Conclusions: Molecular analysis of circulating tumor cells from the blood of patients with lung cancer offers the possibility of monitoring changes in epithelial tumor genotypes during the course of treatment.

Figure 1. Correlation between the Presence of T790M Mutations in Tumor-Biopsy Specimens and Decreased Progression-free Survival

In patients with non–small-cell lung cancer with EGFR mutations who were receiving therapy with gefitinib or erlotinib, the presence of the drug-resistance mutation T790M before initiation of treatment was associated with decreased progression-free survival.

Figure 2. Serial Analyses of Circulating Tumor Cells during Therapy

Panel A shows serial analyses of the numbers of circulating tumor cells (CTCs) per milliliter (red curve) and the radiographic tumor burden in centimeters (blue curve) in four patients with non–small-cell lung cancer with EGFR mutations, as measured at multiple time points during the course of treatment with gefitinib, another chemotherapy agent (chemo), or an experimental agent (exp). The duration of each therapy is indicated by the gray bars. The genotypes of circulating tumor cells are shown for various time points. Mutations in brackets are those that were present at low allele frequencies. In Panel B, SARMS analysis of EGFR genotypes in Patient 9 shows an increased abundance of the T790M drug-resistance allele during disease progression. Arrows denote the cycle of threshold for amplification cycles (Ct) required for detection of the primary mutation (Del or Deletion, referring to the grouped exon 19 deletions) and the T790M mutation, as compared with the exon 2 control. ΔCt reflects the difference in allele frequency between the primary mutation and T790M in the tumor-biopsy specimen, the circulating tumor cells that were isolated at the time of response to gefitinib therapy, and the circulating tumor cells that were isolated at the time of disease progression. Luminescence was measured quantitatively in relative light units.

Figure 3. Tracings of EGFR Nucleotide Sequencing from the Tumor and Circulating Tumor Cells of Patient 2.<

br>Analysis of a tumor-biopsy specimen from Patient 2 shows a T751_I759delinsS mutation that is distinct from the Del 746_750 mutation present in the analysis of circulating tumor cells (CTCs). The 27 nucleotides that were deleted in the tumor (black box) are present in the DNA in the CTCs (red box), whereas the 15-nucleotide deletion in DNA in the CTCs (black box) is present in the tumor DNA (red box). The tracing of CTCs represents direct nucleotide sequencing of DNA lysed from cells captured on the CTC-chip, indicating a high degree of captured tumor-cell purity.