Intratumor heterogeneity of epidermal growth factor receptor mutations in lung cancer and its correlation to the response to gefitinib

Abstract

Somatic mutations introduced into the epidermal growth factor receptor (EGFR) gene in non-small-cell lung cancer (NSCLC) are important factors to determine therapeutic responses to gefitinib. The current diagnostic test measures the overall EGFR mutation status of the cancer tissue, and may ignore the presence of non-mutated, gefitinib-unresponsive cancer cells. Twenty-one NSCLC patients with EGFR mutations were recruited for the study. All patients were treated with gefitinib after surgical treatment. Fifty to sixty areas of NSCLC tumors were sampled from each tissue, and their EGFR mutation states were determined by a primer extension assay. This assay discriminates between EGFR mutation-positive and -negative cancer cells within a single tumor tissue. Fifteen tissues consisted only of cells with EGFR mutations, but the remaining six tissues contained both mutated and non-mutated cells. Time to disease progression and overall survival after gefitinib treatment were significantly shorter in those patients with EGFR heterogeneity (P = 0.009 and P = 0.003, respectively). A considerable proportion of NSCLC contains a heterogeneous population of both EGFR mutated and non-mutated cancer cells, resulting in a reduced response to gefitinib. The intratumor genetic heterogeneity of a target molecule such as EGFR would be an important factor to consider when treating patients with molecular target agents.

Figure 1

Outline of the analysis method. EGFR, epidermal growth factor receptor; LCM, laser capture microdissection.

Figure 2

An example of the intratumor heterogeneity of the epidermal growth factor receptor mutation. The tissue is patient no. 17 in Table 1. (Left) A microscopic view of a lung tumor section. Red circles indicate regions excised by laser‐capture dissection. (Right) Electropherograms of the SNaP shot assay of the corresponding regions. Blue peak, amplified fragment with ddG; green peak, amplified fragment with ddA.

Figure 3

Intratumor heterogeneity of the epidermal growth factor receptor (EGFR) gene. Vertical axis indicates the percentage of the mutated EGFR gene. Horizontal axis areas ordered by the percentage of the mutated gene. Blue regions correspond to the mutated genes, and purple regions correspond to the non‐mutated genes. Columns only consisting of purple regions represent areas consisting of non‐mutated cells. Each panel corresponds to the following patients: (a) 1; (b) 4; (c) 5; (d) 13; (e) 14; (f) 15; (g) 16; (h) 17; (i) 18; (j) 19; (k) 20; and (l) 21.

Figure 4

Kaplan–Meier analysis of group I (straight line) and II patients (dotted line). (a) Time to progression after gefitinib treatment. (b) Overall survival after gefitinib treatment. Vertical axis, fraction of patients with (a) no disease progression or (b) patient survival (b). Horizontal axis, time in months.