Epidermal growth factor receptor variant III mutations in lung tumorigenesis and sensitivity to tyrosine kinase inhibitors

Abstract

The tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) have shown anti-tumor activity in the treatment of non-small cell lung cancer (NSCLC). Dramatic and durable responses have occurred in NSCLC tumors with mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). In contrast, these inhibitors have shown limited efficacy in glioblastoma, where a distinct EGFR mutation, the variant III (vIII) in-frame deletion of exons 2-7, is commonly found. In this study, we determined that EGFRvIII mutation was present in 5% (3/56) of analyzed human lung squamous cell carcinoma (SCC) but was not present in human lung adenocarcinoma (0/123). We analyzed the role of the EGFRvIII mutation in lung tumorigenesis and its response to tyrosine kinase inhibition. Tissue-specific expression of EGFRvIII in the murine lung led to the development of NSCLC. Most importantly, these lung tumors depend on EGFRvIII expression for maintenance. Treatment with an irreversible EGFR inhibitor, HKI-272, dramatically reduced the size of these EGFRvIII-driven murine tumors in 1 week. Similarly, Ba/F3 cells transformed with the EGFRvIII mutant were relatively resistant to gefitinib and erlotinib in vitro but proved sensitive to HKI-272. These findings suggest a therapeutic strategy for cancers harboring the EGFRvIII mutation.

Fig. 1.

Identification of EGFRvIII in human NSCLC at both RNA and genomic DNA levels. (A Left) Detection of WT EGFR (929 bp) and EGFRvIII (128 bp) in NSCLC by RT-PCR. Lanes are as follows: M, marker; 1, 97-19-tumor; 2, 4040-tumor; 3, 4050088A2-tumor; 4, 54943-tumor; 5, 0119-tumor; 6, 0119-normal tissue; 7, 088V-tumor; 8, 3811-tumor; 9, H₂O. Lanes 7 and 8 serve as negative controls. (A Right) A representative sequencing data of the 128-bp band identifies the EGFRvIII transcript shown from 0119 tumor. (B) Long-range PCR products within the EGFR locus identified the EGFRvIII breakpoint in tumors 0119 (Left, lane 2) and 97-19 (Left, lane 4) but not in normal corresponding genomic DNA controls (Left, lane 1 for 0119 and lane 3 for 97-19, respectively). DNA sequence analyses of the PCR products reveal 79,583-bp and 49,785-bp interstitial deletions of EGFR locus in 0119 tumor (Center) and 97-19 tumor (Right), respectively. (C) FISH analysis (as described in Materials and Methods) confirmed the presence of EGFRvIII in 4040 frozen tissue sections. The EGFR probe spanning the entire locus was labeled with Cy3 (green), and the EGFR exon 2–7 probe was labeled with Cy5 (red). Statistical analysis showed that 60.4% of 4040 tumor cells analyzed have only one copy of exon 2–7 in comparison (Left) with 9.7% in normal tissue (Right). P < 0.0001 using Student's exact t tests.

Fig. 2.

Immunostaining analyses of the lung SCC tumor samples positive for EGFRvIII. Immunostaining of EGFRvIII-specific antibody DH8.3 was preformed as described in Materials and Methods. (A) Glioblastoma sample positive for EGFRvIII from both RT-PCR and FISH analysis serves as positive control. (B) The three SCC tumors identified from the RNA and genomic DNA for the presence of EGFRvIII were positive for EGFRvIII staining. Representative immunostaining is shown for the 97-19 tumor. (C) The SCC tumor confirmed to be negative for EGFRvIII serves as negative control. (D) These SCC samples positive for EGFRvIII staining are also stained positive for phospho-EGFR (Y1068). Representative immunostaining photo is shown from a 97-19 tumor.

Fig. 3.

Expression of EGFRvIII in mouse lungs leads to the development of lung cancer in vivo. (A) Monotransgenic Tet-op-EGFRvIII mice had normal lungs with no lesions observable after 4 months of doxycycline administration. (B and C) Bitransgenic mice and Tet-op-EGFRvIII/CCSP-rtTA mice develop atypical adenomous hyperplasia (AAH) in the lungs after ≈6–8 weeks of doxycycline administration. (D and E) Large lung adenoma after 12 weeks of doxycycline treatment. (F and G) Adenocarcinomas after 16 weeks of doxycycline induction. The higher-magnification image (G) shows multiple giant and pleomorphic nuclei (arrows). (H and I) Tumor regression after doxycycline withdrawal. After 12 weeks of doxycycline administration, Tet-op-EGFRvIII/CCSP-rtTA mice with documented lung tumors by MRI were given fresh water without doxycycline for 1 week before lung harvest.

Fig. 4.

Treatment of the lung tumors in the Tet-op-EGFRvIII/CCSP-rtTA, Ink4A/Arf^−/− mice with EGFR inhibitors. The Tet-op-EGFRvIII/CCSP-rtTA, Ink4A/Arf^−/− mice given doxycycline water for >8 weeks were imaged with MRI (see Materials and Methods) before treatment with vehicle (A), erlotinib (B), or HKI-272 (C). The same mice were imaged again after 1 week of daily gavage with vehicle (D), erlotinib (E), and HKI-272 (F). All mice were given fresh water with doxycycline throughout the experiment. The red arrowheads point to the heart of each mouse. The green arrowheads point to the individual tumor nodules.

Fig. 5.

HKI-272 is effective in growth inhibition of Ba/F3 cells transformed with EGFRvIII, EGFR-L858R, and EGFR-L858R-T790M but fail to inhibit the Ba/F3 cells transformed with EGFRvIIIC530S. The sensitivity of various EGFR mutant-transformed Ba/F3 cells to EGFR inhibitors in the absence of IL-3 was determined by the MTS assay. The percentage of cell viability is shown relative to untreated controls. Results are indicated as mean ± SD. IC₅₀ shown in the text was determined from these dose-response curves by using the xlfit 4 software package (IDBS). (A and B) EGFRvIII mutant (IC₅₀ = 432 and 576 nM) is more resistant than EGFR-L858R mutant (IC₅₀ = 10.8 and 12.5 nM) to gefitinib (A) or erlotinib (B), respectively. (C) HKI-272 inhibits growth of both EGFRvIII- and L858R-T790M-transformed Ba/F3 cells (IC₅₀ = 9.4 nM and 179 nM, respectively). (D) EGFRvIII-transformed Ba/F3 cells are more sensitive to HKI-272 than to gefitinib or erlotinib treatment. (E) EGFRvIIIC530S-transformed Ba/F3 cells became resistant to HKI-272 treatment (IC₅₀ = 327 nM).