Mechanisms of acquired resistance to EGFR-tyrosine kinase inhibitor in Korean patients with lung cancer

We reviewed the medical records of patients with NSCLC with EGFR mutations and acquired resistance to EGFR-TKI between 2007 and 2010. All patients fulfilled the definition of acquired resistance to EGFR-TKI [10], which was defined as having received treatment with a single agent EGFR-TKI, exhibiting objective clinical benefit from treatment, and then experiencing disease progression while under continuous treatment with EGFR-TKI. At the time drug resistance developed, some patients underwent post-resistance biopsy for evaluation of the mechanisms of resistance. We selected patients from whom the tissues obtained both before EGFR-TKI treatment and after resistance were sufficient to assess EGFR, KRAS, BRAF, and PIK3CA mutations by “Asan-Panel” analysis, perform fluorescence in situ hybridization (FISH) to identify MET amplification, and examine AXL status, EMT and neuroendocrine markers by immunohistochemistry. All patients provided informed consent, and the study was approved by the Institutional Review Board of the Asan Medical Center (Approval Number: 2011–0526).

A mass spectrometric genotyping technology, called the “Asan-Panel”, was used for genetic analysis. First, DNA was extracted from paraffin-embedded tissues using QIAamp DNA FFPE tissue kit (#56404; Qiagen, Hilden, Germany) according to the manufacturer’s protocol. DNA quantity was measured using the Quant-iT™ PicoGreen® dsDNA Assay kit (Invitrogen, Carlsbad, CA) andbrought to a final concentration of 5 ng/μl. Mutation analysis using the Asan-Panel was performed under the SequenomMassARRAY technology platform with iPLEX-Pro chemistry (Sequenom, San Diego, USA). The protocols that were previously performed as “OncoMap” [11‐13] were followed with minor modifications. In brief, specific assay pools were designed using AssayDesignersoftware in MassARRAY Typerpackage software (v4.0) with filters for proximal single nucleotide polymorphisms (SNPs) and assessment of the specificity of PCR amplification and the subsequent primer extension reaction. To decrease the number of multiplex PCR tubes, manual modification of some PCR primers and extension probes was conducted. A total of 59 amplicons were amplified in eight different multiplex pools with an average of 8-plex. After multiplex PCR, residual deoxynucleotides were inactivated by incubation with Shrimp Alkaline Phosphatase (Catalog No. 10142–2, Sequenom). Single-base extension (SBE) reaction products using a mixture of mutation site-specific probes were then spotted onto a 384-format SpectroCHIP II with the MassARRAY Nanodispenser. Mass determination was performed with the MassARRAY Analyzer Compact MALDI-TOF mass spectrometer, and MassARRAY Typer 4.0 software was used for data acquisition and analysis. Genotypes were called after cluster analysis using the default setting of the Gaussian mixture model. Genotype calls were then reviewed manually to identify any uncertain calls due to clustering artifacts. A total of 87 genetic mutations located in EGFR, KRAS, BRAF and PIK3CA genes were examined by Asan-Panel analysis.

For FISH, 2 μm-thick sections from each paraffin block were prepared. Deparaffinization, pre-treatment and protease digestion procedures were performed following the Abbott Vysis D7S522/CEP 7 FISH probe kit protocol (Abbott Laboratories, Abbott Park, Des Plaines, IL, USA). Probe mixtures were hybridized at 37°C for 14 to 18 hours. After hybridization, slides were washed in 2× SSC/0.3% NP-40 at 72°C for 2 min, air dried, and counterstained with 4,6-diamidino-2-phenylindole (DAPI). The slides were examined under a fluorescence microscope (Olympus, Tokyo, Japan) equipped with Spectrum Orange/Green dual and DAPI single filters. The slides were stored at -20°C until examination. A c-met/CEP7 ratio was established on the basis of a count of at least 60 cells by enumerating both orange (c-met) and green (chromosome 7, CEP7) signals. Samples with a c-met/CEP7 ratio greater than 2 were considered to have MET amplification.

All biopsy specimens underwent histologic review after H&E and immunohistochemical staining for specific markers, such as thyroid transcription factor 1 (TTF-1). For immunohistochemical analysis, paraffin sections (4 μm thick) were deparaffinized with xylene, rinsed thoroughly with ethanol, and then soaked in 0.03% hydrogen peroxide in methanol to inactivate the endogenous peroxidase activity. The sections were incubated with either 10% goat serum or 10% rabbit serum, and then incubated with the primary antibodies. The sections were washed with phosphate-buffered saline (PBS) and processed using a DAKO EnVision kit (DAKO, Los Angeles, CA), as directed by the manufacturer. The color was developed with 3,3′-diaminobenzindine (DAB) containing 0.3% H₂O₂. Primary antibodies against the following antigens were used: CD56, synaptophysin and chromogranin (Santa Cruz Biotechnology, Santa Cruz, CA) for SCLC transformation; E-cadherin and vimentin (Calbiochem, San Diego, CA) for EMT; AXL and p-AXL (R&D Systems, Minneapolis, MN) for AXL status.

Twenty-six patients were eligible for this study; of these, 10 patients (38.5%) were male and 16 (61.5%) were female. The median age was 58-years-old. All patients except one were diagnosed with adenocarcinoma of the lung with EGFR mutation at initial diagnosis. One patient had squamous cell carcinoma with a deletion mutation on exon 19 of EGFR. The deletion mutation on exon 19 of EGFR gene was present in 16 patients (61.5%), while the L858R point mutation on exon 21 was noted in 10 (38.5%). All patients were treated with gefitinib and showed a partial response. The secondary biopsy sites were lung (65.4%), mediastinal or cervical lymph nodes (19.2%), liver (7.7%), malignant pleural effusion (3.8%), and bone (3.8%). The biopsy site after resistance was same as the initial site in 15 patients (Table 1).

Secondary T790M mutation was detected in 11 patients (42.3%), four of which had additional resistance mechanisms: MET amplification was observed in two patients, increased AXL expression in one patient, and PIK3CA mutation in one patient. Increased AXL expression (Figure 1) was seen in 5/26 patients (19.2%), while MET gene amplification was noted in 3/26 patients (11.5%). One patient acquired an H1047L point mutation in the PIK3CA gene, which was accompanied by the T790M mutation. No patient exhibited evidence of EMT, whereas increased CD56 expression suggesting neuroendocrine differentiationwas observed in two patients. However, the morphologic change and expression of synaptophysin and chromogranin was not evident in these patients (Figure 2). Interestingly, conversion from L858R-mutant to wild-type EGFR occurred in one patient (Figure 3). Seven of the patients (26.9%) did not exhibit any known EGFR-TKI resistance mechanisms. The frequency of resistance mechanisms is shown in Figure 4.

Median progression-free survival (PFS) following gefitinib treatment was 11 months, and the median overall survival (OS) time was 32.3 months. PFS was significantly better in patients with secondary T790M mutation than in those without T790M (p = 0.009, Figure 5), while OS was not statistically different (p = 0.617, Figure 5).