Amplification, mutation and loss of heterozygosity of the EGFR gene in metastatic lung cancer

Dear Sir, We read with interest the recent report of Matsumoto et al. on the high prevalence of EGFR gene mutation in brain metastases of lung adenocarcinoma. Although the authors also investigated KRAS gene mutation, there was no information on the copy number of EGFR gene. An association between EGFR gene amplification and mutation has been documented in patient samples as well as lung cancer cell lines, and predicts for clinical response to gefitinib therapy, although survival may not be improved in patients having these genetic disease markers. We report the molecular characterization of combined EGFR gene amplification, mutation and loss of heterozygosity (LOH) in a patient who suffered from nonsmall cell lung cancer (NSCLC) with brain metastasis. A 50-year old previously well Chinese female presented with on and off dizziness for a week prior to hospital admission. There was no headache, nausea or vomiting. She was a smoker and social drinker. Physical examination showed cerebellar signs in the form of dysdiodokokinesia and left past pointing. Computed tomography revealed a 2-cm heterogeneous rim-enhancing mass at the left cerebellar hemisphere with surrounding edema. A chest radiograph showed a nodule in the lower lobe of the left lung. The carcinoembryonic antigen (CEA) level was elevated to 85.9 lg/L, while the upper limit for smokers being 5 lg/L. A positron emission tomography (PET) scan showed a hypermetabolic lung mass with SUVmax of 12.1 suggestive of malignancy, and a hypodense lesion in the left cerebellum with a rim of FDG uptake and central photopenia. No metastatic lesion was detected elsewhere. The radiological diagnosis was lung cancer with solitary cerebellar secondary. The patient underwent surgical resection of both lesions. Left lower lobectomy showed moderately differentiated invasive adenocarcinoma with bronchioloalveolar morphology. Pleural involvement was noted. The brain lesion was confirmed to be metastatic adenocarcinoma. The patient was subsequently put on gefitinib as neoadjuvant therapy as tumor cells harbored EGFR mutation. A follow-up PET scan 1 year after operation showed postoperative changes only in the lung and brain. There was no evidence of residual tumor or recurrence. The latest CEA level was normal at 2.4 lg/L. Detection of EGFR tyrosine kinase (TK) domain mutations was performed on paraffin-embedded tissue enriched for tumor cells by microdissection. Genomic DNA was extracted with QIAGEN DNA Mini Kit (QIAGEN, Hilden, Germany) according to instructions of the manufacturer. Mutation analysis was performed by PCR amplification of exons 18, 19, 20 and 21 together with flanking intronic sequences, followed by direct nucleotide sequencing of PCR product. PCR amplification was performed in accordance with the published protocol. Amplicons were purified using QIAquick PCR Purification Kit. Bidirectional nucleotide sequencing was performed using BigDye Terminator v3.1 Cycle Sequencing Kit and ABI 3130xl genetic analyzer (Applied Biosystems, Foster City, CA). Sequences of PCR products were compared with the cDNA sequence and amino acid sequence of EGFR gene (NM 005228.3) using SeqScape software (Applied Biosystems, Foster City, CA) and manual review. Although DNA extracted from peripheral blood leucocytes showed normal EGFR gene sequence (Figure 1a), there was deletion of 18 nucleotides (c. 2240_2257del) at exon 19 (Figure 1d) in tumor samples from both the brain (Figure 1b) and lung (Figure 1c), resulting in the deletion of 7 amino acids and insertion of 1 additional serine residual (p. Leu747_Pro753delinsSer). Unexpectedly, the wild-type allele was absent in both specimens, suggesting homozygous deletion pattern. To investigate whether the absence of wild-type sequence was because of the gene deletion, the copy number of EGFR gene and chromosome 7 was determined by FISH using Vysis LSI EGFR/CEP 7 dual color probe system, labeled with fluorophores SpectrumOrange and SpectrumGreen, respectively (Vysis, IL). Sections of formalin-fixed paraffin-embedded brain and lung tissues of 4 lm thick were cut and mounted on positively charged slides. The slides were treated with the Vysis Paraffin Pretreatment Reagent Kit II before hybridization. Blood smear was prepared on untreated slides and processed with the Vysis FISH Pretreatment Kit. After the pretreatment steps, 10-lL probes mix was added and incubated at 37 C for 16 hr. All denaturation and hybridization steps were performed with Vysis HYBrite system. Unbound probe was removed with rapid wash procedure. Nucleus was counterstained with DAPI reagent. Hybridization result was visualized by fluorescence microscopy and the probe signals within 40 tumor cell nuclei were enumerated. The ratio of fluorescence signals from EGFR gene to the chromosome 7 centromeric probe was then determined. Leucocytes from peripheral blood (Figure 2a) showed 2 orange signals for EGFR and 2 green signals for chromosome 7, consistent with normal pattern. All tumor cells in brain (Figure 2b) analyzed showed 2 green signals for chromosome 7 but a large cluster of more than 20 orange signals for the EGFR gene, morphologically consistent with the deletion of 1 allele and amplification of the other