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* From the Thoracic Center (Drs. Horiike, Ohyanagi, Satoh, Okumura, Nakagawa, Horai, and Nishio) and Department of Pathology (Dr. Ishikawa), Cancer Institute Hospital, Japanese Foundation for Cancer Research; and Shien-Lab (Drs. Kimura and Nishio), National Cancer Center Hospital, Tokyo, Japan.
Correspondence to: Makoto Nishio, MD, Cancer Institute Hospital, Ariake 3–10-6, Koto-ku, 135-8550, Tokyo, Japan; e-mail: mnishio{at}jfcr.or.jp
Abstract
Background: Somatic mutations of epidermal growth factor receptor (EGFR) are closely associated with an objective response to EGFR tyrosine kinase inhibitors. However, it is difficult to obtain sufficient tumor samples from patients with non-small cell lung cancer (NSCLC), so these diagnoses are often made using cytology procedures alone. The aim of this study was to detect EGFR mutations in transbronchial needle aspiration (TBNA) samples using both direct sequencing and a highly sensitive assay (Scorpions Amplified Refractory Mutation System; DxS; Manchester, UK) [ARMS], and to compare the sensitivity of these methods.
Methods: We enrolled 94 patients (63 men and 31 women) with NSCLC in this study. Cytologic diagnoses were adenocarcinoma (n = 58), squamous cell carcinoma (n = 24), and other types of NSCLC (n = 12). We extracted DNA from the TBNA samples, and EGFR mutations were analyzed using both direct sequencing (exons 19 and 21) and the Scorpions ARMS method (E746 A750del and L858R).
Results: Mutations were detected in 31 patients (33%; 14 women and 17 men). Of these, 23 patients had adenocarcinoma, 4 had squamous cell carcinoma, and 4 had other types of NSCLC. Direct sequencing detected 13 mutations (14%) in 13 patients (E746-A750del, n = 6; L858R, n = 7), and the Scorpions ARMS method detected 27 mutations (29%) in 27 patients (E746 A750del, n = 16; L858R, n = 11 patients).
Conclusions: Both methods detected EGFR mutations in TBNA samples, but Scorpions ARMS is more sensitive than direct sequencing.
Key Words: epidermal growth factor receptor • epidermal growth factor receptor mutation • epidermal growth factor receptor tyrosine kinase inhibitor • Scorpions Amplified Refractory Mutation System • transbronchial needle aspiration
Lung cancer is among the most common malignancies worldwide and one of the few types of cancer with an increasing incidence. Advanced non-small cell lung cancer (NSCLC) is treated with a combination of chemotherapy and radiotherapy, but the outcome remains poor. Gefitinib and erlotinib are inhibitors of the tyrosine kinase activity of epidermal growth factor receptor (EGFR), and have recently been used to treat advanced NSCLC.1 These agents are dramatically effective in some patients yet completely ineffective in others. The response rate to gefitinib is high among individuals with an Asian background.2
In May and June of 2004, two independent groups reported an association between somatic EGFR mutations and a dramatic clinical response to gefitinib, respectively.34 Thereafter, EGFR mutations were extensively investigated.567891011121314151617 The mutations consist of small, in-frame deletions or substitutions clustered around the adenosine triphosphate-binding site in exons 18, 19, and 21 of the EGFR gene, and approximately 90% of patients with EGFR mutations have one of two major mutations. One is a 15-base pair nucleotide in-frame deletion (E746 A750del) in exon 19, and the other is a point mutation involving the replacement of leucine with arginine at codon 858 (L858R) in exon 21.18 The above studies included genetic analyses of surgical tissues or biopsy specimens. However, to obtain sufficient amounts of tumor samples from inoperable NSCLC patients is often difficult. Some studies1920 of patients with advanced NSCLC have found a correlation between clinical manifestations and EGFR mutation status obtained from small tumor samples, such as those obtained using standard transbronchial lung biopsy (TBLB). All of the above studies are limited by the fact that the rate of usable samples obtained from enrolled patients is very low. Therefore, a method is required to detect mutant EGFR, especially the two major mutations, using samples other than surgical tissues from NSCLC patients. We addressed this problem using a sensitive technique for actual tumor sampling, and a highly sensitive assay for detecting EGFR mutations.
Pulmonary lesions are most often clinically diagnosed using flexible bronchoscopy. Common bronchoscopic sampling techniques used for pulmonary lesions are transbronchial needle aspiration (TBNA) and TBLB. One report has indicated that TBNA is superior to TBLB in diagnosing pulmonary lesions: Gasparini et al21 found that the diagnostic sensitivity of these techniques is 50.0% for TBLB, 70.1% for TBNA, and 76.0% for TBLB and TBNA together. We thus presumed that TBNA is a highly sensitive means of tumor sampling, and that DNA obtained from such specimens might provide useful information about the mutation status of the EGFR gene.
We postulated that Scorpions Amplified Refractory Mutation System (ARMS) [DxS; Manchester, UK] technology would enhance the sensitivity of detecting EGFR mutations. Scorpion primers are used with a fluorescence-based method that specifically detects polymerase chain reaction (PCR) products.22 A "scorpion" consists of a specific probe sequence held in a hairpin loop configuration by complementary stem sequences on the 5' and 3' ends of the probe. A scorpion can be combined with ARMS to enable the detection of single-base mutations.2223 The ARMS method is used for allele discrimination, and additional mismatches have been introduced near the 3' termini of the primers to enhance specificity. The ARMS method is superior to both direct sequencing and the WAVE method (Transgenomic; Omaha, NE) for detecting EGFR mutations.24 Here, we aimed to detect major EGFR mutations in TBNA specimens and to verify the sensitivity of these methods for detecting EGFR mutations.
Materials and Methods
Patients
We studied patients with NSCLC diagnosed using specimens obtained by TBLB and/or TBNA. Tumors in saline solution were not collected from enlarged lymph nodes only. After obtaining written informed consent from the patients to participate in all study protocols approved by the Institutional Review Board of the Cancer Institute Hospital, tumor tissues, tumors in saline solution obtained using TBNA, and clinical data were collected. We recorded age at diagnosis, gender, cytologic diagnosis of NSCLC, clinical stage, and smoking status. Cytologic diagnoses were based on the World Health Organization pathology classification. Clinicopathologic staging was determined according to the International Union Against Cancer TNM classification of malignant tumors. Nonsmokers were defined as those who had smoked < 100 cigarettes in their lifetime. We obtained detailed information about smoking history, including age at first cigarette, packs per day, and number of smoking and smoke-free years (after quitting). Patients were categorized as follows: never smoked (< 100 lifetime cigarettes), former smokers (quit 
1 year ago), or current smokers (quit < 1 year ago).
TBNA Sampling
Four experienced operators performed standard flexible bronchoscopy (Olympus P260F; Olympus; Tokyo, Japan) using 21-gauge cytology needles and aspirated for 10 s in the standard fashion.25 Paired samples consisted of two aspirates that were obtained in immediate succession in an identical manner, with the needle insertion points ideally 1 mm apart. At least four aspirates (two pairs) were obtained from each site. For cytologic analysis, the aspirate was immediately placed onto a glass slide, covered with a second slide, and the slides were drawn apart under continuous gentle pressure. The smear was spray-fixed using ethanol, processed routinely and visualized by Papanicolaou staining. The second aspirate was mixed into 2 mL of saline solution and stored at – 80°C until DNA extraction.
DNA Extraction
Samples obtained by TBNA in saline solution were digested with proteinase K, and then DNA was extracted with phenol-chloroform and precipitated with ethanol. Precipitated DNA was eluted in 50 µL of sterile, double-distilled water. The concentration and purity of the extracted DNA were determined by spectrophotometry and then the DNA was stored at – 20°C.
PCR Amplification and Direct Sequencing
Genomic PCR was performed in 25-µL volumes using 50 ng of template DNA, 0.75 U of AmpliTaq Gold DNA polymerase (Perkin-Elmer; Roche Molecular Systems; Branchburg, NJ), 2.5 µL of PCR buffer (Perkin-Elmer), 0.8 µmol/L deoxynucleotide triphosphate (Perkin-Elmer), 0.5 µmol/L of each primer, and various concentrations of MgCl2, depending on the polymorphic marker. Exons 19 and 21 were amplified by nested PCR. Primer sequences were obtained as described by Lynch et al.3 Initial PCR analyses proceeded in a volume of 25 µL as follows: 35 cycles of denaturation at 94°C for 45 s, primer annealing at 58°C for 30 s, and elongation at 72°C for 30 s. A final extension proceeded at 72°C for 10 min. Nested PCR was performed using 20 cycles under the same conditions as the initial PCR. The bands of PCR products were visualized using a 2100 bioanalyzer and the DNA 500 Labchip kit (Agilent Technologies; Palo Alto, CA). Each sample was sequenced in duplicate in both forward and reverse directions using the BigDye Terminator kit (Applied Biosystems; Foster City, CA) and an ABI prism 310 (Applied Biosystems) according to manufacturer instructions. The sequences were then compared with the GenBank-archived human sequence for EGFR (accession number AY588246).
Scorpions ARMS for the Detection of E746 A750del and L858R
We used the EGFR Scorpions kit, which combines two technologies, namely ARMS and Scorpions, to detect mutations in real-time PCR reactions. All reactions proceeded in 25-µL volumes using 1 µL of template DNA, 7.5 µL of reaction buffer mix, 0.6 mL of primer mix, and 0.1 mL of Taq polymerase. Real-time PCR was performed using a SmartCycler II (Cepheid; Sunnyvale, CA) under the following conditions: initial denaturation at 95°C for 10 min, 50 cycles of 95°C for 30 s, and 62°C for 60 s with fluorescence reading (set to FAM, which allows optical excitation at 480 nm and measurement at 520 nm) at the end of each cycle. Data were analyzed using Cepheid SmartCycler software (Version 1.2b). The cycle threshold (Ct) was defined as the cycle at the highest peak of the second derivative curve that represented the point of maximum curvature of the growth curve. Both Ct and maximum fluorescence were used for interpretation of the results. Positive results were defined as Ct 
45 and maximum fluorescence intensity 30. When only the curve that indicated the wild-type increased, the sample was considered wild-type with respect to EGFR. When both wild- and mutant-type curves increased, the sample was considered mutant-type with respect to EGFR. These analyses were performed in duplicate for each sample.
Statistical Analysis
The rates of EGFR mutation between the two groups were compared using 
2 or Fisher exact tests. The latter test was applied to five or fewer observations in a group. We used logistic regression models to further explore observed differences and to identify baseline factors that might independently predict an EGFR mutation. Probability values of < 0.05 were defined as being statistically significant. All statistical tests were two sided.
Results
Patient Characteristics
Ninety-four patients were enrolled in this study (63 men and 31 women; median age, 66 years) [Table 1
]. Among these, 58 patients had adenocarcinoma, 24 patients had squamous cell carcinoma, 5 patients had large cell carcinoma, 2 patients had other classifications of NSCLC, and 5 patients had unclassified NSCLC. Disease in 70 patients was diagnosed from both TBNA and TBLB samples, disease in 23 patients was diagnosed using only TBNA samples, and disease in 1 patient was diagnosed using TBLB samples alone (Table 2
). The DNA from TBNA samples in all 93 patients was extracted at a median concentration of 8.7 ng/µL (range, 0.1 to 39.0 ng/µL).
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Correlation With Responsiveness to Tyrosine Kinase Inhibitors
Only two patients received gefitinib, one of whom was a 63-year-old woman with a cytologic diagnosis of adenocarcinoma who had never smoked (patient 70). She had partially responded to gefitinib administered from September 2005 to August 2006. Her mutation status according to both direct sequencing and the Scorpions ARMS methods was L858R (Table 3). The other patient was a 69-year-old woman with a cytologic diagnosis of adenocarcinoma who had also never smoked (patient 94). Her condition had stabilized in response to gefitinib that had been administered from August 2005 to October 2005. We determined her mutation status as wild-type in exons 19 and 21.
Discussion
We demonstrated the feasibility of detecting EGFR mutations in DNA from TBNA samples from NSCLC patients. Furthermore, we showed that the diagnostic sensitivity of TBNA in our patients was higher than that of TBLB, which agreed with reported findings. The volume of DNA extracted from TBNA samples was measurable by spectrophotometry using our methods and was sufficient to analyze EGFR mutation status. Therefore, TBNA samples are apparently suited to such analysis. The mutation rate in this study was lower (33.3%) than that found by other studies of Japanese NSCLC patients.1112 However, in line with previous results, we detected EGFR mutations at a higher frequency in women, adenocarcinoma patients, and nonsmokers.69 We did not find a relationship between EGFR mutation status and response to EGFR tyrosine kinase inhibitors such as gefitinib. Only two patients had already received gefitinib at the time the study was implemented, and the others were to receive gefitinib as a second-line (or later) treatment. The relationship between EGFR mutation status and response to gefitinib will be determined in the near future.
The results of this study suggest that the Scorpions ARMS method is more sensitive than direct sequencing for detecting the two major EGFR mutations. Direct sequencing is currently the routine method of detecting EGFR mutations in tumor samples, and a standard method for detecting EGFR mutations in tumor specimens other than surgical tissues has been established. Our results indicated that the EGFR Scorpions Kit is superior to direct sequencing for detecting EGFR mutations, especially the major deletion mutations in exon 19 and L858R. We previously showed that EGFR mutation status in serum DNA detected using the Scorpions ARMS method is a useful predictive marker of the response to gefitinib. That study showed that Scorpions ARMS is more sensitive than direct sequencing for detecting EGFR mutations in a mixture of normal and mutant DNA.26 We inferred from these results that the differences in the determined mutation status for the 18 patients who tested positive using Scorpions ARMS and negative using direct sequencing are due to the density of tumor cells in the sample. However, the reason for the differences in the determined mutation status for those patients who tested negative using Scorpions ARMS and positive using direct sequencing remains obscure. The two methods detected different mutations in the same patient (patient 58), indicating that the primer for the deletion mutation of exon 19 can detect not only E746 A750del but also E746 S752del insA in the Scorpions ARMS method. The differences were frequent in patients with L858R in exon 21 (21.4% of patients with L858R, 5.9% of patients with other mutations). The sensitivity of Scorpions ARMS for detecting L858R was approximately equivalent to that for the detection of E746 A750del in our previous study. Some reports1927 have indicated that the presence of EGFR gene amplification is more predictive of responses than EGFR mutation. However, this does not alter the fact that an EGFR mutation is one predictor of response. To detect EGFR gene amplification from cytology samples is complicated by the difficulty of defining fluorescent in situ hybridization. Because there were few cancer cells in cytology samples, and these samples did not yield interpretable signals (data not shown).
Some investigators have tried to improve the sensitivity of detecting EGFR mutations. The novel peptide nucleic acid-locked nucleic acid PCR clamp method28 and the mutant-enriched PCR assay29 are both rapid and sensitive. Although the minimum detectable mutation volumes were not evaluated in these studies, the sensitivity of these methods seems to be comparable to that of Scorpions ARMS and thus sufficient for clinical use. Since the Scorpions ARMS method is simple and very fast, it might be suitable for mutation screening. However, one limitation of the EGFR Scorpions kit is that it can detect only mutations targeted by the designed Scorpions primers. Not all EGFR mutations are found at the two targeted sites, as some are clustered around the adenosine triphosphate-binding site in exons 18, 19, and 21.3456910 Minor variations of deletional mutations in exon 19, such as E747 P753del insS and L747 T751del, and point mutations other than L858R cannot be detected using Scorpions ARMS. Although approximately 90% of NSCLC-associated EGFR mutations comprise the two major EGFR mutations,18 others might be missed using Scorpions ARMS. Moreover, a secondary mutation, a substitution of methionine for threonine at position 790, leads to gefitinib resistance in NSCLC patients with EGFR mutations that are responsive to gefitinib.3031 These mutation states may also be critical factors for gefitinib therapy. Scorpions primers need to be designed to detect these mutations, and further study using these primers is required. In conclusion, both direct sequencing and Scorpions ARMS can detect EGFR mutations in DNA extracted from TBNA samples obtained from NSCLC patients, but the latter method is more sensitive.
Acknowledgements
We thank Dr. Stephan Little (DxS, Manchester, UK) for providing the EGFR Scorpions kit and for technical support.
Footnotes
Abbreviations: ARMS = Amplified Refractory Mutation System; Ct = cycle threshold; EGFR = epidermal growth factor receptor; NSCLC = non-small cell lung cancer; PCR = polymerase chain reaction; TBLB = transbronchial lung biopsy; TBNA = transbronchial needle aspiration
This work was performed at Cancer Institute Hospital, Japanese Foundation for Cancer Research.
This work was partially supported by funds for the Third Term Comprehensive 10-Year Strategy for Cancer Control and a Grant-in-Aid for Scientific Research.
The authors have no conflicts of interest to disclose.
Received for publication July 18, 2006. Accepted for publication January 9, 2007.
References
Related Editorial
This article has been cited by other articles:
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  D. S. Zander Transbronchial Fine-Needle Aspiration, Epidermal Growth Factor Receptor Mutations, and the Scorpion's Touch Chest, June 1, 2007; 131(6): 1619 - 1620. [Full Text] [PDF]
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