[Frontiers in Bioscience 16, 2961-2969, June 1, 2011]
Detection of EGFR and K-ras mutations for diagnosis of multiple lung adenocarcinomas
Teruo Iwata1, Kenji Sugio1,2, Hidetaka Uramoto1, Sohsuke Yamada3, Takamitsu Onitsuka1, Naohiro Nose1, Kenji Ono1, Mitsuhiro Takenoyama1, Tsunehiro Oyama4, Takeshi Hanagiri1, Kosei Yasumoto1
1 Second Department of Surgery, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan, 2 Department of Thoracic Oncology, National Kyushu Cancer Center, Fukuoka, Japan, 3 Department of Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan,4Department of Environmental Health, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
TABLES OF CONTENTS
1. ABSTRACT
The incidence of multiple primary lung adenocarcinoma (MPLA) is increasing, and it is important to distinguish MPLA from intrapulmonary metastasis (IPM) in order to determine the therapeutic strategy. However, there is no reliable method to differentiate between the two. The purpose of this study was to distinguish MPLA from IPM based on the gene status of EGFR and K-ras and the morphological Noguchi classification system. Sixty-eight tumors from 34 cases of clinical MPLA were evaluated. Of them, 11 cases (32.4%) were diagnosed as biological MPLA (bMPLA) by EGFR/K-ras mutation analyses, and 12 cases (35.3%) by morphological analysis. In all, 23 of the 34 cases (67.6%) were diagnosed as bMPLA. The remaining 11 cases were diagnosed as biological IPM (bIPM). The 5-year survival rates of bMPLA and bIPM were 90.9% and 63.6%, respectively (p = 0.04). These findings suggest that the combination method including gene mutation and morphological analysis can guide treatment decisions and that there is a need for systemic chemotherapy, and surveillance monitoring.
2.INTRODUCTION
Lung cancer is the leading cause of cancer mortality in developed countries including Canada, France, Italy, UK, USA, and Japan (1). Lung tumors are classified into 2 broad classes, namely, small cell lung cancer (SCLC), accounting for about 15% of cases, and non-small cell lung cancer (NSCLC), which is the most common form of lung cancer, accounting for up to 85% of all cases (2, 3). The frequency of occurrence of the adenocarcinoma subtype of NSCLC has increased in recent years (4). New developments, such as high-resolution CT, have enabled the detection of small lung tumors and synchronous multiple primary lung cancers (MPLC) (5, 6). The incidence of a second lung cancer in patients who survive after resection of NSCLCs is also increasing (7).
Multiple genetic and epigenetic alterations, such as oncogene and tumor suppressor gene mutations, DNA repair gene variants, microsatellite instability, hypermethylation, and potential inherited predispositions are involved not only in tumor carcinogenesis and tumor progression (8-10) but also in preneoplastic lesion (11). Mutations in K-ras or in the epidermal growth factor receptor (EGFR) gene arise early in the pathogenesis of lung adenocarcinoma and are related with the appearance of the malignant phenotype (12, 13).
Recently, EGFR has emerged as an attractive therapeutic molecular target in patients with adenocarcinoma harboring EGFR mutations (14, 15). In addition, mutations in K-ras, a downstream effector of EGFR signaling, are mutually exclusive with EGFR mutations, and are associated with resistance to EGFR tyrosine kinase inhibitors (16, 17).
Noguchi et. al. histologically classified small peripheral adenocarcinomas measuring 2 cm or less at their greatest dimension into 6 types (types A-F) on the basis of tumor growth patterns (i.e., Noguchi type) (18, 19). Noguchi types A, B, and C exhibit replacement growth of alveolar-lining epithelial cells and are classified as bronchioloalveolar carcinomas (BAC). Noguchi types A and B are considered noninvansive carcinomas in situ (CIS) and should be diagnosed as a primary cancers, and not metastatic cancers.
In patients with multiple lung adenocarcinomas, discrimination between multicentric lung adenocarcinoma and intrapulmonary metastasis (IPM) is important for determining treatment strategies although it is sometimes difficult to distinguish between these 2 malignancies (20). The aim of this study was to differentiate multiple primary lung adenocarcinomas (MPLA) from IPM in 34 cases by determining the mutational status of the EGFR and K-ras genes and by histological classification into Noguchi types A and B.
3. MATERIALS AND METHODS
3.1. Patients
A total of 944 patients with primary lung cancer who had undergone surgical resection between April 1994 and March 2007 at the University of Occupational and Environmental Health, Kitakyushu, Japan were included in the study. Of the 944 patients with primary lung cancer, 42 (4.4%) were diagnosed as synchronous or metachronous clinical multiple primary lung adenocarcinomas (cMPLA) according to the criteria proposed by Martini and Melamed (21). Eight patients were excluded from this study because the corresponding tumor DNA was unavailable. Finally, 68 adenocarcinoma samples from 34 patients with cMPLA (24 patients: synchronous cMPLA, 10 patients: metachronous cMPLA) were examined for EGFR and K-ras mutations and evaluated according to the histological classification of Noguchi types A and B.
The characteristics of the patients with cMPLA are shown in Table 1. The patients consisted of 22 men and 12 women, with an average age of 68.3 years (range, 46 to 81 years old). There were 13 never-smokers, and 21 ever-smokers, including 7 former and 14 current smokers and smoking status was evaluated as pack-years (PY; pack/day � years). Among the 24 synchronous tumors, a tumor larger than the other tumors in the greatest dimension was defined as the first tumor. Ten patients were metachronous with a median interval of 59.6 months (range, 26.3 to 144.6 months). The tumor stage was classified according to the Revisions in the International System for Staging Lung Cancer (22). All of the second tumors were evaluated as stage IA. The study was approved by the institutional review board and informed consent for the use of the tumor specimens was obtained from all the patients.
3.2. Histological classification of tissues into Noguchi types A and B
All tissues were formalin-fixed and processed in the same manner according to standard histological practices. A 3 μm thick formalin-fixed paraffin-embedded tissue section was prepared from each of the 68 tumors from 34 patients. All of the sections were stained with hematoxylin and eosin and then examined by light microscopy. Each specimen was diagnosed histologically, using World Health Organization criteria (2) and the Noguchi classification (19). Pulmonary adenocarcinomas 20 mm in diameter or less are defined in the Noguchi classification as follows. Type A: Localized bronchioloalveolar carcinoma (Figure 1), Type B: Localized bronchioloalveolar carcinoma with foci of alveolar structure collapse, Type C: Localized bronchioloalveolar carcinoma with foci of active fibroblastic proliferation, Type D: Poorly differentiated adenocarcinoma, Type E: Tubular adenocarcinoma, Type F: Papillary adenocarcinoma with a compressive growth pattern (Figure 2).
3.3. Detection of EGFR and K-ras mutations
All tissues were formalin-fixed and processed in a similar manner according to standard histological practices. Mutations in the EGFR and K−ras genes were examined in 2 cancer tissue specimens from each of the 34 patients. Formalin-fixed paraffin-embedded tissue sections of 3 μm were prepared from 68 samples obtained from 34 patients. The relevant areas of adenocarcinoma were precisely microdissected from hematoxylin and eosin-stained slides of paraffin-embedded sections. Genomic DNA was extracted from microdissected adenocarcinoma cells using a QIAamp DNA Micro Kit (QIAGEN, Valencia, CA). Mutational analysis of the exon 19 deletion and exon 21 L858R point mutation of the EGFR gene was performed as previously described (23). As shown in Figure 3, both mutations were easily detectable. A modification of the designed RFLP method using nested PCR reactions was used to detect point mutations in codon 12 of the K-ras gene (13, 24).
3.4. Statistical analysis
Surgical curves were plotted according to the Kaplan-Meier method, and differences between the curves were analyzed by the log-rank test. A statistical difference was considered to be significant if the P value was less than 0.05. All data were analyzed with the use of Abacus Concepts, Survival Tools for StatView (Abacus Concepts, Inc., Berkeley, CA).
4. RESULTS
4.1. Morphological analysis based on histological classification into Noguchi types A and B
The tumor sizes ranged from 3 to 68 mm in diameter. Tumors measuring 2 cm or less in their greatest dimension were observed in 50 cases. Seventeen tumors were classified as Noguchi's type A or B (Table 2). Three tumors were identified as Noguchi type A and 7 tumors were designated as Noguchi type B among the first tumor. Three tumors were determined to be Noguchi type A and 4 tumors as Noguchi type B among the second tumors. One patient was diagnosed as Noguchi type A in both the first and the second tumors. Fifteen patients were diagnosed as Noguchi type A or B either in the first tumor or in the second tumor. These 16 (47.1%) patients were diagnosed as having biological multiple primary lung adenocarcinomas (bMPLA), because Noguchi types A and B tumors were considered as non-invasive CIS.
4.2. EGFR and K-ras mutations in the first and the second tumors
EGFR mutations were detected in 25 of 68 tumors (36.7%) from 34 patients. The EGFR mutations were detected in 13 of 34 first tumors (38.2%) and in 12 of 34 second tumors (35.3%) (Table 1). An L858R mutation in EGFR was detected in both the first and the second tumors in 4 patients. A deletion (2236-2250) in exon19 of EGFR in both the first and the second tumors was detected in 2 patients (Table 3). The EGFR mutations detected in the second tumor were the same as the EGFR mutations of the first tumor in these 6 patients. The EGFR genotypes of the second tumor were different from the EGFR genotypes of the first tumor in 9 patients (Table 3).
K-ras mutations were detected in 4 out of 68 tumors (5.9%) in 3 patients (Table 1). The K-ras mutation detected in the second tumor was the same as the K-ras mutation of the first tumor in 1 patient. The codon 12 mutation of K-ras was detected in the first tumor but not in the second tumor in 1 patient. The codon 12 mutation of K-ras was not detected in the first tumor, but was detected in the second tumor in 1 patient (Table 3).
4.3. Combined analyses by mutational status of EGFR and K-ras genes and according to Noguchi types A and B
EGFR or K-ras mutations were detected in 18 patients. The genotypes of the second tumor were different from the genotypes of the first tumor in 11 of 18 patients and these 11 patients were confirmed to have bMPLA (Table 3 and Figure 4). The mutations of the second tumor were the same as the mutations of the first tumor in 7 patients (Table 3 and Figure 4). Patients were confirmed to have bMPLA by histological classification according to Noguchi types A and B in 4 of the 7 patients (Figure 4). On the other hand, there were no mutations in EGFR or K-ras in 16 patients. A total of 8 out of 16 patients were confirmed to have bMPLA based on histological classification into Noguchi types A and B (Figure 4). In total, 23 of 34 (67.6%) patients were confirmed to have bMPLA through combined analysis by mutational status of EGFR and K-ras genes and by Noguchi types A and B classification. Another 11 cases were confirmed as biological intrapulmonary metastasis (bIPM). The 5-year survival rate for cMPLA was 81.7% (Figure 5A). The 5-year survival rates in the case of bMPLA and bIPM were 90.9% and 63.6%, respectively (P = 0.04, Figure 5A). The 5-year survival rates for bMPLA with a different genotype (n = 11) and bMPLA with Noguchi's A or B at least in 1 tumor (n = 12) were 90.0% and 91.7%, respectively (P = 0.58, Figure 5B).
5. DISCUSSION
The incidence of clinical multiple primary lung cancers (cMPLC) has been reported to be 4% (25). Retrospective data have shown an increased risk of developing a second lung cancer following the diagnosis of the first lung neoplasm: patients with NSCLC have a 1-2% per year risk of developing a second primary lesion (7, 26). With the progress of imaging diagnostic technology, the detection of a synchronous combination of well differentiated adenocarcinomas in cMPLA is increasing (6). Lung cancers of the same pathological type are found in 50-68% of patients with cMPLC (27-30), while multiple adenocarcinomas of the same subtype are present in 61-68% of patients with cMPLC (30, 31), suggesting that the majority of cMPLAs have the same etiology.
Mutations in the EGFR kinase domain are thought to be an early event in the development of adenocarcinoma that is generally unrelated to smoking (32, 33), while K-ras mutations may occur relatively early in the development of smoking-related adenocarcinoma (13, 34). Therefore, these 2 mutations are useful biomarkers to identify different etiologies in lung adenocarcinoma. EGFR and K-ras mutations were mutually exclusive in this study, as previously reported in several studies (23, 35-37). The KRAS-MAPK pathway is 1 of the EGFR downstream signaling pathways, indicating that at least 1 activating mutation in the EGFR-KRAS-MAPK signaling pathway may be sufficient for the carcinogenesis of lung adenocarcinoma.
Patients who have been clinically diagnosed with multiple primary lung cancers sometimes show poor 5-year survival rates (38). These results suggest that some of the cMPLCs might be metastatic lesions (bIPM). In addition, bMPLC and bIPM may show different biological behaviors. Because some of the patients with clinical intrapulmonary metastasis (cIPM) show poor prognosis, it is necessary to discriminate bIPM from cMPLC (39). The distinction between cMPLC and cIPM is often difficult because these 2 malignancies can show the same histological type. A multiple gene analysis has been proposed to identify the clonality in a combination of mutations in genes such as p53, K-ras and chromosomal LOH (40-42). Ono et al. showed that the protein expression profile of cancer-related genes (p53, p16, p27, and c-erbB2) is a useful tool to distinguish between bMPLA and bIPM and to determine the appropriate biological staging of lung cancer (39). The criteria for the molecular diagnosis of bMPLC include a different type of mutation in the first and the second tumor, and a somatic mutation of a particular gene only in the first tumor. However, it is difficult to make a diagnosis of bMPLC when the first tumor shows a wild-type phenotype and the second tumor shows a mutated-type. In such a case, the second tumor may acquire the mutation during the metastatic process, or it may be ascribed to clonal expansion from a minor clone in the first tumor. Although EGFR and K-ras mutations occurred early in the process of carcinogenesis in the cases included in the present study, the genotypes of the second tumor were different from those of the first tumor in 11 of 18 patients and these 11 patients were confirmed to have bMPLA. The 5-year survival rates for bMPLA and bIPM were 90.9% and 63.6%, respectively, which was considered a statistically significant difference between these 2 groups (P = 0.04). One of the factors that may explain why bMPLA showed a better prognosis is the Noguchi type, i.e., types A or B are thought to show a good prognosis (19). In particular, the 5-year survival rate for 4 bMPLA patients who had different genotypes and Noguchi type A or B at least in 1 tumor was 100.0%.
Small peripheral adenocarcinomas measuring 2 cm or less in their greatest dimension were histologically classified into 6 types (Noguchi types A-F) on the basis of tumor growth patterns. Noguchi types A and B were considered noninvasive CIS, which are diagnosed as primary cancers. In total, 23 of 34 (67.6%) patients were confirmed as bMPLA by combined analysis of mutational status of EGFR and K-ras genes and by histological classification into Noguchi types A and B.
Among 18 patients with EGFR or K-ras mutations, 6 of 12 (50%) synchronous tumors exhibited the same mutation in EGFR or K-ras and 1 of 6 (17%) metachronous tumors exhibited the same mutation in EGFR or K-ras. These results suggest that the same mutation in EGFR or K-ras might be triggered by the same carcinogens as field cancerization (43).
The molecular mechanism underlying the development of lung adenocarcinoma with EGFR mutations is still poorly understood. EGFR mutations do not coexist with the G-to-T transversion in p53 and K-ras mutations that are related with tobacco smoke. EGFR mutations may not be associated with tobacco smoke. Matsuo et al. showed that female gender was a risk factor for lung adenocarcinoma with an EGFR mutation (33). Recently, we demonstrated an association between estrogen receptor (ER) expression and EGFR mutations in lung adenocarcinoma, i.e. strong ER beta expression was more frequently detected in adenocarcinomas with EGFR mutations (44). These data may suggest that endogenous gonadal hormones could possibly induce a field cancerization effect in lung adenocarcinomas. In conclusion, the genetic analyses of EGFR and K-ras genes and Noguchi's histological classification are useful in to distinguishing between bMPLA and bIPM among cMPLAs.
6. ACKNOWLEDGMENT
We thank Masako Fukumoto, Yukiko Goto, and Kanako Sasaki for technical assistance, and also thank Dr. Brian Quinn for critical comments in preparing this manuscript. This work was supported in part by Grand-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (NEXT), Japan.
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doi:10.1200/JCO.2008.18.3251
Abbreviations: SCLC, small cell lung cancer; NSCLC, non-small cell lung cancer; MPLC, multiple primary lung cancers; EGFR, epidermal growth factor receptor; BAC, bronchioloalveolar carcinoma; CIS, carcinoma in situ; IPM, intrapulmonary metastasis; MPLA, multiple primary lung adenocarcinomas; cMPLA, clinical multiple primary lung adenocarcinomas; PY, pack-years; bMPLA, biological multiple primary lung adenocarcinomas; bIPM, biological intrapulmonary metastasis; cMPLC, clinical multiple primary lung cancers; cIPM, clinical intrapulmonary metastasis
Key Words: Multiple primary lung cancers, Multiple primary lung adenocarcinomas, Intrapulmonary metastasis, EGFR, K-ras, Bronchioloalveolar carcinoma, biological multiple primary lung adenocarcinoma, biological intrapulmonary metastasis, Noguchi classification
Send corerspondence to: Kenji Sugio, Department of Surgery, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan, Tel: 81936917442, Fax: 81936924004, E-mail:kensugio@med.uoeh-u.ac.jp