Analysis of EGFR, KRAS and P53 mutations in lung cancer using cells in the curette lavage fluid obtained by bronchoscopy

Lung Cancer 78 (2012) 201–206

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Analysis of EGFR, KRAS and P53 mutations in lung cancer using cells in the curette lavage fluid obtained by bronchoscopy Fumihiro Yamaguchi a,b,∗ , Satoshi Kugawa a , Hidetsugu Tateno a,b , Fumio Kokubu b , Kunihiko Fukuchi a a b

Department of Clinical Pathology, Showa University, School of Medicine, 1-5-8, Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan Department of Respiratory Medicine, Showa University Fujigaoka Hospital, 1-30, Fujigaoka, Aoba-ku, Yokohama 227-8501, Japan

a r t i c l e

i n f o

Article history: Received 12 June 2012 Received in revised form 16 August 2012 Accepted 26 August 2012 Keywords: EGFR KRAS P53 Mutation Direct sequencing Curette lavage fluid L747S

a b s t r a c t Histopathological samples are commonly used for molecular testing to detect both oncogenes and tumorsuppressor genes in lung cancer. The purpose of this study was to determine the efficacy of using curette lavage fluid for molecular testing to detect EGFR, KRAS and P53 mutations in lung cancer patients. Samples were obtained from 77 lung cancer patients by bronchoscopy at the time of diagnosis, collected by scraping the site of the primary tumor lesion with a curette. DNA was extracted from cells in the curette lavage fluid, and PCRs were performed to amplify mutation hot spot regions in the EGFR, KRAS and P53 genes. The PCR products were direct-sequenced to detect mutations of each gene. The reference sequence of each gene was obtained from GenBank. Overall, 27% (21 of 77) were found with EGFR mutations, 1% (1 of 77) with KRAS mutations, and 36% (28 of 77) with P53 mutations. KRAS mutations were not detected in patients harboring mutations in either EGFR or P53. P53 mutations were identified in 38% (8 of 21) of the patients with EGFR mutations, all of who had advanced lung cancer. Of these patients, a 62-year-old female current smoker was given EGFR-TKI as third-line therapy, with no improvement in clinical symptoms or results of radiographic examination. Multivariate analysis indicated that P53 mutation rates in advanced-stage lung cancer were significantly higher than those in early-stage lung cancer (P = .017). In contrast, EGFR mutation rates were not significantly associated with staging. L747S in EGFR, described as a mutation associated with secondary resistance to EGFR-TKI, was detected in three patients who had never received EGFR-TKI, including one SCLC patient. It is possible to analyze EGFR, KRAS and P53 mutations using curette lavage fluid collected from lung cancer patients. This is useful when sufficient amounts of tumor samples cannot be obtained. Data from the current study suggest that EGFR mutations in concert with P53 mutations accelerate cancer development and lead to evolution of therapeutic resistance. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The importance of gene mutations in lung cancer patients is well established. Various gene alterations have been uncovered in the carcinogenesis of lung cancer. In particular, it has been revealed that approximately 50% of non-small cell lung cancer (NSCLC) patients have mutations in some oncogenes [1]. There have been reports of an association between EGFR mutations and a significant clinical response to EGFR tyrosine kinase inhibitor (EGFR-TKI), and of the efficacy of EGFR-TKI as a first line treatment for NSCLC patients with such EGFR mutations [2,3]. Hence, the presence of EGFR mutations can be used to determine whether to administer EGFR-TKI to

∗ Corresponding author at: Department of Clinical Pathology, Showa University, School of Medicine, 1-5-8, Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan. Tel.: +81 3 3784 8577; fax: +81 3 3788 4927. E-mail address: f [email protected] (F. Yamaguchi). 0169-5002/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lungcan.2012.08.014

NSCLC patients. In contrast, EGFR-TKI is likely to be ineffective in NSCLC patients with KRAS mutations [4–6]. Since EGFR mutations do not occur concurrently with KRAS mutations [7–9], the ability to detect both types becomes important in making treatment decisions. Lung cancer arises as a result of accumulations of gainof-function mutations in oncogenes and loss-of-function mutations in tumor-suppressor genes [10]. P53 is a tumor-suppressor gene that regulates cell-cycle arrest, senescence and apoptosis. Loss of P53 function in cells through mutation might therefore be expected to lead to unchecked proliferation, tumor growth and therapeutic resistance [11,12]. Because activation of EGFR promotes tumor cell proliferation, invasion, and survival, P53 could play a role in determining EGFR-TKI sensitivity in lung cancer patients. Thus, mutation analyses of individual lung cancer patients should provide useful information for determining the optimal treatment regimen and establishing a prognosis. Originally, mutation analyses in many human cancers were carried out primarily on surgical specimens or biopsy samples.

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Likewise, in lung cancer histopathological samples are commonly used for molecular testing of both oncogenes and tumorsuppressor genes [7,13,14]. However, in clinical practice it is often difficult to obtain sufficient amounts of tumor samples from inoperable patients. When sufficient tissue samples for pathology cannot be collected by bronchoscopy, cytology is substituted to establish a diagnosis. Compared to surgical specimens or biopsy samples, collecting cytological materials is a less-invasive method that could also be beneficial in detecting mutations. In this manner, cytological specimens may be used for molecular biological analyses. Recently several studies have used cytological specimens to detect EGFR mutations in lung cancer patients [15,16]. However, there have been no studies investigating mutation status in oncogenes or tumor-suppressor genes using cytological specimens. In this study, EGFR, KRAS and P53 mutations in cells attached to the curette were analyzed by collecting and studying lavage fluid. 2. Methods 2.1. Samples Samples were obtained from 77 lung cancer patients receiving treatment from April 2009 to October 2010 at the Department of Respiratory Medicine, Showa University Fujigaoka Hospital. Official approval for the study was obtained in advance from the Ethics Committee for Genomic Research at Showa University (approved number 113). All patients gave their written informed consent. All samples were gathered by bronchoscopy at the time of diagnosis and were collected by scraping the site of the primary tumor lesion with a curette, then flushing with 5 ml of saline. Half of the lavage fluid so obtained was sent for cytological evaluation while the remaining fluid was stored at −80 ◦ C, until further needed. Cytological findings were not considered in selecting the patients. There were 40 patients for whom adequate histopathological and cytological specimens were both available, including 18 adenocarcinoma, 12 squamous cell carcinoma, one unclassified NSCLC, and nine small cell lung carcinoma (SCLC) patients. 2.2. Molecular testing The lavage fluid was centrifuged at 8000 rpm for 5 min at room temperature. DNA was extracted from cell pellets using the QIAmp DNA Mini kit (Qiagen, Valencia, CA) and stored at −20 ◦ C. The amount of DNA extracted from cells in the curette lavage fluid ranged from 600 to 3000 ng. Primers for nucleic acid amplification were designed as indicated in Table 1. PCRs were performed to amplify mutation hot spot regions in the EGFR, KRAS and P53 genes, using Gene Amp PCR System 9700 (Roche, Basel, Switzerland). All PCR assays were carried out in a 25 ␮l volume that contained 50 ng of template DNA, 0.1 unit of Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA), 25 pmol of each primer and 5 nmol of dNTP. Cycling parameters were 30 s at 95 ◦ C, 30 s at 60 ◦ C, and 60 s at 72 ◦ C for 35 cycles. PCR products were separated on a 5% polyacrylamide gel or 1.5% agarose gel. The gel was stained with ethidium bromide and photographed under UV illumination. The PCR products were direct-sequenced using the BigDye terminator kit and ABI Prism 3130 xl (Applied Biosystems, Foster, CA), and the mutations confirmed by sequencing with different primers. When frameshift was suspected in the gene sequences, the PCR products were ligated into a pGEM T easy vector (Promega, Madison, WI), which was then transfected into JM109 cells, as reported previously [17]. Multiple clones were selected and the plasmid DNA samples were sequenced. The reference sequence of each gene was obtained from GenBank (accession number NG 007726: EGFR; NG 007524: KRAS; NC 000017: P53).

Table 1 Primer design of EGFR, KRAS and P53. EGFR exon18F EGFR exon18R EGFR exon19F EGFR exon19R EGFR exon20F EGFR exon20R EGFR exon21F EGFR exon21R KRAS exon2F KRAS exon2R P53 exon2,3F P53 exon2,3R P53 exon4F P53 exon4R P53 exon5,6F P53 exon5,6R P53 exon7F P53 exon7R P53 exon8,9F P53 exon8,9R P53 exon10F P53 exon10R P53 exon11F P53 exon11R

5 -TCCAAATGAGCTGGCAAGTG-3 5 -TCCCAAACACTCAGTGAAACAAAA-3 5 -GTGCATCGCTGGTAACATCC-3 5 -TGTGGAGATGAGCAGGGTCT-3 5 -ATCGCATTCATGCGTCTTCA-3 5 -ATCCCCATGGCAAACTCTTTG-3 5 -GCTCAGAGCCTGGCATGAA-3 5 -CATCCTCCCCTGCATGTGT-3 5 -TGTGTGACATGTTCTAATATAGTCA3- 5 -GAATGGTCCTGCACCAGTAA-3 5 -TTTCCTGCTCCACAGGAAGCCG-3 5 -AACCCTTGTCCTTACCAGAACGTTG-3 5 -GGGACTGACTTTCTGCTCTTGTC-3 5 -GCCAGGCATTGAAAGTCTCATGG-3 5 -TTCCTCTTCCTACAGTACTC-3 5 -AGTTGCAAACCAGACCTCAG-3 5 -GTGTTATCTCCTAGGTTGGC-3 5 -CAAGTGGCTCCTGACCTGGA-3 5 -CCTATCCTGAGTAGTGGTAA-3 5 -CCAAGACTTAGTACCTGAAG-3 5 -CAACAGAGTGAGACCCCATCTC-3 5 -AGCTGCCTTTGACCATGAAGGC-3 5 -TGTGATGTCATCTCTCCTCCCTGC-3 5 -GGCTGTCAGTGGGGAACAAGAAGT-3

F: forward primer; R: reverse primer.

2.3. Statistical analysis Fisher’s exact test or Pearson’s Chi-square test was used for univariate analysis of the association between two categorical variables. The adjusted effects of multiple variables on EGFR or P53 mutations were evaluated by using a logistic regression model, and the results were described as an odd ratio with 95% confidence interval. P < .05 was considered significant. All analyses were performed using Dr.SPSS II (SPSS, Chicago, IL). 3. Results 3.1. Detection of deletion in EGFR exon19 and its sensitivity It was not possible to measure the content of cancer cells in the curette lavage fluid. However, it should be noted that when cytological specimens are properly diagnosed with carcinoma, the specimens are reported to have a malignant cell content of more than 1% [18]. The sensitivity of PCR to detect the deletion of EGFR was assessed by carrying out 2-fold serial dilutions of DNA from PC9 cells, which have a deletion in EGFR exon19, and then adding DNA from healthy human lymphocytes with the wild type EGFR gene. The results indicated that deletion of exon19 in EGFR was detectable at a 256-fold dilution. Assuming that all samples in this study had a cancer cell content of more than 1%, all 77 cases should contain a sufficient number of tumor cells to detect mutations. 3.2. Distribution of EGFR, KRAS and P53 mutations Table 2 shows the patient characteristics. There were 52 males and 25 females with an age at diagnosis ranging from 43 to 89 years, all of who were Japanese. Results were obtained for all patients, 54% (42 of 77) of whom harbored alterations of at least one gene. Overall, 27% (21 of 77) were found with EGFR mutations, 1% (1 of 77) with KRAS mutations, and 36% (28 of 77) with P53 mutations. Eight patients were found with EGFR mutations in combination with P53 mutations. KRAS mutations were not detected in patients harboring mutations in either EGFR or P53. As shown in Table 3, EGFR mutations were identified in 44% (11 of 25) of females, compared to 19% (10 of 52) among males (P = .044). P53 mutation rates of males (24 of 52; 46%) differed from

F. Yamaguchi et al. / Lung Cancer 78 (2012) 201–206 Table 2 Clinical characteristics of lung cancer patients. Characteristic

were not significantly associated with EGFR mutations. P53 mutations were frequently associated with staging (OR for the group of stage III–IV versus stage I–II, 5.726; 95% CI, 1.373–23.880; P = .017) and cytological diagnosis (OR for the group of NSCLC versus SCLC, 0.082; 95% CI, 0.008–0.793; P = .031) after removing the influence of other variables. Neither age (P = .086) nor sex (P = .171) nor tobacco consumption (P = .472) was significantly associated with P53 mutations.

Patients No. (%)

Age Mean Standard deviation Sex Male Female Stage I II III IV Cytological diagnosis Non-small cell lung carcinoma Adenocarcinoma Squamous cell carcinoma Unclassified Small cell lung carcinoma Tobacco consumption (pack-years) 0 1–50 51–100 100<

70 ±9.7 52(68) 25(32)

3.4. Mutation status of EGFR

19(25) 7(9) 24(31) 27(35) 68(88) 43(56) 16(20) 9(12) 9(12) 23(30) 29(38) 22(28) 3(4)

those of females (4 of 25; 16%; P = .02). When grouped according to lung cancer stage, the prevalence of EGFR mutations was similar between stage I–II (6 of 26; 23%) and stage III–IV (15 of 51; 29%; P = .749). In contrast, the difference in P53 mutation rates between stage I–II (3 of 26; 12%) and stage III–IV (25 of 51; 49%) was especially evident (P = .003). EGFR mutations were found frequently among NSCLC patients (20 of 68; 29%; P = .43) and never-smokers (12 of 23; 52%; P = .004), while P53 mutations were identified mainly in SCLC patients (8 of 9; 89%; P = .001) and ever-smokers (24 of 54; 44%; P = .046), respectively. 3.3. Multivariate analysis of gene mutations and clinicopathological features

Table 3 Association of each variable with EGFR and P53 Mutations. EGFR mutation

P53 mutation

Positive

Positive

No. (%) Sex Male Female Stage I–II III–IV Cytological diagnosis Non-small cell lung carcinoma Small cell lung carcinoma Smoking history Never Evera a

P-value

No. (%)

P-value

52 25

10 (19) 11 (44)

0.044

24 (46) 4 (16)

0.02

26 51

6 (23) 15 (29)

0.749

3 (12) 25 (49)

0.003

68 9

20 (29) 1 (11)

0.43

20 (29) 8 (89)

0.001

23 54

12 (52) 9 (17)

0.004

4 (17) 24 (44)

0.046

Including current and former smokers.

The details of mutation status are shown in Table 5. Among the EGFR mutation-positive patients, 2 were found with double EGFR mutations: one patient with E709G and L858R, and another with G719S and L747S. Among the 23 EGFR mutations, nineteen were associated with drug sensitivity (E709G, G719S, deletion in exon19, L858R), and 4 with drug resistance (L747S, insertion in exon20). EGFR mutations in exon19 and 21 were detected most frequently in the current study, accounting for 87% (20 of 23) of the EGFR mutations and for 95% (20 of 21) of the patients with EGFR mutations. Among the 18 adenocarcinoma patients for whom adequate histopathological and cytological specimens were both available, 12 were analyzed for EGFR mutation status using the histopathological samples provided by the clinical laboratory. Three patient samples were found with a deletion in exon19, three with L858R, and 6 with no mutations, and all of these results were consistent with the mutation status of the cytological specimens. L747S, described as a mutation associated with secondary resistance to EGFR-TKI, was detected in 3 patients who had never received EGFRTKI, including one SCLC patient. This patient was an 82-year-old male who had been diagnosed with hyponatremia due to the syndrome of inappropriate secretion of antidiuretic hormone. Among the patients harboring L747S, 2 were found with P53 mutations, including one SCLC patient described above.

3.5. Mutation status of P53

Multivariate logistic regression models were employed to control for the potential confounding effects of variables such as age, sex, staging, cytological diagnosis and tobacco consumption (packyears). Table 4 shows the logistic regression models of EGFR and P53 mutations. Along with increased tobacco consumption, the frequency of EGFR mutations decreased significantly (odds ratio [OR], 0.963; 95% CI, 0.934–0.993; P = .017). In contrast, age (P = .679), sex (P = .627), staging (P = .402) and cytological diagnosis (P = .479)

No.

203

As shown in Table 6, P53 mutations were identified in proteincording regions with the exception of exons3 and 11. Among the P53 mutation-positive patients, 1 was found with three mutations, including a deletion in intron 3. Alterations in intronic regions might affect mRNA synthesis. Of the 30 mutations in P53, 73% (22 of 30) were present in codon 102 of exon4 through codon 292 of exon8, which encode a sequence-specific DNA-binding domain, and 63% (19 of 30) of these were missense mutations. One mutation was located in a splice site. As for P53 mutations, an increased incidence of G:C to T:A (G to T) transversions is characteristic of lung cancer tissue from smokers [8,19]. Also, the methylated CpG sequences are very likely to be affected by tobacco exposure [19]. In this study, the transversions were detected in 6 patients, including 5 smokers and 1 never-smoker. P53 mutations in the methylated CpG site were also identified in 6 smokers and in 2 never-smokers, respectively.

3.6. Multiple mutations of EGFR and P53 P53 mutations were identified in 38% (8 of 21) of patients with EGFR mutations, all of whom, interestingly, were diagnosed as clinical stage III or IV. Three patients were found with a deletion in exon19, two with L747S, and 3 with L858R, including 1 patient with E709G. Of these patients, a 62-year-old female current smoker was given EGFR-TKI as third-line therapy, with no improvement in clinical symptoms or results of radiographic examination.

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Table 4 Logistic regression models of EGFR and P53 mutations. Category

Age Sex Stage Cytological diagnosis Tobacco consumption (pack-years)

Male/female III + IV/I + II NSCLC/SCLC

EGFR mutation

P53 mutation

OR

95% CI

P-Value

OR

95% CI

P-value

0.989 1.473 1.675 2.315 0.963

0.936–1.044 0.309–7.007 0.502–5.592 0.226–23.690 0.934–0.993

0.679 0.627 0.402 0.479 0.017

0.948 3.157 5.726 0.082 1.007

0.893–1.008 0.610–16.345 1.373–23.880 0.008–0.793 0.988–1.027

0.086 0.171 0.017 0.031 0.472

OR: odds ratio; CI: confidence interval; NSCLC: non-small cell lung carcinoma; SCLC: small cell lung carcinoma.

Table 5 Genomic alterations in EGFR. Patient no.

69 72 12 77 66 79 57 64 4 43 82 81 36 21 2 9 25 28 42 55 62

Sex

Male Male Female Female Female Male Male Female Male Female Male Male Male Female Female Male Male Female Female Female Female

EGFR Exon

Nucleotide

Amino acid

Exon18,21 Exon18,19 Exon19 Exon19 Exon19 Exon19 Exon19 Exon19 Exon19 Exon19 Exon19 Exon19 Exon20 Exon21 Exon21 Exon21 Exon21 Exon21 Exon21 Exon21 Exon21

c.2126A>G, c.2573T>G c.2155G>A, c.2240T>C c.2235 2249del c.2235 2249del c.2236 2250del c.2236 2250del c.2237 2255delinsT c.2237 2256delinsTG c.2239 2253del c.2240 2257del c.2240T>C c.2240T>C c.2310 2311insGGGTTT c.2573T>G c.2573T>G c.2573T>G c.2573T>G c.2573T>G c.2573T>G c.2573T>G c.2573T>G

E709G, L858Rb G719S, L747S E746 A750del E746 A750del E746 A750del E746 A750delb E746 S752delinsVb E746 S752delinsV L747 T751del L747 P753delinsSb L747Sb L747Sb D770 N771insGFa L858Rb L858Rb L858R L858R L858R L858R L858R L858R

del: deletion; ins: insertion. a Novel mutation. b EGFR mutations in concert with P53 mutations.

4. Discussion In this study, EGFR, KRAS and P53 mutations were analyzed using cells in curette lavage fluid. Overall, 27% patients were found with EGFR mutations, 1% with KRAS mutations and 36% with P53 mutations. EGFR mutations have been reported in around 30% of NSCLC patients in East Asia [20,21], KRAS mutations in up to 10% of Japanese lung adenocarcinoma patients [22,23], and P53 mutations in 38% of lung cancer patients, according to the database of somatic mutations established by the International Agency for Research on Cancer [24]. Although KRAS mutation rates obtained here were lower than those found in previous studies, EGFR or P53 mutation rates were comparable to those reported previously by others. EGFR mutations occurred in concert with P53 mutations, whereas KRAS mutations were not detected in patients harboring mutations in either EGFR or P53. These data are in agreement with previous reports [7–9], suggesting that the coexistence of EGFR and P53 mutations promotes cancer development in some cases. It is generally believed that EGFR mutations are more common in females than males [20], consistent with this study. However, multivariate analysis indicated that the difference in EGFR mutation rates between the sexes were not significant. Meanwhile, EGFR mutations were significantly associated with tobacco consumption. The high frequency of EGFR mutations in never-smokers with lung cancer has been a consistent finding across different parts of the globe [9,14]. In the current study, almost all the smokers were males (49 of 54; 91%), while most of the never-smokers were females

(20 of 23; 87%). Hence, the differences in EGFR mutation rates between the sexes could be due to smoking status. There have been few reports of EGFR mutations in SCLC, while this study showed one SCLC patient with EGFR mutations. SCLC patients harboring EGFR mutations have been described mainly as histological combined subtype of adenocarcinoma component [25]. Thus, EGFR-TKI could be a treatment option for a subset of SCLC [26,27]. In any case, the mutation status in SCLC needs to be further explored. L747S has been described as a mutation associated with secondary resistance to EGFR-TKI [28], but this mutation was also detected in EGFR-TKI naive patients [29]. The present study revealed 3 smokers with L747S who had never received EGFR-TKI, suggesting that the mutation is also related to primary resistance. This mutation should therefore be taken into consideration particularly for smokers with activating EGFR mutations, because dose escalation of EGFR-TKI could potentially overcome the resistance due to the secondary mutation [30,31]. An insertion in exon20, reported in association with a primary resistance to EGFR-TKI [1], was identified in 1 smoker. In brief, all of these resistant mutations were detected in smokers with more than 30 pack-years. Thus, tobacco smoking could lead to mutations associated with resistance to EGFR-TKI. There have been some reports of sex differences in P53 mutations [32], and many studies have indicated that P53 mutations occur more frequently in smokers than in neversmokers [19,33–35]. However, this study revealed that neither sex nor tobacco consumption was significantly associated with

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205

Table 6 Genomic alterations in P53. Patient no.

Sex

45 69 49 30 41 73 3 19 26 53 63 71 47 79 31 48 82 40 10 74 81 21 2 76 44 18 57 43

Male Male Male Male Male Male Male Female Male Male Male Male Male Male Male Male Male Male Male Male Male Female Female Male Male Male Male Female

P53 Exon

Nucleotide

Amino acid

Exon2 Exon4 Exon4 Exon4 Exon4 Exon5 Exon5 Exon5 Intron3, exon5,8 Exon5 Exon5 Exon6 Exon6 Exon6 Exon6 Exon7 Exon7 Exon7 Exon7 Exon8 Exon8 Exon8 Exon8 Exon8 Exon9 Intron9 Exon10 Exon10

c.31G>C c.145G>C c.211del c.310C>T c.352del c.378delinsTT c.461G>T c.469G>T c.96+16 96+31del, c.473G>T, c.823T>C c.488A>G c.527 528insC c.578A>T c.638G>T c.641del c.659A>G c.711G>A c.734G>T c.774A>C c.776A>T c.794T>C c.824G>T c.835del c.839G>C c.845G>C c.991C>T c.994-1G>A c.1024C>T c.1024C>T

E11Q D49Hb P71fs Q104stop T118fsa Y126fsa G154V V157F R158L, C275R Y163C H178fs H193L R213L H214fsb Y220C M237I G245Vb E258D D259V L265P C275Fb G279fsb R280Tb R282P Q331stop R342stopb R342stopb

del: deletion; ins: insertion; fs: frameshift; stop: stop codon. a Novel mutation. b P53 mutations in concert with EGFR mutations.

P53 mutations. This discrepancy might be due to the limited number of patients enrolled in the present study, since there was little difference between P53 mutation rates in smokers versus neversmokers. More specifically, P53 mutations have been reported in 26–71% of lung cancer cases due to smoking, but the rates in lung cancer from never-smokers have ranged up to 47% [7,19,33]. Types and distributions of P53 mutations seem to differ according to smoking status [8,19]. Tobacco smoking is associated with a dose-dependent increase in G to T transversions, and methylated CpG sequences are frequent targets for mutagenesis by the ultimate carcinogenic metabolite of tobacco smoke, including polycyclic aromatic hydrocarbon [19]. In the present study however, among the P53 mutation-positive patients, the frequency of G to T transversions did not differ according to smoking status. Also, tobacco smoking did not correlate with an increased risk of P53 mutations in the CpG site. P53 mutations have been reported in around 60% of SCLC patients [24]. In a mouse model, P53 deletion produces tumors with SCLC features, while KRAS activation leads to NSCLC-like tumors [36]. Thus, the characteristics of SCLC tumors seem to be quite distinct from those of NSCLC tumors, and alterations in P53 could play a crucial rule in tumorigenesis of SCLC. Similarly, this study indicated that P53 mutations occurred more frequently in SCLC patients than in NSCLC patients. Unlike conventional tumor-suppressor genes that are typically affected by nonsense or frameshift mutations, approximately 75% of P53 mutations found in tumors are missense mutations [19,37]. In addition, almost all the mutations are located within the sequence-specific DNA-binding domain, leading to loss of P53 function as a transcription factor [19,38]. Most of the P53 mutations in this study were missense (19 of 30; 63%) and mapped to this domain (22 of 30; 73%). There was no significant association of EGFR mutations with the clinical stage, while P53 mutation rates in patients with stage III or

IV were significantly higher than those in patients with stage I or II. These findings suggest that the interpretation of each mutation differs according to the role of each gene. EGFR mutations rates seem to have no relationship to clinical stage [7,9,39], whereas the proportion of P53 mutations remains controversial in relation to cancer progression [7,19,40]. Assuming that the accumulation of P53 mutations in cancer cells over time leads to an increasing loss of P53 functionality as a transcription factor, the alterations might accelerate cancer development, and they could lead to evolution of therapeutic resistance, including acquired resistance to EGFR-TKI. P53 mutations were detected in 38% of patients with EGFR mutations, making our results comparable to figures reported elsewhere [7]. Interestingly, all of these patients were diagnosed in clinical stage III or IV. Of these, one female patient was given EGFR-TKI, but this did not provide any clinical benefit. These results suggest that prognosis of lung cancer is influenced by the presence of mutations in the EGFR and P53 genes. Aberrations of the tumorsuppressor gene P53 are frequently associated with drug resistance [11,12]. The theory of an ARF-P53 circuit explains that several oncogenes such as RAS and MYC can induce stabilization of the P53 protein by enlisting the activity of ARF [41,42]. In particular, the KRAS protein is activated by EGFR signaling. ARF then functions by binding directly to MDM2, a protein that inhibits the ubiquitination of P53. As a result, ARF promotes P53 functions, including cell-cycle arrest, senescence and apoptosis, although P53 mutations inhibit these functions. According to this theory, P53 mutations are likely to decrease the efficacy of EGFR-TKI in the treatment of NSCLC patients. Indeed, a few studies have reported that EGFR-TKI induced apoptosis through a P53-dependent signaling pathway, such as BIM [43] or FAS [44]. The efficacy of EGFR-TKI in the treatment of lung cancer patients with both EGFR and P53 mutations has not yet been well studied. Data from the current study indicated that P53 mutations tend to occur in more advanced cancer stages. It has been reported that most patients who initially respond to treatment

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eventually manifest tumor recurrence following EGFR-TKI treatment [8,29]. Hence, the loss of P53 function though mutations, in combination with EGFR mutations, could contribute to acquired resistance to EGFR-TKI and a poor prognosis. 5. Conclusion To date, histopathological samples have been commonly used for molecular analyses in lung cancer. The present study demonstrates that it is possible to analyze EGFR, KRAS and P53 mutations using curette lavage fluid collected from lung cancer patients. This is useful when sufficient amounts of tumor samples cannot be obtained. It is not clear whether EGFR mutations in concert with P53 mutations actually contribute to acquired resistance to EGFRTKI and accelerate cancer progression. In the future, large clinical studies should be designed with these issues in mind. Disclosure This study was supported by a grant from Eli Lilly Japan K.K. We declare that we have no conflict of interest in connection with this paper. We have already presented this work in abstract form, “Curette lavage fluid analysis of EGFR, KRAS and P53 mutations in lung cancer patients” for poster discussion at the European Respiratory Society Annual Congress 2011. Conflict of interest None. Acknowledgements This study was supported by a grant from Eli Lilly Japan K.K. We would like to thank NAI, Inc. (http://www.nai.co.jp/) for proofreading this manuscript. References [1] Pao W, Girard N. New driver mutations in non-small-cell lung cancer. Lancet Oncol 2011;12:175–80. [2] Maemondo M, Inoue A, Kobayashi K, Sugawara S, Oizumi S, Isobe H, et al. Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med 2010;362:2380–8. [3] Mitsudomi T, Morita S, Yatabe Y, Negoro S, Okamoto I, Tsurutani J, et al. Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial. Lancet Oncol 2010;11:121–8. [4] Pao W, Wang TY, Riely GJ, Miller VA, Pan Q, Ladanyi M, et al. KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLoS Med 2005;2:e17. [5] Riely GJ. KRAS mutations in non-small cell lung cancer. Proc Am Thorac Soc 2009;6:201–5. [6] Massarelli E, Varella-Garcia M, Tang X, Xavier AC, Ozburn NC, Liu DD, et al. KRAS mutation is an important predictor of resistance to therapy with epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer. Clin Cancer Res 2007;13:2890–6. [7] Kosaka T, Yatabe Y, Endoh H, Kuwano H, Takahashi T, Mitsudomi T. Mutations of the epidermal growth factor receptor gene in lung cancer: biological and clinical implications. Cancer Res 2004;64:8919–23. [8] Subramanian J, Govindan R. Molecular genetics of lung cancer in people who have never smoked. Lancet Oncol 2008;9:676–82. [9] Shigematsu H, Lin L, Takahashi T, Nomura M, Suzuki M, Wistuba II, et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst 2005;97:339–46. [10] Toyooka S, Mitsudomi T, Soh J, Aokage K, Yamane M, Oto T, et al. Molecular oncology of lung cancer. Gen Thorac Cardiovasc Surg 2011;59:527–37. [11] Levine AJ, Oren M. The first 30 years of p53: growing ever more complex. Nat Rev Cancer 2009;9:749–58. [12] Brown CJ, Lain S, Verma CS, Fersht AR, Lane DP. Awakening guardian angels: drugging the p53 pathway. Nat Rev Cancer 2009;9:862–73. [13] Lim EH, Zhang S-L, Li J-L, Yap WS, Howe TC, Tan BP, et al. Using whole genome amplification (WGA) of low-volume biopsies to assess the prognostic role of EGFR, KRAS, p53, and CMET mutations in advanced-stage non-small cell lung cancer (NSCLC). J Thorac Oncol 2009;4:12–21.

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