KRAS oncogene substitutions in Korean NSCLC patients: Clinical implication and relationship with pAKT and RalGTPases expression

Accepted Manuscript Title: KRAS oncogene substitutions in Korean NSCLC patients; Clinical implication and relationship with pAKT and Ral GTPases expression Author: Eun Young Kim Arum Kim Se Kyu Kim Hyung Jung Kim Joon Chang Chul Min Ahn Jae Seok Lee Hyo Sup Shim Yoon Soo Chang PII: DOI: Reference:

S0169-5002(14)00183-4 http://dx.doi.org/doi:10.1016/j.lungcan.2014.04.012 LUNG 4594

To appear in:

Lung Cancer

Received date: Revised date: Accepted date:

22-12-2013 10-4-2014 23-4-2014

Please cite this article as: Kim EY, Kim A, Kim SK, Kim HJ, Chang J, Ahn CM, Lee JS, Shim HS, Chang YS, KRAS oncogene substitutions in Korean NSCLC patients; Clinical implication and relationship with pAKT and Ral GTPases expression, Lung Cancer (2014), http://dx.doi.org/10.1016/j.lungcan.2014.04.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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KRAS oncogene substitutions in Korean NSCLC patients; Clinical implication and relationship with pAKT and Ral GTPases expression

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Eun Young Kim, 1, 2Arum Kim, 1Se Kyu Kim, 1Hyung Jung Kim, 1Joon Chang, Chul Min Ahn, 3Jae Seok Lee, 3Hyo Sup Shim, 1Yoon Soo Chang

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Biomedical Research Center, 3Department of Pathology,

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Department of Internal Medicine and

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Yonsei University College of Medicine, Seoul, Korea

Address for correspondence: YSC, Dept. of Internal Medicine, Yonsei University College of Medicine, 8th Floor Annex Bldg. 211 Eonju-ro, Gangnam-gu, 135-720, Republic of Korea. Phone: +82-2-2019-3309, Fax: +82-2-3463-3882. E-mail: [email protected]

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Abstract Objectives: Since different conformation of each KRAS mutant leads to inherent downstream signaling, its distribution, influence on the clinical outcome, and effect on the signaling mediators were investigated in the Korean NSCLC patients whose tumor have KRAS mutation.

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Materials and Methods: Mutation at KRAS codons 12 and 13 was evaluated in 1,420 Korean NSCLC by direct sequencing and expression of RalA, RalB, and pAKT-Ser473 was evaluated by

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immunohistochemistry in 30 cases whose KRAS mutant tumor tissues were available.

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Results: Eighty-two (5.8%) out of 1,420 patients harbored a KRAS mutation either in codon 12 or 13. Gly12Asp was the most frequent (34.1%), followed by Gly12Cys (22.0%) and Gly12Val (13.4%).

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Transversion at codons 12 and 13, which includes Gly12Cys, Gly12Val, Gly12Ala, Gly13Cys, and Gly12Phe was detected in 45 cases (54.9%) and transition, including Gly12Asp, Gly12Ser, and

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Gly13Asp was detected in 37 cases (45.1%). Male and smoking history were associated with transversion (p=0.001 and 0.006, respectively; χ2-test), and multivariate analysis showed that gender

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was an independent influencing factor (p=0.026; Cochran–Mantel–Haenszel test). Multivariate analysis on survival revealed that KRAS mutation subtype did not influence overall survival of the

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patients with KRAS mutations after adjustment for age, gender, performance status, and stage. There were no differences in the nuclear and cytoplasmic expression of pAKT-Ser473 between transversion and transition mutants. Expression of Ral-GTPases, RalA and RalB, did not differ between transversion and transition mutants, however, strong expression of RalB in the tissue of patients with

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KRAS mutants was associated with advanced stages (p-value=0.020, χ2-test). Conclusions: In this study population, not only the frequency of KRAS mutation but the distribution its subtypes differed from those of Western studies, with unique influencing factors. Clinical outcome and expression of pAKT-Ser473, RalA, and RalB did not differ among subtypes.

Key words: KRAS, pAKT, Ral GTPases, NSCLC

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Introduction

Lung cancer is the second most common malignancies, with approximately 226,160 new cases diagnosed in the US for 2012 [1]. Despite the unremitting efforts toward development of targeted

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drugs and biomarker discovery, it is by far the leading cause of cancer death and, overall, a cancer with poor prognosis. Identification of biology and development of new therapeutic strategies based on

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the specific subtype of lung cancer are needed to improve its clinical outcome.

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The v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) is one of the most frequently activated oncogenes in human cancer, and is detected in approximately 30% of human cancers. Since

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the discovery of KRAS in 1982 from a gene originating from the genomic DNA of LX-1 lung carcinoma cells [2], there have been many trials for the treatment of malignancies harboring this

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mutation, with most of them eventually found to be not effective. KRAS mutations are limited to a few sites; most mutations occur in codon 12, followed by mutations in codons 13, 10, and 61 [3]. KRAS

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mutations in codons 12 or 13 result in base changes that lead to amino acid substitutions that lock the KRas protein in an active state [4].

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The frequency and spectrum of KRAS mutations in codons 12 and 13 differ among cancer types. The most common KRAS mutation in colorectal cancer is a G to A transition, accounting for 92% of mutations. G to A transition at codon 12 and/or codon 13 results in KRas proteins in which a Gly residue is replaced by an Asp (found in approximately 50% of tumors), Val (28%), or a Cys (9%) [5].

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In pancreatic cancer, where 90% of tumors have activating mutations in the KRAS oncogene, transitions and transversions were observed equally with a prevalence of G to A changes among transitions in codon 12 [3]. In Western NSCLC patients who smoke, the most common KRAS mutation is a G to T transversion (84% of mutations), and the most common amino acid replacements at codon 12 and/or codon 13 are Cys (47% of tumors), Val (24%), Asp (15%), and Ala (7%) [6]. However the frequency of KRAS mutations also shows significant ethnic differences. In Western countries, KRAS mutations are found in ~25% of lung adenocarcinomas, and a transition mutation was observed significantly more frequently in never smokers than in former or current smokers [7]. In 3

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Asian studies, the frequency of KRAS mutation in NSCLC was 3.4-13%, which is less than the rates found in Western studies. KRAS mutations were mainly found in codons 12 and 13, and transition mutations were commonly observed in lung cancers from smokers [8-12]. Recent reports indicating that each KRAS mutant has inherent roles in propagating oncogenic

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signaling, as well as the existence of subtypes of KRAS, should be taken into consideration in therapeutic intervention. These matters lead us to revisit the subtype of KRAS mutation [13]. In this

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study, we examined the distribution of KRAS subtypes among 1,420 Korean NSCLC patients and

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evaluated the effect of these subtypes on clinical outcome. The effect of KRAS subtypes on signaling

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pathways was evaluated by immunostaining for pAKT-Ser473, RalA, and RalB.

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Materials and Methods

Study design and subjects. Between June 2005 and June 2012, a total of 1,420 patients who were

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diagnosed as having NSCLC were enrolled at a university-affiliated tertiary care referral hospital (Severance Hospital, Seoul, Republic of Korea). All patients were Korean (East Asian) and underwent genetic analysis of KRAS oncogene substitutions (codons 12 and 13) and their medical records were

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reviewed retrospectively. Patients’ smoking histories were classified into three groups; never, ex-, and current smokers. Never smokers were defined as those who smoked less than 100 cigarettes in their lifetime and ex-smokers had previously smoked cigarettes, but quit smoking more than 1 year prior to

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diagnosis of lung cancer. Patients’ performance status was assessed according to the Eastern Cooperative Oncology Group (ECOG) scale. Tumor histopathology was classified by World Health Organization criteria and tumor stage was described according to the 7th edition of the American Joint Committee on Cancer (AJCC) cancer staging manual. We examined survival outcomes to perform survival analysis. Overall survival (OS) was defined as the time from diagnosis to the date of death due to any cause. Progression-free survival (PFS) was defined as the time from diagnosis to the date of progression or death due to any cause, whichever occurred first. Clinical responses were classified according to Response Evaluation Criteria in Solid 4

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Tumor (v. 1.1). Patients’ survival records were censored on July 21, 2012. This study was approved by the Institutional Review Board of Yonsei University College of Medicine.

KRAS mutation analysis. After separating genomic DNA in tumor tissue, PCR amplification of

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codons 12 and 13 of the KRAS gene and gene sequencing of the purified PCR product was performed

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using an ABI Prism® 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA).

Immunohistochemistry (IHC). Expression of p-Akt Ser473, RalA, and RalB were analyzed by IHC.

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IHC was performed using the LABS®2 system (Dako Corp., Carpinteria, CA, US) according to the

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manufacturer’s instructions. Briefly, sections were deparaffinized, rehydrated, and then antigen retrieval was performed using high pH citrate buffer (Dako Cytomation, Carpinteria, CA, US) for 30

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minutes by microwave heating. Sections were then immersed in H2O2-methanol solution before incubating overnight with primary anti-pAkt-Ser473 (1:50, Cell Signaling Technology, Danvers, MA,

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US), RalA (1:100, BD Transduction Laboratory, San Jose, CA, US), and RalB (1:100, Santa Cruz Biotech, Santa Cruz, CA, US). Sections were incubated for 10 min with biotinylated linker and

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processed using avidin-biotin IHC techniques. For pAkt-Ser 473, 3,3'- diaminobenzidine (DAB) was used as a chromogen in conjunction with the Liquid DAB substrate kit (Novacastra, UK) and horseradish peroxidase was used as a chromogen for RalA and RalB. As a positive control of ralA and ralB immunostaining, rat forebrain was used and that of pAkt-ser473 human breast ductal cancer

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tissues were used. When more than 25% of cancer cells show staining as strong as positive control, either at nucleus or cytoplasm, it was considered for pAKT-Ser473 positive. Expression of ralA and ralB were evaluated by scoring system using product of staining intensity and percentage of positive cells. Staining intensity was classified as 0, 1, 2, 3 where intensity 2 means equal as that of positive control. Frequency was classified as 0, 1 (trace <5%), 1 (-10%) and 2 (-30%), and 3 (>30%). When the product of intensity and frequency was ≥6 it is considered as overexpression.

Statistical analysis. Clinically significant differences in the characteristics of KRAS mutation subtype 5

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were analyzed using the χ2 test and unpaired Student’s t-test. Predictive factors for overall survival (OS), disease free survival (DFS), and progression-free survival (PFS) were calculated using the Kaplan-Meier Estimator and Cox proportional hazards model. All tests of significance were twotailed and p-values of less than 0.05 were interpreted to indicate statistical significance. SPSS

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software (v. 18; SPSS, IL, USA) was used for statistical analysis.

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Results

A. Demographic characteristics Of 1,420 NSCLC patients, KRAS mutations in codon 12 and/or 13 were found in 82 (5.8%) patients. KRAS mutations were more frequently found in male patients and

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those who were currently or had previously been smokers. According to previous reports on Korean lung cancer patients, the frequency of KRAS mutation ranges from 3.9% to 5.2% of NSCLCs [8, 12]

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and 7.0% to 9.6% of lung adenocarcinomas [9, 11]. In a comparison of our results and other reports

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on Koreans, there were no significant differences in the age and gender of study patients. When comparing lung cancer patients with KRAS mutation and patients with wild-type KRAS, those

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harboring KRAS mutations were older, predominantly male, and had increased exposure to cigarette

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smoke (Table 1).

B. Gly12Asp was the most frequent KRAS mutant in Korean NSCLC. Because oncogenic

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substitution of KRAS influences the survival of lung cancer patients [13], we first analyzed the distribution of KRAS substitutions in Korean NSCLC patients (Table 2). Among the patients who

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were tested for KRAS mutation, 82 (5.8%) of 1,420 patients harbored a KRAS mutation in either codon 12 (73 of 82, 89.0%) or 13 (9 of 82, 11.0%) and transversions (45 cases, 54.9%) were more common than transitions (37 cases, 45.1%). Gly12Asp was the most frequent KRAS oncogene substitution (28 of 82, 34.1%), followed by Gly12Cys (18 of 82, 22.0%) and Gly12Val (11 of 82, 13.4%). Also,

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Gly12Asp accounted for the majority (15 of 27, 55.6%) of substitutions in never-smoker patients. Males and subjects who had a history of smoking exhibited a higher rate of transversion than females and never smokers (p=0.001 in gender analysis, p=0.006 in smoking analysis; χ2-test). When controlling for smoking status, transitions in codons 12 and 13 were significantly more frequent than transversions in women (p=0.026; Cochran–Mantel–Haenszel test). This finding suggests that distribution of KRAS mutations in a Korean lung cancer population differs from that of a Caucasian population, which showed Gly12Cys as the most frequent substitution [6]. However, KRAS mutation subtype did not affect patient survival. OS of all KRAS-mutant NSCLC patients, DFS of patients who 7

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underwent curative resection (n=29) and PFS of advanced stage patients (n=53) according to the KRAS mutation subtypes did not differ according to the KRAS mutation subtype (p=0.568 in OS, p=0.860 in DFS, p=0.426 in PFS; Kaplan-Meier estimations) (Fig. 1). In a Cox regression model adjusted for age, gender, performance status, smoking status, KRAS mutation subtype and tumor stage,

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only tumor stage (HR=7.56, 95% CI=2.97-19.25, p<0.001; Cox regression test) and smoking status (HR=3.37, 95% CI=1.35-8.42, p=0.009) were significant predictors of shortened overall survival in

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advanced NSCLC patients with KRAS mutation (Table 3).

C. pAkt-Ser473 is not related to the expression of KRAS mutation subtypes. Because pAkt-

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Ser473 is inhibited more strongly by p70S6K in the KRAS Gly12Cys mutant than in Gly12Asp [13], we postulated that the expression of pAkt-Ser473 is different in tissues expressing Gly12Asp and

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Gly12Cys mutations of KRAS. The expression of pAktSer473 in either cytoplasm or cytoplasm and nucleus of cancer tissues was evaluated by immunohistochemistry (Fig. 2). pAkt-473 was frequently

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detected both in the nucleus and cytoplasm of normal appearing bronchial epithelial cells. pAkt-473 was also detected in the nucleus and cytoplasm of KRAS mutant NSCLC tissues, but it was more

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frequently detected in the nucleus of the cancer cells. There was no difference in the pAkt-Ser473 expression between transversion and transition mutations, and pAktSer473 overexpression did not affect the survival of patients with KRAS mutant NSCLC (p=0.127 in cytoplasmic expression and

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p=0.063 in cytoplasmic and nuclear expression; χ2 test) (Table 4).

D. RalB expression is associated with poor prognosis in KRAS mutant non-small cell lung cancer. Because either Gly12Cys or Gly12Val mutations induce MEK activation through a RalGTPase-dependent pathway [13], expression of RalA and RalB was evaluated in NSCLC patients with KRAS mutants by immunohistochemistry (Fig. 2). RalA was expressed ubiquitously; it was detected in the membranous and the submembranous region of cancer cells, the bronchial epithelial cells, and type I and II pneumocytes. On the other hands, RalB was expressed in membrane and cytoplasm of the cancer tissues only. RalA was strongly expressed in the membranous region of 24 8

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(80.0%) of 30 tumor tissues whereas RalB was overexpressed in the membranous and cytoplasm of 17 (56.7%) of 30 cancer tissues. When RalA and RalB expression was analyzed according to KRAS substitution, there was no difference in protein expression between transversion and transition. Although expression of RalA was not differ among stages, RalB overexpression was more frequent in

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the advanced stage of lung cancer (p-value=0.020, χ2-test). However, the relationship between RalB (+) and OS did not reach statistical significance (p-value=0.179, Log-Rank test). These findings

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suggest that KRAS mutation substitution did not influence the expression of RalA, RalB and pAkt-

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Ser473, though RalB overexpression is associated with disease status in KRAS mutant NSCLC

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Discussion Similar to the distribution of EGFR mutations, oncogenic mutation of KRAS in NSCLC shows significant ethnic differences in prevalence. The KRAS mutation was detected in less than in 10% of lung adenocarcinoma of Far East Asians [8, 9, 11, 12]. Reports on Korean populations have used

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limited numbers of NSCLC patients, which makes it difficult to draw conclusions on the clinical implications of KRAS mutation subtypes [8, 9]. Reports that specified substitution mutation of KRAS

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in Korean NSCLC are very limited as well [12]. In this study, by recruiting a large number of NSCLC

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patients, we were able to show that the prevalence of KRAS mutations at codons 12 and 13 is 5.8%, and to indicate specific substitution subtypes in Korean NSCLC patients with influencing factors.

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In the early era of KRAS mutation test when Sanger sequencing was routinely applied, the frequency of KRAS mutation in the Caucasian lung adenocarcinoma was around 30% [14, 15]. Recent report at

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2013 by Shepherd et al. showed KRAS mutation positive rate was still 33.7% (204 out of 605) in pooled early-stage resected lung adenocarcinoma from Western countries [16], showing the detection

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rate was not significantly differ between Sanger sequencing and currently adopted methods. Another meta-analysis by Meng et al. also showed that the difference in the detection rate among mutation

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tests (PCR-MSOP, PCR-DGGE, PCR-RFLP, PCR-seq) is not significant [17]. Many scientists propose that recently developed sensitive tests such as PCR-clamping could be applied for NSCLC samples with low percentage of tumor cells such as unprocessed bronchial biopsies or after neoadjuvant chemotherapy[18, 19].The cause of KRAS mutation is not clear, but a relationship with

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smoking has been repeatedly reported by many investigators. G>T transversions are the most frequent subtype of substitutions among NSCLC patients with smoking histories, accounting for 84% of total mutations. Our research showed that the frequency of transversions among those with smoking history reached 65.5%, which is lower than that of reports from the US [13]. Gly12Asp at codon 12, which originated from a transition substitution, showed the same frequency as Gly12Cys, reaching 23.6% of mutations. This rate is quite different from that of a report from the US showing Gly12Cys at a rate of 47%, Val at 24%, Asp accounting for 15%, and Ala for 7% [13]. Gender also influenced the distribution of KRAS subtypes. Even when the smoking status was compensated for, male patients 10

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had a higher chance of having a transversion mutation. This finding indicates that additional research, including epidemiologic studies on occupational exposure to carcinogens, is required to identify the difference in the mutation subtype between the genders. KRAS transversion mutations resulting in Gly12Cys and Gly12Val was found to be related to poor

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prognosis in lung cancer patients and are related to the inhibition of pAKT-Ser473 via IRS-1 inhibitory phosphorylation [13]. However, the relationship between expression status of pAkt-Ser473

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and KRAS substitution has not been verified in clinical samples and the clinical implication of pAkt is

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still elusive [20]. In this study, we evaluated both the clinical implications of and relationships to KRAS subtypes. Similar to previous reports, we found that pAkt expression was not only in the

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nucleus, but also in the cytoplasm of normal-appearing adjacent tissues and NSCLC cells. When the relationship of overexpression of pAkt in the cytoplasm and both nucleus and cytoplasm was

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evaluated, we could not find any clinical implication and relationship with subtypes of KRAS mutation. These findings may originate from (1) the small number of KRAS mutations found in the study

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population and limited number of available clinical samples, (2) the dynamics of pAkt-Ser473 expression, which is highly susceptible to diet, nutritional status, fasting, and other disease statuses,

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and (3) the possibility that there is indeed no relationship between subtype and oncogenic substitution. A recent report, indicating that there was no difference in the clinical outcome among lung cancer patients with different types of oncogenic substitution, suggested this option [21]. In spite of their high degree of similarity, the downstream effectors of RAS, the Ral small GTPases,

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have distinct functions. RalA has been implicated in epithelial cell polarity [22], insulin secretion [23], GLUT4 translocation [24, 25], neurite branching, and neuronal polarity [26, 27], while RalB is involved in tumor cell survival [28], migration/invasion [29-31], TBK1 activation [32], and autophagy [33]. Still, reports on the expression statuses and clinical significances of RalA and RalB in KRAS mutant lung cancer are very limited. In a recent report on null and conditional RalA and RalB knockout mouse models, RalB null mice were viable and did not show any phenotypic abnormality but mice that were RalA-null showed exencephaly and embryonic lethality. When these models were crossed with a KRAS-driven lung cancer mouse model, the mouse model that had either RalA or RalB 11

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was sufficient for tumor growth, suggesting that RalA and RalB act in a redundant fashion in KRASdriven lung cancer formation and proliferation [34]. Interestingly, in our samples, expression of RalA was detected in the normal-appearing adjacent lung tissues and majority of KRAS mutant lung cancer tissues, whereas RalB was detected in 56.7% of cancer tissues and was related to advanced stage.

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These findings suggest that RalB would be more relevant to any clinically significance and warrant further studies, including identification of the relationship with Ral GTPase activity in KRAS mutant

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lung cancer tissues.

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This study does have some limitations. The limited number of study tissues, because of the low frequency of KRAS mutations in these study populations, is one of the factors that lessened the ability

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to draw conclusions. Another limitation is an inability to measure Ral GTPase activity in these tissues and the use of pAkt as the only surrogate biomarker for comparing biologic activity of transversion

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and transition. The findings could be further influenced by the retrospective design of the study and data that were obtained from patients who had provided informed consent and were willing to pay for

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genetic analysis of KRAS mutation.

In conclusion, Korean NSCLC patients with oncogenic KRAS mutation had distinctive characteristics

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with lower frequency. We could not find differences between subtypes of KRAS substitution regarding clinical outcome or expression of pAKT-Ser473, RalA, and RalB. To identify the biologic and clinical significance of subtypes of KRAS mutation, a large prospective study may be required.

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Conflict of interest: There are no conflicts of interests. Acknowledgements: This study was supported by a faculty research grant of Yonsei University College of Medicine for 2012 given to EYK (6-2012-0129). The role of funding source: The funding source did not involve in the study design, data collection and interpretation, and writing.

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aPKC. J Cell Sci 2009; 122: 1499-1506.

Lalli G, Hall A. Ral GTPases regulate neurite branching through GAP-43 and the

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Chien Y, White MA. RAL GTPases are linchpin modulators of human tumour-cell

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Lim K-H, O'Hayer K, Adam SJ, Kendall SD, Campbell PM, Der CJ, Counter CM.

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Divergent Roles for RalA and RalB in Malignant Growth of Human Pancreatic Carcinoma Cells. Curr Biol 2006; 16: 2385-2394. Rossé C, Hatzoglou A, Parrini M-C, White MA, Chavrier P, Camonis J. RalB Mobilizes

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Phosphorylation of RalB Is Important for Bladder Cancer Cell Growth and Metastasis. Cancer Res 2010; 70: 8760-8769. 32.

Chien Y, Kim S, Bumeister R, Loo Y-M, Kwon SW, Johnson CL, Balakireva MG,

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Romeo Y, Kopelovich L, Gale M, Yeaman C, Camonis JH, Zhao Y, White MA. RalB GTPase-Mediated Activation of the IºB Family Kinase TBK1 Couples Innate Immune Signaling to Tumor Cell Survival. Cell 2006; 127: 157-170.

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Bodemann BO, Orvedahl A, Cheng T, Ram RR, Ou Y-H, Formstecher E, Maiti M, Hazelett CC, Wauson EM, Balakireva M, Camonis JH, Yeaman C, Levine B, White MA. RalB and the Exocyst Mediate the Cellular Starvation Response by Direct Activation of Autophagosome Assembly. Cell 2011; 144: 253-267.

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Marshall Christopher J. Genetic Deletion of RALA and RALB Small GTPases Reveals Redundant Functions in Development and Tumorigenesis. Curr Biol 2012; 22: 2063-

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2068.

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*Manuscript_Marked Click here to view linked References

KRAS oncogene substitutions in Korean NSCLC patients; Clinical implication and relationship with pAKT and Ral GTPases expression

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Eun Young Kim, 1, 2Arum Kim, 1Se Kyu Kim, 1Hyung Jung Kim, 1Joon Chang, Chul Min Ahn, 3Jae Seok Lee, 3Hyo Sup Shim, 1Yoon Soo Chang

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Biomedical Research Center, 3Department of Pathology,

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Department of Internal Medicine and

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Yonsei University College of Medicine, Seoul, Korea

Address for correspondence: YSC, Dept. of Internal Medicine, Yonsei University College of Medicine, 8th Floor Annex Bldg. 211 Eonju-ro, Gangnam-gu, 135-720, Republic of Korea. Phone: +82-2-2019-3309, Fax: +82-2-3463-3882. E-mail: [email protected]

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Abstract Objectives: Since different conformation of each KRAS mutant leads to inherent downstream signaling, its distribution, influence on the clinical outcome, and effect on the signaling mediators were investigated in the Korean NSCLC patients whose tumor have KRAS mutation.

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Materials and Methods: Mutation at KRAS codons 12 and 13 was evaluated in 1,420 Korean NSCLC by direct sequencing and expression of RalA, RalB, and pAKT-Ser473 was evaluated by

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immunohistochemistry in 30 cases whose KRAS mutant tumor tissues were available.

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Results: Eighty-two (5.8%) out of 1,420 patients harbored a KRAS mutation either in codon 12 or 13. Gly12Asp was the most frequent (34.1%), followed by Gly12Cys (22.0%) and Gly12Val (13.4%).

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Transversion at codons 12 and 13, which includes Gly12Cys, Gly12Val, Gly12Ala, Gly13Cys, and Gly12Phe was detected in 45 cases (54.9%) and transition, including Gly12Asp, Gly12Ser, and

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Gly13Asp was detected in 37 cases (45.1%). Male and smoking history were associated with transversion (p=0.001 and 0.006, respectively; χ2-test), and multivariate analysis showed that gender

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was an independent influencing factor (p=0.026; Cochran–Mantel–Haenszel test). Multivariate analysis on survival revealed that KRAS mutation subtype did not influence overall survival of the

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patients with KRAS mutations after adjustment for age, gender, performance status, and stage. There were no differences in the nuclear and cytoplasmic expression of pAKT-Ser473 between transversion and transition mutants. Expression of Ral-GTPases, RalA and RalB, did not differ between transversion and transition mutants, however, strong expression of RalB in the tissue of patients with

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KRAS mutants was associated with advanced stages (p-value=0.020, χ2-test). Conclusions: In this study population, not only the frequency of KRAS mutation but the distribution its subtypes differed from those of Western studies, with unique influencing factors. Clinical outcome and expression of pAKT-Ser473, RalA, and RalB did not differ among subtypes.

Key words: KRAS, pAKT, Ral GTPases, NSCLC

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Introduction

Lung cancer is the second most common malignancies, with approximately 226,160 new cases diagnosed in the US for 2012 [1]. Despite the unremitting efforts toward development of targeted

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drugs and biomarker discovery, it is by far the leading cause of cancer death and, overall, a cancer with poor prognosis. Identification of biology and development of new therapeutic strategies based on

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the specific subtype of lung cancer are needed to improve its clinical outcome.

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The v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) is one of the most frequently activated oncogenes in human cancer, and is detected in approximately 30% of human cancers. Since

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the discovery of KRAS in 1982 from a gene originating from the genomic DNA of LX-1 lung carcinoma cells [2], there have been many trials for the treatment of malignancies harboring this

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mutation, with most of them eventually found to be not effective. KRAS mutations are limited to a few sites; most mutations occur in codon 12, followed by mutations in codons 13, 10, and 61 [3]. KRAS

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mutations in codons 12 or 13 result in base changes that lead to amino acid substitutions that lock the KRas protein in an active state [4].

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The frequency and spectrum of KRAS mutations in codons 12 and 13 differ among cancer types. The most common KRAS mutation in colorectal cancer is a G to A transition, accounting for 92% of mutations. G to A transition at codon 12 and/or codon 13 results in KRas proteins in which a Gly residue is replaced by an Asp (found in approximately 50% of tumors), Val (28%), or a Cys (9%) [5].

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In pancreatic cancer, where 90% of tumors have activating mutations in the KRAS oncogene, transitions and transversions were observed equally with a prevalence of G to A changes among transitions in codon 12 [3]. In Western NSCLC patients who smoke, the most common KRAS mutation is a G to T transversion (84% of mutations), and the most common amino acid replacements at codon 12 and/or codon 13 are Cys (47% of tumors), Val (24%), Asp (15%), and Ala (7%) [6]. However the frequency of KRAS mutations also shows significant ethnic differences. In Western countries, KRAS mutations are found in ~25% of lung adenocarcinomas, and a transition mutation was observed significantly more frequently in never smokers than in former or current smokers [7]. In 3

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Asian studies, the frequency of KRAS mutation in NSCLC was 3.4-13%, which is less than the rates found in Western studies. KRAS mutations were mainly found in codons 12 and 13, and transition mutations were commonly observed in lung cancers from smokers [8-12]. Recent reports indicating that each KRAS mutant has inherent roles in propagating oncogenic

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signaling, as well as the existence of subtypes of KRAS, should be taken into consideration in therapeutic intervention. These matters lead us to revisit the subtype of KRAS mutation [13]. In this

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study, we examined the distribution of KRAS subtypes among 1,420 Korean NSCLC patients and

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evaluated the effect of these subtypes on clinical outcome. The effect of KRAS subtypes on signaling

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pathways was evaluated by immunostaining for pAKT-Ser473, RalA, and RalB.

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Materials and Methods

Study design and subjects. Between June 2005 and June 2012, a total of 1,420 patients who were

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diagnosed as having NSCLC were enrolled at a university-affiliated tertiary care referral hospital (Severance Hospital, Seoul, Republic of Korea). All patients were Korean (East Asian) and underwent genetic analysis of KRAS oncogene substitutions (codons 12 and 13) and their medical records were

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reviewed retrospectively. Patients’ smoking histories were classified into three groups; never, ex-, and current smokers. Never smokers were defined as those who smoked less than 100 cigarettes in their lifetime and ex-smokers had previously smoked cigarettes, but quit smoking more than 1 year prior to

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diagnosis of lung cancer. Patients’ performance status was assessed according to the Eastern Cooperative Oncology Group (ECOG) scale. Tumor histopathology was classified by World Health Organization criteria and tumor stage was described according to the 7th edition of the American Joint Committee on Cancer (AJCC) cancer staging manual. We examined survival outcomes to perform survival analysis. Overall survival (OS) was defined as the time from diagnosis to the date of death due to any cause. Progression-free survival (PFS) was defined as the time from diagnosis to the date of progression or death due to any cause, whichever occurred first. Clinical responses were classified according to Response Evaluation Criteria in Solid 4

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Tumor (v. 1.1). Patients’ survival records were censored on July 21, 2012. This study was approved by the Institutional Review Board of Yonsei University College of Medicine.

KRAS mutation analysis. After separating genomic DNA in tumor tissue, PCR amplification of

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codons 12 and 13 of the KRAS gene and gene sequencing of the purified PCR product was performed

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using an ABI Prism® 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA).

Immunohistochemistry (IHC). Expression of p-Akt Ser473, RalA, and RalB were analyzed by IHC.

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IHC was performed using the LABS®2 system (Dako Corp., Carpinteria, CA, US) according to the

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manufacturer’s instructions. Briefly, sections were deparaffinized, rehydrated, and then antigen retrieval was performed using high pH citrate buffer (Dako Cytomation, Carpinteria, CA, US) for 30

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minutes by microwave heating. Sections were then immersed in H2O2-methanol solution before incubating overnight with primary anti-pAkt-Ser473 (1:50, Cell Signaling Technology, Danvers, MA,

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US), RalA (1:100, BD Transduction Laboratory, San Jose, CA, US), and RalB (1:100, Santa Cruz Biotech, Santa Cruz, CA, US). Sections were incubated for 10 min with biotinylated linker and

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processed using avidin-biotin IHC techniques. For pAkt-Ser 473, 3,3'- diaminobenzidine (DAB) was used as a chromogen in conjunction with the Liquid DAB substrate kit (Novacastra, UK) and horseradish peroxidase was used as a chromogen for RalA and RalB. As a positive control of ralA and ralB immunostaining, rat forebrain was used and that of pAkt-ser473 human breast ductal cancer

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tissues were used. When more than 25% of cancer cells show staining as strong as positive control, either at nucleus or cytoplasm, it was considered for pAKT-Ser473 positive. Expression of ralA and ralB were evaluated by scoring system using product of staining intensity and percentage of positive cells. Staining intensity was classified as 0, 1, 2, 3 where intensity 2 means equal as that of positive control. Frequency was classified as 0, 1 (trace <5%), 1 (-10%) and 2 (-30%), and 3 (>30%). When the product of intensity and frequency was ≥6 it is considered as overexpression.

Statistical analysis. Clinically significant differences in the characteristics of KRAS mutation subtype 5

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were analyzed using the χ2 test and unpaired Student’s t-test. Predictive factors for overall survival (OS), disease free survival (DFS), and progression-free survival (PFS) were calculated using the Kaplan-Meier Estimator and Cox proportional hazards model. All tests of significance were twotailed and p-values of less than 0.05 were interpreted to indicate statistical significance. SPSS

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software (v. 18; SPSS, IL, USA) was used for statistical analysis.

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Results

A. Demographic characteristics Of 1,420 NSCLC patients, KRAS mutations in codon 12 and/or 13 were found in 82 (5.8%) patients. KRAS mutations were more frequently found in male patients and

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those who were currently or had previously been smokers. According to previous reports on Korean lung cancer patients, the frequency of KRAS mutation ranges from 3.9% to 5.2% of NSCLCs [8, 12]

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and 7.0% to 9.6% of lung adenocarcinomas [9, 11]. In a comparison of our results and other reports

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on Koreans, there were no significant differences in the age and gender of study patients. When comparing lung cancer patients with KRAS mutation and patients with wild-type KRAS, those

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harboring KRAS mutations were older, predominantly male, and had increased exposure to cigarette

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smoke (Table 1).

B. Gly12Asp was the most frequent KRAS mutant in Korean NSCLC. Because oncogenic

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substitution of KRAS influences the survival of lung cancer patients [13], we first analyzed the distribution of KRAS substitutions in Korean NSCLC patients (Table 2). Among the patients who

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were tested for KRAS mutation, 82 (5.8%) of 1,420 patients harbored a KRAS mutation in either codon 12 (73 of 82, 89.0%) or 13 (9 of 82, 11.0%) and transversions (45 cases, 54.9%) were more common than transitions (37 cases, 45.1%). Gly12Asp was the most frequent KRAS oncogene substitution (28 of 82, 34.1%), followed by Gly12Cys (18 of 82, 22.0%) and Gly12Val (11 of 82, 13.4%). Also,

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Gly12Asp accounted for the majority (15 of 27, 55.6%) of substitutions in never-smoker patients. Males and subjects who had a history of smoking exhibited a higher rate of transversion than females and never smokers (p=0.001 in gender analysis, p=0.006 in smoking analysis; χ2-test). When controlling for smoking status, transitions in codons 12 and 13 were significantly more frequent than transversions in women (p=0.026; Cochran–Mantel–Haenszel test). This finding suggests that distribution of KRAS mutations in a Korean lung cancer population differs from that of a Caucasian population, which showed Gly12Cys as the most frequent substitution [6]. However, KRAS mutation subtype did not affect patient survival. OS of all KRAS-mutant NSCLC patients, DFS of patients who 7

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underwent curative resection (n=29) and PFS of advanced stage patients (n=53) according to the KRAS mutation subtypes did not differ according to the KRAS mutation subtype (p=0.568 in OS, p=0.860 in DFS, p=0.426 in PFS; Kaplan-Meier estimations) (Fig. 1). In a Cox regression model adjusted for age, gender, performance status, smoking status, KRAS mutation subtype and tumor stage,

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only tumor stage (HR=7.56, 95% CI=2.97-19.25, p<0.001; Cox regression test) and smoking status (HR=3.37, 95% CI=1.35-8.42, p=0.009) were significant predictors of shortened overall survival in

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advanced NSCLC patients with KRAS mutation (Table 3).

C. pAkt-Ser473 is not related to the expression of KRAS mutation subtypes. Because pAkt-

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Ser473 is inhibited more strongly by p70S6K in the KRAS Gly12Cys mutant than in Gly12Asp [13], we postulated that the expression of pAkt-Ser473 is different in tissues expressing Gly12Asp and

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Gly12Cys mutations of KRAS. The expression of pAktSer473 in either cytoplasm or cytoplasm and nucleus of cancer tissues was evaluated by immunohistochemistry (Fig. 2). pAkt-473 was frequently

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detected both in the nucleus and cytoplasm of normal appearing bronchial epithelial cells. pAkt-473 was also detected in the nucleus and cytoplasm of KRAS mutant NSCLC tissues, but it was more

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frequently detected in the nucleus of the cancer cells. There was no difference in the pAkt-Ser473 expression between transversion and transition mutations, and pAktSer473 overexpression did not affect the survival of patients with KRAS mutant NSCLC (p=0.127 in cytoplasmic expression and

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p=0.063 in cytoplasmic and nuclear expression; χ2 test) (Table 4).

D. RalB expression is associated with poor prognosis in KRAS mutant non-small cell lung cancer. Because either Gly12Cys or Gly12Val mutations induce MEK activation through a RalGTPase-dependent pathway [13], expression of RalA and RalB was evaluated in NSCLC patients with KRAS mutants by immunohistochemistry (Fig. 2). RalA was expressed ubiquitously; it was detected in the membranous and the submembranous region of cancer cells, the bronchial epithelial cells, and type I and II pneumocytes. On the other hands, RalB was expressed in membrane and cytoplasm of the cancer tissues only. RalA was strongly expressed in the membranous region of 24 8

Page 25 of 42

(80.0%) of 30 tumor tissues whereas RalB was overexpressed in the membranous and cytoplasm of 17 (56.7%) of 30 cancer tissues. When RalA and RalB expression was analyzed according to KRAS substitution, there was no difference in protein expression between transversion and transition. Although expression of RalA was not differ among stages, RalB overexpression was more frequent in

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the advanced stage of lung cancer (p-value=0.020, χ2-test). However, the relationship between RalB (+) and OS did not reach statistical significance (p-value=0.179, Log-Rank test). These findings

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suggest that KRAS mutation substitution did not influence the expression of RalA, RalB and pAkt-

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Ser473, though RalB overexpression is associated with disease status in KRAS mutant NSCLC

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patients.

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Discussion Similar to the distribution of EGFR mutations, oncogenic mutation of KRAS in NSCLC shows significant ethnic differences in prevalence. The KRAS mutation was detected in less than in 10% of lung adenocarcinoma of Far East Asians [8, 9, 11, 12]. Reports on Korean populations have used

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limited numbers of NSCLC patients, which makes it difficult to draw conclusions on the clinical implications of KRAS mutation subtypes [8, 9]. Reports that specified substitution mutation of KRAS

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in Korean NSCLC are very limited as well [12]. In this study, by recruiting a large number of NSCLC

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patients, we were able to show that the prevalence of KRAS mutations at codons 12 and 13 is 5.8%, and to indicate specific substitution subtypes in Korean NSCLC patients with influencing factors.

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In the early era of KRAS mutation test when Sanger sequencing was routinely applied, the frequency of KRAS mutation in the Caucasian lung adenocarcinoma was around 30% [14, 15]. Recent report at

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2013 by Shepherd et al. showed KRAS mutation positive rate was still 33.7% (204 out of 605) in pooled early-stage resected lung adenocarcinoma from Western countries [16], showing the detection

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rate was not significantly differ between Sanger sequencing and currently adopted methods. Another meta-analysis by Meng et al. also showed that the difference in the detection rate among mutation

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tests (PCR-MSOP, PCR-DGGE, PCR-RFLP, PCR-seq) is not significant [17]. Many scientists propose that recently developed sensitive tests such as PCR-clamping could be applied for NSCLC samples with low percentage of tumor cells such as unprocessed bronchial biopsies or after neoadjuvant chemotherapy[18, 19].The cause of KRAS mutation is not clear, but a relationship with

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smoking has been repeatedly reported by many investigators. G>T transversions are the most frequent subtype of substitutions among NSCLC patients with smoking histories, accounting for 84% of total mutations. Our research showed that the frequency of transversions among those with smoking history reached 65.5%, which is lower than that of reports from the US [13]. Gly12Asp at codon 12, which originated from a transition substitution, showed the same frequency as Gly12Cys, reaching 23.6% of mutations. This rate is quite different from that of a report from the US showing Gly12Cys at a rate of 47%, Val at 24%, Asp accounting for 15%, and Ala for 7% [13]. Gender also influenced the distribution of KRAS subtypes. Even when the smoking status was compensated for, male patients 10

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had a higher chance of having a transversion mutation. This finding indicates that additional research, including epidemiologic studies on occupational exposure to carcinogens, is required to identify the difference in the mutation subtype between the genders. KRAS transversion mutations resulting in Gly12Cys and Gly12Val was found to be related to poor

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prognosis in lung cancer patients and are related to the inhibition of pAKT-Ser473 via IRS-1 inhibitory phosphorylation [13]. However, the relationship between expression status of pAkt-Ser473

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and KRAS substitution has not been verified in clinical samples and the clinical implication of pAkt is

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still elusive [20]. In this study, we evaluated both the clinical implications of and relationships to KRAS subtypes. Similar to previous reports, we found that pAkt expression was not only in the

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nucleus, but also in the cytoplasm of normal-appearing adjacent tissues and NSCLC cells. When the relationship of overexpression of pAkt in the cytoplasm and both nucleus and cytoplasm was

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evaluated, we could not find any clinical implication and relationship with subtypes of KRAS mutation. These findings may originate from (1) the small number of KRAS mutations found in the study

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population and limited number of available clinical samples, (2) the dynamics of pAkt-Ser473 expression, which is highly susceptible to diet, nutritional status, fasting, and other disease statuses,

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and (3) the possibility that there is indeed no relationship between subtype and oncogenic substitution. A recent report, indicating that there was no difference in the clinical outcome among lung cancer patients with different types of oncogenic substitution, suggested this option [21]. In spite of their high degree of similarity, the downstream effectors of RAS, the Ral small GTPases,

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have distinct functions. RalA has been implicated in epithelial cell polarity [22], insulin secretion [23], GLUT4 translocation [24, 25], neurite branching, and neuronal polarity [26, 27], while RalB is involved in tumor cell survival [28], migration/invasion [29-31], TBK1 activation [32], and autophagy [33]. Still, reports on the expression statuses and clinical significances of RalA and RalB in KRAS mutant lung cancer are very limited. In a recent report on null and conditional RalA and RalB knockout mouse models, RalB null mice were viable and did not show any phenotypic abnormality but mice that were RalA-null showed exencephaly and embryonic lethality. When these models were crossed with a KRAS-driven lung cancer mouse model, the mouse model that had either RalA or RalB 11

Page 28 of 42

was sufficient for tumor growth, suggesting that RalA and RalB act in a redundant fashion in KRASdriven lung cancer formation and proliferation [34]. Interestingly, in our samples, expression of RalA was detected in the normal-appearing adjacent lung tissues and majority of KRAS mutant lung cancer tissues, whereas RalB was detected in 56.7% of cancer tissues and was related to advanced stage.

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These findings suggest that RalB would be more relevant to any clinically significance and warrant further studies, including identification of the relationship with Ral GTPase activity in KRAS mutant

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lung cancer tissues.

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This study does have some limitations. The limited number of study tissues, because of the low frequency of KRAS mutations in these study populations, is one of the factors that lessened the ability

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to draw conclusions. Another limitation is an inability to measure Ral GTPase activity in these tissues and the use of pAkt as the only surrogate biomarker for comparing biologic activity of transversion

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and transition. The findings could be further influenced by the retrospective design of the study and data that were obtained from patients who had provided informed consent and were willing to pay for

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genetic analysis of KRAS mutation.

In conclusion, Korean NSCLC patients with oncogenic KRAS mutation had distinctive characteristics

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with lower frequency. We could not find differences between subtypes of KRAS substitution regarding clinical outcome or expression of pAKT-Ser473, RalA, and RalB. To identify the biologic and clinical significance of subtypes of KRAS mutation, a large prospective study may be required.

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Conflict of interest: There are no conflicts of interests. Acknowledgements: This study was supported by a faculty research grant of Yonsei University College of Medicine for 2012 given to EYK (6-2012-0129). The role of funding source: The funding source did not involve in the study design, data collection and interpretation, and writing.

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Accepted Manuscript Title: KRAS oncogene substitutions in Korean NSCLC patients; Clinical implication and relationship with pAKT and Ral GTPases expression Author: Eun Young Kim Arum Kim Se Kyu Kim Hyung Jung Kim Joon Chang Chul Min Ahn Jae Seok Lee Hyo Sup Shim Yoon Soo Chang PII: DOI: Reference:

S0169-5002(14)00183-4 http://dx.doi.org/doi:10.1016/j.lungcan.2014.04.012 LUNG 4594

To appear in:

Lung Cancer

Received date: Revised date: Accepted date:

22-12-2013 10-4-2014 23-4-2014

Please cite this article as: Kim EY, Kim A, Kim SK, Kim HJ, Chang J, Ahn CM, Lee JS, Shim HS, Chang YS, KRAS oncogene substitutions in Korean NSCLC patients; Clinical implication and relationship with pAKT and Ral GTPases expression, Lung Cancer (2014), http://dx.doi.org/10.1016/j.lungcan.2014.04.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Figure 2 Click here to download high resolution image

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Figure Legends

Figure Legends

Figure 1. Kaplan-Myer estimation of overall survival (A) of all KRAS-mutant NSCLC patients, disease free survival (B) of patients who underwent curative resection (n=29) and

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progression free survival (C) of advanced stage patients (n=53) according to the KRAS

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mutation subtypes. KRAS mutation subtype did not affect patients’ survival.

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Figure 2. Expression of Ral-GTPases, RalA and RalB, and pAkt-Ser473 in the adjacent normal-appearing lung tissues (A), and NSCLC tissues for case #6 (B) and case #8 (C). For

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immunohistochemistry of RalA and RalB, HRP was used as a chromogen. DAB was used as

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a chromogen for pAKT-Ser473. All images are shown at x400.

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Table

Table 1. Demographic characteristics of study participants according to KRAS mutation

KRAS mutation Wild type

Mutant

756 (95.3)

37 (4.7)

≥65

582 (92.8)

45 (7.2)

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<65

Gender (No (%)) 769 (92.9)

Female

569 (96.1)

23 (3.9)

665 (96.1)

27 (3.9)

Current or ex-smoker

673 (92.4)

55 (7.6)

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Never smoker

Total pack years (mean ± S.D.)

35.9 ± 22.2

Histologic subtypes (No (%))

Squamous cell carcinoma

II III IV Total

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I

0.002† 0.037* 0.024†

71 (6.3)

201 (98.5)

3 (1.5)

83 (91.2)

8 (8.8)

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Others

42.5 ± 25.0

1054 (93.7)

ed

Adenocarcinoma

†

59 (7.1)

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Smoking history (No (%))

0.006†

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Male

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0.044†

Age

Stage

P-value

0.046† 157 (90.8)

16 (9.2)

140 (91.5)

13 (8.5)

297 (95.8)

13 (4.2)

744 (94.9)

40 (5.1)

1338 (94.2)

82 (5.8)

2

P-value was obtained by χ -test.

*

P-value was obtained by independent sample t-test.

1

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Table 2. KRAS oncogene substitution as a function of smoking history

KRAS mutation substitution

Current or

Never smoker

Male

Female

ex-smoker 5

17

1

Gly 12Val

10

1

8

3

Gly 12Ala

6

3

7

Gly 13Cys

4

0

4

Gly 12Phe

3

0

Gly 12Asp

13

15

Gly 13Asp

4

1

Gly 12Ser

2

2 0.006*

P-value Total

55

*

2

cr

0

3

0

14

14

59

4

1

2

2 0.001* 23

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P-value obtained from χ2-test.

27

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13

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Transition

Gly12Cys

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Transversion

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Table 3. Multivariate analysis of the variables affecting survival of NSCLC patients with KRAS mutation Variables

Numbers

Events

Multivariate HR

95% CI

P-value*

37

16

1

≥ 65

45

25

0.833

Male

59

26

1

Female

23

15

2.41

ECOG 0-1

78

38

ECOG 2

4

3

Never smoker

27

12

Current or ex-smoker

55

Performance status

Transversion

Stage Resectable Unresectable

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0.056

0.55-6.61

0.31

1.35-8.42

0.009

0.70-3.04

0.32

1

3.37

45

21

1

37

20

1.46

29

10

1

53

31

7.56

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Transition

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KRAS mutation subtype

29

0.98-5.94

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Smoking status

0.59

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Gender

0.43-1.63

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< 65

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Age

2.97-19.25 <0.001

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* P-value was obtained by Cox regression test.

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Table 4. Relationship between KRAS mutation subtype and pAKT-Ser473 expression

pAkt-Ser473

Transversion

Transition

P-value 0.127*

Positive

4

9

Negative

10

7

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Cytoplasmic expression

0.063*

Nuclear and cytoplasmic expression

Negative

10

Total

14

P-value obtained by χ2 test.

6

16

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*

10

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4

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Positive

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*Conflict of Interest statement

Conflict of Interest Statement

None of the authors disclosed any financial and personal relationships with other people or

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organizations that could inappropriately influence this work.

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