Abstract
Purpose
This study aimed to determine the dose effect of smoking on the mutational frequency and spectrum of epidermal growth factor receptor (EGFR) gene in Korean non-small cell lung cancer (NSCLC).
Methods
Detailed smoking histories were obtained from 324 consecutively enrolled Korean NSCLC patients. Mutational status of EGFR (exon 18–21) was determined using nested polymerase chain reaction amplification.
Results
A total of 108 EGFR mutations (33.3%) were identified in 107 patients. Decreased EGFR mutation rate with increased smoking dose was observed, with 48.0% (82 of 171) in never smokers, 23.1% (15 of 65) in former smokers, and 11.4% (10 of 88) in current smokers. The incidence of EGFR mutation was significantly lower in patients who smoked for more than 25 pack-years (P < 0.0001) or who stopped smoking cigarettes less than 10 years ago (P < 0.0001). Mutations in exon 19 or 21 were associated with fewer total smoke years (5.0 vs. 25.0 years in exon 20, P = 0.024), fewer total pack-years (6.3 vs. 38.9 pack-years in exon 20, P = 0.079), and more smoke-free years (11.1 vs. 3.6 years in exon 20, P = 0.027), compared with those in exon 20. Mutations in exon 19 or 21 were associated with female (P < 0.0001), never smoker (P < 0.0001), and adenocarcinoma (P < 0.0001), whereas those in exon 20 were not.
Conclusions
Smoking dosage affects the incidence of EGFR mutations. EGFR mutations in exon 19 or 21 are associated with low exposure to cigarette smoke, whereas EGFR mutation in exon 20 is more common in smokers.
Keywords: Non-small cell lung cancer, Smoking, Epidermal growth factor receptor (EGFR), Mutation
Introduction
Tobacco smoking is the primary risk factor for lung cancer (Wynder and Graham 1985). Most genetic alterations observed in lung cancer, including point mutations of p53 and K-ras gene or chromosomal deletions, are strongly associated with tobacco smoking (Pfeifer et al. 2002; Le Calvez et al. 2005). However, lung cancer also develops in patients who have never smoked. It has recently been reported that 15% of men and 53% of women with lung cancer worldwide are a lifetime never smoker (Parkin et al. 2005). However, genetic alterations closely linked to this type of lung cancer have rarely been known. Mutations in the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) gene has been the first molecular characteristic of lung cancer in never smokers (Kosaka et al. 2004a; Shigematsu et al. 2005).
The EGFR pathway plays a central role in the initiation and progression of non-small cell lung cancer (NSCLC) and attracts considerable interest as a development target of an anticancer drug (Castillo et al. 2004). Small molecules of EGFR–TK inhibitors (TKIs) such as gefitinib and erlotinib have demonstrated a significant promising efficacy for advanced NSCLC patients through recent large-scaled clinical trials (Shepherd et al. 2005; Kim et al. 2008; Mok et al. 2009). Moreover, these drugs show a dramatic response in a specific subset, such as patients with adenocarcinoma histology, never smoker, women, East Asian patients, and patients carrying EGFR mutations (Tsao et al. 2005). Among these predictive factors, mutations in the EGFR gene have been known to be the most potent biological predictor of EGFR–TKIs sensitivity (Pao et al. 2004; Lynch et al. 2004; Mulloy et al. 2007). EGFR mutations including small in-frame deletions and point mutations within the ATP-binding pockets cause significant abnormalities in the signaling behavior of EGFR receptor (Lynch et al. 2004; Paez et al. 2004). Therefore, EGFR mutations leave tumors dependent on the EGFR pathway for growth and survival, and sensitize them to ATP-like small molecule inhibitors (Gazdar et al. 2004; Sharma et al. 2007).
EGFR mutations arise in four exons as follows: substitutions for G719 in the nucleotide-binding loop of exon 18, in-frame deletions within exon 19, in-frame insertions within exon 20, and substitutions for L858 or L861 in the activation loop within exon 21 (Shigematsu et al. 2005). Of these EGFR mutational types, exon 19 point deletion and exon 21 missense mutation (L858R) are the most frequent mutations, representing 85–90% of EGFR mutations (Shigematsu et al. 2005). These mutations have been associated with female gender, adenocarcinoma histology type, and East Asian ethnicity (Shigematsu et al. 2005). A history of never smoking, in particular, has a very strong association with these mutations. Two classes of EGFR mutations are observed in 40–60% of lung cancers in never smokers, whereas they are in 10–20% of lung cancers in smokers (Pao et al. 2004; Kosaka et al. 2004b; Shigematsu et al. 2005). Furthermore, several reports suggest that the cumulative dose of tobacco smoking was inversely related to the occurrence of EGFR mutations (Tokumo et al. 2005; Sugio et al. 2006; Pham et al. 2006). In a study of US patients with lung adenocarcinoma, Pham et al. (2006) showed the likelihood of these EGFR mutations decreases as the number of pack-years increases. The former study indicated that classic EGFR mutations were less frequently found in patients who smoked for more than 15 pack-years or stopped smoking less than 25 years ago compared with those who never smoked (Pham et al. 2006). On the other hand, these effects of tobacco smoking on EGFR mutations also need to be determined in East Asian patients because the ethnic differences also influence the incidence of EGFR mutations. However, there was little known about the dose effect of tobacco smoking on EGFR mutations, which is investigated based on detailed smoking history obtained by a trained interviewer in East Asian cohort.
Therefore, we investigated to determine the dose effect of tobacco smoking on the incidence and type of mutations in the EGFR genes in Korean NSCLC patients. We also characterized the clinicopathologic features of lung cancer according to the type of EGFR mutations.
Materials and methods
Patients and data collection
We consecutively enrolled patients with primary lung cancer who were newly diagnosed, histologically or cytologically proven, and untreated NSCLC between June 2006 and December 2008 at Yonsei Cancer Center. The patients whose tumor tissue samples were available for genetic test were eligible for the study. Pathologic tumor stage was determined using the 7th the American Joint Committee on Cancer/International Union Against Cancer TNM staging system (Rami-Porta et al. 2009). Tumor typing and grading were independently performed by a pathologist according to the WHO criteria for classification of tumors of the lung (Travis et al. 1999). Sample collection for the research was approved by the Institutional Review Board of the hospital, and informed written consent was obtained from each patient.
Smoking histories were obtained by trained interviewers according to a standard protocol, at their first hospital admission. Detailed smoking histories, including age at first cigarette and packs per day, and the numbers of smoke years and smoke-free years (after quitting) were collected. Based on smoking status, patients were categorized as never smokers (<100 lifetime cigarettes), former smokers (quit ≥1 year ago), or current smokers (quit <1 year ago).
Mutational analysis
The sample for analysis was obtained from biopsy (n = 164, 50.6%) or surgical specimens (n = 160, 49.4%) before front chemotherapy. Nucleotide sequencing of the kinase domain of EGFR gene (exons 18–21) was performed using nested PCR amplification of individual exons. Details of sequencing have been described previously (Kang et al. 2007).
Statistical analysis
The Pearson χ2 and Fisher’s exact tests were used for categorical variables to compare the incidence of EGFR mutations among different clinicopathologic groups. The Wilcoxon rank-sum test was used for continuous variables (age at first cigarette, packs per day, number of smoke years, number of years smoke free, and pack-years) for the comparison of median values between mutant and wild-type tumors. The logistic regression analysis using a forward stepwise method was done, and odds ratios (ORs) were calculated in order to investigate the effects of covariates on gene mutations. The receiver operating characteristic (ROC) curve methodology was used to assess the ability of smoking history parameters to predict the likelihood of mutations on EGFR gene. ROC analysis allowed the determination of the optimal cutoff for smoking parameters to maximize accuracy. The diagnostic accuracy of smoking history parameters for predicting the incidence of EGFR mutations was summarized with the area under the ROC curve. The two-sided significance level was set at P < 0.05.
Results
Patient characteristics
The baseline characteristics of all patients are described in Table 1. The median patient age was 61 years. Approximately half of the patients were female, and adenocarcinoma was the major histological type. The majority of the patients had stage III/IV diseases at the time of diagnosis. Patients who had never smoked made up 52.9% of the sample population.
Table 1.
Patient characteristics
| Characteristics | N | EGFR mutations (%) | P valueb |
|---|---|---|---|
| Age, years | 0.721 | ||
| ≤60 | 159 | 51 (32.1) | |
| >60a | 165 | 56 (33.9) | |
| Median (range) | 61 (16–87) | 61 (34–79) | |
| Sex | <0.0001 | ||
| Female | 146 | 68 (46.6) | |
| Male | 178 | 39 (21.9) | |
| Histology | <0.0001c | ||
| Adenocarcinoma | 233 | 100 (42.9) | |
| Squamous cell carcinoma | 71 | 4 (5.6) | |
| Large cell carcinoma | 7 | 0 (0.0) | |
| Mucoepidermoid carcinoma | 2 | 1 (50.0) | |
| Pleomorphic carcinoma | 6 | 1 (16.7) | |
| NSCLC, NOS | 5 | 1 (20.0) | |
| Pathologic grade | <0.0001d | ||
| Well differentiated | 65 | 39 (60.0) | |
| Moderately differentiated | 142 | 42 (29.6) | |
| Poorly differentiated | 117 | 26 (22.2) | |
| Stage | 0.063 | ||
| I | 47 | 24 (51.1) | |
| II | 36 | 10 (27.8) | |
| III | 109 | 36 (33.0) | |
| IV | 132 | 37 (28.0) | |
| Cigarette smokinge | <0.0001 | ||
| Never | 171 | 82 (48.0) | |
| Former | 65 | 15 (23.1) | |
| Current | 88 | 10 (11.4) |
aOne patient had mutations in both exon 21 L858R and exon 20 T790M
bBy Pearson χ2 or Fisher’s exact tests
cAdenocarcinoma versus others
dWell-differentiated versus others
eFormer smokers are defined persons who stopped smoking more than 1 year previously
NSCLC non-small cell lung cancer, NOS not otherwise specified
A total of 108 EGFR mutations were detected in 107 (33.0%) of 324 patients. One patient had a threonine-to-methionine substitution at amino acid position 790 (T790M), along with the activating L858R mutation in exon 21 (Fig. 1). Of patients with EGFR mutations, 68 were female and 39 were male. Thus, EGFR mutations were detected in 46.9% (68 of 145) of females and in 21.9% (39 of 178) of males (P < 0.0001). However, there was no gender difference in the incidence of EGFR mutations in the 171 never smokers (47.5% in female vs. 50% in male, P = 0.805). The presence of EGFR mutations was significantly associated with adenocarcinoma histology (42.9 vs. 7.7% in others, P < 0.0001). Of never smokers, the difference remained statistically significant (50.6 vs. 20.0% in others, P = 0.023). The differentiation grade of the tumors also correlated significantly with the incidence of EGFR mutations. Thus, the incidence of EGFR mutations was higher in well-differentiated tumors compared to moderately to poorly differentiated tumors (60.0 vs. 26.3%, P < 0.0001). Mutations were inversely correlated with tobacco consumption, with only ten mutations (11.4%) in 88 current smokers, 15 (23.4%) in 64 former smokers, and 82 (50.0%) in 171 never smokers (P < 0.0001). The presence of EGFR mutations was independent of patient age or disease stage.
Fig. 1.
Representative nucleotide sequence of the EGFR gene in tumor specimen with both T790M exon 20 and L858R exon 21 mutations. a The arrow indicates the mutation at nucleotide 2,369 (C → T), which leads to the substitution of methionine (ATG) for threonine (ACG) at position 790. b The arrow indicates the mutation at nucleotide 2,574 (T → G), which leads to the substitution of arginine (CGG) for leucine (CTG) at position 858
EGFR multivariate analysis
To define which variables were predictive of EGFR mutation, those factors that were statistically significant in the univariate analysis (sex, histology, histological grade, and never smoking history) were included in the multivariable model. Never smoking history (odds ratio [OR] = 1.74, 95% confidential interval [CI] = 1.20–2.53; P = 0.004), adenocarcinoma (OR = 4.67, 95% CI = 2.04–10.69; P < 0.0001), and well-differentiated histology (OR = 2.86, 95% CI = 1.53–5.35; P = 0.001) remained significantly associated with incidence of EGFR mutation.
The incidence of EGFR mutation and its relationship to smoking history
EGFR mutations were detected in 48.0% of never smokers and in 83.3, 25.0, 21.4, 27.3, and 11.0% of patients with smoke years of 1–10, 11–15, 16–20, 21–25, and 26 years or more, respectively. The incidence of EGFR mutations decreases with an increasing number of pack-years of smoking (Table 2). Patients with smoking histories of more than 20 pack-years had a 12.2% incidence of EGFR mutations compared to 45.8% among individuals with a smoking history of 20 or fewer pack-years (P < 0.0001).
Table 2.
Incidence of EGFR mutations by pack-years
| No. of pack-years | Patients | P valuea | ||
|---|---|---|---|---|
| Total No. | No. with mutations | % With mutations | ||
| 0 (never smokers) | 171 | 82 | 48.0 | NA |
| 1–10 | 15 | 6 | 40.0 | 0.554 |
| 11–20 | 15 | 4 | 20.0 | 0.113 |
| 21–25 | 17 | 2 | 11.8 | 0.004 |
| 26–30 | 18 | 4 | 22.2 | 0.037 |
| 31–35 | 9 | 0 | 0.0 | 0.004 |
| 36–40 | 23 | 4 | 17.4 | 0.006 |
| 41–45 | 13 | 0 | 0.0 | 0.001 |
| 46–50 | 13 | 0 | 0.0 | 0.001 |
| ≥51 | 30 | 5 | 16.7 | 0.001 |
aBy Pearson χ2 or Fisher’s exact tests to compare the incidence of EGFR mutations between smoking pack-years categories and never smokers
NA not applicable
The presence of EGFR mutations by smoke-free years is listed in Table 3. The incidence of mutations in EGFR for 0–10 smoke-free years was 12.6%, whereas that for patients who were smoke free for more than 10 years was 44.4% (P < 0.0001). Age at first cigarette was not associated with EGFR mutation (P = 0.425). A comparison of the mean values of cigarette smoking duration and intensity by EGFR mutation status is shown in Fig. 2.
Table 3.
Incidence of EGFR mutations by smoke-free years
| Smoke-free years | Patients | P valuea | ||
|---|---|---|---|---|
| Total No. | No. with mutations | % With mutations | ||
| Never smokers | 171 | 82 | 48.0 | NA |
| 0 (current smokers) | 88 | 10 | 11.4 | <0.0001 |
| 1–5 | 28 | 3 | 10.7 | <0.0001 |
| 6–10 | 19 | 4 | 21.1 | 0.025 |
| 11–20 | 9 | 4 | 44.4 | 1.000 |
| ≥21 | 9 | 4 | 44.4 | 1.000 |
aBy Pearson χ2 or Fisher’s exact tests to compare the incidence of EGFR mutations between smoke-free years categories and never smokers
NA not applicable
Fig. 2.
Comparison of median values of cigarette smoking duration and intensity by EGFR mutation status
The area under the ROC curve for smoking history parameters is 0.71 for smoking status at diagnosis, 0.56 for age at first cigarette, 0.70 for number of packs per day, 0.67 for years smoke free, 0.73 for number of smoke years, and 0.72 for total pack-years. Among these parameters, total pack-years and number of smoke years were chosen to derive a predictor of EGFR mutation. A smoking history of less than 20 total pack-years was 85% sensitive and 50% specific for predicting EGFR mutation. Similarly, a total of less than 25 smoke years was 91% sensitive and 50% specific for EGFR mutation. The categories of less than 20 total pack-years and less than 25 smoke years had 86% sensitivity and 53% specificity.
The type of EGFR mutation and its relationship to smoking history
Of the 108 EGFR mutations, there were 75 (69.4%) deletions in exon 19, 26 (24.1%) missense mutations (L858R) in exon 21, six (5.6%) duplications and/or insertions in exon 20, and one (0.9%) T790M in exon 20. One case had both the L858R mutation in exon 21 and the T790M mutation in exon 20. All of the 108 EGFR mutations have been frequently described, with the exception of the T790M mutation in exon 20. Interestingly, the T790M mutation occurred in a male former smoker with stage III squamous cell carcinoma. This patient had not been treated with EGFR–TKIs. The somatic nature of the T790M mutation in this patient was confirmed by the mutational analysis of peripheral blood leukocytes as well as primary tumor tissue.
Next, we compared various clinicopathologic features and smoking dose among different types of EGFR mutations (Table 4). Because there was no significant difference between patients with exon 19 deletion and exon 21 L858R in all parameters examined, we categorized the types of mutations as mutations in exon 19/21 and exon 20. Although mutations in exon 19/21 were positively associated with adenocarcinoma (OR, 5.61; P < 0.0001) and never smokers (OR, 2.31; P = 0.001) by logistic regression analysis, mutations in exon 20 did not show a significant association with such parameters. Furthermore, patients with mutations in exon 19/21 had histories of significantly fewer smoke years (5.0 vs. 25.0 in exon 20 mutations, P = 0.024) and more years smoke free (11.0 vs. 3.6 in exon 20 mutations, P = 0.027). There were, also, trends toward fewer total pack-years (P = 0.079) and packs per day (P = 0.070).
Table 4.
Comparison of clinicopathologic features and smoking dose among different types of EGFR mutations
| Characteristics | Exon 19 deletion | Exon 21 L858Ra | P value | Exon 20 insertion/duplication or T790M | P value |
|---|---|---|---|---|---|
| n = 75 (%) | n = 26 (%) | n = 7 (%) | |||
| Sex | 0.33b | 0.099d | |||
| Male | 28 (37.3) | 7 (26.9) | 5 (71.4) | ||
| Female | 47 (62.7) | 19 (73.1) | 2 (28.6) | ||
| Histology | 0.64b | 0.084d | |||
| Adenocarcinoma | 71 (94.7) | 24 (92.3) | 5 (71.4) | ||
| Others | 4 (5.3) | 2 (7.7) | 2 (28.6) | ||
| Cigarette smoking | 0.13b | 0.009d | |||
| Never | 59 (78.6) | 21 (80.8) | 2 (28.6) | ||
| Former | 8 (10.7) | 5 (19.2) | 3 (42.8) | ||
| Current | 8 (10.7) | 0 (0.0) | 2 (28.6) | ||
| Packs per day, median (95% CI) | 0.2 (0.2–0.3) | 0.2 (0.1–0.4) | 0.83c | 1.1 (0.5–5.3) | 0.070e |
| Pack-years, median (95% CI) | 5.5 (4.1–7.8) | 8.8 (5.4–16.8) | 0.47c | 38.9 (16.2–188.6) | 0.079e |
| Smoke years, median (95% CI) | 5.0 (3.7–7.1) | 5.2 (3.2–9.9) | 0.95c | 25.0 (10.4–121.2) | 0.024e |
| Smoke-free years, median (95% CI) | 10.8 (8.0–15.3) | 11.6 (7.1–22.1) | 0.86c | 3.6 (1.4–17.5) | 0.027e |
aOne case had mutations in both exon 21 L858R and exon 20 T790M. b By Fisher’s Exact Test and c by Wilcoxon rank-sum test in exon 19 deletion versus exon 21 L858R. d By Fisher’s Exact Test and e by Wilcoxon rank-sum test in exon 20 insertion/duplication or T790M versus exon 19 deletion/exon 21 L858R
CI confidential interval
Discussion
In the current study, we examined the influence of cigarette smoking on the spectrum of EGFR mutations in Korean patients with NSCLC. We demonstrated a high prevalence of EGFR mutations in our study population (33.0%), which was consistent with several other studies showing high incidence of EGFR mutation in East Asian patients. East Asian patients have a higher prevalence of EGFR mutation (30–60%) than Caucasian patients (10–20%) (Lynch et al. 2004; Paez et al. 2004; Pao et al. 2004). Our results confirmed and extended previous observations that there is a strong inverse relationship between cigarette smoking and incidence of EGFR mutations, suggesting that EGFR mutations may play a key role in the development of NSCLC in patients with a low exposure to cigarette smoking. In addition, this study demonstrated that the duration and intensity of cigarette smoking has potential predictive value for the presence of EGFR mutations in a Korean population. The most important observation from this study was the appreciation of the uniqueness of individual types of EGFR mutations. This was realized by comparing the distribution of specific EGFR mutations in various types of NSCLC, according to the duration and intensity of cigarette smoking and clinicopathologic characteristics.
It has been suggested that the prevalence of EGFR mutations is particularly high in patients with adenocarcinoma histology, never smokers, and women (Shigematsu et al. 2005). In a multivariate analysis, we evaluated the independent contribution of each of these factors to the occurrence of EGFR mutations. Although EGFR mutations were more frequently present in women, the patient’s gender was found to be a confounding factor related to the predominance of never smokers in Korean female patients.
The presence of EGFR mutations has been correlated in response to EGFR–TKIs, and patients with a high likelihood of response to these treatments can be identified by molecular analysis of tumor tissue to detect sensitive mutations (Pao et al. 2004; Lynch et al. 2004; Mulloy et al. 2007). However, acquiring adequate tissue for EGFR mutational analysis is often not feasible, particularly in patients with advanced disease. Therefore, the identification of clinical parameters that can serve as surrogates for EGFR mutation may prove useful when mutational analysis is not feasible. In our study, we found that patients with a cigarette smoking history of fewer than 20 total pack-years or fewer than 25 smoke years had a higher incidence of EGFR mutations comparable to never smokers. Combination of these two parameters could predict the presence of EGFR mutations with 86% sensitivity and 53% specificity. These data demonstrate that a universally available clinical characteristic can estimate the incidence of EGFR mutation in patients with NSCLC and can assist the clinician in choosing patients who are most likely to benefit from treatment with EGFR–TKIs.
Tumors harboring EGFR mutations were not always sensitive to EGFR–TKIs. Differential sensitivity to EGFR–TKIs, which depends on the type of EGFR mutation, has been recently shown by the identification of an exon 20 insertion mutant that is resistant to these inhibitors (Greulich et al. 2005). Although they were not widely reported, a total of 18 (5.1%) exon 20 insertions were observed among 350 EGFR mutations identified in 1,108 NSCLC cases from recent five large-sized studies (Kosaka et al. 2004b; Shigematsu et al. 2005; Mitsudomi et al. 2005; Chou et al. 2005; Huang et al. 2004). In our study, we showed that clinicopathologic features and, most importantly, cigarette smoking parameters in exon 20 mutations were significantly different from those in exon 19 or exon 21 mutations. In our study, four of six patients with exon 20 insertion mutations had been treated with gefitinib or erlotinib. Three of them showed stable disease for a short duration (3 months, 3 months, and 4 months), and one patient showed progressive disease; these findings were consistent with previous studies (Greulich et al. 2005). We have little data regarding TKI sensitivity for patients with insertion mutations to date. However, based on the results from our study and other in vitro data, they may not benefit from the conventional TKIs, despite the fact that their tumors have EGFR mutations. Interestingly, the irreversible EGFR inhibitor has been reported to be more effective than gefitinib for the inhibition of tumors harboring exon 20 insertion mutants, indicating a need for a novel strategy in the treatment of this distinct subset of NSCLC (Greulich et al. 2005).
Patients who responded to TKIs and subsequently relapsed were found to have T790M secondary mutations (Kobayashi et al. 2005; Pao et al. 2005). Although the T790M mutation is mostly associated with acquired resistance to TKIs, such resistance has also been reported in patients who have not taken TKIs, indicating that this mutation is not uniquely associated with acquired drug resistance (Kwak et al. 2005). EGFR-activating mutations (L858R or deletion mutations) preferentially occurred with T790M mutations. Similarly, a T790M mutation in our study was found, together with an L858R mutation, in a patient that had not been treated with a TKI. Contrary to exon 19 deletions and the L858R mutations, T790M mutations, which were also an alteration in exon 20, showed no bias for sex, smoking status, or histology (Inukai et al. 2006). Given the fact that the rate of T790M mutation was significantly higher in advanced tumors (Inukai et al. 2006), which was also seen in our case, compared to early-stage tumors, the T790M mutation may not be involved in tumor initiation; rather, it may occur during the development of NSCLC initiated by some other mechanism, such as an EGFR-activating mutation or a K-ras mutation.
In conclusion, our results suggest that the dose of cigarette smoke, based on patients smoking history, may significantly influence not only the prevalence, but also the types of EGFR gene mutations. There is clearly a need to appreciate the uniqueness of individual types of EGFR mutations, according to the duration and intensity of cigarette smoking. The information on the exact type of mutation in an individual tumor will assist clinical decision-making, namely in regard to the likely sensitivity of the cancer to EGFR–TKI therapy.
Acknowledgments
This study was supported by a faculty research grant of Yonsei University College of Medicine for 6-2009-0066. We thank the members of Lung Cancer Clinic Severance Hospital for thoughtful discussion and review of the manuscript.
Conflict of interest statement
None declared.
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