k-ras Mutation and Occupational Asbestos Exposure in Lung Adenocarcinoma

Introduction

Lung cancer is the most common cancer worldwide, and nearly all lung cancer is environmentally induced and thus preventable. Although smoking contributes most dominantly to lung cancer occurrence, exposure to asbestos synergistically enhances lung cancer risk and accounts for a significant fraction of bronchogenic carcinomas (1) . Asbestos use continues to be widespread and is increasing in many third world countries. In addition, considerable debate remains about its carcinogenic mechanism and the magnitude of cancer risk from exposure to asbestos that does not induce interstitial fibrosis (asbestosis; Refs. 2 and 3 ). Fibrosis of the lung from many causes has been associated with an increased cancer risk, and scar formation in the peripheral lung has also been associated with adenocarcinoma of the lung (4, 5, 6) . This is of interest because some data show asbestos to be associated with the occurrence of adenocarcinoma (7 , 8) . Hence, this has led some authors to posit that only in the setting of asbestosis can a lung cancer be considered truly asbestos-related (2) .

Molecular epidemiologic studies have associated asbestos exposure with somatic mutations at the tumor suppressor loci p53 (9 , 10) and FHIT (11) and with increased p53 expression measured using immunohistochemistry (12) in primary lung tumors. These studies suggest that asbestos exposure augments the mutagenic and carcinogenic potential of tobacco smoke carcinogens. However, they have not established the mechanism of action of asbestos, or whether there is a dose-response relationship between asbestos exposure and mutation induction or whether the induction of mutations is independent of the induction of interstitial fibrosis. The association of asbestos exposure, interstitial asbestosis, and oncogene mutation has been investigated in a cohort of Finnish lung cancer patients; a nonsignificant trend was observed, suggesting that those with higher asbestos exposure are more likely to develop k-ras mutant cancers (13 , 14) . However, this study did not address the questions of k-ras mutation and the timing of asbestos exposure, the potential dose-response relationship, or the complex issue of the role of interstitial fibrosis in carcinogenesis. Here we present data from a large case series of non-small cell lung cancer in New England, characterizing the relationship between k-ras mutation, chest radiographic findings, and the parameters associated with levels of occupational asbestos exposure.

Patients and Methods

Study Population.

Cases consisted of all newly diagnosed, resectable lung cancer patients who received treatment at the Massachusetts General Hospital from November 1992 through December 1996. There were 458 cases enrolled in this time period, which represents approximately 90% of all eligible patients. Twenty cases were excluded because the resected tumor was either a recurrence of lung cancer or a metastatic lesion. Patients with simultaneous multiple tumors were also excluded. For 83 cases, a tissue specimen was not retrieved, either because information was missing from the case file so an appropriate specimen could not be identified or the necessary tumor block was not found in the pathology archives at the hospital. Thus, a total of 355 cases were included for the study of somatic mutations.

Detailed information describing the tumors was available from pathology reports. Information regarding patient demographics and asbestos occupational exposure histories was obtained using an administered questionnaire. Radiographic changes were extracted from chest radiographs taken at the time of surgery.

Asbestos Exposure Assessment.

Subjects were asked whether they had ever been occupationally exposed to asbestos. If so, they were asked about the type of work, corresponding dates and duration of exposure, and the frequency of contact with asbestos. In addition, an exposure index was calculated that integrates these parameters to estimate the total amount of asbestos exposure (15 , 16) . Briefly, an intensity factor was assigned depending on the occupation in which the exposure occurred, and a dose category was assigned depending on the dates of exposure to account for the decreased exposures after the adoption of OSHA regulatory standards. The intensity factor, dose category, number of years exposed, and the fraction of the year exposed were multiplied to obtain an exposure rating for occupational exposures. Questions were asked about exposure to asbestos (yes or no), employment in jobs with known asbestos exposure (e.g., construction laborer, brake mechanic, insulation, pipefitting, shipyard work, and so forth) or employment in industries with known asbestos exposure (e.g., construction). Exposure to asbestos was scored as positive only when answers were consistent in all categories. All patients as well as the interviewer had no knowledge of the k-ras somatic mutation status of the patient’s tumor.

k-ras Mutation Screening.

Tumor tissue was obtained from archived pathology specimens, and DNA was extracted for the study of somatic alterations as described previously (11) . PCR amplification of k-ras exon 1 was performed under standard PCR conditions for 15 cycles using primers described previously (Ref. 17 ; X1s, 5′-CATGTTCTAATATAGTCACA; X1a, 5′-AACAAGATTTACCTCTATTG). The PCR product was then diluted 1:100 and used as a template for the PCR-RFLP detection of k-ras codon 12 mutations (18) . Positive cell line controls for the k-ras mutation genotype were NCI-H23 (mutated) and TK6 (wild type). When mutations were detected, additional PCR-RFLP protocols were used to determine the specific codon 12 mutation (18) .

Statistical Analysis.

All statistical tests were two-sided. Mutation was scored as a dichotomous variable (present/absent). Smoking status was scored as current, ex-smoker, or never, and years since last smoked was a continuous variable. Crude associations between mutation status and categorical variables were tested using Fisher’s exact test (dichotomous) or the Mantel-Haenszel test for trend (polychotomous), and adjusted ORs 3 were calculated using logistic regression. Associations between mutation status and continuous variables were tested using Wilcoxon’s rank-sum test.

Results and Discussion

In the current study, asbestos exposure was assessed by an interviewer-administered questionnaire and subsequently by chest radiograph. Asbestos exposure was classified as being positive for all self-reported occupational exposures, and any reported nonoccupational exposures were scored as negative for occupational exposure. An index was created to estimate the intensity of occupational exposure received. The patient demographics and clinical pathological characteristics are shown in Table 1 ⇓ . As expected, there was a strong association between k-ras codon 12 mutation and adenocarcinoma histology in the entire case series, comparing adenocarcinoma with all other histologies (n = 355; P < 0.001). There was a low prevalence of mutation in squamous cell carcinomas (2.8%) and an intermediate prevalence for both large cell carcinoma and adenosquamous cell carcinoma (8.3% and 7.7%). There were no mutations detected in the 10 bronchoalveolar carcinomas. The study population was >95% Caucasian. Because there was a relatively low probability of significant asbestos exposure in women, only male adenocarcinoma cases were included in the analysis (n = 88). In this subgroup, asbestos exposure data were available for 84 cases.

There were no significant differences in age or smoking by k-ras mutation status; however, mutations were overrepresented in those with an occupational history of asbestos exposure (Table 2) ⇓ . The association between mutation and asbestos exposure remained significant after adjustment for age and pack-years smoked (Table 3 ⇓ , adjusted OR, 6.9; 95% confidence interval, 1.7–28.6).

There was an apparent exposure-response effect of asbestos dose and the prevalence of k-ras mutation, as assessed by a previously designed index that estimates integrated dose (Ref. 15 ; Fig. 1 ⇓ ). To test whether levels of asbestos exposure were greater among patients with a k-ras mutation in their tumors, we compared the mean asbestos exposure index score of the k-ras mutation-positive group with that in the k-ras mutation-negative group. There was a significantly higher calculated dose in those patients with k-ras mutations (P = 0.02, Wilcoxon’s rank-sum test).

Stratification of the study groups by smoking status and asbestos exposure also suggested that the association of k-ras mutation and asbestos exposure was not the result of confounding by heavy smoking (Fig. 2) ⇓ . When we compared the calendar year of first occupational asbestos exposure by k-ras mutation status, it became apparent that those with mutations were exposed to asbestos much earlier than those whose tumors did not have k-ras mutations (Fig. 3) ⇓ . Similarly, the time from initial asbestos exposure to lung cancer diagnosis was significantly longer in the asbestos-exposed group with k-ras mutations compared with the exposed patients whose tumors were k-ras wild type (P < 0.005; data not shown).

Finally, we examined the chest radiographs of all of the patients who reported occupational exposure to asbestos for evidence of asbestosis. Among those who reported occupational asbestos exposure, only one patient had evidence of asbestos-related interstitial changes on chest radiograph (i.e., 1 of 21 patients). This patient also had pathological evidence of asbestos bodies. There was no association between k-ras mutation and asbestos-related radiographic changes.

The prevalence of occupational asbestos exposure determined by independent questionnaire review (that is, ascertained through an administered questionnaire rather than from medical records or other means) in lung cancer cases in the current case series (21 of 84 cases; 25%) is consistent with that reported previously by Wilkinson et al. (19) , using similar interview methods. Wilkinson et al. (19) reported 5.9% of cases with “definite” exposure and 34.3% with either “definite” or “probable” exposure. As we expected, the prevalence of reported exposure in our case series exceeded the prevalence of radiographic changes, consistent with findings from other groups (20) . It is also important to note that our estimates of asbestos exposure are not likely to be biased by mutation status because the molecular analysis was done after all exposure data were collected, and all participants were therefore obviously blinded to k-ras status.

k-ras mutation has been described as an early event in lung carcinogenesis (21 , 22) . If this is the case, the most biologically relevant carcinogen exposures for induction of this mutation would be those in the distant past. Interestingly, when we compared the time of initial asbestos exposure in patients whose tumors were k-ras mutant to those that were wild type, we found very distant exposure to be associated with the presence of k-ras mutation. It should be noted that estimates of asbestos exposure must consider the fact that distant exposures were undoubtedly heavier than more recent exposures (i.e., exposures that occurred after the promulgation of the OSHA standard in the United States in 1974 were certainly less than those in the 1950s and 1960s). Patients with high cumulative asbestos exposures in the calculated index are predominantly those who were exposed before 1965. Thus, an exposure-response association between asbestos and k-ras mutation also reflects historical exposure. This fact would implicate early asbestos exposure in the development of k-ras mutant tumors. Alternatively, those with high exposure indices are also likely to have the longest duration of asbestos exposure, and continued exposure could select for k-ras mutant clones.

Taken together, our data strongly suggest that the induction of oncogenic changes in the lung by asbestos precedes the induction of radiographically detectable interstitial fibrosis. Little data are available to estimate the sensitivity and specificity of a single chest radiograph for the detection of pneumoconiosis. However, it is unlikely that significant bias exists with respect to the detection of interstitial fibrosis in these workers because a single reader, who was blind to k-ras status, reviewed all radiographs. Hence, it seems clear that k-ras mutations were present in asbestos-exposed individuals who did not have asbestosis. In addition, early exposure was the strongest predictor of mutation in this analysis. This finding is important because some researchers have recently hypothesized that “lung cancer risk is elevated only in humans exposed to asbestos when there is asbestosis” (2) . Our data would indicate that this hypothesis should be reconsidered.

The mechanism responsible for the association of significant asbestos exposure and mutation is unclear. It has been suggested that asbestos enhances the delivery of mutagenic polyaromatic hydrocarbons to the respiratory epithelium (23) . This work has shown that the delivery of cigarette smoke condensate to cells is vastly enhanced by adhesion of this material on asbestos fibers before cellular exposure. If asbestos acts to target and enhance the delivery of polyaromatic hydrocarbon to cells in the lung, then the mutagenic response would be entirely indistinguishable from that of cigarette smoke alone, except that the dose and dose rate of carcinogen are significantly enhanced above the non-asbestos-exposed individuals.

The inhalation of asbestos fibers alone is well known to induce an inflammatory response including the recruitment of alveolar macrophages and polymorphonuclear leukocytes and the attendant cytokine release (reviewed in Ref. 24 ). It has been shown that this inflammatory response includes reactive oxygen species that may induce cell damage and that significant asbestos exposure produces cell death and lung fibrosis. Hence, asbestos fibers are associated with oxygen radical production and mitogenesis that could easily give rise to DNA damage and mutation.

Clearly, further work is indicated to continue to define the molecular epidemiology of asbestos carcinogenesis and precisely understand its mechanism. Such work would be of great value in more accurately estimating the attributable fraction of lung cancer related to asbestos exposure by providing molecular markers of asbestos carcinogenesis. That is, because our results implicate asbestos as a cause of k-ras mutations in the absence of fibrosis, they imply that an upward revision of the contribution of asbestos exposure to lung cancer incidence is warranted. Asbestosis is not likely to be the only marker of asbestos-related lung cancer.