INTRODUCTION

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Idiopathic pulmonary fibrosis (IPF), also known as cryptogenic fibrosing alveolitis, is one of the more common interstitial lung diseases of unknown etiology (1). Various etiopathogenic agents have been implicated in IPF, including environmental and occupational exposures, viral infections, genetic factors, and immune-mediated processes (5). The disease has a variable clinical course, with an average survival from diagnosis of 4 to 6 yr (1). Several kinds of altered epithelial cells are observed in lung tissue specimens obtained from patients with IPF, which show characteristic changes in cytokeratin expression as compared with normal lung specimens (9). Bronchogenic carcinoma is the cause of death in 10% to 13% of IPF patients (10, 11). The basis of this relationship (i.e., the higher incidence of lung cancer in IPF patients) is not yet known. On the molecular level, previous studies have shown that p53 and p21 are expressed especially in hyperplastic bronchial and alveolar epithelial cells of lung tissues from all patients with IPF (12). It is suggested that p53 and p21 are upregulated in association with chronic DNA damage, resulting in either G₁ arrest or apoptosis, with the result that the DNA damage can be repaired in this disease. Tumorigenesis in IPF could be the result of a p53 mutation due to chronic DNA damage and repair leading to p53 upregulation (12).

Information on the molecular pathway of cancer development is provided by the identification of novel tumor-suppressor genes (TSGs). The inactivation of TSGs plays a critical role in multistage carcinogenesis. At present, detection of loss of heterozygosity (LOH), using highly polymorphic microsatellite markers, is the most common methodology used for localizing sites in the genome with high probability for the presence of candidate TSGs (13). Microsatellite DNA consists of very short tandem nucleotide repeats that are found scattered throughout the human genomes of eukaryotes (14, 15). Instability of tandem repeat DNA sequences, or microsatellite instability (MSI), has been correlated with a high mutational rate and DNA repair processes (16, 17).

MSI and LOH have been previously reported in malignancies of various origin, including lung carcinomas (18). We recently found that MSI and LOH are frequent phenomena in patients with pulmonary sarcoidosis (21).

The present study was designed to investigate the genetic alterations at the microsatellite level in blood and sputum- cytologic specimens from patients with IPF, using 10 highly polymorphic microsatellite markers, located on several chromosomal arms, that could be part of the complex genetic basis of the disease and implicated in its etiopathogenesis. To the best of our knowledge, this is the first study of microsatellite alterations in patients with IPF.

	METHODS

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Subjects

Twenty-six patients with clinical and radiologic (high-resolution computed tomography [HRCT]) and/or histologic features consistent with IPF, who were followed at our clinic, were studied (1). The diagnosis of IPF was made by surgical lung biopsy (open lung biopsy or video-assisted thoracoscopic surgery) showing usual interstitial pneumonia (UIP) and by the presence of: (1) persistent bilateral crackles on auscultation; (2) a restrictive ventilatory defect or isolated depression of gas transfer on pulmonary function testing; and (3) the presence of bilateral abnormalities with a peripheral distribution and the absence of bilateral patchy infiltrates on HRCT (1). Patients with environmental exposure to a fibrinogen were excluded. Other exclusion criteria were coexistent chronic disease, lung infection, or malignancy.

The median age of the patients was 72 yr (range: 57 to 82 yr); 20 of the patients were male and six were female. Eighteen of the patients were smokers. The total smoking exposure was calculated as follows: pack-years = (total years of smoking) × cigarette packs/d. The smoking history of the patients was 29 ± 21 (mean ± SD) pack-years. The duration of the disease was 3.3 ± 1.4 yr (Table 1).

Twenty-six normal subjects, matched with the IPF patients for age, sex, and smoking history, without clinical, physical, or laboratory evidence of disease, served as a control group. They had unremarkable physical examinations, normal blood and serum analyses, and normal chest radiographs, pulmonary function tests, and arterial blood gas values (Table 1). All patients and control subjects were white and of Greek nationality, and there were no ethnic differences among them.

All patients underwent routine pulmonary function testing, including spirometry, lung volume measurement, and measurement of diffusion capacity of carbon monoxide and arterial blood gases at rest and after exercise, chest radiography, HRCT of the thorax, and fiberoptic bronchoscopy. Characteristics of the studied subjects are shown in Table 1.

Morning spontaneously expectorated sputum and peripheral blood specimens were collected from both study groups. In order to ensure that sputum specimens originated from the lower respiratory tract, they were microscopically examined, and were considered adequate when squamous epithelial cells were fewer than 10 per low-power field (22). Analysis of the sputum-cytologic specimens showed that the cell differential in our population consisted of (3% to 9%) epithelial cells, with the remaining cells consisting of lymphocytes, neutrophils, and macrophages in various percentages.

Extra care was taken to ensure that the cell content of IPF patients and normal control subjects remained similar in the two groups' morning spontaneously expectorated sputum.

Informed consent was obtained from all patients participating in the study, and the study was approved by the medical research ethics committee of our hospital.

DNA Extraction

DNA was isolated from white blood cells (WBCs) and sputum cells with the IsoQuick Nucleic Acid Extraction kit (ORCA, Research, Inc., Bothell, WA), according to the manufacturer's instructions.

Microsatellite Marker and LOH Analysis

Ten microsatellite markers, located on several chromosomal arms (Table 2), were used to reveal MSI or LOH. These markers were THRA1, D17S579, D17S855, D17S250, ANK1, D9S59, D9S290, HXB, D8S133, and D8S137 (23). Regions 8p, 17q, 9p, and 9q were evaluated for the incidence of LOH and MSI. The selection of chromosome regions to be evaluated was based on previous studies done either with lung cancer specimens (20) or benign lesions of the lung such as sarcoidosis (21) (Table 2).

Polymerase chain reaction (PCR) analysis (24) was performed as previously described (21). A volume of 10 µl of the PCR product was analyzed after electrophoresis in a 10% polyacrylamide gel, and was stained with silver stain.

Gels were inspected visually by three independent viewers, comparing the intensity of alleles from sputum and control (WBC) DNA. LOH was defined as a minimum 50% reduction in the intensity of the pathologic allele as compared with the corresponding normal allele (24). Tests with questionable results were repeated. In such cases, densitometric measurements were made to ensure objective reading of data (25).

MSI was scored by comparing the electrophoretic pattern of the microsatellite markers amplified from the paired DNA preparations (sputum/WBCs) according to a shift of one or both of the alleles in the sputum DNA specimen. The shift was indicated either by an addition or deletion of one or more repeat units, resulting in the generation of novel microsatellite alleles. The appearance of additional novel bands may be explained as the result of alterations in the length of microsatellites, limited only to a cellular subpopulation of the pathologic tissue and creating novel cell clones. Thus, in a microsatellite analysis, one may observe in the total extracted DNA alleles from both affected and unaffected microsatellites.

The reproducibility of the MSI scoring method was examined by double analysis of MSI/LOH-positive cases, in which the results were identical for each case. To assess the sensitivity of our method for LOH and MSI, we prepared samples of various tumors (T) to normal ratios (N) from a tumor specimen with known LOH and MSI, respectively. The analysis showed that LOH was detectable in a 1:16 dilution (T/N), and MSI in a 1: 32 dilution (T/N).

Statistical Analysis

Data analysis was done with SPSS statistical software (SPSS Inc., Chicago, IL). Results are expressed as mean ± SD or median (range) unless otherwise indicated. Differences in the mean values of quantitative measurements were tested with the Student's-t or the Mann- Whitney U test. The chi-square test was used for comparison of percentages. Analysis of covariance (logistic regression) was used for covariates. A p value of < 0.05 was considered statistically significant.

	RESULTS

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Results for MSI and LOH in the studied sputum specimens from IPF patients are shown in Table 2. We found that 13 of 26 (50%) IPF patients showed genetic alterations in their sputum specimens, manifested either as MSI or LOH, in at least one of the studied markers. Five (19%) patients exhibited MSI in at least one microsatellite marker. The most commonly affected microsatellite markers were THRA1 and D8S133, which exhibited MSI in two of 26 (8%) and two of 24 (8%) evaluated specimens at chromosome loci 17q12 and 8p21.3- q11.1, respectively.

LOH in at least one marker was found in 10 (39%) sputum samples from the IPF patients. The most commonly affected microsatellite markers were D8S133, which exhibited LOH in four of 24 (16%) evaluated specimens at chromosome locus 8p21.3-q11.1, and ANK1, which exhibited LOH in three of 26 evaluated specimens (12%) at chromosome locus 9p21.1- p11.2. Three (12%) patients showed LOH in more than one marker (Table 2). Two patients (8%) had both MSI and LOH.

Microsatellite analysis of sputum specimens from matched healthy subjects revealed no evidence of MSI or LOH in any of the genetic loci examined. Representative examples of specimens with MSI and LOH are shown in Figures 1 and 2, respectively.

View larger version (45K): [in this window] [in a new window]   Figure 1.   Representative electrophoretic patterns of three microsatellite markers in patients exhibiting MSI. Arrows indicate the shift in mobility of the alleles. The numbers above the locus name represent the patient numbers. N = normal (blood) DNA; S = sputum DNA.

View larger version (48K): [in this window] [in a new window]   Figure 2.   Representative electrophoretic patterns of four microsatellite markers in patients exhibiting LOH. Arrows indicate a significant decrease in the intensity of presence of the alleles. The numbers above the locus name represent the patient numbers. N = normal (blood) DNA; S = sputum DNA.

In order to assess whether MSI or LOH is an index of the severity of IPF, we defined two subgroups of IPF patients, as follows: MSI- and/or LOH-positive (Group I), and MSI- and LOH-negative (Group II). The results showed that there were no significant differences (p > 0.05) between these two subgroups in terms of duration of illness, arterial blood gases, or lung function test values. This suggests that MSI and LOH are unrelated to the severity of IPF.

	DISCUSSION

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In the present study we investigated genetic alterations at the microsatellite level in 26 patients with IPF. We found that half of the patients showed genetic alterations, consisting either of MSI or LOH. Five (19%) patients exhibited MSI and 10 (39%) exhibited LOH in at least one microsatellite marker.

Genetic alterations such as MSI have been detected in almost all human tumors (18) as well as in benign diseases (26, 27). We recently detected MSI in chronic obstructive pulmonary disease (COPD) (28) and sarcoidosis (21). Patients with IPF have a higher tendency to develop lung cancer (10, 11). An excess relative risk of lung cancer of 14.1 was found in patients with IPF as compared with the general population of comparable age and sex, with allowance for the durations of follow-up of the IPF patients (10).

Previous studies have shown that the rate of spontaneous changes in short tandem repeats of tri- and tetranucleotides is higher than that for dinucleotide microsatellites (29, 30). The use in our study of dinucleotide microsatellites may have led to underestimation of the incidence of genome instability in IPF.

No consensus exists on how many loci should be analyzed and how many of them should show alterations for classification as showing a high degree of MSI. Nevertheless, it is common to classify as showing a low degree of MSI specimens unstable for one genetic locus, and as showing a high degree of MSI specimens unstable for two or more loci (23). Despite the high frequency of MSI in our patients (19%), all showed instability at one locus.

A significant incidence of LOH, in four of 24 (17%) specimens, was found at the locus 8p21.3-q11.1, suggesting that important TSGs for the development of IPF may be located in this chromosomal region. Deletions at 8p21.3-q11.1 occur frequently in lung neoplasms (31). The second most frequent locus of LOH was that at 9p21.1-p11.2, which was found to be affected in three of 26 (12%) specimens. Deletions at 9p21.1- p11.2 are also observed in lung neoplasms (20). Fine mapping of these areas is required in order to establish the precise location of candidate TSG(s) and their role in the pathogenesis of IPF. Sputum specimens contain a high amount of normal DNA, which may eliminate the signal of a mutant allele and may produce false-negative results (21).

LOH is another genetic alteration that occurs in tumor cells and in premalignant cells that progress toward malignancy. This constitutes strong evidence that there may be transformed cells in the fibrotic tissue of IPF. According to Knudson's "two-hit" hypothesis (32), the phenomenon of LOH is correlated with the existence of a TSG implicated in a particular disease.

Our selection of chromosome regions to be evaluated in IPF was based on previous studies done either with lung cancer specimens (20, 31) or with benign lesions of the lung, such as COPD (28) and sarcoidosis (21). Some of the markers of these conditions are located in the vicinity of known TSGs, such as p16 at 9p21 and BRCA1 at 17q21, whereas other markers map regions susceptible to other lung lesions. The markers tested in our study do not represent a subsample of the markers studied.

In a recent study (21) of 30 patients with pulmonary sarcoidosis, we found that 14 (47%) of them showed genetic alterations consisting either of MSI or LOH. Six (20%) patients exhibited MSI and nine (30%) patients exhibited LOH in at least one microsatellite marker. In the present study, the frequency of LOH was slightly higher (39%) than in this earlier study, which could partly explain the higher incidence of lung cancer in IPF patients.

The concept of a hereditary susceptibility to IPF is based on several findings (8, 33). These findings support the idea that genetic factors may predispose to this disorder or determine its clinical expression. Familial IPF, including its description in identical twins, some having been separated geographically for many years, has been reported (34). The lack of a clear genetic pattern suggests that if IPF is indeed a disease with a genetic predisposition, it may be a genetically complex disease, associated with multigenic traits and multiple inherited factors, the genetic determinants of which may be difficult to ascertain (35). Since IPF primarily occurs in adults, it appears that genetic predisposition in combination with aging and intrinsic and extrinsic factors most likely contributes to the phenotypic expression of parenchymal inflammation and fibrosis in IPF (35). In the present investigation, the population studied was white and Greek, and did not exhibit genetic varieties, and because the study was conducted on the island of Crete, all of the subjects were affected by the same environmental factors.

Smoking, however, seems not to be related to the previously described genetic abnormalities in IPF, since smokers among our IPF patients did not exhibit MSI or LOH in greater frequency than did nonsmokers. Additional evidence for this concept is provided by two recent studies (21, 28) in which we found that MSI was present in patients with COPD but not in a matched group of non-COPD smokers (28), and that among patients with sarcoidosis, smokers and nonsmokers show both MSI and LOH in comparable frequencies (21).

We compared subgroups of IPF patients that were respectively positive and negative for MSI or LOH. No statistically significant difference was found between the two subgroups in relation to age, duration of illness, arterial blood gas values, and spirometric indices.

A previous study identified a p53 mutation in seven of 10 sites in bronchial epithelium in a patient with widespread dysplastic changes in the respiratory epithelium (36). However, in that study the biologic material was the entire tracheobronchial tree. In our present study the source of biologic material was sputum coming from the entire respiratory tract which did not allow us to distinguish the origins of cell populations. However, to our knowledge there is no evidence to support either LOH or MSI in widely dispersed areas of respiratory epithelium. We might postulate that the epithelial surfaces are composed of actively growing cell populations, which expand clonally through mechanisms such as loss of adhesion, and that these cells may therefore be more readily shed into the sputum. Even if this is the case, however, the majority of the exfoliated cell clones must harbor the same microsatellite alteration. Since the proportion of inflammatory cells in the sputa of our patients was high and could account for the mutations, microdissection might be used to definitively assign the mutations to epithelial cells.

The precise significance of our findings in the present study is unknown. However, we may speculate that the relatively high mutational rate in IPF patients, as reflected by the instability of the microsatellite sequences that we investigated, indicates a destabilization of the genome, which may affect other genes and result in dysregulation of the cells harboring the mutations. It will be of interest to investigate in future studies the frequency of the observed alterations after sputum or cell sorting of bronchoalveolar lavage fluid cells.

In conclusion, the results of this study show for the first time that MSI and LOH are detectable phenomena in IPF and might be implicated in the etiopathogenesis of the disease. Further studies are needed to evaluate the clinical significance and the prognostic value of these genetic alterations for the possible development of lung cancer.