© 2004 Foundation for Promotion of Cancer Research
Detection of Microsatellite Alterations in Bronchial Washings in Squamous Cell Lung Cancer: the First Study from India
1 Department of Pulmonary Medicine, 2 Department of Cytopathology and 3 Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
For reprints and all correspondence: Puneet Malhotra, Department of Pulmonary Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India–160012. E-mail: dranshupuneet{at}yahoo.com
Received March 4, 2004; accepted May 15, 2004
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Abstract |
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Background: In recent times, the possibility of detecting lung cancer using microsatellite alterations (microsatellite instability and loss of heterozygosity) in DNA of bronchial washings has been explored. However, no data regarding the presence of microsatellite alterations in lung cancer are available from India, a country which contributes significantly to the lung cancer burden of the world.
Methods: Bronchial washings as well as tumor specimens obtained on bronchoscopy were analyzed for the presence of loss of heterozygosity (LOH) and microsatellite instability (MSI) using the D3S1300 microsatellite marker on chromosome 3p and the TP53 marker on chromosome 17p.
Results: The sensitivities of the TP53 and D3S1300 loci in bronchial washings were 35% and 45% (combined 50%), respectively, which was significantly better than conventional cytology (positive for malignant cells in 15%). The presence of these microsatellite alterations was not related to the age, cumulative smoking exposure or smoking status (current or former) of patients.
Conclusion: Microsatellite alterations, particularly LOH, occur in a significant proportion of Indian patients with squamous cell carcinoma of the lung.
Key Words: squamous cell carcinoma • bronchial washings • microsatellite instability • loss of heterozygosity
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INTRODUCTION |
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In India, a country which contributes significantly to the lung cancer burden of the world, 33 300 new cases of lung cancer are diagnosed in males per year (1). This figure is likely to be an underestimation of the true incidence because of the lack of a uniform system of cancer registration in this country. Efforts at early diagnosis of lung cancer based on conventional screening procedures such as chest X-ray, CT and sputum cytology have so far been unable to decrease the lung cancer mortality (2,3). In recent times, the possibility of earlier diagnosis of lung cancer through the detection of microsatellite alterations (microsatellite instability and loss of heterozygosity) has been explored (4–6).
Microsatellites of DNA are tandem repeats of 1–6 nucleotides found scattered throughout the human genome. In tumor cells, expansions and contractions of these tandem repeats occur, creating alleles not found in normal cells. This phenomenon is called microsatellite instability (MSI). MSI is an early event promoting the occurrence of genetic alterations necessary for carcinogenesis. Loss of heterozygosity (LOH) refers to the deletion of one allele of a pair at specific chromosomal loci. MSI and LOH correlate with high mutational rates in tumor cells and have been reported in various malignancies including lung cancer (7).
However, no data regarding the presence of microsatellite alterations in lung cancer are available from India. An astonishing variety of modes of tobacco inhalation exist in this country, although bidi smoking is the most common (8). Bidis are hand rolled in small factories or cottage industries by wrapping a small amount of sun-cured Indian tobacco in non-porous leaves of the ‘Tendu’ (Diospyrus melonoxylon) tree. The finished product resembles a narrow, brown, tapered, unfiltered cigarette. In comparison to US cigarettes, the mainstream smoke of bidi contains much higher concentrations of several toxic agents such as hydrogen cyanide, carbon monoxide, ammonia, other volatile phenols, and carcinogenic hydrocarbons such as benz(a)anthracene and benzopyrene (9). Bidis also deliver more nicotine than manufactured Indian cigarettes. Furthermore, since bidis lack added burning agents, they have to be puffed repeatedly to be kept alight. As a result, bidi smokers tend to take almost five puffs per minute compared with cigarette smokers who average two puffs per minute (10). It is not surprising, therefore, that the risk of lung cancer is reported to be higher in bidi smokers compared with cigarette smokers. In contrast to the West, squamous cell carcinoma is the most common histologic type of lung cancer in India (11–14). The present study was planned to evaluate the possibility of using LOH and MSI as molecular markers for the detection of squamous cell carcinoma of the lung.
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SUBJECTS AND METHODS |
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STUDY DESIGN
The study was a prospective analysis of 20 patients with histologically proven squamous cell carcinoma of the lung treated at the Chest Clinic of the Postgraduate Institute of Medical Education and Research, Chandigarh, India. All cases were newly diagnosed and had not received any form of therapy. Informed consent was obtained from the patients on the proforma prescribed by the institutional ethics committee.
CLINICAL PROFILE
Twenty patients with histologically proven squamous cell carcinoma of the lung were studied. There were 18 males and 2 females with ages ranging from 36 years to 75 years (mean = 55). All patients were either current or former smokers. Former smokers (defined as those who had quit smoking for at least a year) numbered five and the number of years since they had quit smoking ranged from 1 to 40. The most common mode of tobacco inhalation was through bidis (n = 14), followed by cigarettes (n = 3) and hookah (a tobacco casket with a long flexible tube which draws smoke through water in a bowl) (n = 3), respectively. The smoking index (the product of the number of cigarettes or bidis smoked per day and the number of years smoked) ranged from 90 to 1250 (mean = 442.35). Since three of the patients were hookah smokers, their smoking indices could not be calculated. Fiberoptic bronchoscopy findings reflected the well-known central location of squamous cell lung cancer. One patient had a growth in the right lower end of the trachea. Left main and right main bronchial lesions were seen in four and two patients, respectively. The right upper lobe bronchus was involved in six patients, while two patients each had right middle, right lower and left upper lobe bronchus involvement. A growth in the left lower lobe bronchus was seen in one patient. The TNM stages of tumors were as follows: stage IB, one patient; stage IIA, four patients; stage IIB, three patients; stage IIIA, four patients; stage IV, eight patients.
The presence of MSI and LOH was studied in samples of bronchial washings and biopsy specimens obtained from tumors visualized by fiberoptic bronchoscopy. Bronchial washings were also analyzed for the presence of malignant cells using conventional cytologic techniques. Peripheral blood was obtained from patients as a source of normal/control DNA. The study involved molecular analysis of DNA from: (i) bronchial washings; (ii) tumor; and (iii) peripheral blood lymphocytes from each patient. Using a flexible fiberoptic bronchoscope (Olympus P20), bronchoscopy was performed and the presence of tumor(s) was identified. Then 100 ml of pyrogen-free 0.9% saline solution were instilled in the bronchus identified on bronchoscopy to contain the tumor. Bronchial washing was performed before obtaining biopsy samples to avoid contaminating them with sloughed off tumor tissue, which would occur during endobronchial biopsy. The fluid was recovered with the help of a suction trap, strained through gauze to remove excess mucus and transported to the cytology laboratory on ice. The fluid was centrifuged at 1800 g for 10 min at 4°C and conventional cytologic smears made from the sediment. The remaining cell pellet was stored at –80°C for molecular analysis.
Under similar conditions as above, bronchoscope-guided biopsy specimens were obtained from the tumor(s). The specimens were submitted to the Department of Histopathology of this institute for routine histopathological examination. After confirmation of the diagnosis, three serial 10 µm sections were cut and by comparison to the hematoxylin and eosin (H&E) stained section the portions of the specimen not representative of the tumor were carefully dissected out. The rest of the tissue was deparaffinized and passed through graded alcohols (100%, 70%, 50%) and brought to water. The tissue was then incubated in lysis buffer (containing 100 mM NaCl, 10 mM Tris–HCl pH 8.0, 25 mM EDTA, 0.5% SDS, 0.3 mg/ml proteinase K) overnight at 55°C. Fresh proteinase K was added the next morning and further incubation was carried out for 6 h. DNA was then extracted with equal volumes of phenol:chloroform:isoamylalcohol, ethanol precipitated, dried and resuspended in 30 µl of ddH2O. For peripheral blood, 10 ml of EDTA anticoagulated blood were collected from each patient as a source of normal/control DNA, which was extracted as described previously (15).
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POLYMERASE CHAIN REACTION |
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The primers used were as follows:
TP53:
- Forward primer: 5'-ACT GGA TGA TTT GAT GCT GTC CC-3'
- Reverse primer: 5'-CGT GCA AGT CAC AGA CTT GGC-3'
- Reverse primer: 5'-CGT GCA AGT CAC AGA CTT GGC-3'
- Forward primer: 5'-AGC TCA CAT TCT AGT CAG CC-3'
- Reverse primer: 5'-AGC TGT TTA TTC TTC GTC GA-3'
- Reverse primer: 5'-AGC TGT TTA TTC TTC GTC GA-3'
Genomic DNA derived from normal peripheral blood lymphocytes, tumor tissue and bronchial washing sediment from each patient was subjected to PCR using each of the primer pairs as described above. The PCR products were then separated by 6% non-denaturing PAGE, the gel stained and visualized by silver staining. In each case, the PCR products of the normal/control DNA, tumor and bronchial washings were compared. LOH was defined on the basis of visual inspection as >50% reduction in the intensity of one of the two bands in comparison with the normal control signals (Figures 1 and 2). MSI was defined by a shift in the mobility of one or more alleles (Figure 3).
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RESULTS |
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MICROSATELLITE ALTERATIONS
The results are depicted in Tables 1 and 2. Loss of heterozygosity was seen in 8 out of 20 (40%) tumors and 6 out of 20 (30%) samples of bronchial washings using the TP53 marker on chromosome 17p. MSI was seen only in two tumors (10%) and one (5%) bronchial washings sample. Using the D3S1300 marker at the FHIT locus on chromosome 3p14.2, LOH was found in 11 out of 20 (55%) tumors and 9 out of 20 (45%) bronchial washings specimens. None of the tumors and bronchial washings samples exhibited MSI at this locus. The sensitivity rose even higher (11/20; 55%) when the presence of LOH and MSI in bronchial washings was analyzed using a combination of both the chromosomal loci (17p and 3p 14.2).
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CYTOLOGIC EXAMINATION
Cytologic examination of bronchial washing fluid was positive for malignant cells in three patients, yielding a sensitivity of 15% (3/20) (Table 1).
AGE AND MICROSATELLITE ALTERATIONS
The ages of the 16 patients who exhibited positivity for either LOH or MSI ranged from 36 years to 75 years with a mean of 56.81 years. The four patients who were negative for these microsatellite alterations ranged in age from 42 to 60 years with a mean of 51.75 years. The difference was not statistically significant.
SMOKING INDEX AND MICROSATELLITE ALTERATIONS
The smoking indices of patients who exhibited positivity for LOH and/or MSI ranged from 90 to 1200 with a mean of 319.67. There were four patients who did not test positive for LOH and/or MSI at both the chromosomal loci. Two of these patients were hookah smokers and their smoking indices could not be calculated. There was no significant association between a high smoking index and the presence of microsatellite alterations.
CURRENT VERSUS FORMER SMOKERS AND MICROSATELLITE ALTERATIONS
Eleven out of the 15 (76%) current smokers exhibited positivity for LOH and/or MSI. There were only five former smokers and four (80%) of them tested positive for either LOH or MSI. The number of years since quitting ranged from 1 to 40. The smoking status (current or former) did not significantly influence the presence of microsatellite alterations.
TNM STAGE AND MICROSATELLITE ALTERATIONS
There were four patients who did not demonstrate LOH or MSI in either tumor or bronchial washings. Two of these patients were in stage IIB while one each was in stage IIIA and IV. Of the 16 patients with LOH or MSI in either tumor or bronchial washings, nine were in stage IIIA or less, i.e. potentially resectable, while seven were in stage IIIB and IV. There was no statistically significant difference with regard to the presence of LOH/MSI in resectable versus non-resectable tumors.
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DISCUSSION |
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Of the various chromosomal loci identified as microsatellite markers in lung cancer, LOH and MSI have been most frequently demonstrated at 3p14.2 (FHIT gene), 9p21-22 and 17p13 (16–21). In order to increase sensitivity, various authors have used a large number of microsatellite markers (ranging from 2 to 17 in different studies). This was a pilot study and financial constraints restricted us to the use of one microsatellite marker each at chromosomes 3p and 17p.
In the present analysis, 40% of cases of squamous cell carcinoma of the lung exhibited LOH using the TP53 marker. This compares favorably with previously published reports of LOH at this locus ranging from 25% to 57% (17–19). On the other hand, 55% of tumors in this study exhibited LOH at the 3p14.2 locus using the D3S1300 polymorphic marker, which maps to the FHIT gene in an intragenic location. Similar results have been published by Sozzi et al. (20) who demonstrated LOH at this locus in 9 out of 17 (52.9%) cases of squamous cell carcinoma. However, a much lower sensitivity in the range of 25% has been reported in previous studies despite using as many as two and nine microsatellite markers at this locus, respectively (18,21).
The sensitivity of LOH in this study as a marker for squamous cell carcinoma of the lung therefore compares favorably with that reported by several authors. The sensitivity of bronchial washings LOH was 30% using the TP53 markers at chromosome 17p and 45% for the D3S1300 marker at the FHIT locus on chromosome 3p14.2. If both markers were combined, 10 out of 20 cases were positive for LOH, yielding an overall sensitivity of 50%.
There was no correlation between smoking index and the presence of LOH in the present study. The FHIT gene on chromosome 3p and the p53 gene on 17p are well-known targets of tobacco-related carcinogens and studies have shown a lower incidence of microsatellite alterations at these loci in non-smokers who develop lung cancer compared with smokers (17,20); however, no study so far has linked high smoking indices with LOH.
LOH occurred at both loci in this study independently of the smoking status of the patients, whether current or former. This is supported by previous work by Mao et al. (22) and Wistuba et al. (23) who demonstrated that clonal alterations persisted in the lungs of smokers several years after quitting. However, these authors also found that current smokers had a significantly higher frequency of LOH at 3p14 than former smokers, while the frequencies of LOH at 9p and 17p13 were similar among current and former smokers. These results suggest that genetic loci differ in their sensitivity to the mutagenic effects of cigarette smoke. Since the overall number of former smokers (five) in the present study was small, our data need further validation with a larger number of samples from former smokers.
Results with analysis of MSI at chromosomes 3p and 17p were not as gratifying as those with LOH. Only one bronchial washings sample was positive for MSI at the 17p locus while none exhibited MSI at chromosome 3p. Tumor-specific MSI was detected in only 3 (14%) of 22 bronchial washings samples from patients with similar alterations in the primary tumors in spite of using a panel of 15 microsatellite markers in a study by Arhendt et al. (16). They postulated that a neoplastic cellularity of at least 5–7% in bronchial washings was the threshold for detecting MSI and the percentage of tumor cells in bronchial washings was several times lower than this threshold. Moreover, while previous studies on MSI have been on bronchial washings solely obtained for molecular analysis, the specimens in our study were divided equally between cytologic and molecular analysis, further decreasing the tumor cell percentage.
Thus, in this pilot study, the first of its kind from India, we have demonstrated that microsatellite alterations are sensitive markers of the presence of squamous cell carcinoma of the lung. They can be effectively employed on cytologic material to enhance the detection of malignancy in a sample even by using just two microsatellite makers.
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CONCLUSION |
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TOPAbstractINTRODUCTIONSUBJECTS AND METHODSPOLYMERASE CHAIN REACTIONRESULTSDISCUSSIONCONCLUSIONReferences |
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This pilot study has demonstrated that microsatellite alterations, particularly LOH, occur in Indian patients with squamous cell carcinoma of the lung. The presence of these microsatellite alterations is not related to the age, tumor stage, cumulative smoking exposure or smoking status (current or former) of patients. Since this was a pilot study on a small number of patients, our data need further validation with a larger number of samples.
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References |
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1 Rao DN, Ganesh B. Estimate of cancer incidence in India in 1991. Indian J Cancer 1998;35:10–18.[Medline]
2 Henschke CI, McCanley DI, Yankelvitz DF, et al. Early lung cancer action project: overall design and findings from baseline screening. Lancet 1999;354:99–105.[CrossRef][Web of Science][Medline]
3 Salomaa ER, Liippo K, Taylor P, Palmgren J, Haapakoski J, Virtamo J, et al. Prognosis of patients with lung cancer found in a single chest radiograph screening. Chest 1998;114:1514–18.
4 Arvanitis DA, Papadakis E, Zafiropoulos A, Spandidos DA. Fractional allele loss is a valuable marker for human lung cancer detection in sputum. Lung Cancer 2003;40:55–66.[CrossRef][Medline]
5 Petmitr S, Nuchfaong S, Chaksangchaichot P, Pongstaporn W, Sutinont P, Limsila T, et al. Microsatellite alterations in non-small cell lung cancer. J Exp Clin Cancer Res 2002;21:31–5.[Web of Science][Medline]
6 Miozzo M, Sozzi G, Musso K, Pilotti S, Incarbone M, Pastorino U, et al. Microsatellite alterations in bronchial and sputum specimens of lung cancer patients. Cancer Res 1996;56:2285–8.
7 Coleman WB, Tsongalis GJ. The role of genomic instability in human carcinogenesis. Anticancer Res 1999;19:4645–64.[Web of Science][Medline]
8 Jindal SK, Malik SK, Datta BN. Lung cancer in Northern India in relation to age, sex and smoking habits. Eur J Respir Dis 1987;70:23–8.[Web of Science][Medline]
9 Gupta PC, Murti PR, Bhonsle RB. Epidemiology of cancer by tobacco products and the significance of TSNA. Crit Rev Toxicol 1996;26:183–98.[Web of Science][Medline]
10 Jayant K, Pakhale SS. Toxic constituents in bidi smoke. In: Sanghavi LD, Notani P, editors. Tobacco and Health: The Indian Scene. Bombay: Tata Memorial Centre, 1989, pp. 101–10.
11 Jindal SK, Behera D. Clinical spectrum of lung cancer: review of Chandigarh experience of 10 years. Lung India 1990;8:94–8.
12 Dikshit RP, Kanhere S. Tobacco habits and risk of lung, oropharyngeal and oral cavity cancer: a population-based case–control study in Bhopal, India. Int J Epidemiol 2000;29:609–14.
13 Thippanna G, Venu K, Gopalakrishnaiah V, Reddy PN, Charan BG. A profile of lung cancer patients in Hyderabad. J Indian Med Assoc 1999;97:357–9.[Medline]
14 Sharma CP, Behera D, Aggarwal AN, Gupta D, Jindal SK. Radiographic patterns in lung cancer. Indian J Chest Dis Allied Sci 2002;44:25–30.[Medline]
15 Lahiri DR, Bye S, Nurnberger JI, Hodes ME, Crisp M. A non-organic and non-enzymatic extraction method gives higher yield of genomic DNA from whole blood sample than other methods tested. J Biochem Biophys Methods 1992;25:193–205.[CrossRef][Medline]
16 Arhendt SA, Chow JT, Yang SC, et al. Molecular detection of tumour cells in bronchial washings from patients with early stage lung cancer. J Natl Cancer Inst 1999;91:332–9.
17 Zienolddiny S, Ryberg D, Arab MO, Skaug V, Haugen A. Loss of heterozygosity is related to p53 mutations and smoking in lung cancer. Br J Cancer 2001;84:226–31.[CrossRef][Web of Science][Medline]
18 Kohno H, Hiroshima K, Toyozaki T, Fujisawa T, Ohwada H. p53 mutation and allelic loss of chromosome 3p, 9p in preneoplastic lesions in patients with non small cell lung cancer. Cancer 1999;85:341–7.[CrossRef][Web of Science][Medline]
19 Froudarakis ME, Bouros D, Spandidos DA, Siafakas NM. Microsatellite instability and loss of heterozygosity at chromosome 9 and 17 in non small cell lung cancer. Chest 1998;113:1091–4.
20 Sozzi G, Sard L, DeGregorio L, et al. Association between cigarette smoking and FHIT gene alterations in lung cancer. Cancer Res 1997;57:2121–3.
21 Shridar V, Siegfried J, Hunt J, del Mar Alonso M, Smith DI. Genetic instability of microsatellite sequences in many non small cell lung cancer. Cancer Res 1994;54:2084–7.
22 Mao L, Hraban RH, Boyle JO, Tockman M, Sidransky D. Detection of oncogene mutations in sputum precedes diagnosis of lung cancer. Cancer 1994;54:1634–7.
23 Wistuba II, Lam S, Behrens C, Virmani AK, Fong KM, LeRiche J, et al. Molecular damage in the bronchial epithelium of current and former smokers. J Natl Cancer Inst 1997;89:1366–73.
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