The development and progression of cancer is a complex and multistep process in which a series of changes culminates in deregulation of cell proliferation in somatic cells. Although the specific genetic and epigenetic events leading to cancerous growth differ among tumor types, a number of affiliated cellular pathways have now been identified.
Genes that may be involved in these processes include those that influence proliferation rate, apoptosis, genomic stability, differentiation, adhesion, and angiogenesis. Alterations in these genes may act in a dominant or recessive fashion and are caused by amplifications, point mutations, deletions, or structural rearrangement. Recent data suggest that some abnormalities occur at an earlier stage than others, and these initial markers may be important for prognosis and early detection.
Identified more than 15 years ago, one of the first abnormalities found in lung cancer was the loss (deletion) of chromosomal material on the short arm of chromosome 3. Since then, an increasing number of genetic changes, including loss of cellular regulation, have been discovered. In non-small-cell lung cancer (NSCLC), changes have been demonstrated in signal transduction (K-ras gene), nuclear transcription factors (myc, fos), receptor protein kinases (HER-2/neu), cell-cycle regulation (Rb gene, cyclins), and genes involved in the prevention and accumulation of DNA damage by cell-cycle control and apoptosis (p53). Other genes like bcl-2, BAX, and the newly identified FHIT (fragile histidine triad) gene (on chromosome 3p) also have been shown to play a role in pulmonary carcinogenesis.
Dominant Oncogenes (Proto-Oncogenes)
The K-ras proto-oncogene encodes the p21 ras protein, which is located on the cytoplasmic side of the plasma membrane and is involved in transmitting proliferation signals from receptors on the cell surface to the nucleus. Mutations of K-ras may be identified in approximately 30% of adenocarcinomas, but are seldom found in other types of non-small-cell lung cancer.[2,3] There is a correlation between specific K-ras mutations (G-C to T-A transversions at codon 12) in adenocarcinomas of the lung associated with a history of smoking. This type of mutation may be a direct result of exposure to carcinogens in tobacco smoke.
Amplification of members of the myc family has been found in 10% or more of patients with non-small-cell lung cancer (myc, 8%; L-myc, 3%). Expression of myc mRNA is detected in about one third of the non-small-cell lung cancers studied, whereas immunohistochemical studies show that myc protein can be detected in about 50% of non-small-cell lung cancer patients. The information on the fos family of proto-oncogenes in non-small-cell lung cancer is sparse. The fos and fos-related genes encode nuclear proteins that form a dimeric complex activating protein-1, which acts as a transcription factor. In small-cell lung cancer cultures, fos mRNA expression increases quickly following the addition of growth factors. In non-small-cell lung cancer, fos proteins were detected in about 50% of the cancer cases studied.
The epidermal growth factor receptor (HER-1) and the protein product of the HER-2/neu (c-erbB-2) are transmembrane glycoproteins with intrinsic tyrosine kinase activity. Several studies have shown that HER-2/neu is expressed in normal as well as transformed epithelial cells. Amplification of the HER-2/neu oncogene is an infrequent event in lung cancer. On the other hand, mRNA and the protein product (p185 neu) have been found consistently in non-small-cell lung cancers, with some studies suggesting a higher percentage of overexpression in adenocarcinomas than in squamous cancers.
The protein product of the bcl-2 proto-oncogene inhibits programmed cell death (apoptosis). Since BAX has been recognized as an inhibitor of bcl-2, it is assumed that bcl-2/BAX ratios influence the cell fate for apoptosis or survival.[11,12] When bcl is overexpressed, apoptotic signals are ignored, permitting neoplastic transformation. The bcl-2 protein has been detected by immunohistochemical analysis in up to 60% in non-small-cell lung cancers, as well as in the basal cells of normal bronchial epithelium.
Recessive Oncogenes (Tumor Suppressor Genes)
As mentioned earlier, deletion of the short arm of chromosome 3 was one of the first genetic abnormalities found in small-cell lung cancer. Most non-small-cell lung cancers also exhibit this abnormality. Mapping studies have shown that three separate regions of 3p (3p14, 3p21, and 3p25) are frequently lost. Recent investigations have pointed out that some of the losses of genetic material recognized in established lung cancer may already exist in bronchial epithelium at risk; ie, in bronchial biopsies from smokers and ex-smokers who have not yet developed carcinomas of the lung. Loss of heterozygosity has been found at 3p14, 9p21, and 17p13 in bronchial biopsies from current and former smokers. Interestingly, the loss of heterozygosity at chromosome 3p14 was identified more frequently in current (88%) than in former (25%) smokers, indicating that genetic loci may differ in their sensitivity to the mutagenic compounds in cigarette smoke. All three loci correspond with tumor suppressor genes: The p53 tumor suppressor gene resides at 17p13; 3p14 contains the FHIT gene; and 9p21 contains the p16 gene, also known as MTS-1 (multiple tumor suppressor 1), which encodes p16, a protein that inhibits cyclin- dependent kinase 4.[16,17] Apart from loss of heterozygosity at the 17p13 (the p53 locus), overexpression of mutant p53 protein has been shown in dysplastic bronchial epithelium. These observations suggest that loss of normal p53 function may play a role in the first stages of carcinogenesis of the bronchial epithelium. This is supported by data showing that p53 mutations associated with lung and head and neck cancers are strongly related to cigarette smoking and by the fact that benzo(a)pyrene adducts have been found to be distributed along the exons of the p53 gene.[18-20] The p53 gene encodes a nuclear protein with specific DNA-binding activity, and dimers of wild type p53 activate the transcription of genes with a specific (p53) binding sequence. The p53 protein is able to arrest the cycling of cells in response to DNA damage and is therefore regarded as a guardian of genomic integrity. In addition, the activity of p53 is required for the activation of apoptosis.
The first tumor suppressor gene discovered was the Rb gene, which predisposes to familial retinoblastoma. The Rb gene encodes a nuclear DNA-binding protein that is important in controlling the cell cycle. This protein is present in an underphosphorylated form in resting cells. Phosphorylation is mediated by cyclins of the D type and is required for cell-cycle progression. Mutations in Rb may thus interfere with normal Rb cyclin D interaction, resulting in a loss of proliferative control. It has been estimated that Rb gene disruption is present in 10% to 25% of non-small-cell lung cancer cases.
The first reports that genetic characteristics may influence lung cancer susceptibility appeared in the early 1980s and showed that first degree nonsmoking relatives of lung cancer patients had an increased risk for lung cancer compared with nonsmokers with no affected relatives.[23,24] There is increasing evidence that the mechanism responsible for familial aggregation of lung cancer must be sought among those genes that code for carcinogen metabolism. Cytochrome p450 enzymes are most commonly involved in the metabolic activation of carcinogens from cigarette smoke. Polymorphisms in p450 genes, leading to increased metabolic activation of carcinogens, have been associated with lung cancer. Similarly, cytochrome p450 2D6, which is responsible for metabolism of the drug debrisoquine, has been studied in relation to an increased risk for lung cancer. A recent case-control study, however, failed to show an increased lung cancer risk among subjects identified as extensive debrisoquine metabolizers.
Another potentially important group of enzymes is the glutathione S transferase (GST) family, which is involved in the detoxification of polycyclic aromatic hydrocarbons. The deficient phenotype of GST 1 (present in 40% to 50% of the Caucasian population) leads to a less efficient detoxification of carcinogens and has been associated with an increased risk for lung cancer. Characterization of individual risk due to variations in metabolic pathways may allow a better selection of high-risk individuals for early detection and intervention (chemoprevention) programs.
The recent understanding that genetic alterations occur at specific chromosomal sites containing putative tumor-suppressor genes in normal, or only slightly altered, bronchial epithelium of smokers, may herald a new approach to screening: identification of individuals at risk by using specific genetic markers. These markers could be applied to sputum, secretions, lavage, or biopsy specimens obtained by bronchoscopy. The use of new endoscopic technology, eg, autofluorescence, might be valuable in identifying, for subsequent biopsy or brush, bronchial mucosa at risk. It would be most advantageous to first select high-risk individuals by examination of sputum and then proceed to bronchoscopy if a certain degree of chromosomal damage is found.
Another strategy might include the use of lung cancer-specific monoclonal antibodies to screen sputum, a technique that has been reported to have a good sensitivity and an even higher specificity for detection of premalignant sputum. Since there may be a significant admixture of normal cells in non-small-cell lung cancer, microdissection techniques have been advocated to increase the sensitivity of screening for molecular genetic abnormalities.
Genes and oncogenes already used as clinical tumor markers are ras and p53. Their prevalence in non-small-cell lung cancer and their nature made them the first candidates for screening archival material (sputum). The results showed that mutations of p53 and ras were present prior to the onset of clinical cancer. The limited success of current treatments of non-small-cell lung cancer, which often presents as advanced disease, points out the need for detection of this disease at an earlier stagepreferably before it becomes invasive. At this earlier stage it may be possible to prevent progression by intervening with chemopreventive agents or localized endobronchial treatment (eg, photodynamic therapy).[32,33] The attraction of using somatic genetic changes as targets for early disease screening is that these alterations may be detected with molecular biologic techniques that display high selectivity and sensitivity.
1. Whang-Peng J, Kao-Shan CS, Lee EC, et al: A specific chromosome defect associated with human small-cell lung cancer: Deletion 3p (14-23). Science 215:181-182, 1982.
2. Rodenhuis S, Van de Wetering ML, Mooi WJ, et al: Mutational activation of the K-ras oncogene: A possible pathogenic factor in adenocarcinoma of the lung. N Engl J Med 317:929-935, 1987.
3. Rodenhuis S, Slebos RJC, Boot AJ, et al: Incidence and possible clinical significance of K-ras oncogene activation in adenocarcinoma of the lung. Cancer Res 48:5738-5741, 1988.
4. Slebos RJC, Hruban RH, Dalesio O: K-ras activation and smoking in adenocarcinoma of the human lung. J Natl Cancer Inst 83:1024-1027, 1991.
5. Johnson BE: The role of MYC, JUN and FOS oncogenes in human lung cancer, in Pass HI, Mitchell JB, Johnson DH (eds): Lung Cancer: Principles & Practice, pp 83-89. Philadelphia, Lippincott, 1996.
6. Ransome LJ, Verma IM: Nuclear proto-oncogenes FOS and JUN. Am Rev Cell Biol 60:539-557, 1990.
7. Volm M, Efferth T, Mattern J: Oncoprotein (c-myc, c-erb B1, c-erb B2, c-fos) and suppressor gene product (p53) expression in squamous cell carcinomas of the lung. Clinical and biological correlations. Anticancer Res 12:11-20, 1992.
8. Ullrich A, Schlessinger J: Signal transduction by receptor with thyrosine kinase activity. Cell 61:203-212, 1990.
9. Sozzi G, Miozzo M, Tagliabue E, et al: Cytogenetic abnormalities and overexpression of receptors for growth factors in normal bronchial epithelium and tumor samples of lung cancer patients. Cancer Res 51:400-404, 1991.
10. Tateishi M, Ishida T, Mitsudomi T, et al: Prognostic value of c-erb B-2 protein expression in human lung adenocarcinoma and squamous cell carcinoma. Eur J Cancer 27:1372-1375, 1991.
11. Pezzella F, Turley H, Kuzu I, et al: Bcl-2 protein in non-small-cell lung carcinoma. N Engl J Med 329:690-694, 1993.
12. Krajewski S, Krajewska M, Shabaik A, et al: Immunohistochemical determination of in vivo distribution of BAX, a dominant inhibitor of bcl-2. Am J Pathol 145:1323-1336, 1994.
13. Fontanini G, Vignati S, Bigini D, et al: Bcl-2 protein: A prognostic factor inversely correlated to p53 in non-small-cell lung cancer. Br J Cancer 71:1003-1007, 1995.
14. Hibi K, Takahashi T, Yamakawa K, et al: Three distinct regions involved in 3p deletion in human lung cancer. Oncogene 7:445-449, 1992.
15. Mao L, Lee JS, Kurie MK, et al: Clonal genetic alterations in the lungs of current and former smokers. J Natl Cancer Inst 89:857-862, 1997.
16. Sozzi G, Veronese ML, Negrini M, et al: The FHIT gene at 3p 14.2 is abnormal in lung cancer. Cell 85:17-26, 1996.
17. Minna JD, Sekido Y, Fong KM, et al: Molecular biology of lung cancer, in: DeVita VT, Hellman S, Rosenberg SA (eds): Cancer: Principles and Practice of Oncology 5th ed, pp 849-857. Philadelphia, Lippincott, 1997.
18. Brennan J, Boyle JO, Koch WM, et al: Association between cigarette smoking and mutation of the p53 gene in squamous cell carcinoma of the head and neck. N Engl J Med 332:712-717, 1995.
19. Westra WH, Offerhaus GJ, Goodman SN, et al: Overexpression of the p53 tumor suppressor gene product in primary lung adenocarcinomas is associated with cigarette smoking. Am J Surg Pathol 17:213-220, 1993.
20. Denissenko MF: Preferential formation of benzo(a)pyrene adducts at lung cancer hotspots in p53. Science 374:430-432, 1996.
21. Levine AJ: p53: The cellular gatekeeper for growth and division. Cell 88:223-331, 1997.
22. Carbone D: Rb1 and p53 genes, in Pass HI, Mitchell JB, Johnson, DH, et al (eds): Lung Cancer: Principles and Practice, pp 107-121. Philadelphia, Lippincott-Raven, 1996.
23. Sellers T, Obi W, Chen VW, et al: Increased familial risk for lung cancer. J Natl Cancer Inst 76:217-222, 1986.
24. Law MR: Genetic predisposition to lung cancer. Br J Cancer 61:195-206, 1990.
25. McLemore TL, Adelberg S, Liu MC, et al: Expression of CYP1A1 gene in patients with lung cancer. J Natl Cancer Inst 82:1333-1339, 1990.
26. Caporaso N, Hayes RB, Dosemeci M, et al: Lung cancer risk, occupational exposure, and the debrisoquine metabolic phenotype. Cancer Res 49:3675-3679, 1989.
27. Shaw GL, Falk RT, Deslauriers J, et al: Debrisoquine metabolism and lung cancer risk. Cancer Epidemiol Biomarkers Prev 4:41-48, 1995.
28. McWilliams JE, Sanderson BJ, Harris EL, et al: Glutathione S-transferase M1 (GSTM1) deficiency and lung cancer risk. Cancer Epidemiol Biomarkers Prev 4:589-594, 1995.
29. Tockman MS, Gupta PK, Myers JD, et al: Sensitive and specific monoclonal antibody recognition of human lung cancer antigen on preserved sputum cells: A new approach to early lung cancer detection. J Clin Oncol 6:1685-1693, 1988.
30. Thiberville L, Bourguignon J, Metayer J, et al: Frequency and prognostic evaluation of 3p21-22 allelic losses in non-small-cell lung cancer. Int J Cancer 64:371-377, 1995.
31. Mao L, Hruban RH, Boyle JO, et al: Detection of oncogene mutations in sputum precedes diagnosis of lung cancer. Cancer Res 54:1634-1647, 1994.
32. Baas P, Van Zandwijk N: Endobronchial treatment modalities in thoracic oncology. Ann Oncol 6:523-531, 1995.
33. Van Zandwijk N, Vant Veer L: Antioxidants and the chemoprevention of lung cancer, in Brambilla C, Brambilla E (eds): Lung tumors: Fundamental Biology and Clinical Management. New York, Marcel Dekker, 1998 (in press).
34. Slebos RJC, Kibbelaar RE, Dalesio O, et al: Kirsten-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung. N Engl J Med 323:561-565, 1990.
35. Rodenhuis S, Boerrigter L, Top B, et al: Mutational activation of the K-ras oncogene and the effect of chemotherapy in advanced adenocarcinoma of the lung: A prospective study. J Clin Oncol 15:285-291, 1997.
36. Top B, Mooi WJ, Klaver SG, et al: Comparative analysis of p53 gene mutations and protein accumulation in human non-small-cell lung cancer. Int J Cancer 64:83-91, 1995.
37. Adachi M, Taki T, Ieki Y, et al: Correlation of KAI1/CD82 gene expression with good prognosis in patients with non-small-cell lung cancer. Cancer Res 56:1751-1755, 1996.
38. Apolinario RM, Van der Valk P, De Jong JS, et al: Prognostic value of the expression of p53, bcl-2 and BAX oncoproteins and neovascularization in patients with radically resected non-small-cell lung cancer. J Clin Oncol 145:2456-2466, 1997.
39. Betticher DC, Heighway J, Hasleton PS, et al: Prognostic significance of CCND1 (cyclin D1) overexpression in primary resected non-small-cell lung cancer. Br J Cancer 73:294-300, 1996.
40. Ogawa J, Inoue H, Koide S: Expression of a-1,3-Fucosyltransferase type IV and VII genes is related to poor prognosis in lung cancer. Cancer Res 56:325-329, 1996.