Genetic and molecular events in the pathogenesis of lung cancer

8. GENETIC AND MOLECULAR EVENTS IN THE PATHOGENESIS OF LUNG CANCER Elizabeth. Rosvold, MD

Work in the field of molecular biology has begun to characterize the molecular and genetic changes that transform normal bronchial epithelium into lung cancer. The process requires accumulation of multiple lesions in a critical group of growth-regulating genes and epigenetic factors that promote cellular proliferation) ONCOGENES IN LUNG. CANCER Protooncogenes are normal cellular genes, which control cellular proliferation and other functions, that can be converted to oncogenes, which are capable of inducing one or more aspects of the neoplastic phenotype through dysregulation of cellular functions. The products of these genes include proteins that act in signal transduction and that function as growth factors, growth factor receptors, and transcription factors. Oncogenes, which are the inappropriately activated form of normal genes, result from mutations or functional alterations through mechanisms such as point mutations, overexpression, or loss of regulation. Such genetic changes in protaoncogenes may represent an important step in the pathogenesis of non-small cell lung cancer (NSCLC). Oncogenes studied for their potential role in lung cancer include the //AS and MYC gene families, as well as EGFR (formerly called ERBB1), ERBB2 (formerly Her2 or NEU), FOS, JUN, and others. //AS Mutations in the//AS family of protooncogenes have beeh observed in many h u m a n tumors. RAS mutations are found in approximately 20% of NSCLC tumors and cell lines, but they have not been observed in small cell lung cancer (SCLC) tumors. 2 Point mutations in the KRAS gene have been observed most commonly. Transversions of G to T in codon 12 account for 70% of t h e KRAS mutations, 3 and these mutations are found more frequently in the tumors of smokers than in those of nonsmokers, suggesting induction by the carcinogens in tobacco smoke. 4.5 Codons 13 and 61 are mutational hot spots Curr Probl Cancer, July/August 1996

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as well? Mutations occur in approximately 12% of squamous cell, large cell, and carcinoid tumors, but in adenocarcinomas, the rate of mutation is approximately 30% (range, 14% to 56%). 6 KRAS muta-~ tions are u n c o m m o n in tile b r o n c h i o l o a l v e o l a r form of adenocarcinoma. 7 KRAS mutations have been associated with poor prognosis in several studies. 8-1~ Preneoplastic lesions from individuals whose lung tumors contained KRAS mutations have been studied. The KRAS mutations were d e t e c t e d in all invasive a n d m e t a s t a t i c lesions a n d in 80% of noninvasive cancers, but only I of 12 dysplastic and no hyperplastic or normal-appearing cells had these mutations. These findings suggest that KRAS mutations occur late in lung cancer pathogenesis, possibly with the appearance of the malignant phenotype. ~3The level of expression of the KRAS g e n e p r o d u c t , p21, is inversely correlated with the survival of patients with NSCLC. ~4 MYC Overexpression of MYC family oncogenes, especiallyMYCL1, is found frequently in SCLC but is not c o m m o n in NSCLC. 15 Restriction fragment length polymorphisms (RFLP) in theMYCL1 gene (formerly called MYCL or L-myc) have been correlated with regional l y m p h node metastases, distant metastases, and poor prognosis in Japanese populations 18.~7but not in Caucasian or African-American populations. 182~ EGFR The p r o d u c t of the EGFR (formerly called ERBB or ERBB1] protooncogene is a sequence homologous to the intracellular portion of the epidermal growth factor receptor. This protein is overexpressed in all types of NSCLC, including approximately 70% of squamous cell cancers and up to 40% of adenocarcinomas, but it is not found in SCLC. 21.22Overexpression of the ERBB2 oncogene is c o m m o n in epithelial cancers such as those from the breast and ovary and is associated with a poor prognosis. 23 Overexpression occurs in 30% to 59% of NSCLC and is common in adenocarcinomas. 24'25The gene encodes p185, and overexpression of the protein in adenocarcinomas has been associated with shortened survival of patients in two studies. 26'27Shi et al. 25 demonstrated that p185 expression correlated with the stage of NSCLC and correlated with lymph node metastasis of squamous cell cancers but not of adenocarcinomas. In animal models, tumors overexpressing p185 are more invasive and have increased metastatic potential compared with tumors having low levels of p185 expression. 28 FOS AND JUN The FOS and ]UN genes, which code for transcription factors, have lower expression at the mRNA and protein levels in NSCLC t h a n in 248

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adjacent normal tissues. 29,3~Downregulation of these genes may be important in the pathogenesis of lung cancer. TUMOR SUPPRESSOR GENES AND OTHER GENETIC INFLUENCES Anti-oncogenes or tumor suppressor genes are genes that normally function to control cellular proliferation and growth regulation. These genes become inactivated or deleted in the process of malignant transformation. Tumor suppressor genes important in the pathogenesis of lung cancer include P53, RB1, and as yet unidentified genes on chromosome region 3p. P53

The P53 gene encodes a protein important in transcription control, apoptosis, and DNA synthesis and repair21 This gene is mutated in many c o m m o n tumors, including breast, ovary, and colon cancers. In NSCLC, mutations are identified in approximately 50% of cases, with the highest incidence found in squamous cell cancers (65%) and the lowest incidence (33%) in adenocarcinomas21 Mutations in P53 may lead to prolongation of the half-life of the protein product and elevation of its steady-state level, enabling detection by i m m u n o h i s t o c h e m i c a l methods. A c c u m u l a t i o n of p53 protein is not seen in normal bronchial mucosa, and it is detected rarely (0% to 7%) in squamous metaplasia. However, it accumulates in up to 30% of early neoplastic lesions (mild dysplasias), and the frequency of detection increases w i t h increasing dysplasia. 32,33P53 protein accumulation persists in the nodal metastases of p53-positive primary lesions, although it is not found in those of p53-negative lesions24 These results suggest a multistage model for the development of squamous cell lung cancer. The s p e c t r u m of the m u t a t i o n s f o u n d in l u n g cancers provides insight into their pathogenesis. The most c o m m o n m u t a t i o n is the G:C to T:A transversion, particularly on the n o n t r a n s c r i b e d DNA strand. 31 This m u t a t i o n is one associated w i t h m a n y of the ~carcinogens f o u n d in tobacco smoke, and the f r e q u e n c y of this type of transversion is positively correlated w i t h lifetime cigarette cons u m p t i o n . 35'36This m u t a t i o n is found with higher frequency in squam o u s and large cell cancers than in a d e n o c a r c i n o m a s , consistent w i t h the weaker association of a d e n o c a r c i n o m a with smoking. 31 P53 m u t a t i o n s have also been associated w i t h a y o u n g e r age of onset of cancer but not w i t h gender27 Alterations in p53 also have been associated with hilar and mediastinal lymph node metastases, 38 and an inverse correlation b e t w e e n p53 protein acc u m u l a t i o n and the degree of histologic differentiation has been described26 C u r t Probl Cancer,

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RB1

The RB1 gene, n a m e d for its role in t h e p a t h o g e n e s i s of retinoblastoma, encodes for a phosphoprotein that is important in cell cycle regulation. Loss of function in the RB1 protein has been implicated in the pathogenesis of many cancers, including more than 90% of SCLC. 1 The RB1 protein is missing or abnormal in approximately 15% to 32% of NSCLC. 39-~1No correlation has been observed between RB1 status and age, sex, stage of disease, smoking history, drug sensitivity, or survival? 9 CHROMOSOME 3

Deletions in a portion of a chromosome or loss of heterozygosity (LOH) for specific chromosomal loci detected frequently in a particular tumor type suggests the presence of a tumor suppressor gene. Deletion of a portion of the short arm of chromosome 3 is common in lung cancer. Loss of one allele can be seen by RFLP analysis in approximately 90% of SCLC cell lines and 50% of NSCLC cell lines. 42 Interstitial or partial deletions were observed in 47% of adenocarcinomas examined by Yokoyama et al., 43 but none were seen in squamous cell carcinomas. Other studies 44~47have shown that 3p losses occur more frequently in squamous cell cancers than in adenocarcinomas. Common regions of deletion include 3p21.3 and 3p14.1-21.1. 43LOH for markers on 3p occur in early stages of lung cancer pathogenesis, including hyperplasia, dysplasia, and noninvasive cancer, and losses were shown to be identical to those in corresponding invasive cancers. 48 Candidate genes on 3p include the VHL gene, which is important in the pathogenesis of the familial renal cell carcinoma associated with the von Hippel-Lindau syndromeS9; RARB, the gene coding for the B r e c e p t o r for r e t i n o i c acid; the RAF1 oncogene; and the phosphotyrosine phosphatase gene. 1 Sekido et al. 49 evaluated the VHL gene in NSCLC and in other thoracic neoplasms and found only rare mutations. Expression of the RARB gene was examined in squamous cell tumors, adenocarcinomas, and in normal lung tissue. 4s Expression was detected in the normal tissue and in the adenocarcinomas. The epidermoid tumors lacked RARB expression, suggesting a role for this gene in the pathogenesis of this tumor type. OTHER CHROMOSOMAL ABERRATIONS

Other chromosomal alterations observed in NSCLC include loss of the long arms of chromosomes 1, 2, 5, 6, 8, 9, 13, 18, 21, and 22 and the short arms of chromosomes 3, 8, 9, and 17. 44.46Chromosomal losses are seen more frequently in squamous cell carcinomas than in adenocarcinomas.47 Loss of the Y chromosome; gains of lq, 7p, and l l q ; isochromosomes; unbalanced exchanges; double minutes; and homogeneously staining regions have also been observed. 44 One can250

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didate gene on 9p is the interferon gene, which is lost in 36% of lung cancers and 43% of lung cancer cell lines. 5~ The APC/MCC gene cluster, associated with predisposition to colon cancer, is a candidate gene on 5q. LOH at the APC/MCC cluster was found in 29% of informative cases of NSCLC and correlated with decreased survival. 51 LOH in the del(27) region, a region proximal to the APC/MCC cluster, has also been demonstrated in 57% of informative lung cancers. 52 LOH at 11p also occurs frequently and has been correlated with advanced T stage and nodal involvement.53 At least one study has correlated allelic losses at loci on 2q, 18q, and 22q with the development of brain metastases. 54

MICROSATELLITES Microsatellites are highly polymorphic, short, tandem repeat sequences found throughout the genome. Mismatch repair errors are thought to cause instability in these loci. Although microsatellite instability is observed in hereditary nonpolyposis colon cancer and in some sporadic cancers, its role in NSCLC is unclear. One study demonstrated such instability in one third of tumors examined, 55but another, larger study observed instability in only 6.5% of cases. 56 GENETIC SUSCEPTIBILITY The existence of an inherited predisposition to lung cancer has been suggested by several studies. 57-59 One study provided evidence that predisposition is inherited in a mendelian codominant fashion, with the genotype determining the age at onset of the cancer. 59 The inheritance of an abnormality in the metabolism of lung carcinogens has been suggested as a factor in susceptibility to lung cancer. Several enzymes involved in the metabolism of potential lung carcinogens exhibit genetic variation, and their role in lung cancer development has been extensively explored. Increased risk was initially demonstrated for individuals inheriting a particular phenotype for the enzyme debrisoquine hydroxylase, a cytochrome P450 enzyme thought to be involved in the metabolism of compounds in cigarette smoke. 6~ Several studies of the genetic polymorphisms associated with this phenotype suggest an increased risk for lung cancer, 6~67 but many studies of phenotype, genotype, or both have shown no association or had equivocal results. 68-74 Two polymorphisms in the CYPIA1 gene, which encodes the enzyme responsible for hydroxylation of aromatic hydrocarbons, have been studied for possible associations with lung cancer. In Japanese populations, there appears to be increased risk for lung cancer in those with certain genotypes. 75~7aHowever, in most of the other populations studied, including Caucasians and African Americans, no association between polymorphisms in this gene and lung cancer risk can be demCurr Probl Cancer, July/August 1996

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onstrated. 79-85 The frequencies of these polymorphisms vary among different ethnic populations, which may account for the differences seen in these studies. A polymorphism in the CYPIA1 gene specific for African Americans has been observed. This polymorphism was associated with an increased risk for lung cancer in one study, 86 but two other studies failed to support this association. 87.8S The cytochrome P4502E1 enzyme is important in the oxidation of carcinogens. Polymorphisms in its gene have been associated with susceptibility to lung cancer in Japanese populations 89'9~but not in Caucasian populations. ~~ The isozymes of glutathione S-transferases are capable of conjugating toxic intermediates of carcinogen metabolism. Lack of activity of GST-pt has been associated with increased risk of lung cancer in Caucasian and Japanese populationsY ,78,92-96although not all studies support such an associationW -99 Several studies have shown an increased risk for squamous cell cancer, rather than adenocarcinoma, in those with the null allele. 93,94.96.97A dose effect of smoking has been demonstrated, with null allele individuals developing s q u a m o u s cell cancers at lower cigarette doses. 78.98 This effect is even more pron o u n c e d for those whose genotypes p r o d u c e low levels of GST-~ and CYPIA1. 7a Lung cancer develops as a result of the interaction between host (i.e., inherited predisposition) and environmental factors in a multistage process. For example, any one defect in carcinogen metabolism or in any oncogene is unlikely to be sufficient to cause lung cancer. Further characterization of these individual events and the sequence of their development may provide opportunities for primary prevention, screening, early diagnosis, and n e w treatment strategies. TUMOR GROWTH FACTORS An important step in the pathogenesis of lung cancer is the stimulation of cellular proliferation by tumor promoters. Several growth factors are thought to be important in this process, including epidermal growth factor (EGF), insulin-like growth factor type I, and gastrin-releasing peptide (GRP33). These growth factors are involved in autocrine regulatory loops in vitro. EGF and a related peptide, transforming growth factoroa, are produced in 60% of adenocarcinomas and s q u a m o u s cell carcinomas, z Monoclonal antibodies directed against these two peptides inhibit growth of lung cancer cell lines in vitro. 1~176176 The growth factor receptors EGFR and ERBB2 are present in a significant percentage of NSCLC. An autocrine loop involving GRP33 has been identified in SCLC, and one study demonstrated a similar system for NSCLC cell linesJ ~ Many other growth factors and their receptors have been detected in NSCLC cells, including plateletderived growth factor, parathyroid hormone-related peptide, hepato252

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cyte growth factor, and colony-stimulating factor, but the significance of these systems remains to be determined. ~~ The growth factors and their receptors represent attractive targets for therapeutic intervention. Studies using monoclonal antibodies directed against specific growth factors and receptors have been initiated.104.~o5 DRUG RESISTANCE NSCLCs are frequently resistant to c h e m o t h e r a p y at the time of diagnosis. 1~ C h e m o r e s i s t a n c e appears to have m u l t i p l e causes. Overexpression of the MDR1 gene, which codes for the energy-dependent drug efflux protein P-glycoprotein, is the best characterized resistance mechanism in cancer cells, but its role in NSCLC is controversial. Few studies examining MDR1 or P-glycoprotein expression in NSCLC detected significantly elevated levels, and no signific a n t c o r r e l a t i o n b e t w e e n the level of e x p r e s s i o n a n d in vitro chemoresistance has been demonstrated. 1~176 One study demonstrated significant correlations among the expression of poglycoprotein, the expression FOS and JUN proteins, and in vitro drug resistance, m Overexpression of GST-~ and downregulation of topoisomerase II activity h a v e also been demonstrated in previously untreated NSCLC with in vitro drug resistance m and in cells with acquired resistance, m The M RP gene codes for the multidrug resistance-associated protein, which has significant homology to P-glycoprotein. m This gene is.expressed in NSCLC cell lines and tumors, 1~4,m and it may be involved in resistance in non-P-glycoprotein multidrug-resistant cells. Overexpression of the ERBB2 gene also correlates with intrinsic multidrug resistance in NSCLC cell lines, n6 Adenovirus-mediated transfer of a wild-type P53 gene into a NSCLC cell line with a homozygous deletion of P53 increased the sensitivity of the cells to cisplatin) ~7 Any of the mechanisms of resistance to chemotherapy are potential targets for intervention and treatment in NSCLC, and such studies are underway2 ~ EARLY DETECTION AND SCREENING Because of the high incidence and poor prognosis of lung cancer, the detection of cancers in earlier, potentially resectable stages is important to improve survival. Several randomized, controlled trials have examined the value of screening m e n at high risk for lung cancer. Between 1970 and 1980, approximately 30,000 m e n were entered into the Early Lung Cancer Cooperative Study, which was performed at three centers in the United States: Johns Hopkins, Mayo Clinic, and Memorial Sloan-Kettering. 9 C u r t Probl Cancer,

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In the Mayo Lung Project, the screened group had chest radiographs and s p u t u m cytologic examinations every 4 months. The control group received standard care. Although in the screened group more cancers were resectable than in the control group (46% versus 32%) and more were detected at earlier stages (stage I cancers, 53% versus 21%), the deaths from lung cancer and the n u m b e r of late-stage cases in the two groups were not significantly different. 118,119 The Memorial Sloan-Kettering Project was designed to determine the effect of adding every-4-month s p u t u m cytologic examinations to annual screening chest radiographs. There was no difference in the proportions of early- or late-stage cancers, operable cancers, survivorship, or mortality rates22~ The Johns Hopkins trial was similar in design to the Memorial Sloan-Kettering study. It also demonstrated that survival and mortality rates did not differ significantly between the two g r o u p s . 121 A randomized, controlled trial was performed in Czechoslovakia. The screened group received chest radiographs and s p u t u m cytologic analysis every 6 months for 3 years, and the controls received chest radiographs only at the conclusion of the 3-year study period. In the screened group, more cancers were diagnosed (36 versus 19 cases), more were resectable (25% versus 15%), and the 5-year survival was better (25% versus 0%); however, more lung cancer deaths resulted (28% v e r s u s 1 8 % ) . 122,123 %vo case-control studies examined the benefit of screening by chest radiographs. In a German study, no reduction in mortality was observed in the screened group? 24 In a study from Japan, a trend, although only of borderline significance, toward a lower risk for lung cancer death was observed for the screened group. 125At this time, there is no scientific justification for screening for lung cancer with chest radiographs or s p u t u m analysis, and no organizations support Such screening. Archived s p u t u m specimens from the Johns Hopkins trial were reevaluated. The specimens were stained with two monoclonal antibodies developed against SCLC and NSCLC. For the subjects with moderate atypical metaplasia, the two stains predicted the development of lung cancer at least 2 years before clinical recognition, with a sensitivity of 91% and a specificity of 8 8 % . 126'127A trial is underway to test this method of detecting changes in s p u t u m samples in an effort to screen for n e w primary lung cancers in individuals after resection of stage I NSCLC. 121 Another technique being used for screening involves the use of a quantitative microscopy system that employs digital imaging. Malignancy-associated changes have been detected i n 8 0 % of apparently normal bronchial biopsy samples from individuals with cancer, and the technique is being employed to evaluate s p u t u m s a m p l e s . 128'129 For some individuals with positive results on sputum analysis, the 254

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chest radiographs do not indicate disease. For example, in the initial screening phase of the Early Lung Cancer Cooperative Study, 81 patients with sqnamous cell cancer were identified, 35 (43%) of w h o m had abnormal sputum cytologic results but normal chest radiograph findings. 13~In most cases, fiberoptic bronch0scopy proved helpful in localizing the tumor. TM The use of fluorescence bronchoscopy improves the detection of these radiologically occult cancers and allows the detection of earlier lesions such as dysplasia and carcinoma in situ. One form of fluorescence bronchoscopy involves administration of a hematoporphyrin derivative that concentrates in malignant tissues and causes them to fluoresce when visualized with special imaging devices. 128 Another technique takes advantage of differences in the autofluorescence of dysplasia and carcinoma in situ compared with normal tissue when illuminated by violet or blue light. The Lung Imaging Fluorescence Endoscope (LIFE), developed in Vancouver, Canada, uses a standard bronchoscope with a helium-cadmium light source attached to an image-intensification camera whose images are digitized and processed for display on a screen. This image allows localization and measurement of suspicious lesions for biopsy. ~32This technique was compared with standard bronchoscopy in the detection of dysplasia and carcinoma in situ. The specificity of the two methods was identical (94%], but the LIFE device was 50% more sensitive (72.5% versus 48.4%). Use of this device may become important in the detection of occult lesions and of premalignant lesions that may be amenable to intervention and prevention? 32 The ability to detect earlier stages in the process of malignant transformation may also be possible with the use of the molecular and genetic changes observed in lung cancer pathogenesis as markers. RAS and P53 mutations are an example of such markers. In the Johns Hopkins Lung Project screening trial, 15 adenocarcinomas were identified. RAS or P53 mutations were detected in 10 of the 15 tumors. S p u t u m samples obtained from these individuals and stored before diagnosis were examined for the same mutations. In 8 of 10 cases, the same mutation was identified in at least one s p u t u m sample, with the earliest change detected more than a year before diagnosis? 33 RAS mutations have also been identified in bronchoalveolar lavage (BAL) fluid. Mutations were detected in 21 samples from patients with known or suspected NSCLC. In 19 of these, the results of cytologic examination of the BAL fluid were negative, and for 9, the resuits of bronchoscopic biopsies and brushings were also negative. Resected tumors contained the identical RAS mutations. Mutations were not detected in 30 cases with diagnoses other than lung cancer. The sensitivity and specificity of KRAS mutation detection in BAL fluid were each 100%. T M P53 mutations also have been detected in bronchial biopsy specimens. 35 Other markers for the early diagnosis of lung cancer and for moniC u r t Probl Cancer, July/August 1996

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toring patients for recurrence and disease progression have been studied. Elevation of serum carcinoembryonic antigen (CEA) correlates with increasing stage of lung cancer 136 and is more sensitive for a d e n o c a r c i n o m a t h a n for s q u a m a u s cell c a r c i n o m a . 137-139 A preoperative level greater t h a n 15 ng/ml indicates t u m o r inoperability. 137 Measurement of CEA is useful for monitoring patients after tumor resection and during other treatment. 136.~37Elevation of the CEA level in BAL fluid appears to be associated with an increase in the diagnostic yield of bronchoscopy, with a positive and negative predictive value of 77% and 94%, respectively, found in one study. ~4~ Squamous cell carcinoma (SCC) antigen is elevated in the serum of patients with NSCLC, particularly those with squamous cell tumors, for which the degree of elevation correlates with the extent of disease. The sensitivity and specificity of SCC antigen does not appear to be adequate for use in the diagnosis of NSCLC, ~38'~41although elevation of SCC antigen levels preoperatively m a y provide a method for monitoring patients after disease resection. 142 Measurement of the SCC antigen in BAL fluid may be useful diagnostically, especially in combination with CEA determinations. 143 Tissue polypeptide antigen (TPA) is produced by proliferating cells, and elevations of this protein have been detected in the serum of patients with various cancers. TM TPA elevation appears to correlate with stage of disease, 145 and elevations have been associated with shortened survival of patients with all histologic types of NSCLC. 1463~7 TPA measurement may be useful in determining operability, with an accuracy approaching that of computed tomography scanning, ~48and it appears to have utility for monitoring patients with advanced disease postoperatively ~39 and during chemotherapy. ~39 Neuron-specific enolase (NSE) is a useful serum marker for monitoring SCLC, but it has little utility in the diagnosis or follow-up of NSCLC? 39NSE levels are significantly higher in the BAL fluid of lung cancer patients than in those with benign lung disease or no disase, ~49 and such measurement may assist in the diagnosis of lung cancer. One m a r k e r for lung c a n c e r being s t u d i e d is CYFRA 21-1, a cytoskeletal protein found in simple and pseudostratified epithelium, such as that in the bronchial tree. Theoretically, this marker should be specific for lung cancers. Measurement of CYFRA 21-1 in serum has been s h o w n in several studies ~5~ to be highly sensitive and specific for NSCLC compared with benign disease. Compared with CEA, SCC antigen, NSE, TPA, and CA 125, CYFRA 21-1 is consistently more sensitive and specific, especially for squamous cell cancer? 5~ CYFRA 21-1 levels correlate with histologic type and with stage of disease. Measurement of CYFRA 21-1 appears to have utility in monitoring patients after disease resection ~s2and during chemotherapy? 5~.~54 The CYFRA 21-1 level has been shown to have independent prognostic significance? 55 256

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Many other markers are being evaluated for the early diagnosis and monitoring of patients with lung cancer, as well as for use as prognostic factors and as intermediate markers for intervention trials, such as chemoprevention studies. These markers include h u m a n chorionic gonadotropin, 158,1s9 ERBB2,16~ m u c i n 1 antigens, 161 rim23,162'~83 ~1" antitrypsin, TM monoclonal antibodies against lung tumor tissue, ~38and soluble interleukin-2 receptors. 1~9.165

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5. 6. 7. 8. 9.

10. 11. 12. 13. 14.

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