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Cytogenetic and molecular aspects of lung cancer
Cancer Letters 239 (2006) 1–9 www.elsevier.com/locate/canlet
Mini-review
Cytogenetic and molecular aspects of lung cancer Anna D. Panani*, Charis Roussos Critical Care Department, Research Unit, Medical School of Athens University, ‘Evangelismos’ Hospital, Ipsilandou 45-47, Athens 10676, Greece Received 18 May 2005; received in revised form 21 June 2005; accepted 24 June 2005
Abstract Lung cancer is one of the most common cancers worldwide and its pathogenesis is closely associated with tobacco smoking. Continuous exposure of smoking carcinogens results in the accumulation of several alterations of tumorigenesis related genes leading to neoplastic bronchial lesions. Lung cancer is divided in two main histological groups, non-small cell lung carcinomas (NSCLCs) and small cell lung carcinomas (SCLCs). It seems that lung tumorigenesis is a multistep process in which a number of genetic events including alterations of oncogenes and tumor suppressor genes have been occurred. Cytogenetic abnormalities in lung cancer are very complex. However, a number of recurrent cytogenetic abnormalities have been identified. Many of these changes are common in both major histological groups of lung cancer while certain chromosomal abnormalities have been correlated with the stage or the grade of the tumors. In addition, several molecular alterations have been constantly found. Some of them are common in different histological subtypes of lung cancer and they appear to play an important role in the pathogenesis of lung cancer. A good understanding of the underlying genetic changes of lung tumorigenesis will provide new perspectives for early diagnosis and screening of high-risk individuals. In addition, a number of genetical prognostic factors have been identified as possibly helpful parameters in the evaluation of lung cancer patients. Further research is required in order to systematically investigate genetical alterations in lung cancer contributing to improvement of lung cancer classification and staging and to development of new molecular targeted therapies. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Lung cancer; Cytogenetics; Molecular changes; Clinical implications
1. Introduction Lung cancer is one of the most common cancers worldwide and its pathogenesis is closely associated with tobacco smoking. Activated carcinogens in tobacco smoke interact with certain genes like p53, * Corresponding author. Tel.: C30 210 7259307; fax: C30 210 7259307. E-mail address: [email protected] (A.D. Panani).
k-ras and FHIT. Thus, chronic exposure of smoking carcinogens results in the accumulation of genetic and epigenetic alterations of these tumorigenesis related genes leading to neoplastic bronchial lesions. However, only a low percentage of the long-term smokers develop lung cancer, since various DNA cellular repair mechanisms appear to prevent DNA damage. Besides cigarette smoking other environmental exposures as well as genetic factors contribute also to the development of lung cancer [1–6].
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.06.030
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Table 1 Frequent cytogenetic changes in lung cancer Chromosome NSCLCs SCLCs
Deletion
Gain
3p, 6q, 8p, 9p, 9q, 13q, 17p, 18q, 19p, 21q, 22q 3p, 4p, 4q, 5q, 8p, 10q, 13q, 17p
1p, 1q, 3q, 5p, 7p, 7q, 8q, 11q, 12q 3q, 5p, 8q, 19q, Xq
Lung cancer is divided in two main histological groups. Eighty percent of the lung cancer are nonsmall cell lung carcinomas (NSCLCs) and 20% are small cell lung carcinomas (SCLCs). In the group of NSCLCs are included adenocarcinomas, squamous cell, large cell and bronchoalveolar carcinomas. It is believed that lung cancer originates from bronchial epithelial cells and that carcinogenesis from a normal cell to an invasive carcinoma is a multistep process preceded by a premalignant lesion, such as hyperplasia, metaplasia or dysplasia of bronchial epithelium. However, only a small number of the premalignant lesions progresses to invasive cancer whereas the majority of them may remain unchanged for a long period of time or even regress. There are poor informative data about the sequence of genetic alterations leading to lung cancer. Several reports suggest that a number of genetic events including alterations of oncogenes and tumor suppressor genes must have occurred before lung cancer becomes clinically evident. Interestingly, studies have shown the occurrence in the premalignant stage of similar genetic changes to those observed in lung cancer [7–12]. Moreover, in the study by Cheng et al. [7] there was evidence showing similar genetic alterations in human bronchial epithelial cell lines immortalized by transfection with viral oncogenes to those observed in the premalignant stage of human lung. Therefore, it seems that many molecular genetic and epigenetic changes are necessary for conversion of the normal bronchial epithelial cells to malignant lung cancer cells. Many of these changes are common in both major histological groups of lung cancer, NSCLC and SCLC (Tables 1 and 2). Interestingly, several studies have investigated the use of certain genetic alterations as potential biomarkers in lung cancer early detection or risk assessment [13–19].
It is thought that a detailed understanding of the underlying genetic events of lung tumorigenesis will contribute to an early diagnosis, prediction of prognosis and to development of novel molecular targeted therapies. In this review, we summarize the most common cytogenetic and molecular genetic abnormalities seen in lung cancer emphasizing also their possible usefulness in the clinical practice.
2. Cytogenetic and molecular cytogenetic analysis In contrast to many hematological malignancies often characterized by simple and balanced chromosomal changes, epithelial tumors usually have complex unbalanced abnormalities. Primary lung tumors often have a low mitotic index making it difficult to obtain sufficient number of metaphases suitable for detailed cytogenetic analysis. Moreover, the chromosomal abnormalities can be extremely complex with many unidentified marker chromosomes complicating the identification of recurrent changes. Nevertheless, recurrent cytogenetic abnormalities have become apparent in lung cancer [20]. The development and application of molecular cytogenetic techniques have proven valuable in resolving some of the above problems of conventional Table 2 Frequent molecular genetic changes in lung cancer Genes
Abnormalities
NSCLCs
SCLSs
FHIT K-RAS ERBB1/EGFR ERBB2/HER2/ NEU MYC Family BCL-2 Cyclins P1bINK4a
Deletion/mutation Mutation Overexpression Overexpression
w40 w20 w60 w20
w80 Rare – –
Overexpression Overexpression Overexpression Inactivation (deletion/ mutation/ hypermethylation) Inactivation (deletion/ mutation) Inactivation (mutation/deletion)
5–10 10–35 ? w70
15–30 75–95 ? Rare
15–30
w90
w50
80–90
Rb P53
Frequency %
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cytogenetics leading to a more accurate cytogenetic analysis. The first recurrent abnormality which was described by Whang-Peng et al. in SCLC was a del(3p) [21]. Loss of 3p is a frequent finding in all histological subtypes of lung cancer. Furthermore, loss of heterozigosity studies have detected allelic imbalances in 3p in almost 100% of SCLCs and NSCLCs. The 3p region is believed to harbor one or more tumor suppressor genes of importance for lung cancer development [20,22–28]. Other recurrent karyotypic abnormalities reported in SCLC include del(5q), del(13q) and del(17p). Comparative genomic hybridization (CGH) studies of SCLCs have detected frequent losses of 3p, 4p, 4q, 5q, 8p,10q, 13q and 17p as well as gains of 3q, 5p, 8q, 19q and Xq. Genomic amplifications are also common in SCLCs recurrently seen as homogeneously staining regions and douple minutes chromosomes. These alterations are usually associated with amplification of the myc oncogene [20,29–34]. In NSCLCs, chromosomal abnormalities are more complex than those in SCLCs. Frequent losses of 3p, 6q, 8p, 9p, 9q, 13q, 17p, 18q, 19p, 21q and 22q have been reported while gains of 1p, 1q, 3q, 5p, 7p, 7q, 8q, 11q and 12q are also common [34–41]. Pei et al. [37] performed CGH analysis on 35 adenocarcinomas and 32 squamous cell carcinomas with the goal of identifying differences in the patterns of genomic imbalance between these histological subtypes. All 67 cases represented untreated patients from whom primary tumor tissues were collected at the time of diagnosis. Many imbalances, such as gains of 1q, 5p and 8q, have occurred at a high frequency in adenocarcinomas as well as in squamous cell carcinomas. However, several statistically significant differences have been found. The most prominent of them was gain of 3q24-qter which was seen in w80% of squamous cell carcinomas but in only 30% of adenocarcinomas. Another prominent difference was gain of 20p13 which was seen in w30% of squamous cell carcinomas versus 6% of adenocarcinomas. Furthermore, loss of 4q was seen at a significantly higher rate in squamous cell carcinomas than in adenocarcinomas while gain of 6p was more common in adenocarcinomas. Several studies have correlated certain chromosomal abnormalities with the stage or the grade of the tumor. Pei et al. [37] found that gains of 7q and 8q
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were associated with higher-stage or higher-grade tumors suggesting that these changes might be indicative of tumor aggressiveness in NSCLCs. Goeze et al. [39] applied CGH technique to screen 83 lung adenocarcinomas of 60 patients for chromosomal imbalances. The results of that study indicated that adenocarcinomas were characterized by a recurrent pattern of chromosomal abnormalities with distinct genetic lesions being associated with the metastatic phenotype. The most common alteration was DNA overrepresentation on chromosome 1q followed by DNA overrepresentation on 8q and 20q and deletion on 3p, 4q, 6q, 9p, 9q, and 13q. Also in that study data suggested that deletion on chromosomes 3p12-p14, 3p22-p24, 4p13-15, 4q21-qter, 6q21-qter, 8p, 10q, 14q21, 17p12-p13, 20p12, and 21q and overrepresentation on chromosomes 1q21q25, 7q11, 9q34, 11q12-q13, 14q11-q13, and 17q25 were associated with the metastatic phenotype. In contrast, losses on chromosome 19 and gains on 3p, 4q, 5p, and 6q were preferentially found in nonmetastasizing tumors. Little is known about the mechanisms behind these chromosomal changes. It was suggested that telomere dysfunction and chromosomal breakage-fusionbridge events may be important mechanisms. The order of the karyotypic events in tumors progression and the possible presence of cytogenetic pathways in lung cancer remain obscured. Interestingly, in a recent study Hoglund et al. [42] identified all published cases of cytogenetically aberrant lung cancers in the Mitelman Database of Chromosome Aberrations in Cancer, altogether 432 cases. Tumors were classified with respect to the presence or absence of the most frequent imbalances and were statistically analyzed to assess the order of appearance of imbalances, possible karyotypic pathways and possible cytogenetic subtypes. They found that the imbalance profiles for adenocarcinomas, squamous cell carcinomas and large cell carcinomas were similar, except for the significantly lower frequency of C20 in large cell carcinomas. The frequent imbalance C7 and 3p- were seen at similar frequencies in all three subtypes. On the other hand the SCLC showed significantly lower frequencies of C7,C11, 1p-, 6q- and 14q- while it showed the highest frequencies of 3p- seen in 65% of the cases. In that study, the analyses suggested that lung cancer may develop through three pathways
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initiated by C7, 3p- and C12, respectively. The 3ppathway is dominated by losses and the C12 pathway by gains. Gain of chromosome 7 was shown to be important in the 3p- pathway while a group of tumors presented C7 and C20 with few additional changes. Also the authors showed that the karyotypic evolution might pass through three different phases. Phase I was characterized by tumors with few changes and by well-separated 3p- and C12 pathways. Phase II cases exhibited less distinct 3p- and C12 pathways while they had an increased number of imbalances. Phase III tumors were polyploid and highly complex. Notably, in that study among morphologic subtypes of lung cancer no marked differences between the karyotypic patterns were found suggesting that mode of karyotypic evolution might be independent of histological classification.
3. Molecular genetic analysis 3.1. Deletions of 3p Loss of heterozygosity at 3p is thought to be an early genetic change in the development of both SCLCs and NSCLCs. Several distinct regions of 3p loss have been identified, such as 3p25-26, 3p24, 3p21, 3p14, and 3p12, suggesting that several tumor suppressor genes may be located at 3p. Homozygous deletions within 3p21, 3p12 and 3p14 regions have been described in primary lung cancer and in lung cancer cell lines. The fragile histidine triad (FHIT) gene is located at 3p14.2 and it appears to play a role in the regulation of apoptosis and in cell-cycle control. There was evidence that FHIT functions as a tumor suppressor gene. FHIT is frequently deleted or mutated in lung cancer, whereas it represents the earliest deletion in bronchial preneoplastic lesions. Abnormal FHIT messenger RNA transcripts have been detected in about 80% of SCLCs and in 40% of NSCLCs. Loss of heterozygosity of the FHIT gene has been observed in a higher percentage of smokers than nonsmokers lung cancer patients, suggesting that FHIT is a target of tobacco smoke carcinogens [1,4,8, 19,23,25–28,43]. In addition, 3p21.3 region has been extensively examined for putative tumor suppressor genes, because as noted earlier homozygous deletions have been found in several lung cancer cell lines and
primary lung tumors. However, none of the putative tumor suppressor genes located at 3p21 found to be consistently mutated in lung cancer [1,23,24]. Dammann et al. [44] reported frequent epigenetic inactivation of a RAS association domain family protein from the lung tumor suppressor locus 3p21.3. There was evidence that the RAS effector homologue, RASSF1, functions potentially as a lung tumor suppressor gene. It was also found RASSF1 promoter hypermethylation in 100% of SCLC cell lines, in 63% of NSCLC cell lines and in 30% of primary NSCLC tumors [1,45]. 3.2. RAS activation Mutation of ras proto-oncogene can be frequently identified in several cancers including lung cancer. K-ras gene mutations were found in about 20% of NSCLCs but are rare in SCLCs. Ras mutations usually include point mutations at codons 12, 13, or 61 and they have been reported to be late events in lung cancer development. Most of the K-ras mutations are detected in lung adenocarcinomas, accounting for about 30% of these tumors and suggesting that K-ras mutations may be important in the development of lung adenocarcinomas. A correlation between cigarette smoking and ras mutations has been reported. Studies showed that the presence of a ras mutation results in shortened survival in NSCLC patients. However, other studies did not confirm this finding showing that ras mutations might not have an influence on survival [1,11,14,18,46–49]. Finally, Fischer et al. [18] suggested that further systematic investigations are needed for a final validation of the prognostic and predictive power of this biologic marker. 3.3. ERBB1/epidermal growth factor receptor (EGFR) The ERBB1 encodes for the EGFR. Overexpression of the epidermal growth factor receptor (EGFR) and transforming growth factor alpha (TGF-a) have been found in about 60% of NSCLCs at both mRNA expression and protein translation levels. Approximately 70% of tumors with squamous histology and 40% with adenocarcinomas overexpress epidermal growth factor receptor (EGFR). Thus, it seems that
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disruption of the EGF signaling pathway is important in NSCLCs. The prognostic value of EGFR for lung cancer patients remains controversial. A metaanalysis study showed that EGFR expression might not represent a significant prognostic factor in NSCLCs [13,18,50–52]. 3.4. ERBB2/HER2/NEU ERBB2 encodes for the receptor HER2/NEU, which belongs to the family of the epidermal growth factor receptors. A correlation between HER2 overexpression and poor prognosis of NSCLC patients has been reported [13,18,53,54]. 3.5. MYC overexpression The family of the myc oncogenes (c-myc, n-myc, l-myc) encodes phosphoproteins involved in the regulation of transcription of other genes responsible for cell proliferation. The usual mechanism of myc activation in lung cancer is gene amplification resulting in overexpression of the gene. Myc amplification occurs in 15–30% of SCLCs and in 5– 10% of NSCLCs. It has been shown that myc amplification affects adversely the survival in SCLC patients [1,31,55–56]. Yakut et al. [56] investigated genetic alterations on the primary tumor tissues and surgical borderline tissues in 51 patients with NSCLCs by using the FISH method with locus specific probes for c-myc oncogene and for p53 tumor suppressor gene. They found c-myc amplification and p53 deletion at the primary tumors as well as at surgical borderlines. The results of that study showed that c-myc amplification was related to the shortening of survival and it was also effective for the presence of metastasis. The authors concluded that the detection of genetic alterations at surgical border tissues might be important for the follow-up of patients for disease recurrence and also for disease genetical staging. 3.6. BCL-2 overexpression The bcl-2 proto-oncogene is located at chromosome 18q21 and it is overexpressed in lung cancers. The bcl-2 gene product is an anti-apoptotic protein and bcl-2 expression is negatively regulated by p53
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gene. Upregulated bcl-2 expression has been found in 75–95% of SCLCs but only in 10–35% of NSCLCs. The prognostic significance of bcl-2 expression in lung cancer has not been well validated. An inverse prognostic correlation of bcl-2 overexpression with mutant p53 has been reported in NSCLCs [1,18,57]. Recently in a study of advanced NSCLCs multivariate analysis showed that expression of p53, Rb and bcl-2 genes were not independent predictors of patients’ survival [58]. 3.7. Cyclin D1/cyclin dependent kinase 4 Overexpression of cyclin D1 is found in NSCLC cell lines and it is mainly caused by abnormal gene amplification. Also in primary NSCLC tumors an overexpression of cyclin D1 was reported. However, only in some of these tumors a cyclin D1 gene amplification has been found. Abnormal expression of cyclin-dependent kinase 4 (CDK4) has also been found in NSCLCs suggesting that it may play a role in lung tumorigenesis [13,59]. 3.8. Cyclin B1 An increased expression of cyclin B1 has been reported in the early stage of NSCLCs. Interestingly, patients whose tumors had expressed a high level of cyclin B1 had a significantly shorter survival time than patients with a low level of cyclin B1 expression. Overexpression of cyclin B1 has been more frequently observed in squamous cell carcinoma and it has been also suggested that it might be an adverse prognostic factor for patients with early-stage squamous cell carcinoma of the lung [13,60]. 3.9. P16INK4a inactivation The tumor suppressor gene p16INK4a is located at chromosomal region 9p21 and it is frequently altered in many types of cancer. This gene is an inhibitor of CDK which in turn inactivate the Rb gene by phosphorylation. Thus, there is a close interaction of p16INK4a with the function of Rb gene. Studies showed that P16INK4a gene is inactivated in up to 70% of NSCLC tumor specimens as well as cell lines but rarely in SCLCs. The inactivation of P16INK4a is mainly caused by homozygous deletions, mutations or
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promoter hypermethylation of the gene. The rate of mutation of P16INK4a is relatively low in primary NSCLC while homozygous deletions of the gene can be found in 10–40% of the tumors. Notably, homozygous deletions or mutations of P16INK4a have only been reported in tumors from smokers whereas in tumors from non-smokers the P16INK4a gene is inactivated through promoter hypermethylation [1,12,61–63]. It has also been shown that aberrant methylation of P16INK4a is an early event in lung cancer and a potential biomarker for early diagnosis. Interestingly, aberrant methylation of P16INK4a can be detected in DNA from sputum in 100% of patients with squamous cell lung carcinoma up to 3 years before clinical diagnosis. Therefore, this aberrant methylation may represent a valuable biomarker for early detection of lung cancer [9,17,18].
protein expression were examined by immunohistochemical analysis [68]. Loss of Rb function (Rb-) was observed in 16% of the cases, whereas expression of a putative mutant p53 protein (p53C) was found in 45% of the tumors. Patients with Rb-/p53C tumors survived for a significantly shorter period compared with those who had RbC/p53- tumors. On the other hand, in a study by Gregorc et al. [58] of advanced NSCLCs a high frequency of Rb loss was detected but it has not been shown to be an independent predictor of survival. Therefore, much more research is required before a clear statement on the prognostic value of Rb gene inactivation can be made. Moreover, the relation of Rb expression with other biological markers such as p16INK4a inactivation, p53 expression and cyclin D1 overexpression must be further investigated.
3.10. Retinoblastoma (Rb) gene inactivation
3.11. P53 inactivation
The Rb gene located at 13q14.1 is involved in the cell cycle control and it is frequently inactivated in lung cancer. Absent or mutant Rb protein has been reported in more than 90% of SCLCs and in 15–30% of NSCLCs [1]. In NSCLCs a higher frequency of Rb loss in patients with stages III and IV tumors was reported compared with those with stages I and II tumors [64]. The prognostic value of loss of Rb expression has not been well defined whereas its synergistic effect with other genetical markers appear to be of major importance [18,65–67]. Jin et al. [59] investigated the association of the immunohistochemical expression of cyclin D1, p16 and the Rb gene product with the prognosis in 106 patients with NSCLCs at stages I and II after a complete resection of the tumor. Cyclin D1-positive patients had significantly poorer survival prognosis than cyclin D1-negative patients, whereas p16-positive patients had significantly better prognosis than p16-negative patients. The survival rate of cyclin D1-positive/p16negative patients was significantly lower than that of cyclin D1-negative/p16-positive patients. The Rb product did not influence significantly the survival rate. These results indicated that cyclin D1 and p16, especially a combination of cyclin D1 and p16, are very useful prognostic markers for NSCLC patients independent of pathological stages I and II. In another study on early stages NSCLCs altered Rb and/or p53
The p53 gene located at 17p13.1 encodes a nuclear protein, which involves in the cell cycle control, DNA repair, cell differentiation and programmed cell death. P53 gene is mutated in 80–90% of SCLCs and in w50% of NSCLCs. Mutations of the p53 gene appear to play an important role in lung tumorigenesis, whereas most of them are missense mutations. P53 mutations also have been found in premalignant lung lesions, suggesting that p53 function is early inactivated in lung carcinogenesis [1]. Yakut et al. [56] found also p53 deletions at surgical border tissues in NSCLC patients. The role of p53 as a prognostic factor for survival in lung cancer patients is controversial. Mutation of the p53 tumor suppressor gene was considered as a possible marker of poor survival among patients with NSCLCs [69,70]. Steels et al. [71] in a systematic review of the literature with a meta-analysis showed that in each group of NSCLCs p53 abnormal status was associated with a poorer survival prognosis. However, data of another meta-analysis of 829 cases from eight published studies did not support a clear role for p53 mutations as a prognostic marker in NSCLCs [72]. Gregorc et al. [58] evaluated the impact of bcl-2, retinoblastoma (Rb) and p53 proteins on overall survival of 102 patients with advanced NSCLCs by using an immunostaining method. In that study, there was no evidence that p53, Rb and bcl-2
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proteins were correlated with patient outcome. Gessner et al. [16] investigated the DNA in exhaled breath condensate of patients with NSCLCs and healthy volunteers. Mutations of the p53 gene were investigated in all exhaled breath condensate samples of NSCLC patients while no mutation was found in volunteer persons. However, different point mutations in exhaled breath condensate samples and tumor tissues were revealed in all cases. The authors concluded that analysis of p53 mutations in exhaled breath condensate samples might be used as a marker of direct tobacco-related DNA damage. Finally, they suggested that in a further study high-risk individuals must be investigated in order to evaluate this diagnostic assay for early lung cancer detection. In fact, molecular genetic alterations in lung cancer, especially in NSCLCs, are diverse and complex. However, different NSCLC types seem to share some characteristic molecular genetic changes. Kettunen et al. [15] conducted a study using cDNA array technology to characterize the expression patterns of cancer-associated genes in squamous cell carcinomas compared with both normal lung tissue and adenocarcinomas of the lung. They revealed marked gene expression differences between squamous cell carcinomas and normal lung tissue while several genes were involved specifically in squamous cell carcinomas. On the other hand, many of the differentially expressed genes were shared with adenocarcinomas. In addition, that study showed that cDNA array technology is a suitable method for screening new candidate genes for further investigation to assess their potential relation with a particular tumor subtype. In conclusion, a variety of genetic alterations seems to play an important role in the pathogenesis of lung cancer. A good understanding of the underlying genetic changes will provide new perspectives for early diagnosis and screening of high-risk individuals. In addition, a number of genetical prognostic factors were identified as possibly helpful parameters in the evaluation of lung cancer patients. Further research is required in order to systematically investigate genetical alterations in lung cancer estimating also their usefulness in clinical practice. Some of these genetical alterations could definitely improve tumor classification and staging while new molecular targeted therapies could be developed.
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Mini-review
Cytogenetic and molecular aspects of lung cancer Anna D. Panani*, Charis Roussos Critical Care Department, Research Unit, Medical School of Athens University, ‘Evangelismos’ Hospital, Ipsilandou 45-47, Athens 10676, Greece Received 18 May 2005; received in revised form 21 June 2005; accepted 24 June 2005
Abstract Lung cancer is one of the most common cancers worldwide and its pathogenesis is closely associated with tobacco smoking. Continuous exposure of smoking carcinogens results in the accumulation of several alterations of tumorigenesis related genes leading to neoplastic bronchial lesions. Lung cancer is divided in two main histological groups, non-small cell lung carcinomas (NSCLCs) and small cell lung carcinomas (SCLCs). It seems that lung tumorigenesis is a multistep process in which a number of genetic events including alterations of oncogenes and tumor suppressor genes have been occurred. Cytogenetic abnormalities in lung cancer are very complex. However, a number of recurrent cytogenetic abnormalities have been identified. Many of these changes are common in both major histological groups of lung cancer while certain chromosomal abnormalities have been correlated with the stage or the grade of the tumors. In addition, several molecular alterations have been constantly found. Some of them are common in different histological subtypes of lung cancer and they appear to play an important role in the pathogenesis of lung cancer. A good understanding of the underlying genetic changes of lung tumorigenesis will provide new perspectives for early diagnosis and screening of high-risk individuals. In addition, a number of genetical prognostic factors have been identified as possibly helpful parameters in the evaluation of lung cancer patients. Further research is required in order to systematically investigate genetical alterations in lung cancer contributing to improvement of lung cancer classification and staging and to development of new molecular targeted therapies. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Lung cancer; Cytogenetics; Molecular changes; Clinical implications
1. Introduction Lung cancer is one of the most common cancers worldwide and its pathogenesis is closely associated with tobacco smoking. Activated carcinogens in tobacco smoke interact with certain genes like p53, * Corresponding author. Tel.: C30 210 7259307; fax: C30 210 7259307. E-mail address: [email protected] (A.D. Panani).
k-ras and FHIT. Thus, chronic exposure of smoking carcinogens results in the accumulation of genetic and epigenetic alterations of these tumorigenesis related genes leading to neoplastic bronchial lesions. However, only a low percentage of the long-term smokers develop lung cancer, since various DNA cellular repair mechanisms appear to prevent DNA damage. Besides cigarette smoking other environmental exposures as well as genetic factors contribute also to the development of lung cancer [1–6].
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.06.030
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Table 1 Frequent cytogenetic changes in lung cancer Chromosome NSCLCs SCLCs
Deletion
Gain
3p, 6q, 8p, 9p, 9q, 13q, 17p, 18q, 19p, 21q, 22q 3p, 4p, 4q, 5q, 8p, 10q, 13q, 17p
1p, 1q, 3q, 5p, 7p, 7q, 8q, 11q, 12q 3q, 5p, 8q, 19q, Xq
Lung cancer is divided in two main histological groups. Eighty percent of the lung cancer are nonsmall cell lung carcinomas (NSCLCs) and 20% are small cell lung carcinomas (SCLCs). In the group of NSCLCs are included adenocarcinomas, squamous cell, large cell and bronchoalveolar carcinomas. It is believed that lung cancer originates from bronchial epithelial cells and that carcinogenesis from a normal cell to an invasive carcinoma is a multistep process preceded by a premalignant lesion, such as hyperplasia, metaplasia or dysplasia of bronchial epithelium. However, only a small number of the premalignant lesions progresses to invasive cancer whereas the majority of them may remain unchanged for a long period of time or even regress. There are poor informative data about the sequence of genetic alterations leading to lung cancer. Several reports suggest that a number of genetic events including alterations of oncogenes and tumor suppressor genes must have occurred before lung cancer becomes clinically evident. Interestingly, studies have shown the occurrence in the premalignant stage of similar genetic changes to those observed in lung cancer [7–12]. Moreover, in the study by Cheng et al. [7] there was evidence showing similar genetic alterations in human bronchial epithelial cell lines immortalized by transfection with viral oncogenes to those observed in the premalignant stage of human lung. Therefore, it seems that many molecular genetic and epigenetic changes are necessary for conversion of the normal bronchial epithelial cells to malignant lung cancer cells. Many of these changes are common in both major histological groups of lung cancer, NSCLC and SCLC (Tables 1 and 2). Interestingly, several studies have investigated the use of certain genetic alterations as potential biomarkers in lung cancer early detection or risk assessment [13–19].
It is thought that a detailed understanding of the underlying genetic events of lung tumorigenesis will contribute to an early diagnosis, prediction of prognosis and to development of novel molecular targeted therapies. In this review, we summarize the most common cytogenetic and molecular genetic abnormalities seen in lung cancer emphasizing also their possible usefulness in the clinical practice.
2. Cytogenetic and molecular cytogenetic analysis In contrast to many hematological malignancies often characterized by simple and balanced chromosomal changes, epithelial tumors usually have complex unbalanced abnormalities. Primary lung tumors often have a low mitotic index making it difficult to obtain sufficient number of metaphases suitable for detailed cytogenetic analysis. Moreover, the chromosomal abnormalities can be extremely complex with many unidentified marker chromosomes complicating the identification of recurrent changes. Nevertheless, recurrent cytogenetic abnormalities have become apparent in lung cancer [20]. The development and application of molecular cytogenetic techniques have proven valuable in resolving some of the above problems of conventional Table 2 Frequent molecular genetic changes in lung cancer Genes
Abnormalities
NSCLCs
SCLSs
FHIT K-RAS ERBB1/EGFR ERBB2/HER2/ NEU MYC Family BCL-2 Cyclins P1bINK4a
Deletion/mutation Mutation Overexpression Overexpression
w40 w20 w60 w20
w80 Rare – –
Overexpression Overexpression Overexpression Inactivation (deletion/ mutation/ hypermethylation) Inactivation (deletion/ mutation) Inactivation (mutation/deletion)
5–10 10–35 ? w70
15–30 75–95 ? Rare
15–30
w90
w50
80–90
Rb P53
Frequency %
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cytogenetics leading to a more accurate cytogenetic analysis. The first recurrent abnormality which was described by Whang-Peng et al. in SCLC was a del(3p) [21]. Loss of 3p is a frequent finding in all histological subtypes of lung cancer. Furthermore, loss of heterozigosity studies have detected allelic imbalances in 3p in almost 100% of SCLCs and NSCLCs. The 3p region is believed to harbor one or more tumor suppressor genes of importance for lung cancer development [20,22–28]. Other recurrent karyotypic abnormalities reported in SCLC include del(5q), del(13q) and del(17p). Comparative genomic hybridization (CGH) studies of SCLCs have detected frequent losses of 3p, 4p, 4q, 5q, 8p,10q, 13q and 17p as well as gains of 3q, 5p, 8q, 19q and Xq. Genomic amplifications are also common in SCLCs recurrently seen as homogeneously staining regions and douple minutes chromosomes. These alterations are usually associated with amplification of the myc oncogene [20,29–34]. In NSCLCs, chromosomal abnormalities are more complex than those in SCLCs. Frequent losses of 3p, 6q, 8p, 9p, 9q, 13q, 17p, 18q, 19p, 21q and 22q have been reported while gains of 1p, 1q, 3q, 5p, 7p, 7q, 8q, 11q and 12q are also common [34–41]. Pei et al. [37] performed CGH analysis on 35 adenocarcinomas and 32 squamous cell carcinomas with the goal of identifying differences in the patterns of genomic imbalance between these histological subtypes. All 67 cases represented untreated patients from whom primary tumor tissues were collected at the time of diagnosis. Many imbalances, such as gains of 1q, 5p and 8q, have occurred at a high frequency in adenocarcinomas as well as in squamous cell carcinomas. However, several statistically significant differences have been found. The most prominent of them was gain of 3q24-qter which was seen in w80% of squamous cell carcinomas but in only 30% of adenocarcinomas. Another prominent difference was gain of 20p13 which was seen in w30% of squamous cell carcinomas versus 6% of adenocarcinomas. Furthermore, loss of 4q was seen at a significantly higher rate in squamous cell carcinomas than in adenocarcinomas while gain of 6p was more common in adenocarcinomas. Several studies have correlated certain chromosomal abnormalities with the stage or the grade of the tumor. Pei et al. [37] found that gains of 7q and 8q
3
were associated with higher-stage or higher-grade tumors suggesting that these changes might be indicative of tumor aggressiveness in NSCLCs. Goeze et al. [39] applied CGH technique to screen 83 lung adenocarcinomas of 60 patients for chromosomal imbalances. The results of that study indicated that adenocarcinomas were characterized by a recurrent pattern of chromosomal abnormalities with distinct genetic lesions being associated with the metastatic phenotype. The most common alteration was DNA overrepresentation on chromosome 1q followed by DNA overrepresentation on 8q and 20q and deletion on 3p, 4q, 6q, 9p, 9q, and 13q. Also in that study data suggested that deletion on chromosomes 3p12-p14, 3p22-p24, 4p13-15, 4q21-qter, 6q21-qter, 8p, 10q, 14q21, 17p12-p13, 20p12, and 21q and overrepresentation on chromosomes 1q21q25, 7q11, 9q34, 11q12-q13, 14q11-q13, and 17q25 were associated with the metastatic phenotype. In contrast, losses on chromosome 19 and gains on 3p, 4q, 5p, and 6q were preferentially found in nonmetastasizing tumors. Little is known about the mechanisms behind these chromosomal changes. It was suggested that telomere dysfunction and chromosomal breakage-fusionbridge events may be important mechanisms. The order of the karyotypic events in tumors progression and the possible presence of cytogenetic pathways in lung cancer remain obscured. Interestingly, in a recent study Hoglund et al. [42] identified all published cases of cytogenetically aberrant lung cancers in the Mitelman Database of Chromosome Aberrations in Cancer, altogether 432 cases. Tumors were classified with respect to the presence or absence of the most frequent imbalances and were statistically analyzed to assess the order of appearance of imbalances, possible karyotypic pathways and possible cytogenetic subtypes. They found that the imbalance profiles for adenocarcinomas, squamous cell carcinomas and large cell carcinomas were similar, except for the significantly lower frequency of C20 in large cell carcinomas. The frequent imbalance C7 and 3p- were seen at similar frequencies in all three subtypes. On the other hand the SCLC showed significantly lower frequencies of C7,C11, 1p-, 6q- and 14q- while it showed the highest frequencies of 3p- seen in 65% of the cases. In that study, the analyses suggested that lung cancer may develop through three pathways
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initiated by C7, 3p- and C12, respectively. The 3ppathway is dominated by losses and the C12 pathway by gains. Gain of chromosome 7 was shown to be important in the 3p- pathway while a group of tumors presented C7 and C20 with few additional changes. Also the authors showed that the karyotypic evolution might pass through three different phases. Phase I was characterized by tumors with few changes and by well-separated 3p- and C12 pathways. Phase II cases exhibited less distinct 3p- and C12 pathways while they had an increased number of imbalances. Phase III tumors were polyploid and highly complex. Notably, in that study among morphologic subtypes of lung cancer no marked differences between the karyotypic patterns were found suggesting that mode of karyotypic evolution might be independent of histological classification.
3. Molecular genetic analysis 3.1. Deletions of 3p Loss of heterozygosity at 3p is thought to be an early genetic change in the development of both SCLCs and NSCLCs. Several distinct regions of 3p loss have been identified, such as 3p25-26, 3p24, 3p21, 3p14, and 3p12, suggesting that several tumor suppressor genes may be located at 3p. Homozygous deletions within 3p21, 3p12 and 3p14 regions have been described in primary lung cancer and in lung cancer cell lines. The fragile histidine triad (FHIT) gene is located at 3p14.2 and it appears to play a role in the regulation of apoptosis and in cell-cycle control. There was evidence that FHIT functions as a tumor suppressor gene. FHIT is frequently deleted or mutated in lung cancer, whereas it represents the earliest deletion in bronchial preneoplastic lesions. Abnormal FHIT messenger RNA transcripts have been detected in about 80% of SCLCs and in 40% of NSCLCs. Loss of heterozygosity of the FHIT gene has been observed in a higher percentage of smokers than nonsmokers lung cancer patients, suggesting that FHIT is a target of tobacco smoke carcinogens [1,4,8, 19,23,25–28,43]. In addition, 3p21.3 region has been extensively examined for putative tumor suppressor genes, because as noted earlier homozygous deletions have been found in several lung cancer cell lines and
primary lung tumors. However, none of the putative tumor suppressor genes located at 3p21 found to be consistently mutated in lung cancer [1,23,24]. Dammann et al. [44] reported frequent epigenetic inactivation of a RAS association domain family protein from the lung tumor suppressor locus 3p21.3. There was evidence that the RAS effector homologue, RASSF1, functions potentially as a lung tumor suppressor gene. It was also found RASSF1 promoter hypermethylation in 100% of SCLC cell lines, in 63% of NSCLC cell lines and in 30% of primary NSCLC tumors [1,45]. 3.2. RAS activation Mutation of ras proto-oncogene can be frequently identified in several cancers including lung cancer. K-ras gene mutations were found in about 20% of NSCLCs but are rare in SCLCs. Ras mutations usually include point mutations at codons 12, 13, or 61 and they have been reported to be late events in lung cancer development. Most of the K-ras mutations are detected in lung adenocarcinomas, accounting for about 30% of these tumors and suggesting that K-ras mutations may be important in the development of lung adenocarcinomas. A correlation between cigarette smoking and ras mutations has been reported. Studies showed that the presence of a ras mutation results in shortened survival in NSCLC patients. However, other studies did not confirm this finding showing that ras mutations might not have an influence on survival [1,11,14,18,46–49]. Finally, Fischer et al. [18] suggested that further systematic investigations are needed for a final validation of the prognostic and predictive power of this biologic marker. 3.3. ERBB1/epidermal growth factor receptor (EGFR) The ERBB1 encodes for the EGFR. Overexpression of the epidermal growth factor receptor (EGFR) and transforming growth factor alpha (TGF-a) have been found in about 60% of NSCLCs at both mRNA expression and protein translation levels. Approximately 70% of tumors with squamous histology and 40% with adenocarcinomas overexpress epidermal growth factor receptor (EGFR). Thus, it seems that
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disruption of the EGF signaling pathway is important in NSCLCs. The prognostic value of EGFR for lung cancer patients remains controversial. A metaanalysis study showed that EGFR expression might not represent a significant prognostic factor in NSCLCs [13,18,50–52]. 3.4. ERBB2/HER2/NEU ERBB2 encodes for the receptor HER2/NEU, which belongs to the family of the epidermal growth factor receptors. A correlation between HER2 overexpression and poor prognosis of NSCLC patients has been reported [13,18,53,54]. 3.5. MYC overexpression The family of the myc oncogenes (c-myc, n-myc, l-myc) encodes phosphoproteins involved in the regulation of transcription of other genes responsible for cell proliferation. The usual mechanism of myc activation in lung cancer is gene amplification resulting in overexpression of the gene. Myc amplification occurs in 15–30% of SCLCs and in 5– 10% of NSCLCs. It has been shown that myc amplification affects adversely the survival in SCLC patients [1,31,55–56]. Yakut et al. [56] investigated genetic alterations on the primary tumor tissues and surgical borderline tissues in 51 patients with NSCLCs by using the FISH method with locus specific probes for c-myc oncogene and for p53 tumor suppressor gene. They found c-myc amplification and p53 deletion at the primary tumors as well as at surgical borderlines. The results of that study showed that c-myc amplification was related to the shortening of survival and it was also effective for the presence of metastasis. The authors concluded that the detection of genetic alterations at surgical border tissues might be important for the follow-up of patients for disease recurrence and also for disease genetical staging. 3.6. BCL-2 overexpression The bcl-2 proto-oncogene is located at chromosome 18q21 and it is overexpressed in lung cancers. The bcl-2 gene product is an anti-apoptotic protein and bcl-2 expression is negatively regulated by p53
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gene. Upregulated bcl-2 expression has been found in 75–95% of SCLCs but only in 10–35% of NSCLCs. The prognostic significance of bcl-2 expression in lung cancer has not been well validated. An inverse prognostic correlation of bcl-2 overexpression with mutant p53 has been reported in NSCLCs [1,18,57]. Recently in a study of advanced NSCLCs multivariate analysis showed that expression of p53, Rb and bcl-2 genes were not independent predictors of patients’ survival [58]. 3.7. Cyclin D1/cyclin dependent kinase 4 Overexpression of cyclin D1 is found in NSCLC cell lines and it is mainly caused by abnormal gene amplification. Also in primary NSCLC tumors an overexpression of cyclin D1 was reported. However, only in some of these tumors a cyclin D1 gene amplification has been found. Abnormal expression of cyclin-dependent kinase 4 (CDK4) has also been found in NSCLCs suggesting that it may play a role in lung tumorigenesis [13,59]. 3.8. Cyclin B1 An increased expression of cyclin B1 has been reported in the early stage of NSCLCs. Interestingly, patients whose tumors had expressed a high level of cyclin B1 had a significantly shorter survival time than patients with a low level of cyclin B1 expression. Overexpression of cyclin B1 has been more frequently observed in squamous cell carcinoma and it has been also suggested that it might be an adverse prognostic factor for patients with early-stage squamous cell carcinoma of the lung [13,60]. 3.9. P16INK4a inactivation The tumor suppressor gene p16INK4a is located at chromosomal region 9p21 and it is frequently altered in many types of cancer. This gene is an inhibitor of CDK which in turn inactivate the Rb gene by phosphorylation. Thus, there is a close interaction of p16INK4a with the function of Rb gene. Studies showed that P16INK4a gene is inactivated in up to 70% of NSCLC tumor specimens as well as cell lines but rarely in SCLCs. The inactivation of P16INK4a is mainly caused by homozygous deletions, mutations or
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promoter hypermethylation of the gene. The rate of mutation of P16INK4a is relatively low in primary NSCLC while homozygous deletions of the gene can be found in 10–40% of the tumors. Notably, homozygous deletions or mutations of P16INK4a have only been reported in tumors from smokers whereas in tumors from non-smokers the P16INK4a gene is inactivated through promoter hypermethylation [1,12,61–63]. It has also been shown that aberrant methylation of P16INK4a is an early event in lung cancer and a potential biomarker for early diagnosis. Interestingly, aberrant methylation of P16INK4a can be detected in DNA from sputum in 100% of patients with squamous cell lung carcinoma up to 3 years before clinical diagnosis. Therefore, this aberrant methylation may represent a valuable biomarker for early detection of lung cancer [9,17,18].
protein expression were examined by immunohistochemical analysis [68]. Loss of Rb function (Rb-) was observed in 16% of the cases, whereas expression of a putative mutant p53 protein (p53C) was found in 45% of the tumors. Patients with Rb-/p53C tumors survived for a significantly shorter period compared with those who had RbC/p53- tumors. On the other hand, in a study by Gregorc et al. [58] of advanced NSCLCs a high frequency of Rb loss was detected but it has not been shown to be an independent predictor of survival. Therefore, much more research is required before a clear statement on the prognostic value of Rb gene inactivation can be made. Moreover, the relation of Rb expression with other biological markers such as p16INK4a inactivation, p53 expression and cyclin D1 overexpression must be further investigated.
3.10. Retinoblastoma (Rb) gene inactivation
3.11. P53 inactivation
The Rb gene located at 13q14.1 is involved in the cell cycle control and it is frequently inactivated in lung cancer. Absent or mutant Rb protein has been reported in more than 90% of SCLCs and in 15–30% of NSCLCs [1]. In NSCLCs a higher frequency of Rb loss in patients with stages III and IV tumors was reported compared with those with stages I and II tumors [64]. The prognostic value of loss of Rb expression has not been well defined whereas its synergistic effect with other genetical markers appear to be of major importance [18,65–67]. Jin et al. [59] investigated the association of the immunohistochemical expression of cyclin D1, p16 and the Rb gene product with the prognosis in 106 patients with NSCLCs at stages I and II after a complete resection of the tumor. Cyclin D1-positive patients had significantly poorer survival prognosis than cyclin D1-negative patients, whereas p16-positive patients had significantly better prognosis than p16-negative patients. The survival rate of cyclin D1-positive/p16negative patients was significantly lower than that of cyclin D1-negative/p16-positive patients. The Rb product did not influence significantly the survival rate. These results indicated that cyclin D1 and p16, especially a combination of cyclin D1 and p16, are very useful prognostic markers for NSCLC patients independent of pathological stages I and II. In another study on early stages NSCLCs altered Rb and/or p53
The p53 gene located at 17p13.1 encodes a nuclear protein, which involves in the cell cycle control, DNA repair, cell differentiation and programmed cell death. P53 gene is mutated in 80–90% of SCLCs and in w50% of NSCLCs. Mutations of the p53 gene appear to play an important role in lung tumorigenesis, whereas most of them are missense mutations. P53 mutations also have been found in premalignant lung lesions, suggesting that p53 function is early inactivated in lung carcinogenesis [1]. Yakut et al. [56] found also p53 deletions at surgical border tissues in NSCLC patients. The role of p53 as a prognostic factor for survival in lung cancer patients is controversial. Mutation of the p53 tumor suppressor gene was considered as a possible marker of poor survival among patients with NSCLCs [69,70]. Steels et al. [71] in a systematic review of the literature with a meta-analysis showed that in each group of NSCLCs p53 abnormal status was associated with a poorer survival prognosis. However, data of another meta-analysis of 829 cases from eight published studies did not support a clear role for p53 mutations as a prognostic marker in NSCLCs [72]. Gregorc et al. [58] evaluated the impact of bcl-2, retinoblastoma (Rb) and p53 proteins on overall survival of 102 patients with advanced NSCLCs by using an immunostaining method. In that study, there was no evidence that p53, Rb and bcl-2
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proteins were correlated with patient outcome. Gessner et al. [16] investigated the DNA in exhaled breath condensate of patients with NSCLCs and healthy volunteers. Mutations of the p53 gene were investigated in all exhaled breath condensate samples of NSCLC patients while no mutation was found in volunteer persons. However, different point mutations in exhaled breath condensate samples and tumor tissues were revealed in all cases. The authors concluded that analysis of p53 mutations in exhaled breath condensate samples might be used as a marker of direct tobacco-related DNA damage. Finally, they suggested that in a further study high-risk individuals must be investigated in order to evaluate this diagnostic assay for early lung cancer detection. In fact, molecular genetic alterations in lung cancer, especially in NSCLCs, are diverse and complex. However, different NSCLC types seem to share some characteristic molecular genetic changes. Kettunen et al. [15] conducted a study using cDNA array technology to characterize the expression patterns of cancer-associated genes in squamous cell carcinomas compared with both normal lung tissue and adenocarcinomas of the lung. They revealed marked gene expression differences between squamous cell carcinomas and normal lung tissue while several genes were involved specifically in squamous cell carcinomas. On the other hand, many of the differentially expressed genes were shared with adenocarcinomas. In addition, that study showed that cDNA array technology is a suitable method for screening new candidate genes for further investigation to assess their potential relation with a particular tumor subtype. In conclusion, a variety of genetic alterations seems to play an important role in the pathogenesis of lung cancer. A good understanding of the underlying genetic changes will provide new perspectives for early diagnosis and screening of high-risk individuals. In addition, a number of genetical prognostic factors were identified as possibly helpful parameters in the evaluation of lung cancer patients. Further research is required in order to systematically investigate genetical alterations in lung cancer estimating also their usefulness in clinical practice. Some of these genetical alterations could definitely improve tumor classification and staging while new molecular targeted therapies could be developed.
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