I. Introduction

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The aim of this review is to examine the current understanding of the influence that the genetic profile of non-small cell lung cancer, the most frequent cause of cancer death in humans in the Western world, may have on the effect of chemotherapeutic agents. The application of the principles of pharmacogenetics by the use of novel techniques may lead to increasing predictability of drug response of the disease, with the aim of targeted therapeutic intervention.

	II. Clinical Relevance and Management of Non-Small Cell Lung Cancer

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Lung cancer is a leading cause of mortality among men and women in the Western world, with 170,000 deaths per year. This exceeds the sum of the next three leading causes of death due to breast, colon, and prostate cancer. There are over one million deaths worldwide due to malignant tumors of the lung, making it an epidemic disease (Jemal et al., 2002). Lung cancer is a deadly illness because of the low proportion of subjects (~15%) that are still alive 5 years after the initial diagnosis. Patients with stage I disease (T1-2, N0, M0) may be cured by optimal treatment, and 70% of them may achieve a 5-year survival; unfortunately, most subjects present with advanced disease, and this condition adversely affects survival (Cortes-Funes, 2002; Ferreira et al., 2002).

From a histological point of view, lung cancer is classified into non-small cell (NSCLC¹) and small cell lung cancer (SCLC); 80% are NSCLCs, including adenocarcinomas, squamous cell (epidermoid), and large cell carcinomas, and 20% are SCLCs (Rom et al., 2000).

Surgery is the recommended treatment; chemotherapy and radiotherapy have a role either as a part of a treatment strategy to cure locally advanced disease or as a palliative therapy for metastatic tumors. In patients with stages I and II NSCLC (T1-2, N1, M0) who cannot be treated by surgery because of nontumor-related comorbidity, radiotherapy is the therapeutic approach of choice. With standard radiotherapy, the survival at 3 and 5 years of patients with stage I disease is about 30 to 40% and 10 to 30%, respectively (Bonnet et al., 2001; Jeremic et al., 2002), while the 2-year survival is 20% for stage II disease. The prognosis of stage IIIA (T3, N0-1, M0 or T1-3, N2, M0) and IIIB (any T4 or any N3, M0) NSCLC is dismal, although still curable in some cases. Survival at 5 years is about 5% in patients with N2 disease (involvement of ipsilateral or subcarinal mediastinal lymph nodes or ipsilateral supraclavicular lymph nodes), and 50% in patients with T3 disease (tumor involving the pleura, chest wall, diaphragm, or pericardium) without lymph node involvement. Patients with N3 (contralateral mediastinal hilar or supraclavicular lymph node involvement) or T4 tumors (invasion of mediastinal organs, malignant pleural effusion) are treated with palliative intent (Malayeri et al., 2001). Postoperative mediastinal radiation therapy has been shown to significantly reduce the risk of local relapse with no or little impact on survival (PORT Meta-analysis Trialists Group, 1998), while adjuvant chemotherapy is ineffective (Souquet and Geriniere, 2001). Preoperative chemotherapy followed by surgery and postoperative radiation therapy, in the case of incomplete resection, seems to be able to prolong disease-free and overall survivals in comparison with surgery alone (Rosell et al., 1994; Roth et al., 1994; Gandara et al., 2001; Rinaldi and Crinò, 2001). Combined chemo-radiotherapy has a strong rationale due to its potential synergistic effects, although the results for locally advanced inoperable disease are still controversial and its feasibility before surgery has been proven only in phase II trials, many of them including stage IIIA-B patients (Lau et al., 2001).

The median survival of patients with metastatic NSCLC treated with chemotherapy is in the range of 8 to 10 months. Standard chemotherapy consists of combination regimens containing cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, vinorelbine, ifosfamide, and etoposide. However, recent randomized studies on more than 2200 patients failed to show major differences in response rates and survival among the combination of cisplatin + gemcitabine, cisplatin + vinorelbine, and cisplatin or carboplatin + paclitaxel or docetaxel (Table 1). According to the results reported, a response rate exceeding 40% cannot be expected, irrespective of what drug combination is administered; in addition to this, the survival rate of patients older than 70 years of age who are treated with chemotherapy not containing cisplatin is similar to that of younger patients (Alberola et al., 2001; Gridelli et al., 2001, 2002; Rodriguez et al., 2001; Scagliotti et al., 2001; Van Meerbeeck et al., 2001) (Table 1).

Chemotherapy has a role in the treatment of locally advanced and metastatic NSCLC, either as a part of a curative strategy or with palliative intent, although clinical response to chemotherapy is still unsatisfactory, particularly with respect to the complete response rate, which is still low. Therefore, a better selection of patients and the identification of predictive factors of sensitivity to chemotherapeutic agents are warranted.

Concerning locally advanced disease (stage IIIA-B), resectability criteria are not uniformly accepted; thus, the distinction between patients with resectable and unresectable tumor may be difficult. Preoperative induction chemotherapy provides a response rate of approximately 60% and a downstaging to resectable disease in 44 to 65% of subjects (Felip and Rosell, 2002). Surgically treated patients achieve a median survival of approximately 20 months and a long-term survival of 20 to 25%. Pathologically complete remissions are low and about 10% of patients will progress under induction chemotherapy (Rosell et al., 1994; Roth et al., 1994; Kumar et al., 1996; Rinaldi and Crinò, 2001). Induction chemotherapy followed by full-dose radiotherapy is suitable for patients with good performance status (Pottgen et al., 2002), and preoperative radio-chemotherapy is suitable for selected patients because of up to 10% morbidity and mortality (Thomas et al., 1999). In patients with incomplete resection, postoperative radiotherapy may be administered (Grossi et al., 2001; Pitz et al., 2002).

Radiotherapy is considered the standard treatment for unresectable tumors; a total dose of at least 60 Gy results in a survival of about 30% and 7% at 1 and 3 years, respectively (Cox et al., 1991; Baumann et al., 2001). A meta-analysis study showed that the addition of chemotherapy to radiotherapy has a marginal impact on survival, with a hazard ratio of 0.94 and a 2% absolute benefit at 2 and 5 years (Non-Small Cell Lung Cancer Collaborative Group, 1995). An RTOG trial provided evidence in support of simultaneous chemotherapy and radiotherapy compared to sequential treatment (median survival 17 versus 14.6 months), although the overall results cannot be considered satisfactory (Werner-Wasik et al., 2000). Finally, patient with stage IIIB disease may be administered chemotherapy for palliative intent.

The vast majority of patients with metastatic NSCLC (stage IV) die from disseminated cancer within two years of follow-up. Since the survival benefit is small and the prognosis is poor, the role of chemotherapy is doubtful and it is not recommended as a standard treatment in subjects in poor general condition (Non-Small Cell Lung Cancer Collaborative Group, 1995; Socinski et al., 2002). Standard chemotherapy mainly consists of combination regimens containing cisplatin; a 27% reduction in the risk of death has been reported, equivalent to an absolute improvement in survival of 10% or an increased median survival of 1.5 months, and a lower incidence of disease-related complications has been observed (Non-Small Cell Lung Cancer Collaborative Group, 1995; Manegold, 2001). Isolated symptomatic lesions, including bone metastases and spinal cord compression, are treated with radiotherapy. Second-line chemotherapy may be effective in some patients; docetaxel produces good results in cisplatin-treated patients (Fossella et al., 1995; Kim et al., 2002), while cisplatin-based chemotherapy after paclitaxel and gemcitabine has been reported to be effective only in 20% of those patients responsive to the first-line treatment and in none with refractory disease (De Pas et al., 2001).

The assessment of the prognosis of patients with lung cancer is essential for the choice of the best therapeutic option. The major clinical prognostic determinant in NSCLC is tumor extension; patients with advanced, unresectable NSCLC have a poor prognosis, with very few 5-year survivors and a median survival of less than 1 year. However, a large variability of clinical outcome characterizes this subset of patients, some of them surviving only a few weeks and some others several years. Many prognostic factors have been recognized and are currently being evaluated to support therapeutic decisions. In patients with surgically resected stage I NSCLC, the prognostic significance of a panel of tumor markers, including ErbB-1/epidermal growth factor receptor (EGFR), HER-2/neu (ErbB-2), Bcl-2, p53, and angiogenesis was evaluated. Statistical analysis demonstrated that tumor extension represented the most powerful prognostic factor for survival and time to recurrence, while increased EGFR expression was significantly associated with a poorer survival (P = 0.02); none of the other immunocytochemical markers was an independent predictive factor for survival (Pastorino et al., 1997). Furthermore, the immunohistochemical analysis of protein expression profiles of 216 patients with NSCLC demonstrated that the expression of nuclear oncoproteins fos and jun and of cyclin A were decreased in carcinomas of patients with long-term survival (Volm et al., 2002).

The subgroup of patients with metastatic NSCLC is heterogeneous, and the differentiation between patients with single or multiple metastases has prognostic relevance. Patients with a single metastasis, particularly in the absence of mediastinal lymph node involvement, have a better prognosis than patients with multiple distant sites of disease. When a single brain metastasis is the only site of first recurrence in patients free of extracranial disease, a surgical approach with brain tumor resection or stereotactic radiosurgery improves the quality of life and offers a chance of long-term survival, with a median survival of up to 27 months (Arbit et al., 1995; Granone et al., 2001). Together with pretreatment stage, performance status and weight loss are important prognostic factors in advanced NSCLC (Paesmans et al., 1995; Buccheri and Ferrigno, 2001). This reflects the tumor biological profile, which in turn translates into the aggressiveness of the disease. Moreover, patients with poor performance status and severe weight loss are unsuitable candidates for antitumor treatment and are more susceptible to severe medical complications. Other factors have a prognostic role in patients with NSCLC, although not always confirmed in retrospective analyses: among the others, it seems to be relevant to the male gender, the presence of clinical symptoms (i.e., cough and hemoptysis), and elevated neutrophil count (Paesmans et al., 1995; Martins and Pereira, 1999). Stages I-IIIA NSCLC are potentially resectable; however, patients belonging to these groups are highly heterogeneous with respect to their prognosis, since the 5-year survival rate is about 80% for stage I and 20 to 30% for stage IIIA. For stage I, important independent prognostic factors are the volume of primary tumor and pretreatment serum lactate dehydrogenase (Feld et al., 1997). Mediastinal lymph node involvement is an important adverse prognostic factor, and the strongest predictor of long-term survival after surgery is the absence of mediastinal neoplastic spread. Patients with metastatic ipsilateral or subcarinal mediastinal lymph nodes or ipsilateral supraclavicular lymph nodes (N2) are nonetheless a heterogeneous subgroup. Moreover, N2 lymph node involvement has a different prognostic value if clinically or pathologically detected. Patients with preoperative evidence of N2 disease have a worse prognosis than patients with clinically undetectable involvement (Andre et al., 2000). Moreover, the number of metastatic mediastinal lymph nodes proved to be an independent prognostic factor and was related to a significant difference in overall survival of surgically treated stage IIIA NSCLC (Andre et al., 2000). Patients with single lymph node metastasis showed a longer median survival than patients with multiple lymph node involvement.

The spreading of tumor cells in the bone marrow of patients with clinically localized NSCLC may be detected by immunohistochemical analysis, and it is associated with a poor prognosis (Pantel et al., 1996; Osaki et al., 2002). Finally, histopathology has no prognostic value and the importance of tumor cell differentiation is controversial, while a poor prognosis is correlated with lymphatic and vascular invasion and the expression of mucin and a high mitotic index of cancer cells (Komaki et al., 1998).

	III. Genetic Instability and Gene Dysfunction in Non-Small Cell Lung Cancer

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Tumor evolution is a multistep process characterized by the loss of function of cellular mechanisms that control normal proliferation and differentiation. It is estimated that 10 to 20 genetic events, including the alteration of oncogenes and tumor-suppressor genes, will have occurred by the time a lung tumor becomes clinically evident (Tran et al., 1998). Genetic instability is the hallmark of cancer as a disease. It may be indicated by a variety of cellular features at the chromosomal and DNA levels. Evidence of DNA instability is represented by the incidence of point mutations, deletions/insertions, recombination, gene amplification, and microsatellite instability, while at the chromosomal level it consists of aneuploidy, translocations, deletions, sister chromatid recombinations, fragile sites, homogeneously stained regions, and double minute chromosomes (Sherbet and Lakshmi, 1997).

Oncogene amplification is frequently detected in human cancer and it is characteristic of solid tumors, including NSCLC. DNA amplification does not occur in normal cells and it is maintained in cancer cells as a result of selection. DNA amplification is observed with cytogenetic methods as double minute chromosomes (DMs) or homogeneously staining regions (HSRs), but more recent technologies, including fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH), have substantially increased the ability to detect such alterations (Imreh et al., 1997; Grompe et al., 1998). DMs are episomal forms of amplified DNA that generally lack centromeres and are unequally distributed between daughter cells at mitosis. In contrast, HSRs are chromosomally integrated forms of amplified DNA. They represent either the replacement of the normal chromosome banding pattern with an extended region of homogeneous staining or the insertion of such a region into an otherwise normally banded chromosome (Grompe et al., 1998). DMs and HSRs tend to be mutually exclusive and are potentially interchangeable manifestations of amplified DNA; therefore, DMs can potentially integrate into distant chromosomal sites to generate heritable HSRs. DMs, and less frequently HSRs or a combination of both, are found in approximately 17% of NSCLC (Imreh et al., 1997).

The vast majority of oncogene amplifications found in human cancers affect the myc family; among them, c-myc is amplified in >50% of NSCLC and it correlates with the extent of lymph node metastasis (Kubokura et al., 2001; Salgia and Skarin, 1998). Less frequent gene amplification involves the cdc25B gene (40%) (Wu et al., 1998), cyclin D1 (5%), and the EGFR gene (5.9%) (Reissmann et al., 1999), and HER-2/neu (<2%) (Cox et al., 2001).

Mutations are DNA sequence alterations that may result in the disruption or abnormal activity of a gene or the encoded protein. These include gene rearrangements, deletions, insertions, and single-base changes. Nonsense mutations result in the appearance of a stop codon and premature termination during protein synthesis, missense mutations are single-base substitutions causing incorporation of an inappropriate amino acid into a protein, and frameshift mutations shift the reading frame of triplet codons in a gene during mRNA translation (Grompe et al., 1998). Mutations that cause the synthesis of structurally aberrant proteins usually occur within the coding region of the gene, while those that result in the production of abnormal amounts of the protein may affect the 1) transcriptional machinery, 2) regulatory regions (i.e., gene promoter), 3) RNA processing (i.e., splicing alterations in the 5' untranslated region or 3' adenylation signals), or 4) translational machinery that controls initiation, elongation, and termination of polypeptide chains (Grompe et al., 1998). At variance with a mutation, a DNA polymorphism is a sequence alteration stably expressed and found at a frequency of >1% in a given population. The simplest type is the single nucleotide polymorphism (SNP), a single base difference between genome sequences that occurs approximately every 1 kb in the human genome (Grompe et al., 1998; Danesi et al., 2001). Additional types of polymorphism are represented by the variable number of tandem repeats (VNTR, minisatellites), multiple copies of short repeats of DNA sequences (0.1-10 kb) distributed along the human genome, and the microsatellite repeats, a simpler but more common variant of minisatellites, in which up to tetranucleotide repeats are reiterated in multiple copies (Grompe et al., 1998).

Although point mutations are more commonly associated with loss of function (i.e., the TP53 gene), there are notable examples of activating point mutations in a cellular proto-oncogene; indeed, in approximately 30% of human NSCLCs, the K-ras oncogene is mutated (Noda et al., 2001). Mutations affecting the tumor-suppressor gene TP53 may be associated with deregulation in telomerase activity, which in turn may be important in the process of lung tumorigenesis and low-grade differentiation in NSCLC (Maniwa et al., 2001). The simultaneous occurrence of TP53 gene mutation and high telomerase activity may be relevant to the grade of malignancy in lung tumors (Maniwa et al., 2001). In NSCLC, inactivation by point mutation of the CDKN2A (cyclin-dependent kinase inhibitor 2A) gene, which encodes p16^INK4a (p16 inhibitor of kinase 4a), is observed in smokers, whereas CDKN2A is inactivated in nonsmokers through promoter hypermethylation (Sanchez-Cespedes et al., 2001). Additional mutations observed in NSCLC involve the tumor necrosis factor-related apoptosis-inducing ligand-receptor 2 (TRAIL-R2) gene (10.6% mutations), mapped to chromosome 8p21-22 and encoding a cell-surface receptor involved in cell death signaling (Lee et al., 1999a), and the lipoprotein receptor-related protein-deleted in tumors (LRP-DIT), a tumor-suppressor gene that is inactivated by homozygous deletion or mutation in at least 40% of NSCLC cell lines and thus may play an important role in lung tumorigenesis (Liu et al., 2000).

Neoplastic cells simultaneously harbor diffuse genomic hypomethylation, more regional areas of hypermethylation, and increased DNA-methyltransferase (DNA-MTase) activity. Each component of this methylation imbalance may contribute to tumor progression. Main targets of the regional hypermethylation are the normally unmethylated CpG (cytidine phosphate guanosine) islands located in gene promoter regions. In particular, methylation of normally unmethylated sites in the promoter regions of tumor-suppressor and DNA-repair genes is correlated with loss of expression of these genes in cancer cell lines and primary tumors (Baylin and Herman, 2000). Methylation of the CpG islands in the O⁶-methylguanine-DNA methyltransferase (MGMT) prevents gene transcription, and cells cannot repair the alkylation of O⁶-methylguanine (Qian and Brent, 1997; Watts et al., 1997; Esteller et al., 1999a; Danam et al., 1999). Furthermore, in vitro treatment with demethylating drugs restores the expression of MGMT (Qian and Brent, 1997; Esteller et al., 2000).

DNA hypermethylation is associated with transcriptional repression and represents an alternative to coding region mutations for inactivation of tumor-suppressor genes, including CDKN2A (p16^INK4a). The hypermethylation of a promoter is an epigenetic phenomenon that leads to its inactivation or down-regulation of gene transcription (Baylin et al., 1998). CpG sequences are located in the promoter regions of about 50% of all human genes; in normal cells, unmethylated CpG islands are protected from methylation on flanking regions, while in neoplastic cells this protection is lost (Baylin et al., 1998; Wistuba et al., 2001). Promoter hypermethylation has relevance in the development of cancer, as it occurs at the level of tumor-suppressor genes (Costello et al., 2000; Wistuba et al., 2001). Promoter hypermethylation is frequently detected in NSCLC, and the number of CpG sequences that are methylated may be very large (up to 4500) (Costello et al., 2000). The hypermethylation of promoter regions of CDKN2A (p16^INK4a), death-associated protein kinase (DAPK), GSTP1 (glutathione S-transferase P1 isoform), and MGMT has been detected in 68% of NSCLC, but not in surrounding normal tissues. Moreover, 73% of patients with abnormal methylation patterns at the level of promoters also showed circulating DNA with abnormal patterns of methylation, while patients without methylation abnormalities did not show the same alterations in circulating DNA, thus suggesting that this finding is specific and may allow genetic testing that may be useful for treatment selection and diagnosis of disease recurrence (Esteller et al., 1999b). DAPK promoter hypermethylation is observed in 44% of patients with stage I NSCLC; this genetic abnormality predicts an adverse prognosis, as the 5-year survival after surgical resection is significantly poorer with respect to those patients without DAPK promoter hypermethylation (Tang et al., 2000). Aberrant methylation of the CDKN2A (p16^INK4a) tumor-suppressor gene has been detected in the early stage of NSCLC. CDKN2A (p16^INK4a) has a relevant role in NSCLC carcinogenesis, and its silencing has been detected at high frequency in invasive and in situ tumors. In particular, CDKN2A (p16^INK4a) methylation is detected in 17% of basal cell hyperplasia, in 24% of squamous metaplasia, and in 50% of in situ carcinomas; this proportion further increases (75%) in in situ carcinoma adjacent to invasive squamous cell cancer (Belinsky et al., 1998). The RAS effector homolog (RASSF1) gene has a putative role as a tumor-suppressor gene in lung cancer; RASSF1 shows promoter methylation at the CpG islands in 40% of NSCLC, and loss of gene expression (Burbee et al., 2001). Finally, promoter methylation also affects the retinoic acid receptor system, which plays an important role in cell differentiation and lung development. Retinoids can suppress carcinogenesis in preneoplastic bronchial lesions and their effects are mediated by nuclear receptors, i.e., the retinoic acid receptors (RAR, RAR, and RAR) and the retinoid X receptors (RXR, RXR, and RXR). Several reports indicate that loss of RAR expression, because of promoter hypermethylation, is associated with increased susceptibility to lung cancer. The RAR gene promoter is hypermethylated in 41% of NSCLC and is almost always unmethylated in control normal samples (Virmani et al., 2001b). Loss of promoter methylation in cell lines by in vitro treatment with 5-aza-2'-deoxycytidine restored RAR gene expression and cell growth (Virmani et al., 2001b).

Another mechanism of gene silencing is represented by histone deacetylation. At variance with hypermethylation of gene promoter regions, histone deacetylation modulates higher-order chromatin structure. The addition of an acetyl group on lysines is catalyzed by histone acetyltransferase, while histone deacetylases remove the acetyl groups. Steady-state histone acetylation is controlled by the balance of both enzymatic activities; hypoacetylated histones increase their positive charge, condense the chromatin, and prevent gene transcription. Conversely, hyperacetylated histones neutralize the electrostatic charge and de-condense chromatin, thus allowing gene transcription to proceed. Transcription repressors, such as pRB, are associated with histone deacetylases. Recent studies have revealed several enzyme isoforms in mammalian cells encoded by histone deacetylase (HDAC) genes: HDAC1, HDAC2, HDAC3, h-HDAC4, h-HDAC5, h-HDAC6, h-HDAC7, and HDAC8 (Hu et al., 2000). There is potential synergy between inhibition of DNA methylation and histone deacetylase activity in restoring silenced gene expression. Indeed, depsipeptide, an inhibitor of histone deacetylase, acts synergistically with 5-aza-2'-deoxycytidine, a hypomethylating agent inhibitor of DNA-MTase, in restoring the expression of CDKN2A (p16^INK4a) (Zhu et al., 2001a) and inducing apoptosis in lung cancer cells (Zhu et al., 2001b). Apoptosis represents a naturally occurring mechanism of cell number regulation by deletion rather than by inhibition of cell division; abnormally triggered apoptosis also occurs after treatment with drugs that induce irreversible cell damage, such as cytotoxic agents, and also after withdrawal of hormones or growth factors or treatment with selected cytokines (Sloviter, 2002). Transforming growth factor (TGF)- strongly inhibits epithelial cell proliferation through interaction with the TGF- type II receptor (TGF-RII). Most NSCLC cell lines have lost the growth-inhibitory response to TGF- because of the loss of TGF-RII expression, which is dependent, at least in part, on histone deacetylation (Osada et al., 2001). Finally, in vitro treatment of cells with the demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A induces the cells to express hTERT (human telomerase reverse transcriptase), suggesting a potential role for DNA methylation and/or histone deacetylation in negative regulation of hTERT (Devereux et al., 1999). Agents that inhibit histone deacetylase in vitro include hybrid polar compounds (Richon et al., 1998), phenylacetate and phenylbutyrate (Samid et al., 1997), and MS-27-275 (Saito et al., 1999). These agents induce terminal differentiation in vitro as well as cell cycle arrest and partial reversion of the malignant phenotype in a variety of neoplasms, including NSCLC. For these reasons, inhibitors of histone deacetylase have been developed and clinically tested. The administration of the investigational agent CI-994 induced a partial response lasting over 2 years in one patient with heavily pretreated adenocarcinoma of the lung and a stable disease in an additional subject. Thrombocytopenia was the dose-limiting toxicity at the maximum tolerated dose of 8 mg/m²/day for 8 weeks. Other toxicities included fatigue and gastrointestinal effects such as nausea, vomiting, diarrhea, constipation, and mucositis (Prakash et al., 2001).

Deletions of specific genes may occur during the development of tumors. This mechanisms of tumorigenesis, called loss of heterozygosity (LOH) or allelic imbalance, consists of the loss of an allele at a specific locus, and it is of obvious importance if this deletion involves a tumor-suppressor gene. Since the deletion involving only one allele may be silent, a second somatic mutation may consist of the loss of the entire chromosome, carrying the residual normal allele, or a large portion of it, or in the elimination of the normal gene by recombination events that duplicate the mutant allele (Black, 1997). In situations in which these events can be traced at the DNA level, for example by monitoring restriction fragment length or a cytidine-adenine repeat-type of polymorphism, the outcome is that the tumor appears to be homozygous or hemizygous for markers in or close to the relevant tumor-suppressor gene. The LOH is part of the two-hit model of carcinogenesis (Knudson, 1971; Black, 1997). Cells containing a pair of chromosomes in which a marker gene (e.g., retinoblastoma [RB]) is either homozygous wild-type or heterozygous have the same normal phenotype, demonstrating the recessive nature of the mutant allele. Tumorigenesis will only ensue if both copies of the gene are mutated (nonfunctioning) or deleted. In an individual who inherits a mutant copy from one parent, only a single somatic mutation is needed to lead to tumorigenesis. In subjects carrying two wild-type alleles, both copies must sustain independent somatic mutations (Black, 1997). The investigation of clinical implications of allelic deletions at three common sites of LOH in regions 5q21, 11p15.5, and 11p13 in 86 patients with NSCLC demonstrated that LOH frequency at 5q21 was 20%, whereas LOH frequencies in 11p15.5 and 11p13 were 31% and 19%, respectively (Sanchez-Cespedes et al., 1997). There was a significant correlation between 5q21 LOH and mediastinal lymph node involvement (P = 0.03); however, no significant differences were observed in median survival times in patients with 5q21 LOH as compared to the remainder (26 versus 37 months, P = 0.33) or in patients with 11p LOH (38 versus 32 months, P = 0.72) (Sanchez-Cespedes et al., 1997).

LOH for a locus on human chromosome 11q22-23 containing a putative tumor-suppressor gene is observed at high frequency in patients with NSCLC (Pletcher et al., 2001). LOH at the adenomatous polyposis coli/mutated in colonic cancer (APC/MCC) locus, a tumor-suppressor gene associated with both familial and sporadic cancer, was observed in 83% of NSCLC cell lines (Virmani et al., 2001a). It has been previously reported that the incidence of LOH on chromosomes 2q, 9p, 18q, and 22q in advanced-stage NSCLC was significantly higher than that in early stages (Shiseki et al., 1994, 1996). These results indicate that tumor-suppressor genes on chromosomes 2q, 9p, 18q, and 22q play an important role in the acquisition of malignant phenotype in NSCLC. However, the clinical implications and prognostic impact of 2q, 9p, 18q, and 22q LOH have not been established.

Transfer of chromosome 11 into the human A549 NSCLC cell line suppresses tumorigenesis, indicating that LOH may be responsible, at least in part, for the malignant phenotype and suggesting that multiple tumor-suppressor genes are located in this chromosome. A region of 700 kb on 11q23.2 of A549 cells also contains a single gene, TSLC1 (tumor-suppressor lung cancer 1), whose expression is reduced or absent in A549 and several other NSCLC cell lines (Kuramochi et al., 2001). Hypermethylation of the TSLC1 promoter would represent the second hit in NSCLC with LOH (Kuramochi et al., 2001). A highly significant association between TP53 mutations and deletions on 3p, 5q, 9p, 11p, and 17p is found in lung cancer (Zienolddiny et al., 2001). Furthermore, 86% of the tumors with concordant deletions in the four most involved loci (3p21, 5q11-13, 9p21, and 17p13) had TP53 mutations as compared to only 8% of the tumors without deletions at the corresponding loci (Zienolddiny et al., 2001). The frequency of deletions was significantly higher among smokers as compared to nonsmokers. This difference was significant for the 3p21.3 (human MutL homolog-1 [hMLH1] locus), 3p14.2 (fragile histidine triad [FHIT] locus), 5q11-13 (human MutS homolog-3 [hMSH3] locus), and 9p21 (D9S157 locus). Deletions were more common in squamous cell carcinomas than in adenocarcinomas. Covariate analysis revealed that histological type and TP53 mutations were significant and independent parameters for predicting LOH status at several loci (Zienolddiny et al., 2001). In a study designed to identify the major tumor-suppressor gene loci involved in the pathogenesis of lung cancer, 22 different regions with more than 60% LOH were identified: 1) 13 regions with a preference for SCLC, 2) 7 regions with a preference for NSCLC, 3) 2 regions affecting both SCLC and NSCLC (Girard et al., 2000). The chromosomal arms with the most frequent LOH were 1p, 3p, 4p, 4q, 5q, 8p, 9p (p16), 9q, 10p, 10q, 13q (RB), 15q, 17p (TP53), 18q, 19p, Xp, and Xq (Girard et al., 2000). In addition, new homozygous deletions were found at 2p23, 8q24, 18q11, and Xq22. On average, 36% of markers showed allele loss in individual NSCLC tumors, with an average size of subchromosomal region of loss of five to six markers. SCLC and NSCLC had different regions of frequent LOH (hot spots), and NSCLC had more of these regions (n = 22) than SCLC (n = 17) (Girard et al., 2000). Finally, in lung cancer cell lines, at least 17 to 22 chromosomal regions with frequent allele loss are involved, suggesting that the same number of putative tumor-suppressor genes is inactivated. In addition to this, SCLC and NSCLC frequently undergo different specific genetic alterations, and clusters of tumor-suppressor genes are likely to be inactivated together (Girard et al., 2000). Lung metaplastic and alveolar hyperplastic lesions with atypia show genetic alterations, including LOH of 3p, 9p, and mutations of the TP53 gene. The analysis of microsatellite markers showed that 5 of 35 cases of squamous cell carcinoma and 3 of 26 cases of adenocarcinoma showed LOH in both preneoplastic lesions and synchronous cancers (Kohno et al., 1999). Nine patients (25.7%) with squamous cell carcinoma and 6 patients (23.1%) with adenocarcinoma had mutations involving TP53; in 2 patients with squamous cell carcinoma, the same mutation was observed in both dysplasia and squamous cell carcinoma (Kohno et al., 1999). These findings suggest that several genetic alterations may occur in preneoplastic lesions or in the early stage of squamous cell carcinoma of the lung, whereas they occur relatively late in the pathogenesis of adenocarcinoma (Kohno et al., 1999).

The analysis of surgically resected NSCLC specimens for LOH at 3p25-26, 3p21, 3p14, 5q, 11p, 17q, and 18q demonstrated that, with respect to pRB, p16^INK4a, and p53, the tumors could be grouped into four categories: normal for all three proteins (21%); abnormal for pRB or p16^INK4a and normal for p53 (30%); normal for pRB and p16^INK4a and abnormal for p53 (20%); and abnormal for all three proteins (28%) (Geradts et al., 1999). An aberrant expression of pRB, p16^INK4a, p53, and 3p LOH, either individually or in combination, was not associated with survival differences or any other clinical parameters, with the exception that pRB and p16^INK4a abnormalities were more common in older patients. pRB and p16^INK4a expression showed a strong inverse correlation, whereas there was no relationship between the expression of pRB, p16^INK4a, and p53 (Geradts et al., 1999). An abnormal expression of any of the three genes inversely correlated with K-ras mutations at codon 12 (P = 0.004), but not with LOH at 3p or at other loci. Therefore, NSCLCs show distinct patterns of tumor-suppressor gene inactivation, but no clear clinical correlates exist either alone or in combination for pRB, p16^INK4a, p53, and 3p abnormalities (Geradts et al., 1999).

In an effort to identify regions containing novel cancer genes, chromosome 18p11 was examined for LOH in matched normal and NSCLC tumor samples by using 18p11 and 18q12.3 polymorphic markers (Tran et al., 1998). This analysis revealed two regions of LOH in 18p11 in up to 38% of the tumor samples examined. The regions of LOH identified included a region between D18S59 and D18S476 markers, and a more proximal region of intermediate frequency between D18S452 and D18S453 (Tran et al., 1998). These results provide evidence for the presence of one or more tumor-suppressor genes on the short arm of chromosome 18, which may be involved in NSCLC (Tran et al., 1998). Deletions in the 5q14 region have been described in a variety of neoplasms, including lung cancer. The high frequency of allelic losses observed in this region implies the presence of putative tumor-suppressor genes. In a series of 56 NSCLCs the allelic imbalance within the 5q14 region and its relationship with p53 abnormalities, kinetic parameters, proliferation and apoptotic index, and the ploidy status of tumors revealed that an allelic imbalance at D5S644 was found at a frequency of 51.2% (Gorgoulis et al., 2000). LOH at 5q14 was associated with a low apoptotic index, suggesting the presence of putative tumor-suppressor genes. Simultaneous alterations of both p53 and D5S644 loci were the most frequent pattern observed (37.5%) (Gorgoulis et al., 2000). These findings imply a synergistic mechanism of cooperation between different tumor-suppressor genes. However, proliferation activity was dependent only on p53 status, leading to the assumption that the putative tumor-suppressor genes present at 5q14 may be involved in apoptotic pathways (Gorgoulis et al., 2000). The use of microsatellite markers at 3p14, 9p21, and 10q24 to analyze tumor samples from 91 patients with pathological stage I NSCLC demonstrated that LOH at any single locus was not significantly associated with survival (Zhou et al., 2000). The analysis of LOH on a panel of 102 NSCLC samples with 20 polymorphic markers evidenced two short regions of the overlap of the deletions (SROs): SRO2a (D1S417-D1S57) and SRO2b (D1S450-D1S243). Allelic losses at either region located on 1p32-pter correlated independently with an advanced stage of disease and with postoperative metastasis and relapse, suggesting that crucial genes in these regions are involved in NSCLC progression (Chizhikov et al., 2001). The short arm of chromosome 3 is thought to harbor an oncogenic locus that is important in lung carcinogenesis because of its sensitivity to loss by the action of carcinogens and evidence of frequent deletion in lung cancer. Of 219 lung cancers, 44.2% of squamous cell carcinomas and 30.2% of adenocarcinomas showed 3p21 LOH, its prevalence being higher in p53 mutated cases (Hirao et al., 2001). The analysis for LOH at chromosome 3p24 in samples of normal and tumor tissues from the lungs of 76 patients with NSCLC revealed that RXR, RAR, and RAR gene expression was decreased in 18%, 63%, and 41% of tumor specimens (Picard et al., 1999). LOH at 3p24 was observed in 41% of tumor samples and in 20% of non-neoplastic lesions. Therefore, a large percentage of tumors shows a marked decrease in the expression of RXR, RAR, and RAR, and a high frequency of LOH at 3p24, which is also observed in non-neoplastic lesions (Picard et al., 1999). These data suggest that altered retinoid receptor expression may play a role in lung carcinogenesis (Picard et al., 1999). LOH at chromosome 3p24, which hosts RAR, was observed in 100% (13 of 13) of SCLC cell lines and 67% (12 of 18) of NSCLC cell lines, and the difference was statistically significant (Virmani et al., 2001b). Abnormalities of FHIT, the tumor-suppressor gene located at 3p14.2, have been found in NSCLC. Analysis of a subset of 76 specimens of stage I NSCLC, in which microsatellite analysis at the FHIT locus was performed, did not show a strong association between LOH at 3p14.2 and pFHIT expression, suggesting the presence of complex mechanisms of gene inactivation (Tseng et al., 1999). However, loss of FHIT was significantly higher in bronchial metaplastic lesions (47%) than in histologically normal bronchial epithelium (20%), and pFHIT expression was significantly reduced in a substantial number of early-stage NSCLC and preneoplastic lesions in chronic smokers (Tseng et al., 1999).

In stage I NSCLC, allelic imbalance is observed on 2q, 9p, 18q, and 22q in 22, 38, 29, and 15% of cases, respectively, whereas p53 is mutated in 41% of stage I NSCLCs (Tomizawa et al., 1999). Allelic imbalance on 9p and 22q, and p53 mutations, were significantly associated with shortened survival of the patients (Tomizawa et al., 1999). These results indicate that clinical aggressiveness of early-stage NSCLC is associated, at least in part, with the presence of allelic imbalance on chromosome 9p, which could be a clinically useful prognostic indicator (Tomizawa et al., 1999). Allelotyping studies suggest that allelic losses at one or both arms of chromosome 4 are frequent in several tumor types. The analysis of clinical specimens and NSCLC cell lines by using 16 polymorphic microsatellite markers showed LOH at three nonoverlapping regions: 1) 4q33-34 (R1), 2) 4q25-26 (R2), and 3) 4p15.1-15.3 (R3) in about 20 to 30%, with no differences between tumors and cell lines, the loss of R3 alone being the most frequent pattern (Shivapurkar et al., 1999). LOH may occur in 83% of NSCLCs with chromosomal duplication, suggesting that the duplicated chromosome is homozygous; these findings imply that LOH occurs before chromosomal duplication during lung carcinogenesis (Varella-Garcia et al., 1998). LOH on chromosome 11q23 is observed at high frequency in NSCLC, suggesting the presence of a tumor-suppressor gene (Murakami et al., 1998). Allelotyping of NSCLC and SCLC cell lines demonstrated significant differences in LOH frequencies between NSCLC and SCLC at 13 regions on 8 chromosome arms (3p, 5q, 6q, 9p, 10q, 11p, 13q, and 19p). Eight homozygous deletions were present in seven cell lines at four regions, 3p12, 3p14.2, 9p21, and 10q23-25. In addition to this, there was LOH at 6p21.3 and 13q12.3 in NSCLC (Virmani et al., 1998). The frequent occurrence of 21q deletions in human NSCLC indicates the presence of a tumor-suppressor gene on this chromosome arm. The ANA (abundant in neuroepithelium area) gene, a member of an antiproliferative gene family, is mapped to 21q11.2-q21.1 and was homozygously deleted in the human Ma17 NSCLC cell line. LOH at this locus was detected in 24 of 47 (51.1%) NSCLCs, and the frequency of LOH in brain metastases was significantly higher than that in stage I-II primary tumors. These data suggest that the homozygously deleted region harbors a novel tumor-suppressor gene involved in NSCLC progression (Kohno et al., 1998). The PTEN/MMAC1 (phosphatase and tensin homolog deleted on chromosome 10/mutated in multiple advanced cancers) is a candidate tumor-suppressor gene recently identified at chromosomal band 10q23. Microsatellite analysis revealed LOH at markers near the gene in 50% of 42 primary NSCLCs. These results suggest that PTEN/MMAC1 gene inactivation plays a role in the genesis of some tumor types (Okami et al., 1998). In a cohort of 87 NSCLCs, LOH was investigated by using dinucleotide repeat sequences from chromosomal locations 1p, 3p, 5q, 8p, 9p, 10p, 11p, 13q, and 17q. In 28% (24 of 87) of NSCLCs, LOH in at least one locus was detected. The frequency of LOH differed between the various cell types of NSCLC. The highest frequency was seen in large cell carcinoma (3 of 6, 50%) followed by squamous cell carcinoma (16 of 43, 37%) and adenocarcinoma (5 of 35, 14%), and the most common site of LOH was 3p (Pylkkanen et al., 1997). The TGF-RII gene has been mapped to chromosome 3p, on which LOH was frequently detected in NSCLC; however, TGF-RII mutations were not found in NSCLC with LOH on chromosome 3p (Tani et al., 1997). Deletions involving the chromosome 9p21 region, which also harbors the tumor-suppressor locus CDKN2A, have been reported as frequent events in NSCLC. LOH at a marker proximal to the CDKN2A locus was found most frequently (52%), while LOH at a marker closest (5 kb) to the CDKN2A gene was seen in only 17% of tumors (Mead et al., 1997). A homozygous loss of markers close to CDKN2A was, however, detected in 2 of 3 cell lines and one accompanying tumor sample. Therefore, a tumor-suppressor gene in the region of deletion proximal to the CDKN2A gene within 9p21 may play a significant role in the pathogenesis and progression of NSCLC (Mead et al., 1997). LOH in the TP53 locus was found in 9 of 38 (23.6%) cases. A trend was found between p53-positive immunostaining and a history of heavy smoking, and was inversely correlated with LOH at the TP53 locus (Liloglou et al., 1997). High LOH on chromosome arms 3p, 9p, and 17p is a common event in NSCLC. LOH was observed at a frequency of 38% on 3p, 58% on 9p, and 38% on 17p. Polarization of the LOH on chromosome arms 3p, 9p, and 17p was observed such that 80% showed loss on 3p, 80% on 9p, and 73% on 17p (Field et al., 1996). LOH on chromosome arms 3p, 13q, and 17p was detected frequently (>60%) in both stage I primary lung tumors and brain metastases, whereas the incidence of LOH on chromosome arms 2q, 5q, 9p, 12q, 18q, and 22q was higher than 60% only in brain metastases. In particular, the incidence of LOH on chromosome arms 2q, 9p, 18q, and 22q in brain metastases was significantly higher than that in stage I primary lung tumors (Shiseki et al., 1996). These results indicate that tumor-suppressor genes on chromosome arms 3p, 13q, and 17p are involved in the genesis of NSCLC, whereas those on several chromosome arms, especially on 2q, 9p, 18q, and 22q, play an important role in the progression of NSCLC (Shiseki et al., 1996). High-density polymorphic marker analysis throughout 11p15.5 confirmed the presence of two distinct regions of LOH for NSCLC in 11p15.5. In 9 of 13 (69%) tumors with LOH, allelic deletion was restricted to 11p15.5, indicating that whole chromosome 11 loss is not a common event in NSCLC and suggesting that chromosome band 11p15.5 harbors a minimum of three separate loci (Tran and Newsham, 1996).

3p21 Loss appears, so far, to be the most frequent and the earliest genetic alteration described in NSCLC, but it does not seem to carry significant prognostic information in invasive tumors (Thiberville et al., 1995). The short arm of chromosome 17, which contains the p53 gene, is frequently affected by LOH in lung cancer. The frequency of LOH at 17q is 42%, approaching that at 17p (54%), and two distinct 17q regions are implicated. LOH at D17S4 on 17q is more frequent in adenocarcinomas than in squamous cell carcinomas, whereas squamous cell carcinomas had more LOH at 17p than at 17q, indicating a molecular genetic heterogeneity between the major NSCLC subtypes. In addition, LOH at 17q correlates with higher tumor stages and a significantly worse prognosis. In comparison, 25% of cases have mutations at TP53 exons 5-8, but these are not associated with tumor stage or survival (Fong et al., 1995a). LOH at the APC/MCC gene cluster at chromosome 5q21 occurs frequently in NSCLC; it affects 29% of NSCLC and it is significantly correlated with worse survival. Furthermore, in squamous cell carcinoma, LOH at 5q not only correlated with a short survival, but also with tumor involvement of the mediastinal and/or hilar lymph nodes (Fong et al., 1995b). In contrast, LOH at chromosome 18q was far less frequent, occurring in 14% of NSCLC cases, and it was not associated with advanced stage or adverse prognosis. These data suggest that LOH at 5q has a role in determining tumor progression and survival in NSCLC, and may prove to be a clinically useful prognostic indicator (Fong et al., 1995b).

Microsatellites are repetitive nucleotide sequences of varying lengths, which occur in the human genome, between and within genes (Eshleman et al., 1996; Sherbet and Lakshmi, 1997). Microsatellite sequences (also called microsatellite markers) are unstable because of variations that can occur in repetitive sequence units, resulting in the expansion or shortening of them. The instability of microsatellite loci contributes to the mutator phenotype of cancer and provides an explanation of the high incidence of mutations compared to normal cells (Loeb, 1994). The instability of microsatellites can affect nonrepetitive sequences of the DNA, and the direct consequence is the generation of a ladder-like motif that replaces the normal allele pattern of the human genome (Wistuba et al., 2001). A majority of microsatellite repeats occur outside the coding regions of genes; therefore, microsatellite instability may not directly lead to carcinogenesis, but could destabilize DNA sequences inside and outside the microsatellite repeats and make the genome hypermutable. As a corollary, one should consider the possibility that microsatellite instability might be engendered by exposure to carcinogens. Microsatellite instability was found in about one-third of NSCLC, with a substantial difference between metastatic lesions (55%) and primary disease (12%) (Adachi et al., 1995), thus suggesting a possible direct relationship between microsatellite instability and cancer progression. It has been recently observed, however, that microsatellite instability, defined as the change in the number of short-tandem DNA repeats, is not common in NSCLC, while the microsatellite alteration, where a single band of altered size is found, has been described in 2 to 49% of NSCLC (Sekido et al., 1998; Wistuba et al., 2001). By using 16 markers on chromosomes 3p and 9p, microsatellite alteration is found in 7 of 20 histologically normal lung tissue specimens at a frequency similar to that observed in NSCLC tumor tissue (8 of 20). Five cases showed microsatellite alteration in both normal lung tissue and the corresponding tumor (Park et al., 2000). In 2 of 12 patients microsatellite alteration was detected in normal lung tissue while the tumor was negative. These results indicate that genetic alterations are widely distributed in the lung tissue of patients with lung cancer (Park et al., 2000).

The short arm of chromosome 3 is thought to harbor an oncogenic locus involved in the pathogenesis of NSCLC. The region at 3p21 is believed to contain a distinct locus that is sensitive to loss from the action of tobacco smoke carcinogens, and has been reported to be specifically targeted for deletion in lung cancer. A recent study examined the LOH on chromosome 3 at 3p21 in NSCLC and the microsatellite alteration at the BAT-26 locus because the mismatch DNA repair gene, hMLH1, is found at 3p21 (Hirao et al., 2001). Instability of BAT-26 was not found, while LOH at 3p21 was detected in 44.2% of squamous cell carcinomas and 30.2% of adenocarcinomas and was frequently associated with TP53 mutations (Hirao et al., 2001). Using a panel of 12 markers, microsatellite instability was detected in 24 of 47 (51%) NSCLC and 10 of 18 (56%) head and neck cancers, but was only observed in 8 of 38 (21%) bladder and 3 of 25 (12%) kidney cancers (Xu et al., 2001). The results of this study suggest that about 50% of respiratory tract cancers exhibit microsatellite instability, predominantly at AAAG sequences. This distinct type of instability is termed EMAST (elevated microsatellite alterations at selected tetranucleotide repeats) and the identification of markers with EMAST may prove useful for the molecular detection of respiratory tract cancers (Xu et al., 2001). Microsatellite instability was observed in 5 of 7 NSCLC cell lines and 3 of 21 NSCLC tissues (Kim et al., 2000). Microsatellite instability was highly associated with TGF-RII frameshift mutations (75%), thus supporting the hypothesis that TGF-RII plays an important role in NSCLC carcinogenesis (Kim et al., 2000). Among 91 patients with stage I NSCLC, 32% of subjects whose tumors had microsatellite instability at 10q24 died of the disease within 5 years after surgery, compared with 16% without microsatellite instability at 10q24 (Zhou et al., 2000). Seventy-one percent of patients with lung adenocarcinoma and microsatellite instability at 10q24 died because of disease progression, compared with 12% without microsatellite instability, indicating the presence of distinct mechanisms in tumorigenesis among different subtypes of lung cancer (Zhou et al., 2000). Of 23 patients who had microsatellite instability at 10q24 and 3p14, 39% died of the disease within 5 years as compared with 15% of the patients without such a profile (Zhou et al., 2000). Furthermore, among the 22 patients with no alteration at any loci tested, none died of lung cancer within 5 years after surgery, whereas 28% of the patients outside these profiles died of the disease (Zhou et al., 2000). These results support the hypothesis that microsatellite alterations can be used as biomarkers for the genetic classification of stage I NSCLC, which may in turn influence treatment decisions (Zhou et al., 2000). Microsatellite alteration may also be detected in the DNA of cells in bronchoalveolar lavage fluid from patients with resectable NSCLC; indeed, microsatellite instability was observed in NSCLC tissue in approximately 50% of patients; the identical alteration was shown in the bronchoalveolar lavage fluid of 14% of the corresponding patients (Ahrendt et al., 1999).

Chromosome 3p is consistently deleted in lung cancer, and it is believed to contain several tumor-suppressor genes. The role of chromosome 3 in tumor suppression has been confirmed by isolation of the human homolog of the ribosomal protein L14 gene (RPL14) located at 3p21.3 (Shriver et al., 1998). The RPL14 sequence contains a highly polymorphic trinucleotide repeat that encodes a variable-length polyalanine tract (Shriver et al., 1998). Genotype analysis of RPL14 shows that this locus is 68% heterozygous in the normal population compared with 25% in NSCLC cell lines. Cell cultures derived from normal bronchial epithelium show a 65% level of heterozygosity, reflecting that of the normal population (Shriver et al., 1998). In additional studies, microsatellite instability at one or more loci was observed in 13 (36%) of 36 cases of resected NSCLC (19 cases of squamous cell carcinoma, 15 of adenocarcinoma, and 2 of large cell carcinoma) (Kim et al., 1998a). Six tumors showed instability in a single microsatellite, three tumors had alterations in three of four tested microsatellites, and the microsatellite that showed instability most frequently in these tumors was D3S1340 (31%) (Kim et al., 1998a). Furthermore, microsatellite instability was found in 24% of 17 cancers at stage I, in 17% of 6 tumors at stage II, in 73% of 11 tumors at stage IIIA, and in none at stage IIIB; overall, microsatellite instability was observed in at least one-third of NSCLC (Kim et al., 1998a). A set of 11 microsatellite loci spanning 1p was used to examine the frequency of allelic imbalance in a panel of 58 tumors; 87.9% of 58 cases had somatic allelic loss at one or more loci tested. Two SROs have been identified: SRO1 at 1p13.1 and SRO2 at 1p32-pter (Gasparian et al., 1998). Allelic losses at these regions have been compared among adenocarcinomas and squamous cell carcinomas, and no difference has been found. On the contrary, SRO2 deletions significantly correlated with advanced stage of the disease and postoperative disease recurrence (Gasparian et al., 1998). These data may suggest that SRO1 and SRO2 harbor tumor-suppressor genes involved in different stages of NSCLC development (Gasparian et al., 1998). The comparison of DNA from human tumor and normal bronchial mucosa with respect to microsatellite instability and LOH on chromosome 17p, 17q, 9p, and 9q, using 10 polymorphic markers, was performed on biopsies and tissue specimens obtained from the tumor and paired normal bronchial mucosa in 20 patients with NSCLC (Froudarakis et al., 1998). Sixteen of 20 tumors (80%) displayed genetic alterations; 30% of tumors exhibited microsatellite instability, 25% exhibited LOH, and 25% of tumors showed microsatellite instability and LOH (Froudarakis et al., 1998). No relationship was found between LOH or microsatellite instability and the histologic subtype of NSCLC or disease stage. These results suggest that genetic alterations have a role in carcinogenesis as they exist in all stages and histologic subtypes of NSCLC (Froudarakis et al., 1998). In a cohort of 379 women with NSCLC, microsatellite instability was observed more frequently in patients with three or more relatives with cancer (6 of 9, 67%) than in control patients (5 of 28, 18%; P = 0.011) (Suzuki et al., 1998). Thus, a significantly higher rate of microsatellite instability is associated with familial clustering of malignancy (Suzuki et al., 1998). The replication-error-type instability (RER+) is a frequent genetic alteration in stage I NSCLC. RER+ at one or both chromosomes 2p and 3p was identified in 24 of 35 patients; 9 patients showed LOH (Rosell et al., 1997). A statistically significant correlation was found between RER+ and poor prognosis; furthermore, RER+ proved to be an independent factor that predicted decreased survival (Rosell et al., 1997). These data suggest that RER+ is common in NSCLC, and it may provide important prognostic information in stage I NSCLC (Rosell et al., 1997).

Reversible protein phosphorylation has emerged as the predominant mechanism of control of protein activity in eukaryotic cells in response to environmental signals, mainly related to cell proliferation. The phosphorylation of specific proteins, which is under the control of two families of enzymes known as protein kinases and phosphatases, provides signal amplification. Since more than 10% of proteins in a normal mammalian cell are thought to be regulated through phosphorylation, this aspect of proteomics is gaining significant interest. Abnormal protein phosphorylation is the basis for or the result of major diseases, including cancer. Mutations in protein kinases and phosphatases or in regulatory genes result in a number of hereditary disorders, including leukemias and lymphomas (Shapiro et al., 1995). Excessive activity of kinases under the control of growth-promoting genes is the apparent mechanism responsible for inactivation of tumor-suppressor gene products, including pRB. Indeed, cyclin-dependent kinase (cdk)4-mediated phosphorylation of pRB is stimulated by cyclin D1, an oncogene, and inhibited by p16^INK4a, the product of the tumor-suppressor gene CDKN2A (Shapiro et al., 1995). NSCLC is predominantly pRB-positive and most tumor specimens and cell lines overexpress cyclin D1, indicating that cyclin D1 overexpression and RB inactivation coexist (Shapiro et al., 1995). Furthermore, pRB-positive NSCLC cell lines have absent or low p16^INK4a, and in primary lung resection specimens p16^INK4a was undetectable in 18 of 27 NSCLC samples. These data confirm the dependence of pRB inactivation on p16^INK4a expression (Shapiro et al., 1995).

To evaluate the role of Akt/PKB (AKR mouse T-cell lymphoma/protein kinase B) in the survival of patients with NSCLC, a panel of NSCLC cell lines that differed with respect to tumor histology and p53, pRB, and p21K-ras status were examined. Constitutive Akt/PKB activity was demonstrated in 16 of 17 cell lines (Brognard et al., 2001). Akt/PKB activation was dependent on phosphatidylinositol 3-kinase (PI3K) and promoted survival because wortmannin, a PI3K inhibitor, suppressed Akt/PKB phosphorylation and increased apoptosis only in cells with activated Akt/PKB (Brognard et al., 2001). To test whether Akt/PKB is involved in drug resistance, tumor cells were exposed to conventional anticancer agents in combination with the phosphatidyl-inositol 3-kinase inhibitor LY294002. LY294002 potentiated chemotherapy-induced apoptosis in cells with high Akt/PKB levels, but was ineffective in cells with low Akt/PKB levels (Brognard et al., 2001). Transfecting constitutively active Akt/PKB into cells with low Akt/PKB activity attenuated chemotherapy- and radiation-induced apoptosis (Brognard et al., 2001). Thus, Akt/PKB is a constitutively active kinase that promotes survival of NSCLC cells, and modulation of its activity by pharmacological or genetic approaches alters the cellular sensitivity to chemotherapeutic agents used to treat patients with NSCLC (Brognard et al., 2001).

	IV. Genetic Abnormalities in Non-Small Cell Lung Cancer

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Multistep tumorigenesis is the process by which genetic events accumulate over time and result in malignant transformation. It is estimated that approximately 10 to 20 alterations of tumor-suppressor genes and/or proto-oncogenes are required for lung tumorigenesis. Numerous alterations have been identified that occur frequently in NSCLC. These include RAS proto-oncogene mutations, TP53 gene mutations, inactivation of the RB gene, and alterations in CDKN2A, HER-2/neu, MYC, Bcl-2, and FHIT (Table 2).

The product of the RAS gene (p21ras) regulates transduction of growth-proliferative signals from the membrane to the nucleus, and mutationally activated RAS is found in 25 to 48% of NSCLC. The p21ras proteins bind to and hydrolyze GTP by means of their intrinsic GTPase activity. Point mutations in p21ras impair its GTPase activity and the constitutive presence of the active, GTP-bound form of p21ras (p21ras^GTP) leads to deregulated growth and cellular transformation (MacDonald and McCormick, 1997). To perform its function in cell signaling, p21ras must be farnesylated on the CAAX motif (Cysteine, Aliphatic amino acid, and any amino acid [X]) at the carboxyl terminus of p21ras protein, a reaction mediated by farnesyl protein transferase (Di Paolo et al., 2001). Intracellular effectors of p21ras include raf-1, MEK (mitogen-activated protein kinase [MAPK]/extracellular signal-regulated kinase [ERK] kinase), and MAPKs, which are needed for RAS-mediated DNA synthesis, gene transcription, and eventually malignant transformation (MacDonald and McCormick, 1997). Although this represents the prevalent hypothesis for p21ras signal transduction, recent studies failed to substantiate it (Ramakrishna et al., 2000). In particular, lung tumors do not have more total p21K-ras or p21K-ras^GTP than normal lung tissue, nor are higher levels of these proteins found in tumors with mutant K-RAS. Activated p21K-ras^GTP levels did not correlate with proliferating cell nuclear antigen (PCNA) staining. Furthermore, tumors with mutant K-RAS displayed smaller size compared with tumors lacking this mutation (Ramakrishna et al., 2000). In nontransformed lung epithelial cells in culture both total and activated p21K-ras increased markedly at confluence, but not after serum stimulation, and mRNA analysis indicated an increase in K-RAS expression in confluent cells. These findings indicate that normal p21K-ras activity is associated with growth arrest of normal lung epithelial cells and that the exact contribution of mutated p21K-ras to tumor development is still undetermined (Ramakrishna et al., 2000). To evaluate the association of K-RAS abnormalities with the incidence of NSCLC, 410 surgically resected specimens were analyzed for K-RAS mutations in codons 12, 13, and 61; mutations were found in 33 patients (8%) and all were smokers or ex-smokers (Noda et al., 2001). There were no significant differences in tumor stage between wild-type and mutant K-RAS. The most frequently identified mutation was a G>T transversion (75.8%) that resulted in the substitution of a glycine for a cysteine or a valine (Noda et al., 2001). This study provides evidence of a clear correlation between smoking and G>T transversions affecting the K-RAS gene (Noda et al., 2001). Survival is strongly associated with K-RAS gene mutations in NSCLC (Rosell et al., 1996). The analysis of the relationship between tumor aggressiveness and K-RAS point mutations at codons 12 and 61 was evaluated in 275 consecutively treated stage I-IV NSCLCs. In stage I disease, median survival was 27 versus 41.5 months in patients with or without K-RAS mutations at codon 12, respectively (Rosell et al., 1996). Furthermore, in patients with stage IIIA disease, median survival time was 7 months in those with K-RAS mutations at codon 12 (aspartic acid to serine) and 15 months for those with other K-RAS mutations (P = 0.01) (Rosell et al., 1996). In a multivariate analysis, point mutation at codon 12 of K-RAS was a strong predictive factor for death (hazard ratio, 2.06; P = 0.02) after adjustment for other factors, including stage and histology. Therefore, in patients with NSCLC specific K-RAS point mutations are associated with a significantly increased risk of recurrence and death, independently of tumor stage and histology (Rosell et al., 1996).

Intron 1 of the human H-RAS gene possesses a unique polymorphism consisting of GGGCCT repeats. Analysis of this locus in matched tumor versus normal samples from 38 patients with NSCLC revealed 6.6% LOH and 10.5% hexanucleotide instability (Kotsinas et al., 2001). The same pattern of alterations was also detected in tissues adjacent to lung adenocarcinomas and dysplasias contiguous to squamous cell carcinomas (7.7% LOH, 5.9% hexanucleotide instability), implying that abnormalities at this locus may be early events in lung carcinogenesis (Kotsinas et al., 2001). In view of reports showing that elements in intron 1 of the H-RAS gene potentially influence its transcriptional regulation, the hexanucleotide locus could be an element with possible involvement in expressional regulation of H-RAS (Kotsinas et al., 2001).

The product of the TP53 tumor-suppressor gene is p53, a DNA-binding, sequence-specific transcription factor that activates the expression of genes engaged in promoting growth arrest in the G₁ phase or cell death in response to genotoxic stress. Also, p53 prevents cells from undergoing mitosis when they enter the G₂ phase with damaged DNA (Taylor and Stark, 2001). Part of the mechanism by which p53 blocks cells at the G₂ checkpoint involves inhibition of cdc2, the cyclin-dependent kinase required to enter mitosis. Binding of cdc2 to cyclin B1 is required for its activity, and repression of the cyclin B1 gene by p53 also contributes to blocking entry into mitosis (Taylor and Stark, 2001). The transfer of the wild-type TP53 gene into the p53-null human NSCLC NCI-H358 cells results in a typical senescence-like phenotype, characterized by reduction in cell growth, enlarged and flat cell morphology, cell cycle arrest in the G₁ phase, down-regulation of cyclin B1 and cdc2 expression, and suppression of DNA synthesis (Ling et al., 2000). The ability of p53 to inhibit cellular proliferation or to induce cell death is suppressed by the product of the mouse double minute 2 (MDM2) gene. This property underlies the oncogenic potential of MDM2, which is overexpressed in various human tumors. Similar to other oncogenes, surveillance pathways might counteract the deleterious effects of deregulated MDM2 expression (Daujat et al., 2001). The GML gene (glycosyl-phosphatidyl-inositol-anchored