87 - Pulmonary Tuberculosis and Other Mycobacterial Diseases of the Lung

Editors: Shields, Thomas W.; LoCicero, Joseph; Ponn, Ronald B.; Rusch, Valerie W.

Title: General Thoracic Surgery, 6th Edition

Copyright 2005 Lippincott Williams & Wilkins

> Table of Contents > Volume II > Section XVI - Carcinoma of the Lung > Chapter 102 - Present Concepts in the Molecular Biology of Lung Cancer

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Chapter 102

Present Concepts in the Molecular Biology of Lung Cancer

Gregory P. Kalemkerian

Harvey I. Pass

Over the past 20 years, major advances in cellular and molecular biology have led to an exponential increase in our understanding of the molecular events underlying the development and progression of lung cancer. These events primarily consist of molecular abnormalities that disrupt the complex homeostatic pathways regulating cellular differentiation, proliferation, and death in normal bronchial epithelial cells. Although the means by which these pathways can be disrupted are virtually unlimited, the most common molecular derangements described in lung cancer result in the dysregulation of select groups of protooncogenes, tumor suppressor genes, and cell death regulatory genes. In addition, the aberrant production of a variety of growth factors and angiogenic factors serves as a prime determinant of the aggressiveness and metastatic potential of lung cancer cells.

The identification of the cellular and molecular derangements involved in the pathogenesis and progression of lung cancer has led to the development of a plethora of novel biologically rational diagnostic and therapeutic approaches directed at specific molecular targets. In the past few years, many of these strategies have evolved from theoretical concepts into clinical realities. Thus far, few of these targeted interventions have been shown to offer clinical benefit for patients with lung cancer. However, initial clinical trials have offered a promising glimpse of a future in which the prognosis of patients with lung cancer will be significantly improved through the use of rationally designed therapy. This chapter reviews the most important aspects of the biology of both small cell (SCLC) and non small cell (NSCLC) lung cancer and emphasizes the translation of these concepts into clinical strategies that will undoubtedly have a major impact on the management of patients with lung cancer.

PROTOONCOGENES

Protooncogenes encode for proteins that serve as physiologic components of the signal transduction pathways that regulate cellular growth and differentiation in normal cells, and thereby fall into several discrete functional categories: growth factors, receptors, membrane-associated second-messenger molecules, cytoplasmic second-messenger molecules, and transcription factors. In nonneoplastic cells the expression of these protooncogenes is tightly regulated in order to maintain normal homeostatic control of cellular proliferation and differentiation. Oncogenic events induce mutations, amplifications, and translocations of various protooncogenes, resulting in either the overexpression of a normal protooncogene product or the expression of an overactive protein. These alterations lead to an overall increase in protooncogene function, and, because most protooncogene products are positive mediators of proliferative pathways, this functional enhancement results in inappropriate activation of these pathways and uncontrolled, neoplastic growth. Such protooncogene abnormalities are a common finding in all malignancies, including lung cancer (Table 102-1).

myc Oncogenes

The three functional myc genes, C-myc, L-myc, and N-myc, encode nuclear transcription factors that are important mediators of proliferation, differentiation, and cell death. All three myc genes complement activated ras, resulting in malignant transformation, with C-myc exhibiting the greatest transforming potency. In general, the transforming capacity of the myc genes is directly related to their degree of dysregulation and overexpression. In the normal bronchial epithelium, Broers and associates (1993) reported that C-myc expression is limited to basal cells and alveolar type II pneumocytes, and N-myc and L-myc are not expressed.

In SCLC, overexpression of C-myc, L-myc, or N-myc occurs in up to 80% of cell lines and primary tumors. Takahashi and colleagues (1989a), among others, reported that myc gene amplification occurs in 40% to 50% of SCLC cell

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lines and 20% to 30% of tumors, but the mechanism of myc overexpression remains unknown in the remainder of cases. C-myc is the most commonly dysregulated of the myc genes in SCLC, and several investigators, including Gazdar and co-workers (1985), have reported that C-myc gene amplification is associated with prior chemotherapy and tumors of the variant SCLC phenotype with a relatively low level of neuroendocrine differentiation. Brennan and associates (1991) reported that amplification or overexpression of C-myc, but not of L-myc or N-myc, is associated with a poor prognosis. In addition, amplification was more frequent in patients who had previously received chemotherapy (28%) than in untreated patients (8%), and appeared to correlate with the prior administration of alkylating agents. These results suggest that C-myc gene amplification is not an early event in the development of SCLC, but that it may play an important role in tumor progression. In NSCLC, initial studies reported C-myc overexpression in 50% of samples, despite infrequent gene amplification. However, Mitani and associates (2001) and others have recently reported C-myc amplification in the majority of NSCLC primary tumors.

Table 102-1. Protooncogenes Implicated in Lung Cancer

  Mechanism Histology
Nuclear transcription factors
   C-myc A/OE
   N-myc A/OE S
   L-myc A/OE S
   c-jun IE S, N
   c-fos IE N
   c-erb-A LOH/? S
   c-myb IE S
Membrane-associated G protein
   K-ras M N
   c-erb-B2 (HER2/neu) OE N
   c-fms IE N
   c-kit IE S, N
   c-met OE S, N
Cytoplasmic kinases
   c-raf-1 LOH/? S
   c-src IE S
Apoptosis inhibitor
   bcl-2 IE S, N
A, amplification; IE, inappropriate expression; LOH, loss of heterozygosity; M, mutation; N, non small cell lung cancer; OE, overexpression; S, small cell lung cancer; ?, unknown.

Dysregulation of N-myc gene expression has been reported primarily in neuroendocrine tumors, such as neuroblastoma and SCLC. In SCLC, Ibson (1987) and Takahashi (1989a) and their co-workers found that N-myc overexpression is associated with the classic phenotype and a greater degree of neuroendocrine differentiation. Wong (1986) and Nau (1986) and their colleagues showed that N-myc overexpression can also occur in the presence or absence of gene amplification. However, in contrast to C-myc, N-myc overexpression is seen in a considerable number of samples from untreated patients, and therefore may represent an early oncogenic event. Although some studies have suggested an association between N-myc overexpression and poor prognosis, as is the case in neuroblastoma, the largest such series by Brennan and colleagues (1991) revealed no correlation. The dysregulation of N-myc or L-myc is rare in NSCLC.

The L-myc protooncogene was initially described in SCLC cell lines by Nau and co-workers (1985), and its expression is highly specific for lung tissue. Rygaard and colleagues (1993b) and others have reported that amplification and overexpression of L-myc are seen predominantly in SCLC tumors with a classic phenotype and a relatively low proliferative index, but that these molecular derangements do not correlate with stage or clinical outcome. Analysis of the L-myc locus on chromosome 1 has revealed a restriction fragment length polymorphism (RFLP) resulting in two distinct DNA fragments, S (6.6 kb) and L (10 kb). However, the RFLP phenotype is not tumor specific and is not associated with lung cancer risk. Although some studies have suggested that the L-L and S-S phenotypes predict a poorer prognosis in patients with lung cancer, others, such as that of Yaylim and co-workers (2002), have not, and the relevance of L-myc polymorphisms remains controversial.

The high rate of myc oncogene dysregulation in lung cancer has led to a variety of novel therapeutic strategies targeting the myc genes, including the use of antisense technology. The expression of any gene is dependent on the transcription of gene-specific DNA into single-stranded messenger RNA (mRNA) followed by the translation of this mRNA into the functional protein. Determination of the mRNA nucleotide sequence that encodes for protein, the sense sequence, allows for the synthesis of a complementary antisense oligonucleotide sequence that can specifically bind to the mRNA of interest. The creation of a double-stranded segment of mRNA effectively blocks the progression of the translational complex and inhibits the expression of functional protein. Robinson and colleagues (1995) have shown that C-myc antisense oligonucleotides can block the expression of C-myc and inhibit the proliferation of NSCLC cells in culture, and Van Waardenburg and co-workers (1997) reported that transfection of a C-myc antisense expression construct into a platinum-resistant SCLC cell line inhibited proliferation through the induction of apoptosis and potentiated the activity of cisplatin. Similarly, Dosaka-Akita and associates (1995) found that antisense oligonucleotides targeting L-myc mRNA inhibited L-myc expression and SCLC cell growth.

Although the development of gene therapy has been hampered by suboptimal delivery systems and unexpected toxicity, several gene therapy strategies have achieved success in proof-of-concept studies. One such approach involves the selective transfection of cancer cells with a suicide gene that will interact with a specific pharmacologic agent to induce the death of the transfected cancer cell. Kumagai and colleagues (1996) inserted an expression construct containing a herpes simplex virus thymidine kinase (HSV-TK) gene under the control of a myc-responsive promoter into SCLC cells that overexpressed either C-myc,

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N-myc, or L-myc, resulting in high levels of HSV-TK expression specifically in these myc-overexpressing cancer cells. Subsequent treatment with ganciclovir, an antibiotic that targets HSV-TK, inhibited the growth of both transfected cells and untransfected cells added to the transfected cell cultures, suggesting a therapeutic bystander effect in which dying transfected cells emit signals that activate cell death pathways in adjacent untransfected cells.

Another potential therapeutic strategy involves the direct modulation of specific protooncogene expression by selective pharmacologic agents. The retinoids are vitamin A analogues that are natural mediators of proliferation, differentiation, and death in many normal and malignant cell types. Many epidemiologic and molecular studies have suggested that dysregulation of retinoid signaling pathways may play a role in the development of upper aerodigestive tract cancers, including lung cancer. Based on these findings, Kalemkerian and co-workers (1994) evaluated the effects of all-trans-retinoic acid (RA) in an in vitro model of SCLC and found that RA inhibited C-myc expression and induced L-myc expression, resulting in increased neuroendocrine differentiation along with the inhibition of proliferation and ras-induced progression.

ras Oncogenes

The ras oncogenes, H-ras, K-ras, and N-ras, encode for membrane-associated G proteins that link tyrosine kinase growth factor receptors to the downstream cascade of cytoplasmic second-messenger molecules involved in the transduction of the proliferative signal to the nucleus, primarily through the Raf mitogen-activated protein kinase (MAPK) pathway. Normal ras proteins possess GTPase activity and their activation status is dependent on the binding of either GDP (inactive) or GTP (active), allowing for tight control of signal transduction. The ras proteins become oncogenic through point mutations at codons 12, 13, or 61 that ablate GTPase activity, resulting in constitutive activation of ras and unregulated cellular proliferation.

Activating mutations of the ras oncogenes have been identified in up to 30% to 40% of NSCLCs and primarily involve the K-ras gene. Slebos (1991) and Ahrendt (2001) and their colleagues have determined that ras mutations occur almost exclusively in adenocarcinomas from patients with a history of tobacco use. The most common ras mutation in NSCLC, a G-T transversion at codon 12, is the same mutation that You and associates (1989) identified in benzo[a]pyrene-induced murine adenomas. In addition, Feng and co-workers (2002) reported that exposure to benzo[a]pyrene diol epoxide preferentially induced DNA damage at codon 12 of the K-ras gene in human bronchial epithelial cells, further suggesting a direct molecular effect of cigarette smoke in human lung carcinogenesis.

Studies evaluating the clinical implications of ras mutations in NSCLC have yielded conflicting results. The association between the presence of a K-ras mutation and poor prognosis in patients with NSCLC has been reported by several investigators and is most evident in patients with early-stage adenocarcinoma. In a study of 69 patients undergoing curative resections, Slebos and colleagues (1990) reported a 5-year survival of 37% in patients whose tumors exhibited a ras mutation and 68% in those without a mutation (P = 0.002). However, in larger studies, Siegfried (1997a) and Graziano (1999) and their co-workers reported conflicting data suggesting that K-ras mutations did not portend a poorer prognosis in patients with early-stage disease. Although initial studies in animal cells by Sklar (1988) suggested that ras mutations induced resistance to cytotoxic therapy, subsequent work in human NSCLC cell lines by Tsai and associates (1993) failed to identify a correlation between ras mutations and chemosensitivity. In addition, Rodenhuis and co-workers (1997) reported that ras mutations did not correlate with chemoresistance or survival in patients with advanced-stage adenocarcinoma.

Despite the frequency of activating ras mutations in NSCLC, such mutations have not been detected in SCLC cell lines or tumors. This genetic difference between SCLC and NSCLC was exploited by Mabry and colleagues (1988) to develop an in vitro model of phenotypic transition that mimics clinical SCLC progression. In this model, the introduction of activated v-H-ras into SCLC cells that overexpress C-myc or N-myc resulted in transition to a phenotype consistent with large cell carcinoma, suggesting that cooperation between the ras and myc oncogenes may play a role in the progression of SCLC to a more treatment-resistant phenotype.

A variety of pharmacologic and molecular strategies targeting activated ras have been developed in an attempt to inhibit cancer cell proliferation. Ras (and many other signaling proteins) undergoes extensive post-translational modification, including the attachment of a lipid farnesyl group by the enzyme farnesyl transferase. Farnesylation is required for appropriate localization to the plasma membrane and subsequent activation. Numerous farnesyl transferase inhibitors (FTIs) have been developed that can block farnesylation of ras proteins and inhibit the growth of human NSCLC cells and xenografts. Interestingly, although this effect appears to be relatively selective for malignant cells, it is not dependent on the presence of an activating ras mutation, suggesting that FTIs also target other membrane-associated signaling molecules. Preclinical studies by J. Sun (1999) and Adjei (2001) and their colleagues have shown that FTIs can augment the activity of standard chemotherapeutic agents against NSCLC cell lines and xenografts. Several FTIs have entered clinical trials, and three have progressed beyond phase I studies: lonafarnib (SCH66366, Sarasar), tipifarnib (R115777, Zarnestra), and BMS-214662, with several single-agent and combination trials ongoing in patients with NSCLC. Thus far, as reported by Adjei and co-workers (2000) and others, these agents have been well tolerated, and partial objective responses have been noted in a small number of patients with advanced NSCLC.

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A second anti-ras strategy involves the use of ras-specific antisense molecules. Georges and colleagues (1993) demonstrated that the intratracheal injection of a retroviral construct expressing K-ras antisense significantly inhibited the development of tumors in nude mice that had undergone intratracheal instillation of K-ras mutant NSCLC cells. Zhang and associates (2000a) evaluated another gene therapy approach in which intratumoral injection of an adenoviral vector containing an anti-K-ras ribozyme, an antisense oligonucleotide that can enzymatically cleave the target mRNA, induced the regression of K-ras mutant NSCLC xenografts. An H-ras-specific antisense oligonucleotide, ISIS 2503, which has been shown to inhibit the growth of cancer cells harboring H-ras or K-ras mutations, is currently being evaluated in clinical trials in patients with advanced NSCLC.

Several downstream mediators in the ras signaling pathway are also being evaluated as potential targets for anticancer therapy. Raf-1 is a serine/threonine kinase that is a direct substrate for activated ras. In a phase I clinical trial, Stevenson and colleagues (1999) demonstrated that c-raf-1 expression was downregulated in patients with stable disease during treatment with ISIS 5132, a c-raf-1-specific antisense oligonucleotide, but that this effect was lost upon disease progression. Downstream from raf, the MAPK cascade promotes ras-mediated signals. In an attempt to inhibit the ras pathway at this point, H. Y. Lee and associates (2002a) developed an adenoviral vector expressing a dominant-negative mutant MAPK kinase, MKK4. Aerosolized delivery of this agent to lung cancer prone transgenic mice harboring a mutant K-ras gene inhibited ras-dependent signaling and the development of pulmonary adenocarcinomas.

c-jun and c-fos

The AP1 transcription factor, composed of the products of the c-jun and c-fos protooncogenes, stimulates the expression of numerous genes involved in cellular proliferation. As such, inappropriate expression of c-jun or c-fos or both may result in chronic activation of proliferative pathways and neoplastic growth. The expression of both c-jun and c-fos is controlled by the c-jun N-terminal kinase (JNK) family of mitogen-activated protein kinases that are involved in the transmission of growth-promoting signals through a number of signal transduction pathways.

Szabo and colleagues (1996) and others have reported that c-jun is expressed in 31% to 51% of NSCLCs, as well as many bronchial and alveolar preneoplastic lesions, but not in normal lung epithelium. Similarly, Schuette and associates (1988) found that half of SCLC cell lines express c-jun. Wodrich and Volm (1993) and others showed that 41% to 60% of NSCLCs express c-fos, and that the expression of c-fos and c-jun is more common in lung cancers of smokers than nonsmokers. In addition, Heintz and colleagues (1993) reported that asbestos, a known lung carcinogen, induces the expression of c-jun and increases cellular growth in hamster tracheal epithelial cells. These studies suggest that tobacco carcinogens and asbestos may activate c-jun and c-fos, leading to dysregulated neoplastic proliferation.

Recently, Levresse and associates (2002) identified a direct association between c-jun expression and resistance to cisplatin, a cytotoxic agent commonly used in lung cancer, in SCLC cells. Interestingly, exposure to cisplatin stimulated JNK activity, whereas blockade of the JNK/c-jun pathway enhanced the sensitivity of SCLC cells to platinum compounds.

TUMOR SUPPRESSOR GENES

In contrast to protooncogenes that encode positive regulators of cellular proliferation, the prototypical tumor suppressor genes, p53, RB1, and p16, encode negative regulators of the cell cycle. Therefore, the loss or inactivation of tumor suppressor gene function can lead to uncontrolled proliferation and malignant transformation. This loss of function requires that both alleles of the specific tumor suppressor gene undergo genetic or epigenetic alterations resulting in lack of gene expression or loss of protein function. Mechanisms of tumor suppressor gene inactivation include allelic deletions, inactivating mutations, and promoter methylation. Among the known tumor suppressor genes, some are relatively tumor specific (e.g. RB1, retinoblastoma; WT1, Wilm's tumor), whereas others are involved in a wide variety of human malignancies (e.g., p53, p16). Not surprisingly, a number of tumor suppressor genes are inactivated in both SCLC and NSCLC (Table 102-2).

p53 Family

The p53 tumor suppressor gene encodes a transcription factor with diverse biological functions, including the mediation of cell cycle arrest and cell death after DNA damage. The loss of wild-type (normal) p53 activity is the most common genetic abnormality thus far identified in human cancer and can occur through allelic deletions or point mutations, or both, that result in amino acid substitutions that

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alter protein function. Abnormalities in p53 are also the most frequent genetic alterations in lung cancer, occurring in 90% of SCLCs and up to 60% of NSCLCs. These alterations primarily involve inactivating point mutations, but deletions, rearrangements, and splice mutations have also been described by Takahashi and colleagues (1989b) and others. Interestingly, most of the p53 mutations in SCLC are guanine-to-thymine transversions similar to those found in activated ras genes in NSCLC, but distinct from the usual guanine-to-adenine transitions that affect p53 in non-tobacco-related tumor types. Site-specific carcinogen exposure may account for the high incidence of this particular mutation in oncogenes associated with lung cancer. Denissenko and co-workers (1996) reported that benzo[a]pyrene, a major carcinogen in tobacco smoke, preferentially forms adducts at the guanine positions in p53 that are most frequently mutated in human lung cancer. In addition, Ahrendt and associates (2000) found that p53 mutations occur at a much higher frequency in lung cancers from patients with a history of significant alcohol and tobacco use.

Table 102-2. Tumor Suppressor Genes Implicatedin Lung Cancer

  Mechanism Histology
p53 I/D/M S, N
RB1 I S, N
p16 D/M/HM N
APC LOH/HM S, N
hMLH1 LOH/? N
FHIT LOH/HM S, N
D, deletion; HM, hypermethylation; I, inactivation; LOH, loss of heterozygosity; M, mutation; N, non small cell lung cancer; S, small cell lung cancer; ?, unknown.

A functional role for p53 inactivation in the pathogenesis of lung cancer has been suggested by Takahashi and associates (1992), who reported that the transfection of heterologous wild-type p53 suppressed in vitro and xenograft growth of NSCLC cells that lack endogenous wild-type p53. Further support for the role of p53 mutations in the progression of lung cancer comes from a study by Mabry and colleagues (1990) in which the expression of heterologous mutant p53 in a SCLC cell line resulted in increased growth and decreased neuroendocrine differentiation, changes that mimic those occurring during the clinical course of SCLC.

These and other studies suggest that the manipulation of p53 expression or activity is a rational therapeutic strategy. Fujiwara and co-workers (1993, 1994b) have demonstrated that a retroviral vector expressing wild-type p53 can induce cell death in NSCLC cell lines and orthotopic xenografts. Subsequently, Roth (1996) and Swisher (1999) and their associates tested this approach in patients with NSCLC using intratumoral injections of retroviral or adenoviral vectors expressing wild-type p53. These studies demonstrated that this approach was safe, led to the expression of the wild-type p53 transgene in tumor cells, and induced clinical response in a minority of tumors. However, complications arising from viral-mediated delivery systems in other human trials have slowed the clinical development of gene therapy. Recently, nonviral, liposomal delivery systems have been developed that may allow for safe and efficient systemic gene therapy. Ramesh and colleagues (2001) have reported that systemic, liposome-mediated delivery of wild-type p53 in mice with metastatic human NSCLC resulted in intratumoral transgene expression, inhibition of tumor growth, and prolonged survival.

The central role that p53 occupies in the control of DNA-damage-induced cell death suggests that the loss of p53 function could impair the activity of both radiation therapy and chemotherapy. However, in a clinical trial of neoadjuvant chemotherapy prior to resection of early-stage NSCLC, E. A. Johnson and colleagues (2002) failed to find any correlation between p53-positive immunostaining and response to platinum-based chemotherapy. In contrast, Rodriguez-Salas and associates (2001) have reported that p53 overexpression is associated with lower response rates in patients with SCLC. If the inactivation of p53 is associated with drug resistance, then the restoration of p53 function could serve as a useful adjunct to standard chemotherapy. Indeed, Fujiwara and co-workers (1994a) have reported that the infection of NSCLC cells and xenografts with an adenovirus vector expressing wild-type p53 augments the cytotoxic activity of cisplatin. In addition, Nishizaki and colleagues (2001) found that the same wild-type p53 adenoviral vector induced a synergistic response when combined with radiation therapy and docetaxel to treat NSCLC xenografts. Several clinical trials have explored the use of intratumoral injections of an adenoviral vector expressing wild-type p53 along with systemic chemotherapy. In patients with advanced NSCLC receiving combination chemotherapy, Schuler and associates (2001) noted transgene expression in the majority of injected tumors, but no difference in response rates between tumors that had received intratumoral gene therapy and those that had not. Other trials investigating the potential utility of p53 gene therapy in combination with chemotherapy or radiation therapy in patients with lung cancer are ongoing.

An alternate pharmacologic approach, which may avoid the pitfalls and complications of systemic gene therapy, involves compounds that can alter the conformation of mutant p53 proteins, restoring some wild-type activity. Preclinical results with one such agent, CP-31398, have been reported by Takimoto and associates (2002), who found that this compound enhanced apoptotic cell death induced by some chemotherapeutic agents.

Mutant p53 proteins commonly found in cancerous, but not in normal, tissues may be a useful target for immunotherapy, an approach that requires the identification of tumor-specific antigens to serve as targets for immunologic intervention. Winter and associates (1992) reported that 13% of lung cancer patients produced anti-p53 antibodies and that these patients all harbored tumors containing missense p53 mutations. In a study of 170 patients with SCLC, Rosenfeld and colleagues (1997) detected anti-p53 antibodies in the serum of 16% of patients, but this did not correlate with clinical characteristics or survival. Yanuck and co-workers (1993) demonstrated that immunization of mice with mutant p53 protein from a lung carcinoma induced specific CD8++ cytotoxic T lymphocytes that were capable of killing cells expressing mutant, but not wild-type, p53 protein.

The prognostic significance of p53 abnormalities in lung cancer remains controversial. Since the half-life of mutant p53 protein is usually significantly longer than that of the wild-type protein, the detection of p53 by immunohistochemistry is considered a marker for p53 mutation, although Carbone and associates (1994) found that the

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concordance between positive p53 immunostaining and the presence of a p53 mutation was only 67%. Numerous studies have reported that p53 mutation or overexpression is predictive of poor prognosis, whereas others have found no association between p53 mutations and survival in either NSCLC or SCLC. Two recent meta-analyses by Mitsudomi (2000) and Steels (2001) and their colleagues concluded that the presence of p53 mutation or overexpression does correlate with a poorer prognosis in patients with NSCLC. The former analysis noted this association with adenocarcinoma, but not with squamous cell carcinoma.

Recently, two mammalian homologues of p53 have been identified, p51 and p73. Although their exact biological functions have not been elucidated, both p51 and p73, when overexpressed, can bind to DNA, activate transcription, and induce apoptosis in a manner similar to p53. Both genes yield multiple transcripts through alternative splicing, suggesting a complex system of protein regulation. In normal lung tissue, p73 expression is monoallelic, with transcription occurring only off one preferred allele. However, Mai (1998) and Tokuchi (1999) and their colleagues reported increased expression of p73 in most lung cancers relative to normal lung tissue, with frequent biallelic expression. This overexpression, along with the lack of somatic mutations affecting the p73 gene in lung cancers, as reported by Nomoto and associates (1998) and others, suggests that p73 is not a classic tumor suppressor gene, although its dysregulation may still play a role in lung carcinogenesis. Similarly, very few mutations of the p51 gene have been identified in lung tumors. However, Tani and co-workers (1999) found low-to-absent expression of p51 in 23% of lung cancer cell lines, and all of those with high levels of expression preferentially expressed the nonfunctional N isotype that acts as a dominant-negative inactivator of functional p51 protein. These findings suggest that the loss of p51 function may be a relevant factor in the development or progression of lung cancer.

Fig. 102-1. The cell cycle. RB1 protein remains unphosphorylated and bound to E2F transcription factors in quiescent cells (G0/G1). In response to growth stimulatory signals, cyclin D levels rise and cyclin D associated cyclin-dependent kinases (cdk4, cdk6) mediate RB1 phosphorylation and the release of E2F, allowing entry into the proliferative phases of the cell cycle. Further RB1 phosphorylation by cyclin E:cdk2 complexes commits the cell to DNA replication (S phase). RB1 remains phosphorylated until late in G2, when specific phosphatase activity leads to RB1 dephosphorylation. The cdk inhibitors (p16, p21, p27) repress cdk kinase activity, thereby inhibiting cell cycle progression. Adapted from Carbone D, Kratzke R: RB1 and p53 genes. In Pass HI, Mitchell JB, Johnson DH, et al (eds): Lung Cancer: Principles and Practice. Philadelphia: Lippincott-Raven, 1996, p. 107. With permission.

RB Family

The RB1 tumor suppressor gene encodes a 105-kDa protein that plays a central role in the regulation of cell cycle progression (Fig. 102-1). Unphosphorylated RB1 acts as an inhibitor of cellular proliferation by binding to the E2F transcription factor and inhibiting its ability to stimulate the expression of a variety of factors required for progression through the G1/S boundary. The phosphorylation of RB1 by G1 cyclin:cyclin dependent kinase (cdk) complexes, such as cyclin D1:cdk4, results in the release of E2F and the expression of genes required for cell cycle progression. Therefore, the loss of RB1 expression or function or both during carcinogenesis results in uncontrolled proliferation through the loss of a major cell cycle checkpoint.

The seminal finding by Friend and colleagues (1986) that retinoblastoma is caused by the inactivation of both RB1 alleles provided the first evidence for a human tumor suppressor gene and proved Knudson's two-hit hypothesis. The high frequency of cytogenetic abnormalities at the RB1 locus in chromosomal band 13q14 in lung tumors led investigators to evaluate the integrity of RB1 in lung cancer. Over 90% of SCLC cell lines and tumors exhibit defects in RB1 gene expression or protein function. Although Harbour and colleagues (1988) and others have reported that RB1 rearrangements are rare, Yokota and associates (1988) and others have identified loss of heterozygosity at the RB1 locus in nearly all SCLCs. Several groups, including Mori and co-workers (1990), reported that the majority of SCLCs express aberrant RB1 transcripts, resulting in the expression of inactive RB1 protein or no RB1 protein at all. In a large case series, Kleinerman and colleagues (2000) identified an increased risk of lung cancer, predominantly SCLC, in patients who were cured of hereditary retinoblastoma. In addition, Sanders and co-workers (1989) found that heterozygous relatives of patients with retinoblastoma have a markedly increased risk of developing lung cancer, predominantly of the small cell type.

Abnormalities of RB1 expression and function are far less common in NSCLC than in SCLC. In a review, Carbone and Kratzke (1996) noted that 30% to 40% of NSCLC

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samples failed to express functional RB1 protein due to a variety of defects at the DNA, RNA, or protein levels. Shimizu (1994) and Reissmann (1993) and their associates found aberrant RB1 protein expression in 15% of NSCLC cell lines and 32% of tumors, respectively, but both groups failed to identify any correlation between RB1 expression and clinical response or survival. However, Dosaka-Akita and colleagues (1997) reported that the lack of RB1 expression had a significant negative impact on survival in patients with NSCLC whose tumors also expressed either ras or p53. Kratzke (1993) and Ookawa (1993) and their co-workers noted that transfection of wild-type RB1 into RB1-deficient NSCLC and SCLC cells resulted in partial suppression of growth and tumorigenicity. Interestingly, Radulescu and Jaques (2000) have reported that a peptide fragment of RB1 is cytotoxic to both RB1-negative and RB1-positive NSCLC cells.

The retinoblastoma gene family includes two homologues of RB1, p107 and RB2/p130, which are also involved in cell cycle regulation. However, despite strong structural and functional similarities with RB1, the relevance of p107 and RB2/p130 in human cancer remains controversial. Involvement of RB2/p130 in lung carcinogenesis has been suggested by Claudio and associates (2000), who reported mutations of RB2/p130 in 78% of primary lung cancers, and by Baldi and co-workers (1997), who found low-to-absent RB2/p130 expression in 31% of lung tumors. In addition, loss of RB2/p130 expression was associated with higher histologic grade and the development of metastatic disease. Claudio and colleagues (2000, 2001) have also reported that retroviral-mediated transfection of wild-type RB2/p130 into human lung adenocarcinoma cells inhibited growth and tumorigenesis in nude mice, and that intratumoral injection of this vector induced regression of xenografts through the inhibition of vascular endothelial growth factor expression and angiogenesis. However, the role of RB2/p130 remains unclear due to conflicting data, such as those of Modi and associates (2000), who failed to identify inactivation or loss of expression of RB2/p130 in any of 69 lung cancer samples or to identify RB2/p130 mutations in 11 lung cancer cell lines.

p16

Cytogenetic studies have identified chromosomal region 9p21 as one of the most commonly affected loci in human cancers, including lung cancer. The p16 (INK4a, CDKN2A) gene, located at 9p21, has been characterized as a tumor suppressor gene based on the high frequency of p16 mutations and deletions present in a wide variety of human tumors and the presence of germline mutations in families with hereditary melanoma. The p16 protein is one of a family of cdk inhibitors that regulate progression through the cell cycle by inactivating cdk4 or cdk6, thereby inhibiting the phosphorylation of RB1 by cyclin D:cdk4/6 complexes. The maintenance of RB1 in the unphosphorylated state allows RB1 to remain bound to E2F, resulting in cell cycle arrest at the G1/S boundary. Therefore, the loss of p16 function or the overexpression of cyclin D1 would have the same net effect as the loss of RB1 function, namely, unregulated passage through the G1/S checkpoint with resultant neoplastic proliferation. At least one of these three abnormalities loss of RB1, loss of p16, or overexpression of cyclin D1 occurs in most, if not all, human cancers, suggesting that dysregulation of the RB cyclin D cdk4/6 p16 pathway controlling the G1/S transition may be a necessary step during malignant transformation. In NSCLC, Tanaka and colleagues (1998) reported that over 90% of tumors exhibit inactivation of p16 or RB1 or overexpression of cyclin D1.

Loss of p16 expression has been identified in up to 60% of NSCLCs, but only 5% of SCLCs. This low incidence of p16 inactivation in SCLC can be explained by the fact that over 90% of SCLCs have lost functional RB1 and that the additional loss of p16 would offer no further advantage. In support of this theory, Otterson and associates (1994) reported that all six SCLC cell lines that they identified as p16 negative expressed wild-type RB1, whereas all 48 RB1-negative cell lines had detectable levels of p16. In NSCLC, several mechanisms of p16 inactivation have been identified, including homozygous deletion, point mutation, and promoter methylation. Evaluation of a large series of lung cancer cell lines by Kelley and co-workers (1995) identified homozygous deletions of p16 in 23% of NSCLC lines and only 1% of SCLC lines.

Although p16 inactivation is common in all NSCLCs, Sanchez-Cespedes and associates (2001b) found that the mechanism of inactivation differed between smokers and nonsmokers, with deletions and mutations primarily responsible in the former and only promoter hypermethylation being noted in the latter. However, a link between tobacco carcinogens and gene methylation has been suggested by several investigators, including Belinsky and colleagues (1998), who identified p16 methylation in nearly all NNK-induced adenocarcinomas in rats. These investigators also identified an increasing frequency of p16 promoter methylation in progressive premalignant lesions, from 17% in hyperplasia to 24% in squamous metaplasia to 50% in carcinoma in situ. In addition, Kim and associates (2001) found that the incidence of p16 methylation in NSCLC correlated directly with the duration and degree of tobacco use. Dysregulation of the RB cyclin D cdk4/6 p16 pathway appears to be an early event in lung carcinogenesis. Brambilla and co-workers (1999) noted loss of p16 expression or overexpression of cyclin D1 in 12% and 46% of moderate dysplasias and 30% and 38% of carcinomas in situ, respectively.

The clinical impact of p16 inactivation in early-stage NSCLC has been addressed by several investigators. Kratzke and colleagues (1996) and others have reported that the loss of p16 is associated with a poor prognosis, while Kinoshita and associates (1996) found that inactivation of p16 or RB1 was associated with an increased proliferative index in tumors lacking p53 activity. The potential utility of p16 gene therapy has been explored by Shapiro and co-workers (1995),

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who demonstrated that the expression of heterologous p16 resulted in growth inhibition and G1 arrest only in NSCLC cells that expressed RB1. In an alternate therapeutic approach targeting the RB cyclin D cdk4/6 p16 pathway, Soni and colleagues (2001) identified a small molecule inhibitor of cdk4 that inhibited proliferation in cells lacking an intact upstream cell cycle regulatory pathway.

MOLECULAR GENETICS

Cytogenetic Abnormalities

Complex chromosomal alterations have been described in nearly all human malignancies, including lung cancer, suggesting that genomic instability is an inherent characteristic of cancer cells. Flow cytometry studies have revealed broad variations in DNA content, not only between tumors and normal tissue samples, but also between different cells within the same tumor. Approximately 75% of lung cancers have an abnormal number of chromosomes, or aneuploidy, but studies by Bunn and colleagues (1983) and others have failed to identify any consistent associations between aneuploidy and clinical features or survival. Early cytogenetic and RFLP studies identified numerous nonrandom chromosomal abnormalities in both SCLC and NSCLC that included gains or losses of entire chromosomes, unbalanced translocations, and internal and terminal deletions, suggesting the inactivation of unidentified tumor suppressor genes. In addition, chromosomal structures known as double minutes and homogenous staining regions are commonly seen in SCLC and are associated with amplification of gene copy number. A recent genome-wide analysis of lung cancer cell lines by Girard and colleagues (2000) used 399 microsatellite markers to identify common sites of loss of heterozygosity (LOH), indicative of chromosomal deletion. They reported 22 regions that exhibited LOH in over 60% of samples, and noted significant differences in the pattern of loss between SCLC and NSCLC cell lines. Although some of these regions correlate well with known tumor suppressor genes, such as APC (5q21), p16 (9p21), RB1 (13q14), and p53 (17p13), most do not, and further studies will be required to identify candidate tumor suppressor genes at these sites.

Numerous studies have implicated tobacco use as the primary cause of the high incidence of chromosomal anomalies in lung cancer. For example, Sanchez-Cespedes and associates (2001a) reported that chromosomal alterations were widespread in pulmonary adenocarcinomas from smokers, but infrequent in tumors from nonsmokers. In addition, Hirao and co-workers (2001) noted that LOH at a specific locus on 3p was strongly associated with current or former smoking status and the presence of p53 mutation.

Deletions involving 5q13 21 are a common abnormality in lung tumors of all histologic types. The APC tumor suppressor gene, which is involved in familial adenomatous polyposis coli, localizes to 5p21 and regulates cell cycle progression and apoptosis. Virmani and co-workers (2001) have reported LOH of APC in over 80% of SCLC and NSCLC samples. Although APC mutations are rare in lung tumors, Usadel and colleagues (2002), and others, have noted methylation of the APC promoter in nearly all lung cancers, leading to suppression of expression. Interestingly, they also identified APC promoter methylation in the serum of 50% of lung cancer patients, suggesting potential utility as a diagnostic tumor marker.

Center and colleagues (1993) have described cytogenetic abnormalities of 9p in 85% of NSCLCs but in only a few SCLCs. These abnormalities primarily involve the 9p21 22 region that contains the p16 tumor suppressor gene. Deletions involving 18q have been reported frequently in NSCLC, but appear to be rare in SCLC. Shiseki and associates (1994) found that while allelic losses at 3p, 13q, and 17p were common in both early- and advanced-stage NSCLCs, deletions of 18q were significantly more frequent in the latter, suggesting that loss of a tumor suppressor gene in this locus may be involved in lung cancer progression.

3p Deletions

In the 1980s, karyotypic and RFLP analyses demonstrated that interstitial or terminal deletions of 3p occur in all SCLC, and most NSCLC, cell lines and tumors regardless of phenotype or treatment status, with 3p14 23 being the region of common overlap. Normal lymphoid cells from patients failed to reveal any 3p abnormalities, implying that the loss of 3p is an acquired somatic mutation and not a germline anomaly. Recent data from Wu and colleagues (1998) suggest that 3p is particularly sensitive to the mutagenic effects of benzo[a]pyrene diol epoxide (BPDE), a metabolite of tobacco smoke carcinogens, and that BPDE-induced 3p alterations may be a marker for lung cancer risk.

Extensive mapping has identified three regions of 3p that are likely to house tumor suppressor genes: 3p14, 3p21.3, and 3p25. Strong evidence for the presence of at least one tumor suppressor gene on chromosome 3 comes from the work of Satoh and co-workers (1993), who reported that the introduction of a normal chromosome 3 into a lung adenocarcinoma cell line that exhibited loss of heterozygosity on 3p resulted in a complete loss of tumorigenicity. Although the von Hippel-Lindau (VHL) gene is located at 3p25 and its loss is associated with hereditary renal cell carcinoma, VHL inactivation is not a common event in lung cancer. Several other candidate genes have been mapped to the deleted regions of 3p, including retinoic acid receptor- (RAR ), SEMA3B, FUS1 RASSF1, FHIT, and c-raf-1, but thus far, there is no clear consensus on their role as lung cancer tumor suppressor genes.

The c-raf-1 oncogene maps to 3p25 and encodes a serine/threonine kinase involved in ras-mediated proliferative

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signal transduction pathways. Ravi and colleagues (1998) demonstrated that transfection of SCLC cells with an activated form of c-raf-1 induced growth inhibition through cell cycle arrest, suggesting that c-raf-1 may have tumor suppressor activity. However, Sithanandam and co-workers (1989) reported that although all SCLC cell lines examined had lost one allele at the c-raf-1 locus, these cells continued to exhibit c-raf-1 kinase activity. In addition, Pfeifer and associates (1989) demonstrated that cotransfection of c-raf-1 and C-myc into immortalized bronchial epithelial cells results in transformation to a large cell carcinoma phenotype, suggesting that c-raf-1 has the functional characteristics of a protooncogene, rather than a tumor suppressor gene.

The RASSF1 gene product inhibits DNA synthesis and cell cycle progression through downregulation of cyclin D1. Although the RASSF1 locus exhibits frequent LOH in lung cancers, the gene is rarely mutated. Burbee and associates (2001) reported that expression of one of the gene products, RASSF1A, is lost in all SCLC cell lines and most NSCLC cell lines and tumors, and that this loss is usually due to promoter hypermethylation. In addition, exogenous expression of RASSF1A could inhibit proliferation and tumorigenicity.

The FHIT gene encodes an enzyme involved in adenosine metabolism and has been mapped to 3p14.2, a locus that contains the most fragile breakpoint region in the human genome. Sozzi and colleagues (1996) found allelic loss of FHIT in 79% of primary lung tumors and aberrant FHIT transcripts in 80% and 40% of SCLCs and NSCLCs, respectively. In a follow-up study, Sozzi and co-workers (1997) reported that 73% of SCLCs and 39% of NSCLCs also failed to express FHIT protein, and that such loss was also evident in preneoplastic bronchial lesions. Although subsequent studies have confirmed these findings, they have also shown that point mutations in the FHIT gene are rare, that aberrant transcripts are frequently found in normal as well as malignant cells, and that many tumors express wild-type, in addition to aberrant, FHIT transcripts. These findings suggest that FHIT is not a typical tumor suppressor gene. However, Zochbauer-Muller and associates (2001) reported that 37% of NSCLCs exhibit methylation of the FHIT promoter in association with loss of FHIT expression.

Studies to evaluate the functional role of FHIT in lung cancer by Siprashvili (1997) and Otterson (1998) and their colleagues have failed to settle the question. Although both of these studies reported that heterologous expression of FHIT in cancer cells lacking endogenous FHIT expression did not alter cellular proliferation, the former group found that FHIT expression decreased tumorigenicity, whereas the latter group did not. One explanation for these disparate findings would be that although the loss of FHIT expression does not appear to play a role in the dysregulation of proliferation in lung cancer cells, it may be an important factor in tumor progression. In support of this concept, Burke and co-workers (1998) reported that the inactivation of FHIT is an independent poor prognostic factor in patients with NSCLC.

DNA Repair

Repetitive DNA sequences known as microsatellites are distributed throughout the human genome; instability of these microsatellites is a marker for DNA repair defects that are characteristic of many cancer cells. Several genes, including hMLH1 and hMSH2, encode mismatch repair enzymes that maintain genomic integrity. Shridhar (1994) and Merlo (1994) and their co-workers reported that microsatellite instability is present in at least 34% of NSCLCs and 45% of SCLCs, respectively. Wieland (1996) and Chang (2000) and their associates demonstrated that microsatellite instability correlated with LOH at the hMLH locus at 3p21 and loss of hMLH1 expression, suggesting a direct link between 3p deletions and mismatch repair defects, genomic instability, and malignant transformation. Decreased expression of hMLH1 and hMSH2 was identified in nearly 60% of NSCLCs by Xinarianos and colleagues (2000), with a strong association between hMLH1 downregulation and a history of heavy tobacco use. While impairment of some DNA repair mechanisms has been implicated in carcinogenesis, the increased capacity for nucleotide excision repair has been associated with chemoresistance. In patients with NSCLC who had received chemotherapy, Bosken and co-workers (2002) noted that those with higher levels of DNA repair capacity had poorer survival.

GENOMICS AND PROTEOMICS

The technologic advances that now allow researchers to study thousands of genes at one time have recently been reviewed by Mohr and colleagues (2002). DNA microarrays, single chips containing thousands of fixed DNA fragments from specific genes of interest, can be used to evaluate genome-wide mutational or polymorphism status, gene copy number, or gene expression in selected specimens. Gene expression can be determined through the isolation of mRNA from the sample of interest followed by reverse transcription to complementary DNA (cDNA) that is then fluorescently labeled and used to probe a microarray. Differences in gene expression between an experimental sample (e.g., tumor) and a reference sample (e.g., normal epithelium) can be determined by labeling each set of cDNA with a different fluorescent dye and mixing the samples prior to microarray probing. Automated detection of color differences between gene markers can then identify genes that are over- or underexpressed in one sample relative to the other. Complex informatics programs can then cluster samples with similar gene expression patterns.

In lung cancer studies, such differential expression techniques have been used by Gordon (2002) and Bhattacharjee (2001) and their colleagues to differentiate primary lung cancers from mesotheliomas and pulmonary metastases, respectively. Interestingly, in the former study, the differential expression of only four genes, of the 12,000 evaluated, was

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required to differentiate mesothelioma from adenocarcinoma. Thus far, DNA microarrays have been primarily used in lung cancer research to identify prognostic gene expression profiles. For example, Beer and associates (2002) reported a 50-gene expression panel that stratified patients with early-stage, resected pulmonary adenocarcinoma into high-risk and low-risk subgroups. In theory, such prognostic studies, if validated, could be used to select patient populations who may benefit most from adjuvant therapy or to identify molecular targets for novel therapeutic approaches.

In addition to being able to perform genome-wide evaluations of gene expression on the mRNA level with DNA microarrays, investigators can now determine the expression of thousands of proteins in an individual tissue or fluid sample through the rapidly evolving field of proteomics. Several techniques for separating proteins have been developed that rely on differences in molecular weight, ionic charge, and chemical modification. Subsequent mapping of protein expression patterns and identification of individual proteins requires sophisticated informatics systems and mass spectroscopic analyses. Oh and co-workers (2001) have constructed a huge database incorporating data on protein and mRNA expression in lung cancer that can be used to create novel classification schemes and to identify markers for early diagnosis. Toward this end, Chen and colleagues (2002) have begun to identify sets of proteins that are overexpressed in adenocarcinomas. At this time, our understanding of how to harness the power of genomic and proteomic analyses is in its infancy, and the potential utility of this technology in oncology remains to be determined in future studies.

APOPTOSIS

In 1972, Kerr and colleagues coined the term apoptosis to describe the morphologic and biochemical features of one form of physiologic or programmed cell death that plays a central role in a wide variety of physiologic and pathologic processes. The morphologic criteria of apoptosis include membrane ruffling, cytoplasmic shrinkage, chromatin condensation, and nuclear fragmentation into apoptotic bodies. Since the integrity of the cell membrane is maintained, intracellular contents are not extruded and inflammation is not induced. The biochemical features of apoptosis include new protein synthesis, utilization of cellular energy stores, cytoskeletal disruption, and activation of specific proteases and endonucleases resulting in intracellular proteolysis and DNA cleavage.

Apoptosis is a tightly regulated process that can be triggered by a variety of intra- and extracellular signals that activate a final common cell death pathway, as reviewed by Reed (1999). Stimuli that can activate apoptotic pathways include oxidative stress, growth factor deprivation, cytotoxic cytokines, and DNA damage, such as that induced by radiation or chemotherapy. The molecular pathways that regulate apoptosis are highly conserved and involve complex interactions between numerous stimulators and inhibitors, some of which are also mediators of cellular proliferation. Apoptotic stimuli induce the activation of a series of caspases, enzymes that specifically cleave select proteins and are the effectors of the common cell death pathway, either through the release of cytochrome c from mitochondria or through interactions with specific cytokine receptors of the tumor necrosis factor (TNF) family.

The protein product of the bcl-2 oncogene serves as a major inhibitor of the final common apoptotic pathway, in part by blocking the release of cytochrome c from mitochondria. Numerous bcl-2-related genes have been identified that also function as positive or negative mediators of apoptosis. The bcl-x gene encodes two proteins via alternative mRNA splicing. The long-form, bcl-xL, inhibits apoptosis, whereas the short form, bcl-xS, antagonizes bcl-2 and promotes apoptotic cell death. The bcl-2 homologues bax, bad, and bak encode proteins that promote apoptosis through interactions with bcl-2 and bcl-xL. The products of the p53 and C-myc oncogenes can also act as stimulators of apoptosis under a variety of conditions, with p53 inducing apoptosis in the face of irreparable DNA damage through direct regulation of bcl-2 and bax gene expression, as reported by Miyashita and colleagues (1994).

For many years, cancer was viewed primarily as a disease of increased cellular proliferation. However, since the initial discovery of bcl-2 overexpression as the primary cause of follicular B-cell lymphomas, it has become clear that the disruption of normal cell death pathways is also an integral aspect of the carcinogenic process. In addition, the activation of apoptotic pathways has been recognized as an important mechanism by which gamma irradiation and many chemotherapeutic agents exert their cytotoxic activity, and molecular alterations that disrupt normal apoptotic pathways can precipitate resistance to standard anticancer therapy. This novel mechanism of drug resistance has now been demonstrated in numerous experimental tumor models, suggesting that the modulation of apoptotic pathways in tumor cells is a rational strategy for augmenting the effectiveness of cytotoxic therapy, as recently reviewed by Ferreira and associates (2002).

bcl-2 Family

In the normal bronchial epithelium, bcl-2 expression is limited to basal cells, which constitute the renewable stem cell compartment of the pulmonary mucosa. In NSCLC, most studies, including those of Pastorino (1997) and Cox (2001) and their co-workers, have suggested significant bcl-2 expression in 15% to 40% of primary tumors, with a higher rate of positivity in squamous cell carcinoma than in adenocarcinoma. Although Pezzella and colleagues (1993) and others have suggested that the bcl-2 overexpression is associated with improved survival in patients with resected,

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early-stage NSCLC, a large, multivariate analysis of patients with stage I disease by Pastorino and associates (1997) failed to identify bcl-2 expression as an independent prognostic factor. In SCLC, numerous reports, including those by Ikegaki (1994) and Brambilla (1996) and their co-workers, have noted that 70% to 90% of cell lines and primary tumors overexpress bcl-2.

A role for bcl-2 as a mediator of chemoresistance in SCLC cells was suggested by Ohmori and colleagues (1993) in an in vitro transfection model. However, although a clinical study by Takayama and associates (1996) showed that bcl-2 overexpression resulted in increased treatment resistance, a larger analysis by Kaiser and colleagues (1996) failed to identify bcl-2 expression as an independent prognostic factor in patients with SCLC. Among other known apoptotic mediators, Reeve and co-workers (1996) reported that both bcl-xL and bax are overexpressed in most SCLC and NSCLC cell lines relative to normal lung tissue.

These data suggest that the modulation of apoptotic pathways is a rational therapeutic strategy. One promising approach is the use of antisense oligonucleotides that inhibit the expression of apoptotic inhibitors such as bcl-2. Ziegler (1997) and Zangemeister-Wittke (1998) and their associates demonstrated that bcl-2-specific antisense oligonucleotides can inhibit the growth of SCLC cells through the induction of apoptosis and can augment the activity of chemotherapy in SCLC cell lines and xenografts. Similarly, Koty and colleagues (1999) reported the induction of apoptosis by bcl-2-specific antisense molecules in bcl-2-expressing NSCLC cell lines. Subsequently, Zangemeister-Wittke and associates (2000) developed a bispecific antisense oligonucleotide that inhibited the expression of both bcl-2 and bcl-xL, and induced apoptosis in both SCLC and NSCLC cell lines.

In the clinical setting, G3139 (Genasense), a bcl-2-specific antisense oligonucleotide, has demonstrated single-agent activity in patients with B-cell lymphomas, but Rudin and co-workers (2001) failed to note clinical activity in a trial of G3139 plus paclitaxel in patients with refractory SCLC. In an alternate therapeutic strategy targeting apoptotic stimulators, Pearson and colleagues (2000) reported that adenoviral-mediated expression of wild-type p53 in lung cancer cells resulted in the upregulation of proapoptotic bax and bak protein levels and the rapid induction of apoptosis.

Death-Receptor Pathways

Several members of the TNF family of cytotoxic cytokines, such as Fas ligand and tumor necrosis factor related apoptosis-inducing ligand (TRAIL), can induce apoptosis through interaction with specific receptors containing a cytoplasmic death domain. When activated by ligand binding, these death receptors activate caspase-8, which in turn initiates the apoptotic cascade. Inactivation of these receptor-mediated cell death pathways through a variety of mechanisms has been noted in many types of cancer cells. For example, S. H. Lee and co-workers (1999) identified potentially inactivating mutations in the death domain of the TRAIL receptor gene, DR5/TRAIL-R2, in 11% of NSCLCs, and Fisher and associates (2001) reported mutations in the ligand binding domain of DR5 in 40% of lung cancer cell lines and primary tumors. Further disruption of this apoptotic pathway was reported by Shivapurkar and colleagues (2002), who noted loss of expression of caspase-8 in 79% of SCLC cell lines and 35% of SCLC tumors, but not in NSCLC samples. This loss of expression was frequently associated with methylation of the caspase-8 gene promoter. Kagawa and co-workers (2001) demonstrated that adenoviral-mediated transfer of TRAIL resulted in selective apoptosis in NSCLC cell lines and xenografts, but not in normal human bronchial epithelial cells.

The ability of cancers to avoid immune surveillance may also be dependent on the modulation of apoptotic pathways. In T lymphocytes, the binding of Fas ligand to its receptor, Fas, activates cell death pathways and serves as a major negative regulator of the immune response. Recent studies have shown that the expression of Fas ligand by cells in the eye and testis is responsible for maintaining the immune privileged status of these organs. Several reports have suggested that the production of Fas ligand by tumor cells may also protect cancers from immune attack. Niehans and associates (1997) reported that all SCLC and NSCLC cell lines, 80% of SCLC tumors, and 93% of NSCLC tumors express Fas ligand, and that in coculture experiments, Fas ligand producing lung cancer cells could induce apoptosis in human T lymphocytes. In addition, Nambu and colleagues (1998) found that NSCLC cells fail to express Fas, enabling them to resist autocrine-induced Fas ligand mediated cell death.

Inhibitors of Apoptosis Proteins

Inhibitors of apoptosis proteins (IAPs) bind to and inactivate common effector caspases that are involved in chemotherapy-induced apoptosis. Survivin is an IAP that exhibits very limited expression in normal adult tissues but has been found to be expressed in a number of tumor types. In NSCLC, Monzo and co-workers (1999) reported survivin expression in 86% of primary tumors and correlated its expression with poor survival. Olie and associates (2000) identified an antisense oligonucleotide that downregulated survivin, induced apoptosis, and enhanced the cytotoxic activity of etoposide in NSCLC cells.

Telomerase

Telomeres are a series of tandem hexameric DNA repeat sequences at the ends of chromosomes that maintain chromosomal integrity. Telomere length also appears to be an

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important mediator of cellular aging and survival. In normal mortal cells, the telomere shortens with each cellular division, due to the inability of common DNA polymerases to fully replicate chromosomal ends, until a critical length is reached that signals cell cycle arrest and senescence. Cellular immortalization requires the acquisition of the ability to maintain telomere integrity in the face of ongoing cellular division. Telomerase is a ribonucleoprotein consisting of an RNA template, hTR, and a protein reverse transcriptase, hTERT, that can maintain telomere length in long-lived proliferating cells, such as stem cells. Kim and colleagues (1994) and others have clearly demonstrated that most cancer cells express telomerase and that inappropriate telomerase activity plays a significant role in malignant transformation. Hiyama (1995) and Albanell (1997) and their associates have shown that telomerase activity is present in over 80% of lung cancers, but is limited to basal cells in the normal bronchial mucosa. Amplification of the hTERT gene has been identified in over one-third of lung cancers by Zhang and colleagues (2000b). In patients with stage I NSCLC, Wang and co-workers (2002) and others have identified telomerase activity and hTERT expression as independent predictors of poor prognosis.

In clinical studies by Yahata (1998) and Arai (1998) and their associates, the presence of telomerase activity in bronchoscopic washings identified 80% of patients with known lung cancer and was significantly more sensitive than traditional cytology, suggesting a possible role in screening of high-risk populations. Yashima and co-workers (1997) identified telomerase in most preneoplastic lung lesions, ranging from hyperplasia to carcinoma in situ, suggesting that telomerase dysregulation is an early event in lung carcinogenesis. Building on this finding, Soria and associates (2001) reported that treatment of smokers with fenretinide [N-(4-hydroxyphenyl) retinamide], a synthetic retinoid, in a controlled chemoprevention trial significantly decreased hTERT expression in bronchial biopsy samples. The near ubiquitous and relatively selective expression of telomerase in cancer cells makes it an attractive target for anticancer therapy, and a number of novel approaches targeting telomerase activity, such as reverse transcriptase inhibitors and antisense molecules, are currently being evaluated.

GROWTH FACTOR PATHWAYS

Peptide growth factors act through binding to specific membrane-associated receptors. This interaction activates signal transduction pathways involving a multitude of second-messenger molecules that carry growth stimulatory signals to the nucleus, where cell cycle activation results in cellular proliferation. Many growth factor receptors and second-messenger molecules are kinases that phosphorylate specific amino acid residues on downstream mediators, leading to their classification as either tyrosine or serine/ threonine kinases. Phosphorylation generally activates these downstream mediators, resulting in the promulgation of the growth-promoting signal through the cell. Dysregulation of the expression or function of growth factors, receptors, or second-messenger molecules can result in uncontrolled proliferation and malignant transformation. It is now known that there is substantial cross-talk between proliferative and apoptotic pathways in many cells, with the activation of growth factor pathways frequently serving as a survival signal that can inhibit physiologic cell death. Although most growth factors implicated in cancer are stimulators of proliferation, several growth inhibitory factors have also been identified. Growth factors secreted by cancer cells or tumor stromal cells can participate in autocrine or paracrine loops by interacting with specific receptors on their own cell membranes or on the surfaces of nearby cells, respectively. The ability of malignant cells to produce autocrine growth factors plays an important role in the establishment and survival of both primary tumors and metastatic foci.

A wide variety of growth factors and growth factor receptors have been implicated in the pathogenesis and progression of lung cancer (Table 102-3). Many of these are preferentially expressed by either SCLC or NSCLC cells, and their proliferative effects are similarly cell-type specific. The dependence of many cancer cells on the proliferative and survival signals provided by growth factor pathways has led to the development of many targeted therapeutic strategies aimed at blocking specific steps along the signal transduction cascade. Approaches that interrupt the ligand receptor interaction and those that block the enzymatic activity of receptor and nonreceptor kinases involved in growth factor mediated pathways have shown the most promise in inhibiting cancer cell proliferation and survival,

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and several of these approaches have already been proved to be effective against select cancers in the clinical setting.

Table 102-3. Growth Factors and Receptors Implicated in Lung Cancer

Growth Factor/Receptor Histology
Gastrin-releasing peptide/GRP receptor S
Vasopressin/V1 receptor S
Neuromedin B/NMB receptor S
Neurotensin/NT receptor S
Nerve growth factor/NGF receptors S
Insulinlike growth factors/IGF receptors N, S
Vasoactive intestinal peptide/VIP receptor N, S
Opioids-endorphins/opioid receptors N, S
Nicotine/nicotinic receptors N, S
Stem cell factor/SCF receptor (c-kit) N, S
Hepatocyte growth factor/HGF receptor (c-met) N, S
Transforming growth factor- /TGF receptors N, S
Transferrin/TF receptor N, S
Epidermal growth factor/EGF receptor (c-erb-B1) N
Transforming growth factor- /EGF receptor (c-erb-B1) N
Amphiregulin/EGF receptor (c-erb-B1) N
HER2/neu (c-erb-B2) N
Platelet-derived growth factor/PDGF receptor N
Colony-stimulating factor 1/CSF-1 receptor (c-fms) N
N, non small cell lung cancer; S, small cell lung cancer.

Neuropeptides

The proliferation of most SCLC cells is enhanced by a network of autocrine and paracrine pathways involving multiple neuropeptides and their receptors. Gastrin-releasing peptide (GRP) is the mammalian homologue of the amphibian neuropeptide bombesin. Moody and colleagues (1981) were the first to identify the expression of GRP and high-affinity GRP receptors in SCLC, and Sausville and co-workers (1986) later showed that nearly all SCLC cell lines and tumors produce and secrete GRP. Carney and associates (1987) subsequently reported that GRP selectively stimulates the growth of SCLC, but not NSCLC, cells in vitro. Further evidence that GRP is an autocrine growth factor in SCLC came from Cuttitta and colleagues (1985), who demonstrated that an antibombesin monoclonal antibody blocked the GRP GRP receptor interaction, inhibited the growth of SCLC cell lines, and induced regression of human SCLC xenografts. A role for inappropriate GRP expression in lung carcinogenesis was suggested by Willey and co-workers (1984), who found that GRP induces the proliferation of normal human bronchial epithelial cells, and by Aguayo and associates (1989), who identified high levels of bombesinlike immunoreactivity in bronchoalveolar lavage fluid from smokers.

In addition to GRP, the effects of numerous other neuropeptides on SCLC cells have been examined. Although many of these molecules induce biochemical evidence of activation of signal transduction pathways, including intracellular calcium ion mobilization, relatively few have been shown to enhance the proliferation of SCLC cells. Among this latter group are bradykinin, vasopressin, cholecystokinin, gastrin, galanin, and neurotensin. Recently, Missale and associates (1998) reported that SCLC cells can produce nerve growth factor (NGF) and express NGF receptors, and that NGF inhibits both the growth and tumorigenicity of SCLC cells.

The profound impact of neuropeptides on SCLC proliferation has led to the evaluation of neuropeptide antagonists as therapeutic agents. Building on the work of Woll and Rozengurt (1988), a number of investigators have synthesized substance P analogues that can block signaling through multiple neuropeptide pathways and can inhibit basal and neuropeptide-stimulated growth of SCLC cell lines and xenografts. At least one of these compounds, antagonist G, has entered into clinical trials, but the high concentrations required for growth inhibition and the short biological half-life of substance P analogues have hindered their clinical development. Another therapeutic strategy aimed at interrupting autocrine growth loops in SCLC was reported by Kelley and colleagues (1997), who used an anti-GRP monoclonal antibody to induce a complete response in a patient with SCLC. GRP, and many other peptide hormones, is dependent on -amidation for bioactivation. Iwai and associates (1999) took advantage of this required post-translational modification to identify inhibitors of peptidylglycine -amidating monooxygenase that can inhibit the growth of SCLC cell lines. In an approach targeting the GRP receptor, Langer and co-workers (2002) inhibited SCLC proliferation through exposure to antisense oligonucleotides directed against GRP receptor mRNA. Unlike SCLC-specific GRP receptors, bradykinin receptors are frequently expressed on both SCLC and NSCLC cells. Chan and colleagues (2002) have developed a bradykinin antagonist dimer, CU201, that inhibits the growth of both SCLC and NSCLC cell lines and xenografts and acts synergistically with several standard and targeted cytotoxic agents.

Insulinlike Growth Factors

The insulinlike growth factors (IGF-1 and IGF-2) are polypeptides that are closely related to insulin in both structure and biological activity. IGF-1 and IGF-2 are predominantly produced in the liver, but also appear to be expressed by many cell types and to have paracrine functions in most organs, including the lung. IGF activity is regulated by six specific binding proteins (IGF-BPs) that are differentially expressed by SCLC and NSCLC. The ability of IGF-1 to act as an autocrine growth factor in SCLC cell lines and primary tumor cells has been demonstrated by Macaulay and co-workers (1990). However, Quinn and associates (1996) reported that although nearly all SCLC and NSCLC cell lines express IGF-1, IGF-2, and high-affinity IGF-1 receptors, IGF-2 represents the major secreted product, suggesting that the IGF-2/IGF-1 receptor pathway may be most important. In a case control study, Yu and colleagues (1999) found that serum levels of IGF-1, but not IGF-2, correlated directly with the risk of lung cancer, while high levels of IGF-BP3 predicted a lower risk. Recently, Chang and associates (2002) noted hypermethylation of the IGF-BP3 gene promoter and reduced IGF-BP3 expression in most stage I NSCLCs, with a significant correlation between these findings and a poorer prognosis.

The apparent importance of IGF pathways in lung cancer led C. T. Lee and associates (1996) to develop an adenoviral vector containing IGF-1 receptor antisense that has been shown to inhibit receptor expression and proliferation in NSCLC cells and to improve survival in mice with human NSCLC xenografts. Subsequently, H. Y. Lee and co-workers (2002b) used a similar technique to demonstrate that IGF-BP3, delivered via an adenoviral vector, induced cytotoxicity in NSCLC cell lines and xenografts through the inhibition of IGF-1 pathways and the activation of mediators of apoptosis.

Epidermal Growth Factor Receptor

The epidermal growth factor receptor (EGFR), a tyrosine kinase receptor encoded by the c-erb-B1 oncogene, can be

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activated by several ligands, including epidermal growth factor (EGF) and transforming growth factor- (TGF ). Rusch and colleagues (1993) documented the overexpression of EGFR and TGF in over 80% of NSCLCs when compared with normal lung tissue, but did not detect any expression of EGF. Subsequent studies have reported EGFR expression in 80% to 90% of squamous cell lung cancers and 60% to 70% of adenocarcinomas and large cell carcinomas. Moscatello and associates (1998) have identified in NSCLC a truncated form of EGFR, EGFRvIII, that lacks the ligand-binding domain and is thus rendered constitutively active. In contrast to NSCLC, several series have failed to show significant expression of TGF , EGF, or EGFR in SCLC samples. The prognostic value of EGFR remains unclear despite reports that EGFR expression correlates with drug resistance, short survival, and lymph node metastases. Recent data from Brabender and colleagues (2001) suggest that EGFR expression is not an independent indicator of prognosis in NSCLC, but that its effects may be additive with those of HER2/neu.

Evidence that EGFR and TGF participate in an autocrine growth loop in NSCLC comes from the work of Rabiasz and associates (1992), who reported that the growth of NSCLC cell lines is inhibited by monoclonal antibodies directed against EGFR or TGF . In addition, Hamburger and colleagues (1998) showed that TGF enhances tumorigenicity in lung epithelial cells expressing EGFR and p185HER2. These studies have led to the development of numerous therapeutic approaches that target the EGFR pathway in NSCLC, including a variety of monoclonal antibodies and small-molecule tyrosine kinase inhibitors. Kris (2002), Fukuoka (2002), and Perez-Soler (2001) and their colleagues have recently reported that two EGFR tyrosine kinase inhibitors, gefitinib (ZD1839, Iressa) and erlotinib (OSI-774, Tarceva), have activity in phase II clinical trials in patients with relapsed NSCLC, with objective response rates of 10% to 20%, results that are comparable with those of standard chemotherapeutic agents in this setting. Thus far, the anti-EGFR antibodies that have demonstrated the most promise in preclinical and clinical trials are cetuximab (IMC-C225), a chimeric human-murine monoclonal antibody, and ABX-EGF, a fully human monoclonal antibody. In another therapeutic approach, Cristiano and Roth (1996) took advantage of the high expression of EGFR in NSCLC cells by developing a gene therapy delivery system in which the gene of interest is conjugated to EGF, and then showing that this EGF DNA conjugate is specifically taken up by EGFR-expressing cells, resulting in efficient heterologous gene expression.

HER2/neu

The HER2/neu (c-erb-B2) oncogene encodes p185HER2, a transmembrane growth factor receptor with tyrosine kinase activity that is a member of the EGFR family. In breast cancer and other tumors, gene amplification is the primary mode of HER2/neu dysregulation, leading to very high levels of gene expression in a significant fraction of tumors. However, although Kern and colleagues (1990) and others have reported expression of HER2/neu in 30% to 60% of NSCLCs, gene amplification is a rare event and most tumors exhibit modest levels of expression. Several groups have consistently found that p185HER2 expression is rare in SCLC. Studies with immortalized human bronchial epithelial cells by Noguchi and co-workers (1993) and with transgenic mice by Stocklin and colleagues (1993) have suggested that overexpression of HER2/neu may be involved in the development of preneoplastic lung lesions but is not sufficient for malignant transformation.

HER2/neu has become a popular target for novel therapeutic strategies due to reports by Kern (1990), Tsai (1993), Brabender (2001) and their colleagues that identified associations between HER2/neu overexpression and advanced-stage disease, chemoresistance, and poor prognosis in patients with NSCLC. Several preclinical approaches have resulted in growth inhibition or increased chemosensitivity in lung cancer cells, including antisense HER2/neu cDNA constructs developed by Casalini and colleagues (1997) and tyrphostin AG825, a p185HER2-selective tyrosine kinase inhibitor, reported by Tsai and associates (1996). In addition, the significant clinical activity of trastuzumab (Herceptin7), a monoclonal antibody targeting p185HER2, in patients with breast cancer has led to interest in applying this approach to NSCLC. In vitro studies by Bunn and colleagues (2001) and others demonstrated that trastuzumab could inhibit the growth of NSCLC cells that express p185HER2 and could also enhance the cytotoxic activity of standard chemotherapeutic agents. A number of clinical trials are now evaluating the activity of trastuzumab in patients with NSCLC, although the enthusiasm for this approach has been somewhat dimmed by the relatively modest level of expression of HER2/neu in NSCLC in comparison to that found in breast cancer.

Kit and Stem Cell Factor

The c-kit protooncogene encodes Kit, a membrane-associated tyrosine kinase receptor that is expressed in a variety of malignancies. Rygaard and associates (1993a), among others, have reported that over 70% of SCLC cell lines and tumors coexpress c-kit and its ligand, stem cell factor (SCF). Krystal and colleagues (1996, 1997) demonstrated that the coexpression of SCF and c-kit constitutes an autocrine growth pathway in SCLC cells, and that AG1296, a tyrosine kinase inhibitor that is relatively selective for c-kit, induced apoptosis in these cells.

Imatinib (Gleevec), a small-molecule tyrosine kinase inhibitor, has demonstrated dramatic clinical activity in chronic myelogenous leukemia due to its ability to inhibit the activity of the bcr-abl fusion protein. Imatinib also inhibits

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other tyrosine kinases, including Kit, and has been found to be of significant clinical benefit for patients with gastrointestinal stromal tumors, in which the c-kit gene usually undergoes an activating mutation. Krystal and colleagues (2000) reported that imatinib inhibited SCF-induced growth of SCLC cell lines. However, activating c-kit mutations have not been identified in SCLC cells, and the initial clinical trial reported by B. E. Johnson and associates (2002) failed to demonstrate activity in patients with relapsed SCLC.

Protein Kinase C

The protein kinase C (PKC) family of serine/threonine kinases plays a central role in mediating cellular proliferation and differentiation through a variety of signal transduction pathways. The various PKC isoenzymes exhibit complex patterns of tissue-specific expression, and several, including PKC- , have been reported to be highly expressed in lung cancer cells. Theoretically, inhibition of PKC activity should interfere with cancer cell growth in a more global manner than strategies that target specific growth factor pathways and might thereby overcome the tumor cell heterogeneity that limits the clinical activity of many targeted approaches. In addition, Basu and co-workers (1996) demonstrated that PKC can serve as a determinant of sensitivity to cytotoxic agents.

Among the various strategies that have been developed to interfere with PKC function in malignant cells, the selective inhibition of PKC- through the use of ISIS 3521 (Affinitac), a specific antisense oligonucleotide, has shown the most clinical promise. Yuen (2001), Ritch (2002), and Moore (2002) and their colleagues have reported phase I and II clinical trials of ISIS 3521 in combination with standard chemotherapy in patients with untreated or relapsed NSCLC that suggest relatively high response rates. However, randomized phase III trials to determine the specific benefits of ISIS 3521 have not yet been reported.

PI3K/Akt/PTEN Pathway

Phosphatidylinositol-3-kinase (PI3K) is another central second-messenger molecule involved in the transduction of proliferative and survival signals through a variety of growth factor mediated pathways. PI3K activates Akt/protein kinase B, a serine/threonine kinase that blocks apoptotic signaling through phosphorylation and inactivation of bad, an apoptotic stimulator. SCLC cells have been found to have elevated levels of PI3K activity, presumably due to the activation of multiple autocrine growth loops. In SCLC cell lines, Krystal and colleagues (2002) reported PI3K activation through the IGF-1 and SCF/kit pathways, and demonstrated that the inhibition of PI3K or Akt activity resulted in growth inhibition and the enhancement of etoposide-induced apoptosis. The PI3K/Akt pathway also regulates protein synthesis through the activation of mTOR, the mammalian target of rapamycin, and p70S6K. Seufferlein and Rozengurt (1996) found that rapamycin can block p70S6K activation and inhibit SCLC growth in vitro. In a phase I trial, Hidalgo and associates (2000) have noted clinical response in patients with NSCLC treated with the rapamycin analogue CCI-779.

PTEN, the phosphatase product of the PTEN tumor suppressor gene, negatively regulates the PI3K/Akt pathway through dephosphorylation and inactivation of phosphatidylinositol 3,4,5-triphosphate (PIP-3), a product of PI3K. As such, loss of PTEN activity results in upregulation of Akt signaling and unchecked cellular survival and proliferation. Several investigators have reported that genetic alterations of PTEN occur infrequently in SCLC and NSCLC. However, Soria and co-workers (2002) identified loss of PTEN protein expression in 24% of NSCLCs. In 35% of these tumors, the lack of expression was due to aberrant methylation of the PTEN promoter that could be reversed by 5-azacytidine, a nonspecific demethylating agent. Since the loss of PTEN or the overexpression of PI3K-dependent Akt activity can promote survival, Kandasamy and Srivastava (2002) evaluated the effects of modulation of these mediators on apoptotic death-receptor pathways. They found that the pharmacologic inhibition of PI3K, the molecular inhibition of Akt, or the transfection of wild-type PTEN enhanced the sensitivity of NSCLC cells to TRAIL-mediated apoptosis.

Transforming Growth Factor-

The transforming growth factor- (TGF ) family consists of several growth factors and associated receptors with pleiotropic effects on growth and differentiation in a variety of normal and malignant cell types. Jetten and colleagues (1990) have demonstrated that TGF inhibits growth and regulates normal mucoepithelial differentiation in tracheobronchial epithelial cells. Dysregulation of TGF pathways has been implicated in the pathogenesis of lung cancer by the work of Bottinger and co-workers (1997), who found an increased rate of lung tumors after exposure to chemical carcinogens in transgenic mice expressing dominant-negative mutant TGF . Jakowlew and associates (1995) reported that TGF 1 and TGF 2 are both secreted by some SCLC and NSCLC cell lines, that most of these cells express TGF receptors, and that TGF 1 inhibits lung cancer growth. Inoue and associates (1995), and others, have found that high levels of TGF are associated with better survival in patients with NSCLC, while Raynal and co-workers (1997) noted that TGF 1 enhances the apoptotic effects of DNA-damaging agents in NSCLC cells. However, many lung cancers appear to be unresponsive to the inhibitory effects of TGF . Although Norgaard and colleagues (1996) reported that resistance to TGF correlated with the loss of TGF receptor expression in some SCLC

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cells, the cause of TGF resistance in most lung cancer remains unknown.

Hematopoietic Colony-Stimulating Factors

Although many SCLC cells express receptors for hematopoietic colony-stimulating factors (CSFs), very few proliferate in response to granulocyte CSF (G-CSF) or granulocyte-macrophage CSF (GM-CSF). However, Young and associates (1993) reported that GM-CSF enhanced in vitro motility and invasiveness of murine Lewis lung carcinoma cells. Despite the concerns raised by these findings, clinical trials have not revealed adverse consequences resulting from the use of G-CSF or GM-CSF in patients with lung cancer. CSF-1 (macrophage CSF) and its receptor, CSF-1R, the product of the c-fms oncogene, mediate normal macrophage invasiveness. Filderman and colleagues (1992) reported that CSF-1R is expressed on some NSCLC cells and that exposure of these cells to CSF-1 increased invasiveness, suggesting a role for CSF-1:CSF-1R in metastasis. In contrast, Yano and co-workers (1997) demonstrated that transfection of CSF-1 into SCLC or NSCLC cells inhibited invasiveness in vitro and decreased the metastatic potential of xenografts in an organ-specific manner.

Other Growth Factors

Hepatocyte growth factor (HGF), the ligand for the receptor product of the c-met protooncogene, plays a physiologic role in angiogenesis, cell motility, and invasion. In the lung, HGF is primarily expressed by fibroblasts, while the expression of c-met has been demonstrated in nearly all lung cancer cells by Rygaard and colleagues (1993a). Singh-Kaw and associates (1995) reported that HGF induced proliferation, motility, and invasiveness in c-met-expressing lung cancer and normal bronchial epithelial cells, and Siegfried and co-workers (1997b) showed that high tumor levels of HGF were a strong negative prognostic factor in patients with resected, early-stage NSCLC. Significant upregulation of c-met has been noted in ras-transformed cells, and Furge and colleagues (2001) demonstrated that inhibition of Met signaling suppressed ras-mediated tumorigenicity and metastases. In SCLC, Maulik and colleagues (2002) reported that c-met is expressed by most SCLC cell lines and that HGF stimulates cellular motility. In addition, disruption of the c-met pathway by geldanamycin inhibits cell motility and induces apoptosis in some SCLC cells. Further evidence of the potential benefits of the interruption of Met signaling comes from the work of Furge and associates (2001), who noted that the introduction of a dominant-negative mutant met gene resulted in the inhibition of tumorigenicity, invasion, and metastases in ras-transformed cells.

The production of platelet-derived growth factor (PDGF) was demonstrated in most NSCLCs by Kawai and colleagues (1997) and was associated with a poor prognosis. In contrast, PDGF expression has not been found in SCLC cells. Conflicting data regarding the expression of PDGF receptors in NSCLC cells versus tumor stromal cells have raised the question of whether PDGF serves as an autocrine growth factor or a mediator of stromal reaction.

Many investigators have noted that women with lung cancer have a better prognosis than men, even when controlling for other known clinicopathologic prognostic factors. Recently, Stabile (2002) and Mollerup (2002) and their colleagues have reported the expression of estrogen receptors, both ER and ER , in lung cancer cells. Although these receptors were frequently present only in variant forms, Stabile and associates (2002) did demonstrate biological response to -estradiol, confirming the presence of functional estrogen-mediated pathways, which may begin to explain the gender differences in lung cancer survival.

Maneckjee and Minna (1990) reported that some SCLC and NSCLC cells express opioid and nicotinic acetylcholine receptors, and that exogenous opioids inhibited the growth of these cells. In addition, they found that nicotine reversed opioid-induced growth inhibition and suggested that in normal bronchial epithelium the endogenous opioid system may act as a natural tumor suppressor pathway that can be disabled by chronic exposure to tobacco smoke.

ANGIOGENESIS

Angiogenesis, the directed growth of new blood vessels, is a primary requirement for the development of clinically relevant primary and metastatic tumors. Without the ingrowth of new blood vessels to satisfy their nutritional and respiratory needs, tumors are unable to grow beyond 1 to 2 mm3 in size. Angiogenesis requires tumor cells to develop the ability to direct a number of specialized physiologic processes, primarily involving endothelial cells. First, the basement membrane and extracellular matrix around existing blood vessels must be degraded, a task accomplished by an array of matrix metalloproteases. Then, endothelial cells must migrate into the tumor, proliferate, and form new capillary tubes. Throughout these processes, the endothelial cells must receive signals that allow them to survive despite the pathologic milieu into which they are proceeding.

Most human cancers, including lung cancers, secrete a variety of angiogenic factors that drive these processes by stimulating the proliferation, directional migration, and survival of endothelial cells, leading to the formation of a tumor-specific vascular system. The density of vessels within a tumor is generally considered to be a measure of angiogenic, and perhaps metastatic, potential. In early-stage, resectable NSCLC, many, but not all, reports, such as that of Fontanini and colleagues (1997), demonstrated that

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high microvessel density is an independent indicator of metastatic disease and poor prognosis.

Vascular Endothelial Growth Factor

Among the many angiogenic factors that have been implicated in lung cancer, the most potent appears to be vascular endothelial growth factor (VEGF). VEGF can be produced by a wide variety of cells, and its expression is physiologically induced by hypoxia. Binding of VEGF to its tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), which are expressed almost exclusively on endothelial cells, activates signal transduction pathways leading to endothelial cell proliferation and migration. Nor and associates (1999) have also shown that VEGF can serve as a survival factor for endothelial cells through the induction of bcl-2 expression. The majority of lung cancers express VEGF, and this expression has been associated with high microvessel density, hematogenous and lymphatic metastases, and poor prognosis by many investigators. In patients with SCLC, Salven and co-workers (1998) have reported that high plasma levels of VEGF are associated with chemoresistance and poor survival.

The prominent role of VEGF in regulating angiogenesis in a variety of malignancies, including lung cancer, has resulted in widespread interest in the clinical development of antiangiogenic strategies targeting VEGF-mediated pathways. At least four different approaches are being evaluated in clinical trials: anti-VEGF monoclonal antibodies, anti-VEGF receptor monoclonal antibodies, small-molecule tyrosine kinase inhibitors selective for VEGF receptors, and an anti-Flt-1 ribozyme.

Bevacizumab (Avastin) is a humanized, anti-VEGF monoclonal antibody that inhibited endothelial cell proliferation and tumor growth in preclinical models and reduced plasma VEGF levels in early human trials. Data from a randomized phase II trial by D. H. Johnson and colleagues (2001) recently suggested that the addition of bevacizumab to standard combination chemotherapy may improve survival in patients with advanced nonsquamous cell NSCLC. However, these investigators also noted an apparently excessive rate of severe hemoptysis in patients with central squamous cell carcinomas receiving bevacizumab, raising the concern that antiangiogenic interventions may also affect the integrity of established vascular structures. Further clinical trials of bevacizumab in patients with nonsquamous lung cancer are currently under way.

Several antibodies directed against VEGFR-2/Flk-1 have been developed, including DC101, which has been shown by Kozin and colleagues (2001) to inhibit the growth of SCLC xenografts and to potentiate the effects of radiation therapy in this model system. An alternate approach aimed at VEGFR-2/Flk-1 involves selective small-molecule tyrosine kinase inhibitors, such as SU5416, SU6668, and ZD6474, all of which are being evaluated in clinical trials. All of these agents demonstrated significant antiangiogenic and antitumor activity in a variety of preclinical models. VEGFR-1/Flt-1 has also been evaluated as a therapeutic target through the development of Angiozyme, a ribozyme that selectively degrades Flt-1 mRNA. Preclinically, Pavco and associates (2000) reported the inhibition of murine Lewis lung carcinoma growth and metastatic potential by this anti-Flt-1 ribozyme, and phase II trials are under way.

Other Angiogenic Factors

Nearly all NSCLCs express basic fibroblast growth factor (bFGF) and one of its receptors, FGFR-1. Although conflicting data exist, most investigators, including Ueno and colleagues (2001), found that the plasma level of bFGF is not an independent predictor of survival in patients with NSCLC. Interestingly, these investigators also reported that elevated plasma bFGF is associated with a favorable response to chemotherapy and prolonged survival in patients with SCLC. Koukourakis and co-workers (1997) noted high levels of platelet-derived endothelial cell growth factor (PD-ECGF) expression in a third of early-stage NSCLCs, but not in SCLC. In addition, high expression of PD-ECGF was associated with increased tumor vascularity, but not survival.

Tie1 and Tie2 are endothelial cell specific tyrosine kinase receptors that, along with their ligands, angiopoietin-1 and angiopoietin-2, constitute a major pathway regulating embryonic angiogenesis. Takahama and associates (1999) have reported relative overexpression of Tie2 and angiopoietin-1 in many primary NSCLCs. However, Tanaka and co-workers (2002) noted that the expression of angiopoietin-2, but not angiopoietin-1, was strongly associated with aggressive angiogenesis and poor survival, especially in connection with high VEGF expression, in patients with resected NSCLC.

In NSCLC, Arenberg and colleagues (1997) have determined that some members of the CXC chemokine family, such as interleukin-8 (IL-8) and epithelial neutrophil activating peptide (ENA-78), act as angiogenic factors, whereas others, such as interferon- -inducible protein-10 (IP-10), act as antiangiogenic factors. Smith and co-workers (1994) noted that IL-8 is produced by and induces angiogenesis in lung cancer. This work led to a study by Arenberg and colleagues (1996) in which an IL-8-neutralizing antibody decreased angiogenesis and tumor size in NSCLC xenografts. Recently, Yuan and associates (2002) noted a strong association between aberrant expression of p53 and elevated levels of VEGF and IL-8, suggesting a role for p53 in the regulation of angiogenesis.

The disruption of angiogenic pathways has become a popular experimental strategy for inhibiting the growth and spread of many types of cancer. Angiostatin, a plasminogen fragment that potently inhibits endothelial cell proliferation, was reported by Sim and colleagues (1997) to inhibit 90% of Lewis lung carcinoma metastases. Subsequently, Volm and co-workers (2000) noted that 24% of NSCLCs express angiostatin and that patients with angiostatin-producing

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tumors lived significantly longer than those with angiostatin-negative tumors. Suzuki and associates (2002) reported that patients with NSCLC had elevated plasma levels of endostatin, another natural antiangiogenic peptide. Of great interest, Boehm and colleagues (1997) demonstrated that the repetitive administration of a combination of angiostatin and endostatin resulted in sustained regression and prolonged dormancy of Lewis lung carcinoma xenografts. Both endostatin and angiostatin are being evaluated in early-phase clinical trials, and Herbst and co-workers (2002a) have documented inhibition of tumor blood flow, but no clinical responses, in patients receiving recombinant endostatin.

TNP-470 is a novel analogue of fumagillin that inhibits methionine aminopeptidase, an enzyme that is essential for endothelial cell proliferation. In preclinical studies, TNP-470 appears to enhance the antitumor activity of standard chemotherapeutic agents, and promising clinical results with TNP-470 and paclitaxel in advanced NSCLC have recently been reported by Herbst and associates (2002b).

Squalamine is a natural aminosterol isolated from dogfish shark liver that potently inhibits growth factor mediated endothelial cell proliferation and migration through blockade of the NHE3 Na+/H+ exchanger. Schiller and Bittner (1999) demonstrated that squalamine potentiated the antitumor effects of platinum compounds and reduced new blood vessel formation in NSCLC xenografts. Based on these data and other promising preclinical studies, clinical trials evaluating the use of squalamine in combination with standard chemotherapy in patients with advanced NSCLC are under way.

Cyclooxygenase 2 Inhibitors

Cyclooxygenase (COX), the rate-limiting enzyme in the synthesis of prostaglandins, exists in two isoforms, COX-1 and COX-2. COX-1 is constitutively expressed in most tissues and is involved in homeostatic functions, such as gastrointestinal cytoprotection and platelet activation. COX-2 is an inducible enzyme that is overexpressed in inflammatory and neoplastic tissues. Many stimuli, including a variety of growth factors involved in malignant proliferation, can induce COX-2. COX-2 has been shown to affect the development and progression of cancer through a variety of prostaglandin-mediated processes, including increased cellular proliferation and invasiveness, prevention of apoptosis, impairment of antitumor immunity, and induction of angiogenesis.

The overexpression of COX-2 has been documented in the majority of NSCLC tumors, with greatest frequency in adenocarcinoma, by Hida and colleagues (1998) and others, and is commonly found in premalignant bronchial lesions, such as atypical alveolar hyperplasia. Several studies, including one by Khuri and co-workers (2001), have noted a significant association between COX-2 overexpression and shorter survival in patients with early-stage NSCLC. With regard to angiogenesis, Marrogi and associates (2000) reported that COX-2 expression correlated directly with the level of VEGF expression and microvessel density in resected NSCLCs.

The potential role of COX-2 in lung carcinogenesis and progression provides the rationale for the evaluation of COX-2 inhibitors in lung cancer prevention and therapy. Recently, a number of selective COX-2 inhibitors have been developed and are now widely used in patients with inflammatory conditions, such as arthritis. Celecoxib, a selective COX-2 inhibitor, has been approved for use in patients with familial adenomatous polyposis coli, a condition associated with a high risk of colon cancer, based on the reduction of polyp formation. In preclinical studies, Hida and colleagues (2002) demonstrated that COX-2 inhibition suppressed proliferation of NSCLC cell lines and xenografts through the induction of apoptosis and potentiated the activity of several standard chemotherapeutic agents. In xenograft models, COX-2 inhibitors have induced tumor regression in association with decreased VEGF expression and reduced tumor vascularity, suggesting the importance of their antiangiogenic effects. The potential clinical utility of COX-2 inhibitors in the prevention and treatment of lung cancer is currently being evaluated in a number of clinical trials.

PRENEOPLASIA

The seminal work of Saccomanno and colleagues (1974) evaluating sputum cytology in patients at high risk for lung cancer led to the identification of a series of abnormal, but nonmalignant, epithelial lesions occurring predominantly in the central bronchi. Pathologically, these lesions have been categorized as follows: hyperplasia; squamous metaplasia; mild, moderate, and severe dysplasia; and carcinoma in situ, and are associated with a progressively increasing risk of lung cancer, primarily of the squamous cell subtype. Studies of peripheral airways have also identified histopathologic lesions, such as atypical adenomatous hyperplasia (AAH), that appear to be precursors of adenocarcinoma.

Recently, Keith and associates (2000) coined the phrase angiogenic squamous dysplasia to describe highly proliferative premalignant lesions associated with aberrant rearrangement of the epithelial capillary microvasculature that occurred in 34% of high-risk smokers without cancer, suggesting that the activation of angiogenic pathways may be an early step in lung carcinogenesis. Chronic exposure to tobacco smoke results in both overt and subtle derangements in the proliferative capacity of bronchial epithelial cells. J. J. Lee and colleagues (2001) evaluated the expression of Ki-67, a marker for proliferation, in airway samples from current and former smokers and found that Ki-67 expression correlated directly with the degree of metaplasia and the number of packs of cigarettes smoked per day. Interestingly, although the expression of Ki-67 did decrease in former smokers, it was still significantly detectable for over 20 years after smoking cessation even in the absence of histologic epithelial abnormalities.

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The prognostic significance of preneoplastic lung lesions remains controversial because (a) they can be identified in most smokers, even those who never develop lung cancer; (b) even severe dysplasia can spontaneously regress; and (c) most lung cancers arise in histopathologically normal mucosa. In order to more accurately predict a patient's risk of malignant transformation, intense research has been focused on the molecular events that trigger the progression from preneoplastic lesions to invasive cancer. The identification of specific genetic derangements that can define a highest-risk population will facilitate the development of strategies designed to inhibit premalignant progression or detect lung cancer at an early stage.

Chromosomal abnormalities have been identified not only in cells from preneoplastic lesions but also in normal-appearing bronchial epithelial cells from current and former smokers. Sundaresan and co-workers (1992) reported LOH of 3p in most dysplastic lesions from smokers with and without lung cancer, and Hung and associates (1995) identified similar deletions in 76% of hyperplastic lesions. These findings suggest that 3p deletion is a relatively ubiquitous response to lung injury that probably does not correlate with lung cancer risk. Both Mao (1997) and Wistuba (1997) and their co-workers reported that LOH at 3p, 9p, and 17p is common in normal-appearing bronchial cells from both current and former smokers but is not present in airway epithelial cells from nonsmokers. In addition, they found that some of these genetic abnormalities persisted for many years in former smokers despite continued abstinence. Recently, Wistuba and associates (1999) identified LOH at 8p21 23 in the bronchial mucosa of 65% of current and former smokers, with persistence for up to 48 years after discontinuation of tobacco use. Miozzo and colleagues (1996) detected microsatellite alterations in normal-appearing bronchial mucosa from one-third of patients with early-stage NSCLC, further implicating impaired DNA repair as an early event in lung carcinogenesis.

Molecular derangements have also been detected in a wide variety of specific protooncogenes and tumor suppressor genes in preneoplastic bronchial lesions. Sozzi and associates (1991) reported the overexpression of HER2/neu or EGFR in normal bronchial epithelium from one-half of lung cancer patients, while Rusch and co-workers (1995) found that EGFR expression was common in all degrees of preneoplastic lesions. Several studies, as summarized by Vermylen and colleagues (1997), have reported evidence of p53 mutation in most dysplastic lesions but in only a few samples of normal or metaplastic epithelium. Mutations of p53 have also been frequently identified in AAH. These findings suggest that p53 mutation is a relatively late event that may be associated with progression to both squamous cell and adenocarcinoma. Brambilla and co-workers (1998) supported this concept by finding frequent p53 overexpression in premalignant lesions from patients with lung cancer, but not in those from smokers without cancer. These investigators also noted a progressive propensity for increased bcl-2 expression and decreased bax expression along the spectrum of bronchial preneoplasia. The integrity of the Rb pathway in preneoplastic bronchial lesions was evaluated by Brambilla and colleagues (1999), who reported the progressive loss of p16 expression in premalignant lesions in patients with cancer, but not in those without cancer, and progressive overexpression of cyclin D1 in such lesions in patients with and without frank malignancy. In addition, Kersting and co-workers (2000) identified hypermethylation of p16 promoter sequences in cells from sputum and lavage samples from 28% of chronic smokers without cancer. Finally, K-ras mutations were frequently detected in atypical alveolar hyperplasia from patients with adenocarcinoma by Westra and associates (1996), though others have failed to identify K-ras mutations in samples from chronic smokers without cancer. Although current evidence suggests that many molecular derangements can be detected in preneoplastic and normal bronchial epithelial cells from patients exposed to lung carcinogens, the natural history of these abnormalities has not been well defined and their clinical usefulness as predictors of malignant transformation remains unproved.

RETINOIDS

Retinoids are important mediators of normal bronchoepithelial differentiation, and, in animals, retinoid deficiency results in reversible preneoplastic changes. The biologic activity of retinoids is mediated through nuclear retinoic acid and retinoid X receptors, RARs and RXRs, that act as transcription factors when bound to the appropriate ligand. Therefore, alterations in retinoid receptor expression can simulate retinoid deficiency, allowing affected cells to stray from normal differentiation pathways. Gebert and colleagues (1991) and others have demonstrated that most lung cancer cell lines exhibit abnormal expression of the RAR gene, while Xu and associates (1997) found that only 42% of resected NSCLC tumors expressed RAR . In addition, Houle and co-workers (1993) reported that the transfection of RAR into NSCLC cells lacking endogenous RAR expression inhibited growth and tumorigenicity, suggesting that RAR may possess tumor suppressor activity. One mechanism of RAR inactivation was identified by Virmani and colleagues (2000), who noted methylation of the RAR promoter in 72% of SCLC and 41% of NSCLC samples. Of interest, pharmacologic reversal of methylation restored RAR expression.

Other receptor- and nonreceptor-associated defects in retinoid signaling pathways have also been identified in lung cancer cells, resulting in resistance to the growth inhibitory effects of retinoids. For example, Wan and associates (2001) showed that the transfection of RAR or RXR into retinoid-resistant NSCLC cells that already expressed RAR enhanced retinoid-mediated growth inhibition by interfering with the function of the AP-1 transcriptional activating

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complex. In a preclinical model of lung carcinogenesis, S. Y. Sun and co-workers (1999) reported that the retinoid responsiveness of human bronchial epithelial cells decreased as they progress toward malignancy, and that RAR-selective retinoids were more potent inhibitors of bronchial epithelial cell growth than RXR-selective retinoids. Mechanistically, Boyle and colleagues (1999) noted that retinoid-mediated growth inhibition was associated with increased proteolysis and decreased expression of cyclin D1 in human bronchial epithelial cells.

These findings suggest that derangements of retinoid signaling pathways may represent early, reversible events in lung carcinogenesis, and have led to several clinical trials evaluating retinoids for the prevention of lung cancer. As a whole, these studies have been disappointing, with little evidence that retinoids can reverse premalignant changes or reduce the incidence of lung cancer. However, some studies have demonstrated clinical modulation of intermediate endpoints associated with malignancy. In one such study, Soria and associates (2001) reported the downregulation of hTERT, the enzymatic component of telomerase, in the bronchial mucosa of patients treated with fenretinide, a synthetic cytotoxic retinoid.

CLINICAL IMPLICATIONS

Recent advances in our understanding of the cellular and molecular events involved in the pathogenesis and progression of lung cancer will allow the development of rational diagnostic, prognostic, and therapeutic strategies that will favorably affect the overall survival of patients with this disease. The long-term prognosis of patients with SCLC and NSCLC remains poor, with overall 5-year survival rates of only 5% to 10% and 10% to 15%, respectively. Because most long-term survivors initially present with limited-stage SCLC or stage I to II NSCLC, the development of effective early diagnostic strategies will increase the percentage of patients with potentially curable, early-stage disease. However, because many early-stage patients relapse and die of lung cancer despite undergoing potentially curative therapy, it will also be useful to identify prognostic factors that define subgroups of patients with a relatively poor prognosis who would benefit from aggressive adjuvant therapy. In light of the high incidence of primary and secondary resistance to standard cytotoxic treatment in NSCLC and SCLC, respectively, novel therapeutic strategies will be required to improve the clinical outcome of patients with both advanced and early-stage disease.

Molecular Diagnostics

Conventional screening techniques, including chest radiography and sputum cytology, have not improved the mortality rate in lung cancer, and the potential benefit of newer modalities, such as spiral computed tomography, remains to be proved. However, the identification of tumor-specific molecular abnormalities has led to renewed interest in screening using molecular techniques to identify patients with early-stage disease. Mao and colleagues (1994) retrospectively analyzed sputum samples acquired before the diagnosis of NSCLC from 10 patients whose tumors harbored p53 or ras mutations. They reported that the same mutation was detectable in the sputum from 8 of these 10 patients up to 13 months prior to the clinical diagnosis of lung cancer. Similarly, Palmisano and co-workers (2000) reported the detection of aberrant methylation of the p16 or O6 methyl-guanine-DNA methyltransferase promoter in the sputum of all patients with squamous cell lung cancer up to 3 years prior to clinical diagnosis. Others have reported that a wide variety of oncogene and chromosomal abnormalities can be detected in sputum and bronchoalveolar lavage samples from patients with preneoplastic and malignant lung lesions. However, most of these studies were performed retrospectively in small, preselected populations with known bronchial lesions; thus, they do not directly address the utility of such techniques for mass screening.

In a prospective study, Ahrendt and associates (1999) reported that microsatellite alterations, p16 promoter methylation, or p53 or K-ras mutations were detectable in bronchoalveolar lavage fluid from 53% of patients with early-stage NSCLC whose tumors were subsequently found to harbor at least one of these genetic alterations. Although the low sensitivity is disappointing, this study did demonstrate that molecular techniques can prospectively detect tumor cells in patients with peripheral, early-stage NSCLC. The potential lack of specificity is another concern with this screening strategy, since numerous molecular abnormalities, including ras mutation, promoter hypermethylation, and microsatellite instability, have been identified in sputum or bronchial fluid samples from current and former smokers without cytologic or radiographic evidence of malignancy. Clearly, further follow-up is required to determine if these markers are indicative of the individual's risk of developing lung cancer.

The Lung Cancer Early Detection Working Group has been evaluating techniques for staining exfoliated airway cells that may improve the ability to predict the development of lung cancer in high-risk individuals despite normal sputum cytology. Tockman and colleagues (1997) developed an antibody specific for heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 that could be used to detect overexpression of hnRNP A2/B1 in normal-appearing airway cells by quantitative densitometry of immunostained sputum samples from subjects at high risk for lung cancer. In two ongoing prospective clinical studies, sputum has been collected annually from patients with resected stage I NSCLC at high risk for second primary lung cancer and from Chinese miners at high risk for primary lung cancer. In these two studies, 67% and 69% of subjects with overexpression of hnRNP A2/B1 by sputum immunostaining developed

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lung cancer in the first year of follow-up, suggesting that the overexpression of hnRNP A2/B1 could improve the early diagnosis of lung cancer by identifying individuals who may benefit from more intensive radiographic or bronchoscopic screening.

The surprising finding that the blood of cancer patients is enriched for free DNA compared with that of normal controls has led investigators to assess blood samples for cancer-associated genetic and epigenetic alterations that may serve as early markers of malignancy. Sozzi and co-workers (1999) evaluated two microsatellite markers and identified alterations in the plasma of 40% of patients undergoing resection of stage I to III NSCLC. Interestingly, the detection of tumor-specific DNA alterations did not correlate with stage, an important finding when considering the utility of such an approach for screening. Similarly, Esteller and colleagues (1999) detected aberrant promoter methylation in at least one of four selected genes in plasma from 50% of patients with stage I to IIIA NSCLC, and all abnormalities correlated with those found in the resected primary tumor. The findings of these studies offer the hope that effective screening techniques that will dramatically improve the early detection, and thus curability, of lung cancer will be developed in the near future.

Molecular Prognostics

Many studies have identified tumor stage and performance status as the most important clinicopathologic prognostic determinants in patients with lung cancer. Unfortunately, due to the lack of curative therapy for patients with advanced-stage disease, the clinical utility of prognostic factors in this setting remains limited. However, the ability to select early-stage patients at high risk for relapse and death would be useful in planning future adjuvant treatment trials. Throughout this chapter, numerous studies evaluating the prognostic potential of individual molecular markers, such as C-myc, K-ras, and p53, have been discussed. The fact that most of these studies are retrospective analyses of single biomarkers in relatively small cohorts of patients severely limits their clinical applicability.

Two groups recently sought to develop a prognostic model in patients with stage I NSCLC by evaluating a broader panel of biomarkers and clinicopathologic features in a multivariate manner. In one study, Harpole and associates (1995) from Duke identified the presence of symptoms, HER2/neu overexpression, T2 tumor size, vascular invasion, and p53 overexpression as significant negative prognostic factors in 271 patients with stage I NSCLC. In addition, they reported a 5-year survival rate of 72% in patients with tumors that were negative for both p53 and HER2/neu expression, but only 38% in patients with tumors that were positive for both markers. In the second study, Pastorino and co-workers (1997) evaluated multiple clinicopathologic and immunohistochemical parameters, including p53, bcl-2, EGFR, HER2/neu, and vessel density, in 515 patients with stage I NSCLC and identified T stage as the strongest prognostic variable. However, none of the molecular markers emerged as independent predictive factors for survival.

Subsequently, the Duke group developed a nonanatomic, molecular-based prognostic model by evaluating the expression of 10 molecular markers in tumors from 408 patients with resected stage I NSCLC. As reported by D'Amico and colleagues (1999, 2000, 2001), multivariate analysis demonstrated that the expression status of five markers representing disparate molecular processes correlated with recurrence and death: p53 (apoptosis), factor VIII (angiogenesis), HER2/neu (growth regulation), CD44 (cell adhesion), and RB1 (cell cycle regulation). Subsequent studies revealed that the prognostic significance of these molecular markers differed depending on patient gender and tumor histology, and that the expression of some molecular markers was strongly associated with the development of central nervous system metastases, suggesting a possible role for molecular characterization of tumors as a selection criterion for patients who may benefit from further treatment, such as prophylactic cranial irradiation.

The recent development of array technology has likely rendered studies of the prognostic importance of limited immunohistochemical markers obsolete. Because cancer is a complex, dynamic disease, the optimal stratification of prognostic subgroups will rely on the identification of multiple relevant factors through the use of microarray techniques that allow a comprehensive survey of gene and protein expression. Genomic and proteomic technology also hold promise for identifying targets for future therapy and predicting response, as well as overall outcome. By combining these goals, it is possible to envision the development of targeted therapy that can be directed at specific subgroups of patients who will benefit the most from such treatment.

Several groups are developing prognostic models using genomic and proteomic methods that can analyze the expression of thousands of molecular markers at one time. Garber (2001), Bhattacharjee (2001), and Beer (2002) and their colleagues from Stanford, Harvard, and Michigan, respectively, each reported a panel of molecular markers that could stratify patients with early-stage adenocarcinoma of the lung into good and poor prognostic groups based on hierarchical clustering of gene expression data obtained with microarray technology. Although each of these studies was large and utilized similar experimental and informatics techniques, variations in algorithm development resulted in significant differences in the panels of prognostic markers that each group identified. For example, the expression of ornithine decarboxylase was a poor prognostic factor in the Harvard study, but a good prognostic factor in the Stanford study. In addition, among the molecular markers identified by the Michigan group, only one, cathepsin L, matched with those from the Stanford study, and none matched with

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those from the Harvard study. Thus far, none of these models has been adequately validated for practical use, but clinical trials are under way to prospectively evaluate their predictive utility.

Molecular Therapeutics

Insights gained from basic research into the cellular and molecular events involved in the pathogenesis and progression of lung cancer have led to myriad novel therapeutic strategies, many of which have been discussed in this chapter (Table 102-4). Many of these approaches are currently being evaluated in clinical trials, and several have yielded promising early results, as recently reviewed by Dy and Adjei (2002a, 2002b). However, because of the genetic and phenotypic heterogeneity of malignant cells, it is highly unlikely that any one of these strategies will be the final solution for the treatment of lung cancer. It has also become clear that even the most well-designed targeted therapies are not necessarily cancer specific or target specific, and can result in significant unintended toxicity. Nevertheless, it is now reasonable to envision a scenario in which cancer patients will be effectively treated with a specific combination of molecular therapies tailored to the particular molecular derangements present in their tumors.

In addition to having intrinsic anticancer activity, some molecular modalities also potentiate the activity of traditional cytotoxic agents. For example, although the inhibition of bcl-2 activity may not completely eradicate tumors exhibiting bcl-2 overexpression, it can lower the apoptotic threshold of malignant cells and enhance the activity of standard chemotherapeutic agents. Similarly, combinations of molecular treatments can be devised to take advantage of synergistic interactions resulting in complete inhibition of tumor cell proliferation or metastatic potential. Although traditional cancer treatment approaches clearly benefit some patients with lung cancer, most still die within 1 year of diagnosis. Further advances in the clinical management of these patients will depend on our ability to translate our understanding of the biology of lung cancer into effective therapeutic strategies.

Table 102-4. Molecular Therapeutic Approaches in Lung Cancer

Inhibition of growth factor production or availability (e.g., anti-GRP antibodies)
Antagonism of growth factor binding (e.g., neuropeptide antagonists)
Inhibition of protooncogene expression or function (e.g., ras-directed farnesyl transferase inhibitors)
Reconstitution of tumor suppressor gene function (e.g., wild-type p53 gene therapy)
Immunotherapy targeting mutant oncogene or tumor suppressor gene products (e.g., cytotoxic T cells specific for mutant p53 or ras proteins)
Stimulation of apoptotic pathways (e.g., antisense bcl-2 oligonucleotides)
Manipulation of signal transduction components (e.g., tyrosine kinase inhibitors)
Inhibition of angiogenesis (e.g., anti-VEGF antibodies)
Inhibition of invasion/metastatic factors (e.g., metalloproteinase inhibitors)
GRP, gastrin-releasing peptide; VEGF, vascular endothelial growth factor.

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Fleming MV, et al: Bcl-2 immunohistochemistry in a surgical series of non-small cell lung cancer patients. Hum Pathol 29:60, 1998.

Volm M, et al: Prognostic value of basic fibroblast growth factor and its receptor (FGFR-1) in patients with non-small cell lung carcinomas. Eur J Cancer 33:691, 1997.



General Thoracic Surgery. Two Volume Set. 6th Edition
General Thoracic Surgery (General Thoracic Surgery (Shields)) [2 VOLUME SET]
ISBN: 0781779820
EAN: 2147483647
Year: 2004
Pages: 203

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