Molecular Prognostication of Lung Cancer
Betty C. Tong
David H. Harpole Jr
At present, the best prognostic indicator of long-term survival for patients with lung cancer is tumor stage. The recent proposed revisions to the TNM Classification of Malignant Tumours by the International Association for the Study of Lung Cancer (IASLC) are an analysis of 67,725 cases of non-small cell lung cancer (NSCLC) to further refine the prognostic accuracy of tumor staging (see Chapter 30).1 Regrettably, however, up to 30% of patients who undergo curative resection for stage I lung cancer will have recurrence of their disease. Present conventional wisdom is that the long-term survival in many cancers may be increased if clinicians had the means to identify and treat patients who would benefit from adjuvant therapy that might not otherwise be indicated based on their initial tumor stage. To this end, many investigators have examined molecular and genetic factors that may influence tumor behavior and therefore long-term prognosis.
A number of genetic alterations and abnormal expression of several regulatory genes have been detected and described for lung cancer. These alterations are caused by gene mutation, chromosomal modification, epigenetic silencing, and deregulated messenger RNA (mRNA). In addition, several studies have correlated specific molecular genetic changes with clinical prognosis and survival for patients with lung cancer. Currently, several clinical trials are underway to further define patients’ molecular “signatures” in an effort to predict both overall prognosis as well as response to therapy for lung cancer (http://www.clinicaltrials.gov).
Early studies attemping to comment on prognostic variables in lung cancer are summarized in a comprehensive and systematic review of 887 studies, which identified 169 host-and tumor-related molecular factors associated with prognosis in lung cancer.2 Several factors were significantly associated with survival, independent of stage and reported in more than three studies: p53, p21, Ki-67, and p185 gene status; serum cytokeratin 19 fragments; agryophilic nucleolar organizer region; and markers of angiogenesis such as vascular endothelial growth factor (VEGF) expression and vessel invasion. Other studies have used molecular, immunohistochemical, and clinical-pathologic markers to predict patient prognosis and outcomes.3,4,5
The subsequent development of oligonucleotide and complementary DNA (cDNA) arrays to analyze gene expression on a larger scale has contributed significantly to the understanding of molecular and genetic alterations in lung cancer. These tools allow for the simultaneous analysis of literally thousands of genes such that a genetic profile or signature can be constructed for a particular patient or tumor. In addition to genetic profiling, investigators have identified several proteomic (see Chapter 9) and microRNA (miRNA) profiles for lung tumors. These profiles are correlated to clinical behavior, thereby providing prognostic information for patients with lung cancer.
INDIVIDUAL GENE ALTERATIONS AND PATIENT SURVIVAL
K-ras Members of the ras gene family encode cell membraneassociated G-proteins, which serve as mediators of signal transduction for cellular proliferation (also reviewed in Chapter 5). Up to 30% of NSCLCs are characterized by mutations in the k-ras gene6,7; most of these mutations are found in adenocarcinomas and are associated with a history of tobacco use.8 The most common mutation of k-ras is a G→T transversion in codon 12 that results in constitutive activation and continuous transmission of growth signals to the nucleus. Alterations in k-ras appear to be early events in lung carcinogenesis, having been observed in atypical alveolar hyperplasia lesions that are thought by many to be precursors of lung adenocarcinomas.9 The prognostic significance of k-ras mutations in lung cancer remains controversial. Although many studies report an association between decreased survival and worse prognosis in patients whose tumors exhibit k-ras mutations, others, including a metaanalysis of 881 cases, report no significant link between ras mutation status and prognosis.10,11,12,13
p53 The p53 tumor suppressor gene is mutated in more than half of all human malignancies, and alterations in the p53 gene are the most frequently found in human cancer (also
reviewed in Chapter 5). Approximately 50% of NSCLCs and over 90% of small cell lung cancers (SCLC) harbor mutations or deletions of the p53 gene.10,14 Inactivation of p53 results in diminished efficiency of DNA repair, derangements of cell cycle regulation, and overall increased genomic instability.15 In the normal state, the p53 network is quiet and senescent. In times of cellular injury or stress, however, the p53 network is activated and its downstream effects include cell cycle regulation, induction of apoptosis and DNA repair mechanisms.
reviewed in Chapter 5). Approximately 50% of NSCLCs and over 90% of small cell lung cancers (SCLC) harbor mutations or deletions of the p53 gene.10,14 Inactivation of p53 results in diminished efficiency of DNA repair, derangements of cell cycle regulation, and overall increased genomic instability.15 In the normal state, the p53 network is quiet and senescent. In times of cellular injury or stress, however, the p53 network is activated and its downstream effects include cell cycle regulation, induction of apoptosis and DNA repair mechanisms.
A prospective study by Ahrendt et al.16 demonstrated that p53 gene mutations were independently predictive of decreased survival in stage I tumors but not in stage II or III tumors. Missense mutations were not significant for patient outcome. However, p53 mutations that were truncating, structural, or those abolishing DNA contact were associated with a poorer overall patient outcome among all samples. The relationship between p53 mutational status and adverse survival outcomes has been corroborated by several other studies incorporating NSCLC samples from all tumor stages.17,18,19,20,21,22,23,24,25
Immunohistochemical studies of p53 have been less consistent. In the largest studies examining p53 expression levels, some authors have reported a correlation between abnormal p53 expression and poor prognosis; however, others report no statistically significant relationship.3,26,27,28,29,30,31,32,33 Carbognani et al.34 examined the role of p53 status in long-term survival following resection of NSCLC. Using immunohistochemical analysis of several prognostic markers, p53 status was the only independent predictor of 10-year survival following resection of adenocarcinoma. In another study, Tsao et al.13 observed that p53 protein overexpression was a marker of poor prognosis and shorter overall survival. In addition, patients with tumors containing wild-type p53 had a survival benefit from adjuvant chemotherapy as compared to those with functionally aberrant p53 status. Despite the mixed evidence from immunohistochemical studies, however, a metaanalysis of 56 studies was conducted to further investigate the role of p53 alterations and lung cancer.35 Abnormal p53 status was associated with decreased overall survival in patients with NSCLC across all stages and in both squamous cell and adenocarcinoma histologies.
Cell Cycle Regulation
Rb and p16 The retinoblastoma (Rb) susceptibility gene is a tumor suppressor gene with a key role in human carcinogenesis (also reviewed in Chapter 5). The Rb gene is inactivated in 20% to 30% of NSCLCs and up to 90% of SCLC.10 Despite this, the effect of Rb mutation or abnormal expression on patient prognosis is controversial, with most studies demonstrating no significant relationship between Rb abnormalities and survival.27,36,37,38
However, Burke et al.39 recently demonstrated that the additive effect of concurrent abnormalities in either or both of the Rb and p53 pathways was predictive of patient prognosis in NSCLC. In this study, there was no association between patient survival and isolated abnormalities of the Rb pathway proteins pRb, cyclin D1, and p16INK4A and p53 pathway proteins p53 and p21Waf1. However, certain combinations of abnormalities were predictive of poor prognosis. These included concurrent pRb negative status and cyclin D1 overexpression; concurrent pRb negative, cyclin D1 overexpression, and p53 mutation; concurrent cyclin D1 overexpression and p53 mutation.
The p16INK4A gene, located on chromosome 9p21, is a tumor suppressor gene that encodes a cyclin-dependent kinase (CDK) inhibitor (see also Chapter 14). Normally, p16 binds to the cyclin D/CDK4/6 complexes to inhibit phosphorylation of the Rb protein, thereby inhibiting G1→S progression. In a recent analysis of tumors from patients with histologically proven N2 NSCLCs, the immunohistologic presence of both p16 and p21 protein correlated with improved long-term survival.40
Similarly, dysfunctional or absent p16 expression can result in unchecked progression through the cell cycle. p16 plays a prominent role in NSCLC; inactivation is present in 40% to 70% of NSCLCs. Mechanisms of p16 inactivation include point mutations or deletions in coding regions, as well as epigenetic silencing by hypermethylation of the gene promoter cytosine-guanine-phosphate (CpG) island. Alteration and inactivation of p16 are associated with a number of clinical correlates in NSCLC, including metastases, poor prognosis and overall decreased survival.41,42,43,44
The cyclins, p21WAF1/CIP1, and p27 Other cell cycle regulatory genes of interest include cyclin D1, cyclin E, cyclin B1, p21WAF1/CIP1, and p27. Cyclin D1 plays a role in cell cycle regulation by allowing transition from G1 to S phase. Although overexpression of cyclin D1 occurs in 25% to 47% of NSCLC, its prognostic effects are somewhat controversial. In some studies, overexpression has been correlated with the presence of lymph node metastasis, advanced pathologic stage, and shorter overall survival.38,45 However, other investigators have reported favorable outcomes associated overexpression of cyclin D1.5,46
Cyclin E helps to regulate entry into the S phase of the cell cycle by formation of a complex with CDK2 and subsequent phosphorylation of pRb. High levels of cyclin E expression in NSCLC are found in up to 53% of NSCLCs, and have been correlated with tumor invasion, unfavorable prognosis, and decreased patient survival.47,48 The cyclin B1/CDC2 complex regulates the G2-M phase checkpoint of the cell cycle. In early stage NSCLCs, overexpression of cyclin B1 occurs more commonly in tumors of squamous histology, and high levels of expression have been linked to shorter survival.49
p21 and p27 belong to the Cip/Kip family of CDK inhibitors, which bind to and inactivate CDKs in times of cellular stress, hypoxia, DNA damage, and in response to growth inhibitory signals (also reviewed in Chapters 5 and 14). p21WAF1/CIP1 can inhibit cell cycle progression at multiple sites. Early in G1, p21WAF1/CIP1 binds to the cyclin D/CDK4 and cyclin E/CDK2 complexes. Prior to transition from S phase to G2, p21 WAF1/CIP1 can inhibit the cyclin A/CDK2 complex. Although some authors determined that p21 expression was associated with improved survival, others found no relationship.50,51,52 p27Kip1 interacts with both cyclin D1 and cyclin E to regulate the cell cycle. Several studies have
employed immunohistochemical techniques to determine p27 expression; decreased levels of p27 expression have been uniformly correlated with poor prognosis in NSCLC.53,54,55
employed immunohistochemical techniques to determine p27 expression; decreased levels of p27 expression have been uniformly correlated with poor prognosis in NSCLC.53,54,55
Protein Kinases
EGFR The epidermal growth factor receptor (EGFR) family (also reviewed in Chapters 5 and 49) includes a group of tyrosine kinases whose activation results in a cascade of downstream signals that ultimately enhance cellular proliferation, tumor cell motility and angiogenesis, and decrease apoptosis.56 Although EGFR is overexpressed in many epithelial cancers, including 40% to 80% of all NSCLCs, these aberrations are rare in SCLC.57 Downstream targets of EGFR activation include the ras and raf pathways that directly regulate gene transcription and cellular proliferation. Another gene targeted by EGFR activation is the serine threonine kinase Akt, which acts as a key regulator of cellular survival through suppression of apoptosis.58
EGFR mutations in lung cancer are associated with nonsmokers, women, patients from East Asian countries, adenocarcinoma histology, and, specifically, bronchoalveolar subtype.59,60,61,62,63 In addition, tumors with k-ras mutations (associated with tobacco exposure) and those with EGFR mutations appear to be mutually exclusive.60,63 It was initially thought that EGFR tyrosine kinase inhibitors such as erlotinib and gefitinib might revolutionize the treatment of patients with overexpression of EGFR in NSCLC. However, clinical studies have demonstrated significant responses in only specific subsets of patients, limiting gefitinib to use as a second-or third-line agent and erlotinib as a first-line agent specifically for elderly patients and those with EGFR mutations.64,65
The impact of EGFR mutation and overexpression on lymph node metastasis, patient prognosis, and overall survival is controversial. Gene amplification often occurs with EGFR overexpression (as opposed to transcriptional or translational modification), and this has been associated with lymph node metastasis and advanced pathologic stage.66 Although many have found that EGFR mutation and overexpression correlates with worse survival in NSCLC, other studies report no significant association between the two.59,63 A recent metaanalysis of 16 studies found that immunohistologic expression of EGFR does not correlate with overall prognosis in patients with NSCLC.67
ErbB2/HER-2/neu Another member of the protein kinase gene family is ErbB2/Her2/neu. Screening studies for mutations in the kinase domain of ErbB2/Her2/neu in NSCLCs have revealed that mutations in squamous cell carcinomas are rare, but found in approximately 10% to 30% of adenocarcinomas. 68,69,70 As with mutations of EGFR, mutations of ErbB2/ Her2/neu are more common in nonsmokers than in smokers.63 ErbB2/Her2/neu overexpression has been associated with early tumor recurrence, chemotherapeutic drug resistance, poorer prognosis, and overall shorter survival time.71,72,73,74
Angiogenesis and Growth Factors (see also Chapters 8 and 48) For tumors to grow, they must obtain oxygen and nutrients. Tumors greater than ∽1 mm in size cannot depend on simple diffusion and therefore must create a vascular supply to meet these metabolic demands. VEGF is a potent growth factor for endothelial cells, promoting angiogenesis by increasing vascular permeability and stimulating endothelial cell proliferation. The VEGF receptors, VEGFR-1, -2, and -3, are tyrosine kinases. VEGF expression has been demonstrated in NSCLCs and is stimulated by tissue hypoxia, other growth factors, and cytokines.75,76
The presence of VEGF in NSCLC tumors of all stages has been uniformly correlated with poorer prognosis and impaired survival.77 Bevacizumab (Avastin) is a humanized monoclonal antibody that binds to circulating VEGF and inhibits its interaction with the VEGF receptors. The Eastern Cooperative Oncology Group (ECOG) phase III trial E4599 demonstrated an overall survival benefit for patients with advanced stage adenocarcinoma who received bevacizumab in addition to paclitaxel and carboplatin.78 Another phase III trial, the European AVAstin in Lung cancer (AVAiL) trial, demonstrated a favorable progression-free survival for patients with nonsquamous NSCLC receiving bevacizumab in addition to cisplantin and gemcitabine.79 In addition to other studies of bevacizumab in NSCLC, several multitargeted tyrosine kinase inhibitors are under clinical investigation. Targets of interest include several VEGF receptors, EGFR, platelet-derived growth factor (PDGF), raf, and kit.
Interleukin-8 (IL-8) also has angiogenic properties in NSCLC.80 IL-8 expression in tumors has been correlated not only with angiogenesis and microvessel density, but also with advanced stage, lymph node metastasis and overall patient prognosis.81 Other growth factors of interest include PDGF and basic fibroblast growth factor (bFGF). PDGF increases DNA synthesis, tumor growth, and endothelial cell migration; it has been correlated with decreased 5-year survival for patients with resected primary lung adenocarcinomas.77,82 FGF2 stimulates tumor growth and angiogenesis, and in vitro studies have established a synergistic effect of FGF2 and PDGF.83
The Matrix Metalloproteinase Family The matrix metalloproteinase (MMP) family is a group of proteolytic enzymes associated with degradation of extracellular matrix and penetration of basement membranes, two key elements in the metastasis of tumors. MMP-2 (also known as gelatinase A) has been associated with lymphatic and vascular invasion of NSCLC.84 Overexpression of MMP-2, as measured by immunohistochemical analysis, has been identified as a negative prognostic factor in lung cancer survival.85 Similarly, differential levels of MMP-7 expression were found between resected squamous cell carcinomas and adenocarcinomas, with higher levels in the squamous cell carcinomas.86 MMP-7-positive status was significantly associated with poor prognosis and shorter overall survival. In contrast, the data regarding MMP-9 are controversial. Although some studies suggest a negative prognostic influence of MMP-9, others have found no significant relationship between the two.87,88,89
Recently, Sienel et al.90 described a role for the extracellular matrix metalloproteinase inducer (EMMPRIN) in determining prognosis for lung adenocarcinoma. EMMPRIN is a transmembrane glycoprotein that has been shown to stimulate synthesis of several MMPs, including MMP-1, -2, -3, and -9. EMMPRIN expression was determined in a cohort of NSCLCs using immunohistochemical staining, and a score was assigned to each specimen. Furthermore, investigators recorded either a membranous or cytoplasmic pattern of staining. For patients with adenocarcinoma, a membranous staining pattern was independently associated with poor prognosis, defined as either local recurrence or distant metastasis. These relationships were not significant for other histologic subtypes.
Maspin is a member of the serpin (serine protease inhibitor) family and has been shown to be a suppressor of tumor growth and metastasis in several types of tumors. Maspin can inhibit invasion and metastasis of malignancies, although direct evidence of the clinicopathologic significance of cytoplasmic relative to nuclear expression is limited. Cytoplasmic and nuclear expression patterns of maspin are involved in the cellular differentiation of normal lung tissue and the histogenesis of different lung carcinomas. The cytoplasmic maspin may play an important role in lung carcinomas by regulating apoptosis and thus is a favorable prognostic marker for AD patients, whereas the nuclear location may be linked to promotion of angiogenesis. Immunohistochemistry reveals that maspin expression is virtually universal in NSCLC, but squamous cell carcinoma show almost exclusively a combined nuclear-cytosolic stain. In contrast, nuclear maspin, but not combined nuclear-cytoplasmic maspin, significantly correlates with low histological grade, lower proliferative rate, absence of invasion, and negative p53 stain in ACa. Nuclear localization of maspin may thus stratify subtypes of NSCLC with favorable clinical-pathological features.91,92,93,94,95
GENE EXPRESSION ARRAYS
The development of gene expression microarrays has enabled investigators to move beyond analysis of single genes and not only explore patterns or profiles of gene expression in tissues but also compare these patterns between tissue types. These gene expression profiles have been shown to be consistent between institutions, with good comparability of sample characteristics. 96,97 Examining the expression profiles of tumors allows for novel identification of genes previously not associated with malignancy. In addition, comparing expression profiles between groups of patients has enabled investigators to perform molecular phenotyping and classification of tumors. Unique and characteristic profiles have been identified not only for the histologic types of NSCLC, but also for subgroups within these histologic classes.98,99,100,101
There are other potential uses for data acquired from microarray-derived gene expression profiles. Correlating these profiles with defined tumor behavior may help to elucidate specific processes or pathways involved in carcinogenesis. For example, profiling invasive and metastatic tumors may reveal novel genes or pathways of interest that could subsequently be targeted for anticancer therapeutics. Also, these methods may be used to investigate a tumor’s response (or nonresponse) to therapy, elucidating possible mechanisms of drug resistance and providing a basis for predicting future clinical behavior (see Chapter 47).
Gene expression profiles have also been used to refine and predict prognosis and survival among patients with identical TNM staging but differing clinical outcome,102,103 and is the focus of this chapter. Other uses include identification of novel biomarkers associated with and specific to lung tissue and/or NSCLC.104 In the current era of molecular therapeutics, there is potential to use these gene expression profiles and biomarkers not only to classify tumors with molecular staging techniques, but also to identify targets for therapy and treatment. As these techniques continue to develop, it is possible that lung cancer staging, and therefore treatment, will depend not only on TNM status but also on the genetic profiles generated by these methods.
Lung Cancer Heterogeneity Clinically, lung cancer is classified as small cell (SCLC) and non-small cell (NSCLC), with NSCLC accounting for approximately 80% of all lung cancers. Within each histologic subtype, there is significant heterogeneity such that NSCLCs are further classified as adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and neuroendocrine carcinoma as well as tumors with mixed histology such as adenosquamous tumors.105 Among adenocarcinomas, further variation is present in acinar, papillary, bronchoalveolar carcinoma (BAC), and mucinous carcinoma subtypes. For example, BAC appears to arise from type II pneumocytes and is generally associated with better prognosis compared with invasive adenocarcinomas.106
The heterogeneity among primary lung tumor subtypes likely reflects the potential cell derivation, and these differences may be further increased by the diverse genetic alterations observed in lung cancers.107,108,109,110 Additional tumor heterogeneity may be a result of alterations in gene expression that affect diverse processes such as proliferation, apoptosis, and cellular differentiation, among others.111,112 To this end, gene expression profiling methods have been employed to better understand this heterogeneity and to identify specific pathways or genes that might distinguish tumors of different cellular origin or clinical behavior.
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