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Department of Surgery, Duke University Medical Center, Durham, North Carolina
* Address correspondence to Dr D’Amico, Duke University Medical Center, Box 3496, Durham, NC 27710 (Email: damic001{at}mc.duke.edu).
Presented at the Minimally Invasive Thoracic Surgery Summit, New York, NY, June 8–9, 2007.
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Abstract |
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 Top Abstract IntroductionMolecular Staging of the...Specific Mechanisms for...Genomic Analysis of Non-Small...Molecular Staging: Limitations...SummaryReferences |
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Introduction |
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TopAbstractIntroductionMolecular Staging of the...Specific Mechanisms for...Genomic Analysis of Non-Small...Molecular Staging: Limitations...SummaryReferences |
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Inaccurate staging negatively affects outcomes of patients with lung cancer. When the stage is underestimated, patients do not receive the benefit of induction or other systemic therapy, or may have futile procedures; when stage is overestimated, patients may be denied surgical resection or other curative procedures. Incongruent stage groupings also weaken the power of clinical trials to demonstrate benefit.
The International Association for the Study of Lung Cancer (IASLC) has recently endorsed proposals for amendment of the current TNM staging system based on an international database of more than 100,000 patients [3–5]. To summarize, the proposals recommend the following new subclassifications:
Several reclassifications have also been endorsed:
The inclusion of these proposals awaits adoption by the publication of the forthcoming 7th edition of the TNM Classification For Malignant Tumors by the American Joint Commission on Cancer (AJCC).
The power of these large databases in predicting prognosis is self-evident; nevertheless, there is an inherent inaccuracy of this staging process. According to the TNM system, the predicted 5-year survival after complete resection for T1 N0 M0 NSCLC (stage IA) is only 67% [2–5]; therefore, 33% of patients with stage IA NSCLC are incorrectly staged at presentation. Even with optimal therapy, these patients will succumb to their disease, predominately from the development of metastatic disease not detected at the time of diagnosis and initial therapy, despite the use of standard staging procedures [6]. Similarly, a significant fraction of all patients with Stage Ib or II disease are incorrectly staged, resulting in inaccurate assessment of extent of disease, prognostic stratification, and selection of therapy. Adjuvant chemotherapy has currently been established as beneficial for selected patients with after complete resection [7–9]; however, most patients will not benefit from its administration: substantial fractions will die despite chemotherapy or would have survived even without chemotherapy.
Molecular biologic staging refers to the assessment tumor markers associated with various oncogenic mechanisms to improve the risk stratification provided by conventional TNM staging. Biologic staging may target oncogenes, oncogenic protein products, growth factors, or receptors. The biologic techniques used include analysis of DNA, RNA, or protein products. Molecular biologic staging may potentially be applied to the primary tumor, lymph nodes, bone marrow, or serum, to establish the diagnosis of malignancy at earlier stage, to assess prognosis, to detect occult metastases, to select therapy, and to predict chemotherapy sensitivity or resistance.
The purpose of the assessment of prognostic markers in the primary tumor of those with early-stage disease is to identify patients, or groups of patients, whose risk of recurrence is sufficiently high enough to justify adjuvant therapy. The assessment of the primary tumor may also enable more accurate selection of adjuvant therapy, either cytotoxic chemotherapy or targeted therapy. Assessment of lymph nodes may allow identification of micrometastatic disease; that is, occult metastases not identified on routine pathologic examination. Correct assessment of micrometastatic lymph node involvement improves assessment of extent of disease, prognostic stratification, and choice of adjuvant therapy [10]. Assessment of bone marrow and serum may identify evidence of occult distant metastatic disease (stage IV). Identification of these patients would prevent unnecessary surgical resection and allow patients to receive systemic therapy sooner.
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Molecular Staging of the Primary Tumor |
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TopAbstractIntroductionMolecular Staging of the...Specific Mechanisms for...Genomic Analysis of Non-Small...Molecular Staging: Limitations...SummaryReferences |
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Single oncogenic markers cannot be used to predict patient prognosis because the frequency of aberrant expression of any one marker may not be present in most tumors. For example, p53 protein and epidermal growth factor receptor (EGFR) overexpression are observed in approximately 43% and 52% of NSCLC, respectively [13]. Thus, a panel of molecular markers increases the utility of this approach.
Studies that evaluate molecular prognostic variables must be limited to early-stage disease; the inclusion of patients with advanced-stage disease dilutes the potential prognostic value of the markers because this subgroup of patients will have a dismal prognosis regardless of marker status. In one study of 408 stage I patients who underwent complete resection and no adjuvant therapy, multivariable analysis demonstrated significantly elevated risk for the following molecular markers: p53 (hazard ratio [HR], 1.68; p = 0.004), angiogenesis factor VIII (HR, 1.47; p = 0.033), erbB-2 (HR, 1.43; p = 0.044), CD-44 (HR, 1.40; p = 0.050), and rb (HR, 0.747; p = 0.084) [13]. Each of these factors improved the stratification independently, and as a composite, molecular substaging identified groups of patients with 5-year survival ranging from 37% (5 negative prognostic markers) to 80% (1 negative prognostic marker). The identification of these factors also establishes potential therapeutic strategies, such as blockade of the erbB-2 receptor in patients with overexpression of erbB-2, the administration of normal p53 in patients with p53 mutations, or antiangiogenic therapy in patients with high angiogenesis factor VIII.
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Specific Mechanisms for Therapeutic Intervention |
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TopAbstractIntroductionMolecular Staging of the...Specific Mechanisms for...Genomic Analysis of Non-Small...Molecular Staging: Limitations...SummaryReferences |
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), growth factors (EGFR, erbB-2), and mismatch repair (human mutL homolog 1 [hMLH1], hMLH2). After chemotherapy, patients underwent radiologic evaluation for response measured by standard criteria. Multivariable analysis of marker expression associated overexpression of p53 and low expression of hMSH2 with poor treatment response and cancer death. In addition, there was a significant difference in median survival for patients that expressed none (>60 months), one (18 months), or two (8 months) of the negative prognostic markers (p < 0.003) [16]. Although numerous markers and pathways have been demonstrated to improve prognostic stratification, therapeutic intervention targeting these pathways is more limited. Targeted therapy is proposed as strategy to deliver mechanistically specific therapy, with a side effect profile that is superior to cytotoxic chemotherapy. Some of the pathways that are amenable to targeted therapy are reviewed.
Proto-Oncogenes erbB-1 and erbB-2 (HER-2/neu)
The proto-oncogene c-erbB-1 encodes for EGFR, a tyrosine kinase-type membrane receptor. Ligand-binding to EGFR results in receptor dimerization, autophosphorylation, activation of cytoplasmic proteins, and eventually DNA synthesis [17]. Mutations in erbB-1 can result in constitutive activation of EGFR, despite the absence of ligand, with uncontrolled tumor growth as a result. Elevated levels of EGFR have been shown to be present in NSCLC compared with normal lung tissue. The gene erbB-2 (also known as HER-2/neu) shares extensive homology (80%) with erbB-1 and encodes for a transmembrane tyrosine kinase receptor (p185neu) that also functions as a growth factor receptor. Kern and colleagues [18] found that 10 of 29 patients with adenocarcinoma overexpressed p185neu, and this overexpression was associated with decreased survival.
The class of EGFR-targeted therapies contains several agents in various stages of development. Expression of EGFR is also associated with resistance to chemotherapy and radiotherapy. Two general approaches have been pursued to modify EGFR activity: monoclonal antibodies directed at EGFR or its ligand (EGF) and small molecule inhibitors of the EGFR tyrosine kinase. Both approaches inactivate the EGFR pathway and inhibit tumor activity [19].
Two small molecule inhibitors in particular have been well studied: gefitinib and erlotinib. Of the EGFR-targeted agents, gefitinib is approved for the treatment of NSCLC; erlotinib is currently under United States Food and Drug Administration (FDA) review for an indication in NSCLC. Initial studies of gefitinib demonstrated favorable tolerability and antitumor activity, and the FDA granted an indication for this agent as monotherapy in advanced NSCLC after failure of both platinum-based and docetaxel chemotherapies. Two large-scale clinical trials, Iressa Non-small cell lung cancer Trial Assessing Combination Treatment (INTACT-1) [20] and Gefitinib in Combination With Paclitaxel and Carboplatin in Advanced Non-Small Cell Lung Cancer (INTACT-2) [21] evaluated the use of gefitinib in combination therapy compared with chemotherapy alone. In these two studies, the differences between groups in median survival were not significant.
Erlotinib was evaluated in combination with other chemotherapy agents in two recent studies [22, 23]. Herbst and colleagues [22] compared the combination of erlotinib plus carboplatin–paclitaxel with placebo plus carboplatin–paclitaxel. The study enrolled 1059 patients who had not received previous chemotherapy. There were no statistical differences between groups in the primary outcome measure of overall survival, which was 10.8 months with erlotinib vs 10.6 months with placebo (p = 0.95). The second trial followed a similar design but used cisplatin and gemcitabine rather than carboplatin and paclitaxel. The 1172 study patients had not previously received chemotherapy. Again, there were no statistical differences between groups in overall survival or time to progression [23].
Another study evaluated the efficacy of erlotinib after the failure of first- or second-line chemotherapy [24]. This trial randomized 731 patients to receive erlotinib or placebo. The erlotinib group showed statistically significant improvements in overall survival compared with placebo (6.7 months vs 4.7 months; p < 0.001). This trial indicates that erlotinib is safe and tolerable and can prolong survival in patients after failure of first- or second-line chemotherapy.
Trastuzumab, a humanized monoclonal antibody to erbB-2 (HER-2/neu), is currently being investigated for the treatment of lung cancer. The Eastern Cooperative Oncology Group (ECOG) evaluated combination carboplatin, paclitaxel, and trastuzumab in patients with advanced NSCLC [25]. Toxicity with chemotherapy and trastuzumab was no worse than cytotoxic therapy alone. Overall survival was similar to historical data for carboplatin and paclitaxel used alone; however, patients with 3+ HER-2/neu expression did well in contrast to historical data, suggesting potential benefit for trastuzumab in this subset of patients NSCLC.
A randomized phase II trial examined the effect of adding trastuzumab to a standard chemotherapeutic combination (gemcitabine–cisplatin) in patients with HER-2/neu–positive NSCLC. [26] In this study, 51 patients were treated with trastuzumab plus gemcitabine–cisplatin and 50 with gemcitabine–cisplatin alone. Efficacy was similar in the trastuzumab and control arms: response rate, 36% vs 41%; median progression-free survival, 6.1 vs 7 months. Response rate (83%) and median progression-free survival (8.5 months) appeared superior in the trastuzumab-treated patients with high expression of HER-2/neu (3+ by fluorescence in situ hybridization-positive NSCLC).
The EGFR pathway, including erbB-1 and erbB-2, represent a promising avenue for treatment of NSCLC, either with antibodies or tyrosine kinase inhibitors. Ongoing studies of these agents, either alone or combined with cytotoxic chemotherapy, will determine the ultimate role of this strategy.
Angiogenesis
Tumor-induced neovascularization (angiogenesis) is necessary for both tumor growth and metastatic spread, and a large research effort currently is directed at studying its role in cancer development. Immunohistochemical staining for factor VIII, vascular endothelial growth factor (VEGF), CD-31, and CD-34 can be used to assess microvessels, and number of microvessels in a NSCLC can be used to assess angiogenesis. Vascular endothelial growth factor, strongly induced by hypoxia, promotes vascular permeability, endothelial cell replication, and migration. Metastatic spread can be controlled by inhibiting angiogenesis and tumor growth.
Recombinant humanized anti-VEGF antibodies (rhuMAb VEGF) and VEGF receptor tyrosine kinase inhibitors have been tested in animal models and are being investigated in clinical trials. In animal studies, anti-VEGF antibodies suppressed tumor growth, metastatic spread, and ascites formation in tumor-bearing nude mice but did not cause tumor regression [27].
Hurwitz and colleagues [28] reported that rhuMAb VEGF (bevacizumab) plus chemotherapy resulted in increased survival, progression-free survival, response rate, and duration of response when compared alone in patients with colon cancer, increasing the interest in the study of this agent in patients with other types of cancer, including NSCLC. A randomized study of rhuMAb VEGF was conducted in patients with advanced NSCLC (stage IIIb with pleural effusion, stage IV, or recurrent disease) [29]. Patients were randomized to carboplatin and paclitaxel (CP) alone, CP plus low-dose rhuMAb VEGF (7.5 mg/kg every 3 weeks), or CP plus high-dose rhuMAb VEGF (15 mg/kg every 3 weeks). Sudden and life-threatening hemoptysis occurred in 6 subjects treated with rhuMAb VEGF and was fatal in 4; 4 of 6 patients had squamous cell histology. This study found rhuMAb VEGF (15 mg/kg) combined with CP chemotherapy was associated with improved response rates and prolonged time to disease progression compared with carboplatin/paclitaxel chemotherapy alone. A subset analysis of nonsquamous patients was subsequently performed [30]. Median survival for the nonsquamous population was improved in both rhuMAb VEGF dose groups and compared favorably with that achieved with CP chemotherapy alone. Thus, treatment of selected patients with NSCLC—noncentral, nonsquamous—may improve survival with minimal side effects and may represent an important treatment strategy in the future.
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Genomic Analysis of Non-Small Cell Lung Cancer |
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TopAbstractIntroductionMolecular Staging of the...Specific Mechanisms for...Genomic Analysis of Non-Small...Molecular Staging: Limitations...SummaryReferences |
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Powerful computer software and statistical methods are used to analyze the data generated by microarray analysis of the transcriptome to yield gene expression profiles that can be used for therapeutic targets identification and oncogenic pathway discovery, for subclassification of cancers within the same histopathology to improve diagnostic accuracy, and for correlation with treatment outcomes leading to the development of clinically applicable molecular prognosticators.
Miura and colleagues [32] conducted a study using laser-capture microdissection and cDNA microarray analysis of gene expression in a small group of stage I adenocarcinomas. They identified a set of gene transcripts (20 known genes and 15 expressed sequence tags or genes of unknown function) that separated smokers from nonsmokers and also another set of 27 genes that classified survivors and nonsurvivors.
Another important study of gene profiling in NSCLC using oligonucleotide microarray was reported by Beer and colleagues [33] of the University of Michigan. The authors examined gene expression profiles of 86 primary adenocarcinomas (67 stage I and 19 stage III) and 10 nonneoplastic lung samples using 6800 transcript-containing microarrays. Unsupervised hierarchical cluster analysis performed on 4966 genes revealed 3 clusters of tumors. Segregation of tumors by their gene expression profiles created subgroups that had strong association with tumor stage and differentiation.
The study population was evenly divided by random assignment into a training group and a test group of 43 each. In the training set, the top 10, 20, 50, and 75 genes were used to create risk indices that were evaluated for their association with survival using the 50th, 60th, and 75th percentile cutoff points to categorize patients into high or low groups. The 50-gene risk index had the best overall association with survival in the training set. This 50-gene risk index correctly identified low- and high-risk individuals within the independent testing set. Of more importance was that within the stage I adenocarcinoma of the test group, this 50-gene risk index segregated these early-stage lung cancers into two distinct groups with statistically significant different 5-year survivals of 90% for the stage I low-risk group and about 50% for the stage I high-risk group (p = 0.028).
Hoang and colleagues [34] performed gene expression profile of laser-captured NSCLC cells using a 2400 gene cDNA microarray platform to determine if it would be possible to establish a metastatic genotypes of cancer cells on the basis of lymph node tumor burden. Their cohort comprised 15 patients with stage I to IIIA adenocarcinoma (n = 12) or squamous cell carcinoma (n = 3) with defined clinical outcome, and the tumor burden in the regional lymph node was none, microscopic metastasis, or macroscopic metastasis. The authors identified a 75-gene discriminatory subset that, when used in hierarchical clustering analysis, segregated the tumors into three groups that corresponded to their lymph node tumor burden. Their finding from the training set, unfortunately, was not validated with a larger test set; thus, it is unclear if this molecular signature would be able to predict microscopic lymph node metastasis by the genotyping of the primary tumors.
Potti and colleagues [35] at Duke University identified gene-expression profiles that predicted the risk of recurrence in a cohort of 89 patients with early-stage NSCLC, termed the lung metagene model. They subsequently evaluated the predictor in two independent groups of 25 patients from the American College of Surgeons Oncology Group (ACOSOG) Z0030 study and 84 patients from the Cancer and Leukemia Group B (CALGB) 9761 study. The lung metagene model predicted recurrence for individual patients significantly better than did clinical prognostic factors and was consistent across all early stages of NSCLC. Applied to the cohorts from the ACOSOG Z0030 trial and the CALGB 9761 trial, the lung metagene model had an overall predictive accuracy of 72% and 79%, respectively. The predictor also identified a subgroup of patients with stage IA disease who were at high risk for recurrence and who might be best treated by adjuvant chemotherapy. The lung metagene model provides a potential mechanism to refine the estimation of a patient’s risk of disease recurrence and, in principle, to alter decisions regarding the use of adjuvant chemotherapy in early-stage NSCLC.
These studies not only provide evidence that gene expression profiling can be used to predict treatment outcome and to establish molecular prognosticators but also identify hundreds of genes, both known and unknown, that are differentially expressed in cancers vs normal cells, in adenocarcinomas vs squamous cell carcinomas, and in good outcome vs poor outcome tumors. Such wealth of data, which can only be acquired by gene expression microarray technology, provide a fertile ground for research on the molecular basis of carcinogenesis of lung tumors as well as on the identification of molecular targets for the development of novel therapeutics.
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Molecular Staging: Limitations on Progressing from the Bench to the Bedside |
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TopAbstractIntroductionMolecular Staging of the...Specific Mechanisms for...Genomic Analysis of Non-Small...Molecular Staging: Limitations...SummaryReferences |
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Summary |
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TopAbstractIntroductionMolecular Staging of the...Specific Mechanisms for...Genomic Analysis of Non-Small...Molecular Staging: Limitations...SummaryReferences |
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Molecular biologic staging offers an opportunity to individualize a chemotherapeutic regimen according to the molecular profile of the tumor, thus providing the potential for improved outcomes with less morbidity in patients with NSCLC [36–38]. The ultimate power of molecular biologic staging depends on the ability to alter therapy and improve outcome, which has not yet been demonstrated. With current technology, however, it would be possible to determine the relative prognosis of a patient with clinical stage I NSCLC based on molecular staging of a biopsy specimen. Patients with strong negative prognostic markers and patients with occult metastases in the bone marrow or serum might be treated with induction biologic therapy or chemotherapy; furthermore, the biologic characteristics of the tumor would determine the choice of agents. This strategy will become even more accurate with the development of real-time genetic analysis, such as with reverse transcription polymerase chain reaction, enabling the analysis of genetic mutations at the time of surgery. In the near future, it is possible that patients with NSCLC will be staged and treated according to a TNMB staging system: tumor, nodes, metastases, and biology.
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