Molecular staging of lung and esophageal cancer

Surg Clin N Am 82 (2002) 497–523

Molecular staging of lung and esophageal cancer Christine L. Lau, MDa, Mary-Beth H. Moore, BAb, Kelly R. Brooks, MDa, Thomas A. D’Amico, MDc, David H. Harpole, Jr., MDa,d,* a

General and Thoracic Surgery, Duke University Medical Center, Durham, NC 27710, USA b Lung and Esophageal Cancer Laboratory, Duke University Medical Center, Durham, NC 27710, USA c Division of Cardiothoracic Surgery, Duke University Medical Center, Durham, NC 27710, USA d Cardiothoracic Surgery, Durham Veterans Administration Medical Center, Durham, NC 27710, USA

The most important predictor of survival in both non small-cell lung cancer (NSCLC) and esophageal cancer is the TNM stage at diagnosis, with the best chance for cure remaining complete surgical resection. While both NSCLC and esophageal cancer in advanced stages carry a very poor prognosis, patients with earlier staged tumors may have favorable outcomes when treated aggressively. An understanding of tumor virulence on a molecular level may identify subsets of early stage patients with poorer prognosis. Additionally, markers capable of identifying patients with advanced or metastatic disease would prevent unnecessary surgical procedures in this group. If a definitive set of tumor markers that document early recurrence could be identified, earlier intervention (radiation therapy or re-resection for a local recurrence, chemotherapy or a novel therapy for a distant recurrence) may increase the overall survival rate. Carcinogenesis The change from normal bronchial epithelium to carcinoma is the end result of a series of acquired genetic mutations and alterations in normal cellular proteins. A similar process occurs in the progression of normal esophageal mucosa to metaplasia, then to dysplasia, and finally to carcinoma. This * Corresponding author. E-mail address: [email protected] (D.H. Harpole, Jr.). 0039-6109/02/$ - see front matter Ó 2002, Elsevier Science (USA). All rights reserved. PII: S 0 0 3 9 - 6 1 0 9 ( 0 2 ) 0 0 0 2 4 - 5

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process is stimulated and influenced by a variety of environmental, biologic, and molecular processes. The critical regulatory genes mutated include various proto-oncogenes and tumor suppressor genes. Carcinogenesis can occur with either activation or deletion of these important regulatory genes. Protooncogenes when altered may acquire transforming potential, leading to dysregulation of normal cell growth. Only one of the two alleles needs to be altered for the transforming effect, and therefore, proto-oncogenes areknown as dominant genes. Tumor suppressor genes have been implicated in various cancers, with cellular transformation occurring by either elimination of both alleles or by mutation or functional abnormality of their protein products. The role of molecular biologic markers is being explored as an aide in the diagnosis, staging, and treatment of non small-cell lung and esophageal cancer. The background and techniques involved in the study of the molecular biology of cancer have been reviewed extensively [1–3]. The goal of this molecular biological research is to improve early detection, to formulate a prognostic model of staging, and to discover new therapeutic approaches. Molecular markers in diagnosis and prognosis To be useful, diagnostic molecular markers should have a high prevalence in malignancy, and techniques to measure the markers must have a high sensitivity and specificity. Genetic abnormalities may be detected by identification of the specific mutation, using single strand conformational polymorphism (SSCP) or reverse transcriptase polymerase chain reaction (rt-PCR). Alternatively, mutations may be identified by detection of abnormal gene products (proteins) using immunohistochemical (IHC) antibody binding. Molecular markers used in the diagnosis of NSCLC and esophageal cancer may provide the clinician with the ability to establish the diagnosis of malignancy at an earlier stage than conventional radiographic and histopathologic techniques allow. The impact of an effective molecular marker in distinguishing metaplasia and dysplasia from carcinoma would be an important advance. In addition, the use of molecular markers may allow a lesion to be identified as a primary tumor versus a metastatic lesion. This could be useful in distinguishing a new primary NSCLC from a metastatic melanoma lesion, for example. As the prevalence of most markers is too low, the use of a panel of markers in distinguishing dysplasia from carcinoma might represent a superior strategy, and this strategy is now under investigation. Growth-regulating proteins There are a number of genes that normally control cell growth and development. Alterations in these genes can change the phenotype of a cell, allow the expression of abnormal signal transduction proteins, and activate multiple enzyme cascades in the cell. Growth-regulating genes whose protein products have been implicated in carcinogenesis include ras oncogenes, epidermal growth factor receptor gene, and erbB-2/Her2-neu oncogenes.

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Ras oncogenes Ras oncogenes are a family of genes which code for a protein located on the inner surface of the cell membrane. The protein, known as p21, is involved in signal transduction and has guanosine triphosphatase activity. Mutations of ras genes result in blockage of guanosine triphosphatase activity, allowing a continued signal for proliferation. There are three main members of the ras oncogene family (K-ras, H-ras, and N-ras), and the vast majority of mutations involved in carcinogenesis occur in K-ras at codon 12 (Casson [2] and Kim et al [4] discuss other codons). Mutations in K-ras are found most commonly in adenocarcinomas and are associated with smoking and asbestos exposure [5]. Slebos and colleagues initially reported activation of K-ras oncogene as a strong negative prognostic factor in patients with resected adenocarcinomas of the lung [6]. Fukuyama and colleagues reported NSCLC patients with K-ras mutations at codon 12 had a relative risk of death of 5.6 compared with patients without the mutation [7]. In addition to the prognostic significance of K-ras gene mutations, the overexpression of p21 has similarly been found to correlate with a negative survival. Harada and colleagues found NSCLC patients with p21-negative tumors (stained immunohistochemically using anti-ras p21 monoclonal antibody rp-35) had significantly longer survival times compared with patients whose tumors were p21 positive [8]. More recently however, Nemunaitis and colleagues did not attribute significant negative prognostic value to elevated p21 in adenocarcinoma patients with normal ras-oncogenes [9]. The majority of evidence, albeit in retrospective studies, indicates K-ras mutations correlate with decreased survival, and therefore, little controversy exists that it has value as a molecular marker in lung cancer patients. In the esophagus, K-ras codon 12 mutations appear to be rare events in normal tissue as well as in tissues with metaplasia and low-grade dysplasia. However, these mutations are found in up to 40% of high-grade dysplasias and adenocarcinomas. Therefore, K-ras codon 12 mutation may be a late event in the metaplasia, dysplasia, carcinoma pathway in esophageal cancer and therefore, may be useful as a marker for early cancer detection [10,11]. The investigations of K-ras mutations have resulted in the development of novel chemotherapeutic agents. RAS must be positioned correctly in the cell wall for activation. This requires post-translational modification by farnesyl transferase enzyme. Blockade of this enzymes inactivates K-ras. Synthetic farnesyl transferase inhibitors are in phase II trials [12]. Proto-oncogenes c-erbB-1 (EGFr) & c-erbB-2 (HER-2/neu) The proto-oncogene c-erbB-1 encodes for epidermal growth factor receptor (EGFr), a tyrosine kinase-type membrane receptor. Ligand binding to EGFr results in receptor dimerization, autophosphorylation, activation of cytoplasmic proteins, and eventually DNA synthesis [13]. Mutations in c-erbB-1 can result in constitutive activation of EGFr despite the absence of

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ligand, with the result being uncontrolled tumor growth. In NSCLC, elevated levels of EGFr have been shown to be present compared with normal lung tissue. Additionally, elevated levels of EGFr are found in later stage lung cancers and in lung cancers with mediastinal involvement [14], but these levels have not been found to correlate with prognosis [5,13,15]. However, subset analysis (T1N0) in one study evaluating a panel of immunohistochemical markers in resected stage I NSCLC did find a decreased survival in EGFr overexpressing tumors [16,17]. Although EGFr may play a role in initial tumorigenesis and may be a marker for advanced disease, increased expression of EGFr currently cannot be considered as a significant prognostic molecular marker in NSCLC based on the available data. Iressa (ZD 1839) is a quinazoline tyrosine kinase inhibitor selective for the epidermal growth factor receptor. ZD 1839 inhibits transphorylation of the EGFr, thus blocking the first step in signal transduction (Fig. 1). Its anti-proliferative effects have been demonstrated in preclinical trials and it is currently being evaluated in human trials [18]. In a phase I study 64 patients were treated on an intermittent dosing schedule with escalating doses. 38% of NSCLC patients had partial response or stage downstaging [19]. The National Cancer Institute (Canada Clinical Trials Group) is in the early stages of a prospective randomized, double-blind, placebo-controlled trial of Iressa in patients with completely resected stage IB, II, and IIIA NSCLC. C-erbB-2/HER-2/neu shares extensive homology (80%) with c-erbB-1 and encodes for a transmembrane tyrosine kinase receptor (p185neu) that also functions as growth factor receptor. Kern and colleagues found 10 of 29 patients with adenocarcinoma overexpressed p185neu, and this overexpression was associated with decreased survival [20]. Following this initial report, several additional studies have confirmed these results with adenocarcinomas and NSCLC as a group [17,21–23]. Overexpression of p185neu is currently believed to be an independent negative prognostic factor in NSCLC. Although important in predicting prognosis in NSCLC, overexpression of erbB-2 is less important in the prediction of prognosis in patients with esophageal cancer [24,25]. In one series, 26% of squamous cell carcinomas and 23% of adenocarcinomas overexpressed erb-B2, with no correlation with prognosis [24]. Herceptin (Trastuzumab) is a humanized monoclonal antibody to HER2/neu. Currently there are three ongoing cooperative group trials of its use in lung cancer with enrollment requiring patients to be at stage III, and the tumor must stain positive immunohistochemically for HER-2/neu. Cell-cycle specific proteins Normal epithelium is arrested in the G0 phase of the cell cycle (Fig. 2), with a designated cell life. When it reaches the end of its life, an apoptotic cascade begins, allowing the senescent cell to die. Deletion or mutations in the genes involved in regulating the life span of the cell can cause it to

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Fig. 1. Scheme showing mechanism of action of EGFR (A) and mechanism of action of EGFR tyrosine kinase inhibitors (B).

become resistant to apoptosis (programmed cell death) or ‘‘immortal.’’ Under normal circumstances, apoptosis is involved in the removal of transformed or DNA damaged cells and is therefore a protection mechanism against the development of tumors [26]. Important regulators of apoptosis are the tumor suppressor gene, p53, and the Bcl-2 family of proteins. p53 tumor suppressor gene P53 is a commonly known tumor suppressor gene that encodes for a nuclear phosphoprotein involved in cell growth through regulation of transcriptional events. Overexpression of the wild-type p53 gene product directly

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Fig. 2. Cell-cycle.

initiates cell cycle arrest in the G1 phase in response to DNA damage caused by oncogenic activation, radiation, or chemotherapeutic drugs [27,28]. This allows for DNA repair or, if the damage is too great, destruction of the cell through the activation of the apoptotic pathway. Mutations in the p53 gene are the most common lesions found in many human cancers [29]. In NSCLC p53 is found to be mutated or overexpressed in >50% of cases. Like abnormalities in K-ras, mutations in p53 are associated with smoking [5]. Significant controversy remains over its use as a molecular prognostic indicator in NSCLC. This is because in some studies mutation or protein expression of p53 is associated with decreased survival [17,21,23,30–35]; in some studies there is no association with survival [15,36]; and in some studies there is association with increased survival [37,38]. Reasons for this controversy are unclear, but may at least partially relate to whether gene mutation or protein expression is measured [29,39]. Overexpression does not necessarily mean the p53 gene is mutated, but it is an abnormal finding. Additionally, the group of NSCLC patients assessed may provide some explanation for the controversy. Dalquen and colleagues found p53 expression to be a negative prognostic factor in node negative but not node positive NSCLC patients [40]. Along these lines, a study consisting of 408 stage I NSCLC patients confirmed the negative prognostic value of overexpression of p53 with a 70% five-year survival for NSCLC patients with p53- tumors compared with 52% survival for p53þ tumors (P ¼ 0:001) [17]. Found in 53 to 71% of tumors, mutation of the tumor suppressor gene p53 is also common in esophageal cancer, thus suggesting that the detection of p53 may be useful in the diagnosis of esophageal cancer [25,41,42]. The ability to detect p53 abnormalities has been proposed as a methodology to differentiate columnar-lined esophageal epithelium (CLE) without carcinoma from adenocarcinoma [43,44]. However, the prevalence of the p53 mutation may vary by the method of detection. In one study, the genetic mutation was detected with SSCP in 53% of tumors and the p53 protein

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overexpression found in 51%, with a discordance rate of 31%. Overall, only 27% of patients had no detectable p53 abnormality, but there was no control analysis to evaluate specificity in this study [42]. In one series, the presence of p53 antibodies was detected in 4 of 36 patients with CLE and no evidence of malignancy, compared to 10 of 33 with carcinoma [43]. The expression of abnormal p53 protein appears to correlate with progression of the metaplasia-dysplasia-carcinoma sequence of CLE. In metaplastic epithelium, p53 abnormalities are rare. Abnormal p53 is found in 9 to 14% of patients with low-grade dysplasia, 45 to 55% of patients with high-grade dysplasia, and 50 to 87% of patients with adenocarcinoma [45–47]. Another study, using IHC analysis of 100 surveillance esophageal biopsies, demonstrated that the progression from metaplasia to low-grade dysplasia to high-grade dysplasia was characterized by a progressive increase in p53 protein accumulation, suggesting the use of p53 in determining risk of malignancy [48]. In another series, 204 patients with esophageal cancer were analyzed with respect to p53 overexpression and survival, and no correlation was found [49]. In a series that analyzed 74 patients for p53 protein expression and p53 gene mutation, neither variable was found to be associated with prognosis [42]. In an analysis of 205 patients with esophageal cancer, 68% of squamous cell carcinomas and 66% of adenocarcinomas demonstrated p53 overexpression, with no significant correlation with survival [24]. Mutation of p53 was associated with decreased survival in patients with esophageal carcinoma in two studies [41,50]; however, at this time, the preponderance of evidence supports no definite role of p53 in the prognosis of patients with clinically evident carcinoma of the esophagus. The inability to prove conclusively that p53 alterations have prognostic significance may relate to the biology of esophageal cancer, suggesting superceding mutations. The Bcl-2 family The Bcl-2 family contains proapoptotic (Bax) as well as antiapoptotic proteins (Bcl-2, Bcl-XL). Though the protein-protein interactions between these groups is not fully understood, it is known that Bcl-2 protein binds to other proteins with which it has sequence homology, such as Bcl-XL and Bax, forming hetero-dimers [51–53]. Therefore, it has been suggested that apoptosis is not regulated by proapoptotic proteins alone, but rather, the ratio of the two types of proteins determines the extent of apoptosis in cells where both types are found [54]. The loss of expression of proapoptotic proteins or the overexpression of antiapoptotic proteins in tumor cells may also play a role in altering the apoptosis cascade. Tumor cells that have lost the ability to signal apoptosis may become immortal and thus resistant to therapy. Little data are available which include measurement of more than one to two of these proteins with respect to survival after treatment with chemotherapy in NSCLC and esophageal cancer. Overexpression of Bcl-2

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surprisingly, has been associated with improved survival in NSCLC [55,56] or a trend toward improved survival [17,30]. In squamous cell esophageal cancer, Takayama and colleagues reported a longer mean survival time in patients with tumors with decreased Bcl-X expression. Multivariate analysis revealed Bcl-X expression to be an independent prognostic factor (P ¼ 0:022) [57]. Cyclooxygenase II (COX-2) enzyme Cyclooxygenase enzymes function to convert arachidonic acids to prostaglandins. The cyclooxygenase2 enzyme (COX-2) produces prostaglandin E2 (PGE2), which stimulates Bcl-2 and thus inhibits apoptosis, and induces interleukin 6, which enhances haptoglobin synthesis. This process results in increased tumor invasion, angiogenesis, and metastasis [58]. Increased expression of the COX-2 enzyme is prominent in gastrointestinal tumors, including esophageal adenocarcinomas. Morris demonstrated that while increased expression is found in Barrett’s metaplasia (75%) and low-grade dysplastic tissues (83%), it is found in nearly all tissues with high-grade dysplasia or adenocarcinoma. The level of expression is significantly higher in high-grade dysplasia and adenocarcinoma compared with metaplasia or low-grade dysplasia (P < 0:0001) [59]. This suggests that the level of COX-2 expression may be useful as a diagnostic marker for the progression along the metaplasia-dysplasia-carcinoma pathway. Selective COX-2 inhibitors have resulted in decreased tumor cell growth and increased apoptosis in tumors expressing COX-2, and thus may play a role in the prevention and treatment of esophageal adenocarcinoma [60]. Retinoblastoma(RB) and p16INK4a tumor suppressor genes The RB gene encodes a nuclear protein that interacts with the protein product of p53 in the nucleus and is involved with transcriptional events and cell cycle regulation (keeping the cell quiescent). This tumor suppressor gene is located on a chromosomal region frequently found deleted in the childhood ocular cancer, retinoblastoma. Loss of RB protein expression occurs commonly in NSCLC, most often seen in later stage tumors. Loss of RB protein expression has been associated with either a decrease in survival [17,61] or no correlation with survival [30,62]. Additionally, patients with both loss of RB protein expression and p53 protein overexpression have an even worse prognosis [17,61]. D’Amico and colleagues reported five-year survivals in 404 stage I NSCLC patients of 72%, 62%, and 48% for RB+/ p53-; RB+/p53+; and RB-/p53+ (protein expression) tumors respectively [17]. Therefore the loss of RB is thought to be a negative prognostic factor. A cyclin-dependent kinase inhibitor p16INK4a is involved in keeping a cell in the quiescent state (G0). The mechanism by which the gene product of p16INK4a inhibits entry into the cell cycle is through its inhibition of the cyclin-dependent kinase 4 mediated phosphorylation of the retinoblastoma gene product. Inactivation of p16INK4a results in unregulated cell growth

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and transformation. Worse survival has been seen in NSCLC patients whose tumors do not express p16INK4a. Additionally, an inverse correlation has been appreciated between levels of RB and p16INK4a [63]. Telomerase Telomerase, a ribonucleoprotein that synthesizes telomeric DNA, has been identified as a tumor marker in a variety of malignancies [4]. It has been described as a marker of cell immortilization and suggests a potential role as a diagnostic tumor marker. With each replication and division, cells lose a portion of their telomeric DNA, eventually resulting in senescence and subsequent cell death. Telomerase replenishes telomeric DNA, therefore making cells immortal [64]. In NSCLC, telomerase activity is present in tumor cells and not in noncancerous tissues [65]. However, in esophageal carcinoma, telomerase activity has been found in 87% of tumors and in 23% of normal biopsies [66]. Using a more sensitive methodology, a recent investigation has shown that expression of hTERT (human telomerase reverse transcriptase), which plays a role in telomerase activation, is an early event in the development of Barrett’s esophagus and significantly increases through each progressing phase (ie, metaplasia, dysplasia) in the development of esophageal adenocarcinoma [67]. A recent study in NSCLC demonstrated that both telomerase activity and hTERT are significantly correlated with lymph node metastasis, tumor stage and grade [65]. Thus, telomerase (hTERT) may be useful as a marker for staging in both NSCLC and esophageal cancer. Markers of proliferation Markers of proliferation estimate the proportion of tumor cells nearing mitosis and consist of assessment by light microscopy (mitotic index), flow cytometry (ploidy, S-phase), or immunohistochemical techniques (Ki-67, proliferating cell nuclear antigen- PCNA). Ki-67 The tumor proliferation marker Ki-67, a nonhistone nuclear protein, is expressed by cells near mitosis and can be used to identify rapidly dividing tumors. Several series have noted a decrease in survival in association with higher levels of expression of Ki-67 [21,23,68]. Pence and colleagues evaluated Ki-67 proliferation indexes in 61 predominantly early-stage lung cancer patients (39 stage I patients, 12 stage II patients, and 10 stage III/IV patients) and found patients with Ki-67 proliferation indexes of less than 3.5 had two-year survivals of 54%, compared with 8% for patients with Ki-67 proliferation indexes >3.5 (P ¼ 0:01) [68]. In patients with stage I NSCLC, Harpole and colleagues reported a 52% five-year survival in patients with a Ki-67 index greater than 10%, compared to a 68% five-year

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survival in patients with a Ki-67 index of 0 to 5% (P < 0:02) [21]. Additionally, Ki-67 proliferation had a highly significant correlation with p53 overexpression. In a larger group of patients, D’Amico and colleagues reported a trend that did not reach significance toward decreasedsurvival in high versus low level Ki-67 expression (P ¼ 0:071) [17]. In conclusion, higher Ki-67 proliferation indexes appear to inversely correlate with prognosis. Proliferating cell nuclear antigen (PCNA) Proliferating cell nuclear antigen is a nuclear protein that binds to DNA polymerase. Because of its involvement with DNA replication, its presence is highest in rapidly growing cells, while its level is undetectable in quiescent cells. Ishida and colleagues found positive staining for PCNA correlated with decreased survival in NSCLC patients [29,69]. Mitotic index/ploidy Harpole and colleagues reported that tumors with 15 mitosis per high power field had a significantly worse survival than tumors with a lower number of mitotic figures present [70]. Recently however, Kwiatkowski found no survival differences in stage I NSCLC patients whose tumors had mitotic index of 0 to 10, 10 to 20, 21 to 30, or >30, with five-year survival rates of 68%, 67%, 72%, and 60% respectively [30]. Investigation into DNA content by flow cytometry initially suggested that more biologically aggressive tumors were aneuploid, whereas those associated with a better prognosis appeared to be diploid [71,72]. Subsequent studies, however, have not confirmed this survival advantage with diploid tumors [5,68]. The prognostic significance of the number of cells in the S-phase (S-phase fraction) of the cell cycle has not been extensively studied in NSCLC. High S-phase fraction has been associated either with a decrease in survival [72] or with no correlation to survival [29,73]. Volm and colleagues reported on 187 predominantly advanced stage NSCLC patients and found patients whose tumors had 8% of their cells in the S phase had significantly improved survival compared with patients whose tumors had >8% of their cells in S-phase (P ¼ 0:041) [72]. In comparison, Schmidt and colleagues found no prognostic significance to S-phase percentage in T1N0 tumors [73]. Tumor invasion and metastasis A carcinoma can invade surrounding structures, including blood vessels and lymphatic channels, allowing tumor cells to circulate throughout the body. The formation of metastatic lesions, however, requires that circulating tumor cells adhere to the endothelium of a distant capillary or lymph vessel and transgress the endothelial membrane of the target organ. In 1889, Paget stated that tumors require both the ‘‘correct seed and soil’’ for metastatic spread [74]. Therefore, only tumor cells that express the correct adhesion

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molecules (CD-44, siayl-Tn, blood group A) will be able to successfully attach and invade a new location in the body. Two processes are necessary for a tumor colony to grow and become invasive: angiogenesis and basement membrane degradation (Fig. 3). When a tumor colony expands, it outgrows its blood supply and central necrosis develops. The resultant ischemia stimulates angiogenesis within the cancer, resulting in ingrowth of capillaries and tumor growth. In order for an in situ tumor to invade surrounding tissues, the glycoprotein matrix that surrounds normal tissue must be degraded by enzymes (matrix metalloproteinases, cathepsin B). These two processes allow cancer cells to enter the lymphatic or vascular spaces, thus setting the stage for the next phase: distant metastases. Angiogenesis Tumor-induced neovascularization (angiogenesis) is necessary for both tumor growth and metastatic spread, and a large research effort is currently directed into studying its role in cancer development. The number of microvessels in a NSCLC can be used to assess angiogenesis. Immunohistochemical staining for factor VIII, vascular endothelial growth factor (VEGF), and CD 31 can be used to assess microvessels. Antibody to factor VIII can be used to identify the number of microvessels in a NSCLC, and the degree of angiogenesis as measured by factor VIII staining has been shown to have significant negative prognostic implications [75]. Harpole et al examined angiogenesis in 275 consecutive patients with stage I NSCLC and found angiogenesis was the most significant prognostic factor in stage I NSCLC compared with erbB-2 (HER-2/neu), p53, and KI-67 [23]. Attempts to increase sensitivity and specificity using immunostaining for CD31 (platelet/endothelial cell adhesion molecule) instead of factor VIII did not prove successful, and its use did not correlate with prognosis [76]. Recently, an anti-CD34 monoclonal antibody specific for endothelial cells has been found useful in immunostaining of microvessels [77,78]. In contrast to anti-CD31, anti-CD34 antibody has been found to be more specific and reproducible than

Fig. 3. Biological schema showing progression of tumor from carcinoma in situ to invasive carcinoma.

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staining for factor VIII. Fontanini et al prospectively analyzed a cohort of 407 patients with NSCLC, using anti-CD34 antibody to detect microvessels, and found patients with more vascularized tumors experienced significantly decreased survivals (P < 0:00001) [77]. 50% of patients with stage I lung cancers and highly vascularized tumors had two-year survivals, compared with 95% two-year survival in the group of patients with the lowest microvessel counts. Besides using endothelial cell antigen antibodies to assess microvessel counts, Imoto et al reported on the prognostic significance of vascular endothelial growth factor expression in non small-cell lung cancer and found it to be a significant prognostic indicator. This was most pronounced in squamous cell cancer [79]. In addition, in patients with esophageal cancer, high levels of expression of the angiogenesis marker, factor VIII, have been shown to correlate with decreased survival (P ¼ 0:04) [25] (Fig. 4). Attacking a tumor’s angiogenic abilities is currently being evaluated in phase II/III clinical trials. By inhibiting angiogenesis, tumor growth and metastatic spread can be controlled. VEGF promotes vascular permeability, endothelial cell replication, and migration. It is strongly induced by hypoxia. High levels of VEGF have been found in malignant effusions. High levels have correlated with recurrence rates in ovarian, gastric, lung, and breast cancer [80]. Both anti-VEGF antibodies (RhuMAb VEGF) and VEGFr tyrosine kinase inhibitors (SU5416) have been tested in animal models. In

Fig. 4. Esophageal cancer-specific survival analysis comparing patients with low expression of factor VIII versus those with factor VIII high-level expression, plotted as percent survival versus time in months using the Kaplan-Meier product limit estimator. Number of patients at risk at one-, three-, and five-year intervals is given along the x-axis. (From Aloia TA, Harpole DH Jr, Reed CE, Allegra C, Moore MB, Herndon JE II, D’Amico TA. Tumor marker expression is predictive of survival in patients with esophageal cancer. Reprinted with permission from the Society of Thoracic Surgeons. Ann Thorac Surg 2001;72(3):859–66.)

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animal studies, anti-VEGF antibodies suppressed tumor growth, metastatic spread, and ascites formation in tumor-bearing nude mice but did not cause tumor regression [80]. In a phase II Trial of RhuMAb VEGF in Stage IIIB/ IV NSCLC, 99 patients receiving Carboplatin/Taxol were randomized to additionally receive placebo, low-dose RhuMAb VEGF, or high-dose RhuMAb VEGF. Response rates were 18.8% to 25% for placebo, 28.1% to 21.9% for low dose, and 31.4% to 34.3% for high dose. Time to progression was 129 to 181 days, 131 to 124 days, and 225 to 207 days respectively [81]. In this study, safety concerns for RhuMAb VEGF were reported. Out of 67 patients treated with RhuMAb VEGF, 6 patients (9%) developed massive hemoptysis that was fatal in four cases. The majority of these patients had centrally located squamous cell tumors. Based on these findings, phase III clinical trails will not allow central tumors to be enrolled [81]. Invasion/extracellular matrix degradation Matrix metalloproteinases (MMP) have been implicated in the breakdown of vascular barriers, allowing tumor cells to infiltrate blood vessels. The matrix metalloproteinase stromelysin-3 has received the greatest attention in NSCLC, and has been found to be more abundant in NSCLC than normal lung tissue [82]. Plasminogen activators are members of the serine protease family. They are responsible for converting plasminogen to plasmin. Plasmin can degrade various proteins in the extracellular matrix. Plasminogen activators are regulated by plasminogen activator inhibitors. Yoshino and colleagues have found that decreased levels of plasminogen activator inhibitor 2 correlate with decreased survival and increased lymph node metastases in NSCLC patients [83]. Another means by which basement membrane degradation occurs through the plasminogen activation system is the secretion of urokinase plasminogen activator (uPA) in its inactive form (pro-uPA) by tumor cells. Pro-uPA is converted to its active form (uPA) upon binding to its specific membrane-bound receptor, u-PAR. This activated form of uPA then converts plasminogen into plasmin, which degrades the protein components of the extracellular matrix, such as laminin and fibronectin. Plasmin can also activate pro-enzyme forms of MMPs to further break down the extracellular matrix. U-PAR is expressed on stromal cells as well as tumor cells. Pedersen et al demonstrated a significantly worse survival for patient with NSCLC who expressed u-PAR in their tumors [84]. Pappot et al reported that u-PAR in plasma of patients with NSCLC was significantly higher than in the plasma of healthy controls [85]. These findings suggest a potential role for uPA and u-PAR as markers for the early detection of recurrence. Furthermore, D’Amico et al have shown significantly higher expression of uPA in NSCLC patients who later developed isolated brain metastasis. These results suggest that it may be possible to identify patients with a high risk

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for CNS metastasis, thus identifying a population that would benefit from adjuvant therapy such as prophylactic cranial irradiation [86]. Cathepsin B is a cellular protein located in the lysosome (cysteine proteinase), and it is involved in protein degradation within the cell. In tumor cells it has been implicated in the digestion of the extracellular matrix. Because it can digest the extracellular matrix, it is involved in tumor invasion and metastases. Higher grade expression of cathepsin B has been prognostically correlated with decreased survival in NSCLC [29,87]. Sukoh and colleagues reported that patients with stage I NSCLC whose tumors strongly expressed cathepsin B had decreased survival compared with patients whose tumors did not express cathepsin B [87]. In contrast, Mori and colleagues did not find high expression of cathepsin B in stage I NSCLC patients to correlate with survival [88]. Adhesion molecules Cluster designation 44 (CD-44), an integral membrane glycoprotein, is a receptor for hyaluronan (a component of the extracellular matrix). CD-44 is involved in cell-to-cell and cell-to-extracellular matrix interactions and is correlated with metastatic spread. Expression of CD-44 has been shown to be negative prognostic factor in NSCLC [17]. Hirata and colleagues reported that expression of a variant isoform of CD-44 negatively correlated with survival in stage I NSCLC patients [89]. D’Amico and colleagues reported a five-year survival of 54% in patients with CD-44 expression compared with a 67% survival in patients without CD-44 expression (P ¼ 0:05) [17]. Blood group antigens and precursor antigens The loss of expression of blood group A allows immature blood group antigens to be exposed, and these antigens have been implicated in cell motility and therefore have been speculated to be involved with metastatic spread. Lee and colleagues assessed the presence of blood group antigens immunohistochemically in 164 patients with completely resected NSCLC [90]. In this study, survival of the 28 patients with blood group A or AB whose lung cancers lacked the blood-group A antigen was significantly worse than survivals of patients with the blood-group A antigen on their tumors, or patients with blood group B or O. Miyake and colleagues stained for the precursors of blood group A and B antigens (H/Ley/Leb) using the monoclonal MIA-15-5 (migration-inhibitory antibody; named for its ability to inhibit motility and metastatic ability of cancer cells) and reported MIA positive tumors had a significantly worse prognosis than MIA negative tumors [91]. The MIA positive tumor patients had a five-year survival of 20.9% versus 58.6% for patients with MIA negative tumors (P < 0:001). Additionally, Matsumoto and colleagues found loss of blood group antigens on lung cancer correlated with increased metastatic potential and worse

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prognosis [92]. More recently, however, reports have not shown this prognostic correlation in early stage lung cancers [30,93] and one report even found MIA-15-5 positive tumors had an increased survival [15]. Thus currently blood group antigens and their precursors are not felt to be important in the biologic aggressiveness of NSCLC. CpG hypermethylation Epigenetic inheritance is the acquisition of heritable changes in gene expression that occurs without a change in DNA sequence [94,95]. Methylation of a DNA sequence is limited to the cytosine of the CpG dinucleotide. It is the main epigenetic modification in humans and plays an important role in tumorigenesis [96,97]. About 50 to 60% of human genes contain CpG islands in their promoter region. CpG islands are CpG-rich DNA that are usually unmethylated, regardless of expression status [98,99]. Hypermethylation of these CpG islands within the promoter region results in transcriptional silencing [100–103]. Therefore, eliminating the function of defined genes is considered to be a mechanism for the initiation and acceleration of malignant cell growth [104]. Since hypermethylation consistently occurs at the region where loss of heterozygosity (LOH) is frequently detected, DNA hypermethylation may underlie chromosomal instability [105–107]. Two types of methylation have been reported in association with cancer progression. The first, type A, is age-related [108]. It arises as a function of age in normal cells by affecting the genes that are responsible for the regulation of growth or differentiation [105]. This type of methylation may also result as a predisposition to tumor formation [105]. The second, type C, is cancer-specific [108]. This type of methylation is found in a subset of cancers that exhibit increased methylation in the promoter region of a gene, representing a CpG island methylator phenotype (CIMP) [108]. Recently, there has been an increased interest in the study of the hypermethylation of CpG islands within the promoter region of various genes. Studies in NSCLC and esophageal cancer have investigated aberrant promoter hypermethylation of several genes. These studies have shown that aberrant promoter methylation of the tumor suppressor genes p16INK4a and p15 INK4b, death-associated protein kinase (DAP-kinase), the cellular detoxification gene glutathione S-transferase P1 (GST-p1), the DNA repair gene O6-methylguanine-DNA-methyltransferase (MGMT), tissue inhibitor of metalloproteinase 3 (TIMP-3), retinoic acid receptor, E-cadherin, and adenomatous polyposis coli (APC) have been found in primary tumors [102,103,109–121]. In NSCLC, p16INK4a methylation has been observed in lesions that are considered to be precursors to lung cancer [122]. Tang et al reported that the patients with DAP-kinase promoter methylation had a significantly decreased overall survival five years after surgery (P ¼ 0:007) [114]. In a study of the tumor suppressor gene APC, Brabender et al reported that

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patients with low APC methylation status in their tumor exhibited a significantly longer survival (P ¼ 0:041) [116]. Although, promoter methylation of the tumor suppressor gene p15INKb is a rare event in NSCLC, Kurakawa et al reported a markedly shortened survival in those patients in whom it was found to occur [115]. With increased interest in promoter hypermethylation, recent studies have concentrated on the analysis of the presence of promoter hypermethylation as a marker for cancer cell DNA detection in the plasma and serum of cancer patients. Several studies, in various tumor types, have analyzed the presence of promoter hypermethylation as a marker for cancer cell detection in serum of cancer patients [123,124]. These investigations have yielded promising results. These results have also been tested and reproduced in recent studies in NSCLC and esophageal cancer. In a study in NSCLC in which methylation status of p16, DAP-kinase, GSTp1, and MGMT were analyzed, patients that exhibited methylation within their primary tumor, 11 of 15 samples (73%), also had abnormal methylated DNA in their matched serum samples. Patients who did not exhibit p16 methylation in their tissue samples had no detectable level in their corresponding serum samples [125]. In esophageal cancer, APC promoter methylation was found in the tumor tissue samples of 94% of the adenocarcinomas (n ¼ 51) and in 59% of squamous cell carcinomas (n ¼ 32) [126]. This study further confirmed that if there was no detectable level of APC methylation in the tissue, then none was detected in the serum [126]. Interestingly, a statistically significant correlation was observed between methylated APC and advanced stage of cancer, as well as a correlation of high serum methylated APC levels with reduced survival [126]. This investigation also observed the more frequent occurrence of methylated APC in the serum of patients with recurrent disease (73%), warranting further study for methylated APC as a novel tumor marker [126]. Several imaging techniques that are standard diagnostic tools employed in the detection and monitoring of NSCLC and esophageal cancer (ie, chest radiograph, computed tomography) have their limitations. Additionally, traditional cytological tests using sputum and bronchoalveolar lavage material have remained ineffective tools for cancer screening and monitoring, thus indicating a need for more sensitive and reliable mechanisms of detection and monitoring of NSCLC and esophageal cancer. Detection of hypermethylated genes in serum samples collected from cancer patients may provide a noninvasive and inexpensive diagnostic tool to monitor the effectiveness of treatment protocols and detect cancer recurrence. Multivariate models Because the non small-cell lung cancers are a diverse group of tumors, one molecular marker invariably cannot predict increased biologic virulence 100% of the time. Adenocarcinomas, large cell carcinomas, and squamous cell carcinomas preferentially express various markers. For these reasons,

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molecular model systems comprised of a panel of molecular markers have the ability to optimize the sensitivity of biologic markers across the spectrum of NSCLC. Kwiatkowski and colleagues evaluated 244 patients with stage I NSCLC and identified six independent variables that predicted tumor recurrence: adenocarcinoma solid tumor with mucin subtype, tumor diameter P 4 cm, lymphatic invasion, p53 expression, K-ras mutation, and absence of H-ras expression [30]. This group proposed a molecular pathologic substaging system in stage I NSCLC based on these variables. If patients had 6 two factors they were assigned as grade 1a, if they had three factors they were assigned grade 1b, and if they had P four factors they were assigned grade 1c. This system was based on the detected five-year, cancer-free survival rates of 87%, 58%, and 21% for each proposed substage, respectively. Recently D’Amico and colleagues proposed a molecular model system consisting of five biologic markers effecting various points in the metastatic sequence (growth regulation, cell cycle regulation, apoptosis, angiogenesis, and metastatic adhesion factor) to further risk stratify patients with NSCLC. The five biologic factors found to be significant predictors of survival in multivariable analysis were erbB -2, RB, p53, factor VIII staining for angiogenesis, and CD-44. Combining these factors, patients are stratified into substage 1, zero to one marker, substage 2, two markers, or substage 3, three to five markers, with five year survivals of 77%, 62%, and 49%, respectively (P ¼ 0:0001) [17] (Fig. 5). In esophageal cancer, Aloia and colleagues evaluated the prognostic value of immunohistochemical tumor marker expression in patients with node-negative esophageal cancer treated with complete resection alone. They found by multivariate analysis a significant relationship between cancerspecific death and the high-level expression of p53 (P ¼ 0:04) (Fig. 6). Additionally, the number of involved tumor markers present was strongly predictive of negative outcome (P ¼ 0:0001) [25] (Fig. 7). Detection of micrometastases by molecular biologic markers Occult micrometastatic disease in the lymph nodes and bone marrow not appreciable by routine histologic staining can be detected with the use of immunohistochemical techniques and reverse transcriptase-polymerase chain reaction techniques (rt-PCR). These techniques are able to detect the presence of 1 tumor cell in 105 to 108 normal cells [127]. Rt-PCR may be more sensitive than immunohistochemical staining for detection of micrometastases [127]. The markers used to detect micrometastases include cytokeratins, mucins (cell surface glycoprotein), and various molecular markers such as epidermal growth factor receptor and p53 expression. The use of epithelial elements is particularly appealing because they are directed toward the identification of epithelial elements that are not present in normal lymph nodes and bone marrow. Detection of occult micrometastatic

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Fig. 5. Analysis of overall survival according to involvement of the five molecular markers that were significant in multivariate analysis in lung cancer. (From D’Amico TA, Massey M, Herndon JE II, Moore MB, Harpole DH Jr. A biologic risk model for stage I lung cancer: immunohistochemical analysis of 408 patients using 10 molecular markers. J Thorac Cardiovasc Surg 1999;117:736–4321; with permission.)

Fig. 6. Esophageal cancer-specific survival analysis comparing patients with low expression of p53 versus those with p53 high-level expression, plotted as percent survival versus time in months using the Kaplan-Meier product limit estimator. Number of patients at risk at one-, three-, and five- year intervals is given along the x-axis. (From Aloia TA, Harpole DH Jr, Reed CE, Allegra C, Moore MB, Herndon JE II, D’Amico TA. Tumor marker expression is predictive of survival in patients with esophageal cancer. Reprinted with permission from the Society of Thoracic Surgeons. Ann Thorac Surg 2001;72(3):859–66.)

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Fig. 7. Esophageal cancer-specific survival analysis comparing patient groups determined by the number of negative prognostic markers present (0, 1–2, 3, and 4 markers), plotted as percent survival versus time in months using the Kaplan-Meier product limit estimator (From Aloia TA, Harpole DH Jr, Reed CE, Allegra C, Moore MB, Herndon JE II, D’Amico TA. Tumor marker expression is predictive of survival in patients with esophageal cancer. Reprinted with permission from the Society of Thoracic Surgeons. Ann Thorac Surg 2001;72(3):859–66.)

lymph node or bone marrow disease using rt-PCR or immunohistochemistry potentially may allow selection of a subset of patients who would benefit substantially from induction therapies. Occult micrometastatic disease found using immunohistochemical staining has been shown to correlate with early relapse and worse prognosis [128–131]. Maruyama and colleagues reported on the detection of micrometastases in lymph nodes using an anti-cytokeratin monoclonal antibody and the value of this detection in predicting early relapse in patients with stage I NSCLC [131]. They identified cytokeratin-positive cells in lymph nodes of 31 out of 44 stage I patients (70.5%), restaging 19 of these as having N1 and 12 as having N2 disease. In these patients with micrometastatic disease in the mediastinal lymph nodes, the disease-free survival was significantly shorter than in those with node-negative disease (P ¼ 0:004). Immunohistochemical staining for the presence of EGFr or p53 has also been used in the detection of micrometastatic disease. The presence of these biologic markers in lymph nodes is correlated with decreased survival [132]. Salerno and colleagues used rt-PCR to detect the presence of messenger RNA transcripts for MUC1 in lymph nodes. Using this technique, they found occult metastases in 33 of 88 lymph nodes negative for metastatic disease based on light microscopy [127]. Several groups have been able to identify micrometastases of lung cancer in bone marrow. Using immunohistochemical techniques, Pantel and colleagues identified cytokeratin-positive cells in the bone marrow of patients

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with operable lung cancer and found their presence correlated with cancer relapse (66.7% versus 36.6%, P < 0:05) [130]. This same group later found that 60% with apparently localized disease had micrometastases in their bone marrow. Cote and colleagues, using immunohistochemical techniques to identify bone marrow metastases in 43 NSCLC patients, reported a significantly shorter time to tumor relapse with the presence of micrometastases in the bone marrow (7.3 versus 35.1 months, P ¼ 0:0009) [133]. Additionally, for patients with early stage lung disease the detection of occult bone marrow metastases was associated with higher rate of recurrence. The ability to use molecular markers in the diagnosis of esophageal cancer offers a potential approach in the detection of occult metastases. The evaluation of lymph nodes, bone marrow, and serum for metastatic disease has been limited by conventional histopathologic techniques. Identification of micrometastases using IHC or rt-PCR may be more sensitive, producing superior staging and appropriate treatment [134]. In a study of 30 patients with esophageal cancer, 123 lymph nodes were analyzed using rt-PCR to detect carcinoembryonic antigen (CEA) mRNA [135]. Of 73 histologically negative lymph nodes, 49% were positive for CEA. The prognosis for patients with histologically negative nodes that were positive for CEA was equivalent to patients with histologically positive nodes. In a study of 78 patients, 31% of those with adenocarcinoma and 17% of those with squamous cell carcinoma had occult lymph node metastases, as detected by IHC analysis of cyokeratin markers AE1/AE3 [136]. In this study, the presence of occult metastases was not associated with survival. In another study of 68 patients with esophageal cancer, 17% of lymph nodes were found to have occult metastases, using the epithelial biomarker BerEp4 [137–139]. In this study, the presence of occult lymph node metastases was associated with decreased survival. A reliable indicator of occult metastatic disease has not yet been discovered. Due to the diversity in the pattern of genetic mutations, the identification of a single molecular marker of micrometastases is unlikely, and the development of a panel of markers may represent a superior strategy of molecular substaging. Summary In both esophageal and NSCLC, the TNM stage at diagnosis remains the most important determinant of survival. Significant research to investigate the biology of NSCLC and esophageal carcinoma is ongoing, and the roles of proto-oncogenes, tumor suppressor genes, angiogenic factors, extracellular matrix proteases, and adhesion molecules are being elucidated. While evidence is accumulating that various markers are involved in NSCLC and esophageal tumor virulence, the current studies are compromised by small sample sizes, heterogeneous populations, and variations in techniques. Large prospective studies with homogenous groups designed to evaluate the

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role of these various markers should clarify their potential involvement in NSCLC and esophageal cancer. Identification of occult micrometastases in lymph nodes and bone marrow using immunohistochemical techniques and rt-PCR is intriguing. These techniques are promising as a method to more accurately stage patients, and therefore to predict outcomes and to determine therapies. Perhaps the most promising area of research is the development of novel drugs whose mechanism of action targets the pathways of various molecular markers. Molecular biologic substaging offers an opportunity to individualize a chemotherapeutic regimen based on the molecular profile of the tumor, thus providing the potential for improved outcomes with less morbidity in patients with both NSCLC and esophageal cancer.

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