Clinical relevance of molecular markers in lung cancer

Surgical Oncology 11 (2002) 167–179

Review

Clinical relevance of molecular markers in lung cancer P. Iyengara,b, M.-S. Tsaoa,b,* a

Department of Pathology, University Health Network-Princess Margaret Hospital, Toronto, Ont., Canada M5G 2M9 b Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ont., Canada M5G 2M9

Abstract The last two decades have seen an exponential growth of our knowledge on the molecular biology of cellular processes and neoplastic transformation. There is high expectation that these advances will be translated into further improvement in the care of cancer patients, especially in the areas of prevention, diagnosis and treatment. Realizing that the histopathological classification of lung cancer has reached its limit in providing additional critical information to further improve treatment strategy, numerous molecular aberrations occurring in lung cancers have been explored as potential new diagnostic markers and markers for molecular sub-staging. Despite extensive studies, most results remain largely controversial. This manuscript will briefly review molecular/ genetic changes that have been investigated as candidate diagnostic, prognostic and predictive markers and as biomarkers for early detection in lung cancer. A more concerted and global approach to study the clinical relevance of molecular changes in lung cancers is required in the future. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Molecular pathology; Biomarkers; Diagnosis; Prognostic Markers; Early detection

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2.

Diagnostic markers . . . . . . . . . . . . . . 2.1. Neuroendocrine markers . . . . . . . . 2.2. Cytokeratins . . . . . . . . . . . . . . 2.3. Thyroid transcription factor-1 (TTF-1) 2.4. Mesothelial cell markers . . . . . . . .

3.

Prognostic markers . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Ras oncogene . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. HER/c-erbB (epidermal growth factor receptor family) and their 3.3. Hepatocyte growth factor and its receptor Met/HGFR . . . . . 3.4. Retinoblastoma (Rb) and p16INK4A . . . . . . . . . . . . . . . 3.5. Cyclin E and p27kip1 . . . . . . . . . . . . . . . . . . . . . . . 3.6. p53 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Bcl-2 family genes . . . . . . . . . . . . . . . . . . . . . . . . 3.8. Other marker genes . . . . . . . . . . . . . . . . . . . . . . . . 3.9. Marker interactions . . . . . . . . . . . . . . . . . . . . . . .

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Predictive markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Ras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. p53 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

172 172 173

*Corresponding author. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ont., Canada M5G 2M9. Tel.: +1-416-946-2104; fax: +1-416-946-6579. E-mail address: [email protected] (M.-S. Tsao). 0960-7404/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 7 4 0 4 ( 0 2 ) 0 0 0 4 7 - 6

P. Iyengar, M.-S. Tsao / Surgical Oncology 11 (2002) 167–179

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4.3. 4.4.

HER-2/c-erbB2/neu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Beta-tubulin gene mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

173 173

5.

Early 5.1. 5.2. 5.3. 5.4. 5.5. 5.6.

detection markers . . . . . . hnRNP . . . . . . . . . . . . p16 promoter methylation . . FIHT gene . . . . . . . . . . Ras gene and p53 mutations . Retinoic acid receptors . . . . Other circulating biomarkers .

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173 174 174 174 174 175 175

6.

Discussion and future consideration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Lung cancer is the second most common cancer among both men and women in North America, but is the most common cause of cancer-related mortality in both sexes [1]. Although there is marked heterogeneity in the histopathological appearances of lung cancers as illustrated in the recently revised WHO classification (Fig. 1) [2], the large majority of tumors are broadly classified into small cell carcinoma (SCLC) and nonsmall cell lung carcinoma (NSCLC). This simplified grouping is based on the biology and clinical behavior of the tumors and their treatment options. Like most other human cancers, lung cancers are characterized by extensive and complex chromosomal and genetic aberrations as well as diverse gene expression and biochemical changes [3,4]. Many of these involve the loss of tumor suppressor genes and the activation of dominant oncogenes, but only few genes within these abnormal chromosomal regions had been identified as having direct roles in the pathogenesis and biology of lung cancer. Functionally important genes

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Variants: Papillary, Clear cell, Small cell, Basaloid.

Small cell carcinoma (SCLC) Variants: Combined small cell carcinoma.

Adenocarcinoma (ADC) Acinar Papillary Bronchioloalveolar carcinoma – – –

Non-mucinous (clara cell/type II peumocytes) Mucinous (goblet cell) Mixed mucinous and non-mucinous

Solid Adenocarcinoma with mixed subtypes: Variants: Well-differentiated fetal, Mucinous (“colloid”), mucinous cystadenocarcinoma, Signet ring adenocarcinoma, Clear cell carcinoma

Large cell carcinoma (LCC) Variants: Large cell neuroendocrine, combined large cell neuroendocrine, Basaloid, Lymphoepithelioma-like, clear cell, large cell with rhabdoid

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that are amplified or deleted are usually associated with increased or loss of expression at mRNA and protein levels, but altered expression may also occur without changes in gene copy number, such as by methylation of the promoter or enhancer sequences of the genes [5]. Changes in the activity of some genes may also result from point mutations at critical functional domains. At the functional level, these genes are mechanistically involved as important regulatory genes in one or more of the following mechanistic pathways: growth or cell cycle check-point, cell survival or apoptosis, invasion and motility and differentiation. Since oncogenic transformation results from the cumulative changes in the behavior of these genes, greater understanding on their roles and mechanisms of action will undoubtedly impact on the future management of lung cancer patients. This review is not meant to be exhaustive but will attempt to summarize the current understanding on the roles of selected molecular changes at the clinical level, especially with respect to their use as diagnostic, prognostic and predictive markers as well as early detection biomarkers.

WHO Classification of Pulmonary Carcinomas Squamous cell carcinoma (SQCC)

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Adenosquamous carcinoma (ADSQ) Carcinomas with pleomorphic, sarcomatoid or sarcomatous elements Carcinomas with spindle and/or giant cells Pleomorphic carcinoma Spindle cell carcinoma Giant cell carcinoma Carcinosarcoma Pulmonary blastoma

Carcinoid tumor Typical carcinoid Atypical carcinoid

Salivary gland tumors Mucoepidemoid Adenoid cystic Others

Unclassified carcinoma

Fig. 1. The 1999 WHO classification of malignant lung epithelial neoplasms.

P. Iyengar, M.-S. Tsao / Surgical Oncology 11 (2002) 167–179

2. Diagnostic markers Although the broad classification of lung cancer into SCLC and NSCLC lessens the importance of precise histopathological subtyping of lung cancers, occasional tumors still pose significant histopathological challenge for reaching the correct diagnoses. Heterogeneity within each major histological subtypes of NSCLC especially among poorly differentiated tumors often requires ancillary studies to sort out the differential diagnoses. The need for these special studies especially immunohistochemistry (IHC) may occur on a daily basis.

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neoplasms arising from these cells [10]. In lung neoplasms, TTF-1 expression is expressed in up to 80% of adenocarcinoma but much less commonly (5–40%) in squamous cell carcinoma [9,10]. A very high proportion ðB90%Þ of atypical carcinoids and small cell carcinoma express TTF-1, but immunoreactivity is slightly less frequent in typical carcinoid [11]. In practice, positive nuclear staining for TTF-1 of the tumor cells is almost diagnostic for lung primary following the exclusion of thyroid origin by a negative thyroglobulin immunostaining. 2.4. Mesothelial cell markers

2.1. Neuroendocrine markers Hyperplastic or neoplastic proliferation of neuroendocrine cells give rise to a spectrum of lesions/tumors with variable clinical aggressiveness. They range from the benign tumorlet, carcinoid, atypical carcinoid to the very aggressive SCLC and large cell neuroendocrine carcinoma [6]. Tumors with neuroendocrine phenotype can be confirmed with positive markers such as neuron specific enolase (NSE), chromogranin, synaptophysin and neural cell-adhesion molecule (N-CAM/CD56). The latter is especially useful for in the diagnosis of SCLC by the characteristic strong membranous staining pattern in approximately 90% of SCLC.

The distinction between malignant pleural mesotheliomas and lung adenocarcinoma involving the pleura always presents a diagnostic challenge for pathologists and almost always require additional studies involving IHC. Since there is no single marker is diagnostic of malignant mesothelioma, most pathologists perform a routine panel of marker studies that includes calretinin, cytokeratin 5/6, thrombomodulin and WT-1 as positive markers, and Ber EP4, CEA, Bg8, TTF-1 and Leu-M1 (CD15) as negative markers [12,13]. In general, results that include 3 positive and 2 negative markers are diagnostic for malignant mesothelioma.

2.2. Cytokeratins

3. Prognostic markers

Immunophenotyping the cytokeratin (CK) profile of tumor cells may be useful in cases where there is a need to distinguish a primary lung versus secondary adenocarcinoma. CK is a family of intermediate filaments characteristically found in epithelial cells. They consist of at least 20 family members that can be distinguished not only by their mobility in gel electrophoresis but also by type-specific monoclonal antibodies. CK is especially useful to confirm a diagnosis of carcinoma. These tumors tend to recapitulate CK profiles of the epithelium from which they arise [7]. Chu et al. [8] studied the expression frequency of CK7 and CK20 in 435 carcinomas originating from diverse organs. Virtually all cases of lung adenocarcinomas (ADC) (90%) are CK7 þ =CK20; while greater than 90% of the cases of colorectal ADC are CK7  =CK20 þ : In the appropriate clinical setting, the CK7/CK20 IHC panel is used to distinguish a primary from metastatic colorectal ADC in the lung.

Prognostic factors are defined as patient and tumor factors that predict the survival outcome of the patient. Currently tumor stage is the single most important determinant of survival in NSCLC [14], although other clinical and laboratory variables have also been reported to show prognostic importance [15]. With the discovery of new genes with putative critical regulatory roles in cellular functions, many biological and molecular changes have also been investigated as potential molecular prognostic factors [16,17].

2.3. Thyroid transcription factor-1 (TTF-1) TTF-1 is a nuclear protein expressed after birth in the follicular and parafollicular C cells of the thyroid and in bronchioloalveolar cells of the lung [9]. Expression of TTF-1 was documented to be highly specific for

3.1. Ras oncogene The interaction of a cell with its surrounding microenvironment, including neighboring cells and extracellular substances, is critical for its survival and function within a multi-cellular organism. The ras family genes and proteins are one of the most studied and best understood molecules that transduce biochemical signals from cell surface to the nuclei (Fig. 2). There are three family members of the Ras proto-oncogene: Haras, K-ras and N-ras [18]. These genes are located on separate chromosomes but they produce functionally identical and structurally similar proteins. The activation and deactivation of Ras protein involves conversion between the inactive Ras-GDP and active Ras-GTP

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170 Receptor Tyrosine Kinases (e.g. EGFR, HGFR, IGFR, etc.)

Sos

Grb2

PI3Kinase P

Akt/PKB

Raf

RAS

MEK

others

P

Akt/PKB-P P

P

MAPK

MAPK-P

Caspase-9 Bad GSK-3β NFkB FKHR

Anti-apoptosis

Gene transcription

Fig. 2. A simplified signaling diagram of the receptor tyrosine kinases and its downstream ras mediated pathways.

forms. Oncogenic mutations at one of the critical codons (12, 13 or 61) render the Ras proteins locked into a growth stimulatory GTP-bound state. Ras activation has been linked to various cell processes that are important in cancer biology, including mitogenesis, apoptosis, angiogenesis, invasion and metastasis [19–21]. In lung cancer, ras mutations predominantly involve the K-ras gene and they are found in approximately 35% of lung ADC and 20% of large cell undifferentiated carcinomas (LCUC), but they rarely occur (o5%) in squamous cell carcinomas (SQCC) [22–24]. Since the first report on significant negative impact of ras mutations on the prognosis of lung ADC patients [25], numerous positive and negative studies have been reported with an overall result that indicates the prognostic impact of ras mutations in NSCLC remains indefinite and controversial. 3.2. HER/c-erbB (epidermal growth factor receptor family) and their ligands The Ras protein and the downstream pathways may also be activated by its upstream stimulators that include the receptor tyrosine kinases (RTKs) (Fig. 2). RTKs are transmembrane proteins with extracellular ligand binding domain and intracellular tyrosine kinase domain that is capable of phosphorylating protein tyrosine residues [26]. Although there are many RTK loops, the dominant and most studied ones in lung cancer include members of the HER/c-erbB family receptors: the epidermal growth factor receptor (EGFR) and the HER-2/c-erbB2/Neu [27,28]. High expression of EGFR by IHC has been noted in > 90% of SQCC and approximately 50% of ADC [29,30]. The prognostic value of EGFR overexpression in NSCLC remains controversial [31]. In a few studies, however, EGFR expression has been reported to correlate with poorer

prognosis when seen in stage T1N0 patients and when co-expressed with other markers such as its preferred ligand the transforming growth factor (TGF)-a [33], matrix metalloproteinase (MMP)-9 [34], HER-2/neu [35] or the p53 gene mutations [36]. The second important c-erbB family gene is the HER2/c-erbB2/neu, which has been most extensively studied in breast cancer. Unlike the latter, HER-2 gene amplification rarely occurs in lung cancer [37]. Thus, overexpression is due to increased transcription or posttranslational protein stabilization. Kern et al. [38] were the first to evaluate the frequency and prognostic significance of HER-2 expression in NSCLC. Their initial study demonstrated 38% and 36% expression among ADC and SQCC, respectively. Correlation with poorer survival was found among the ADC patients. Several subsequent studies have reported lower rates of HER-2 overexpression in NSCLC and failed to demonstrate a consistent prognostic value of HER-2 immunostaining in NSCLC patients [39,40]. Pastorino et al. [32] evaluated 515 stage I NSCLC patients that included 217 ADC patients. They found HER-2 positivity in only 4% of tumors and 6% of ADC, and there was no correlation with the overall ðRR ¼ 1:05; p ¼ 0:87Þ as well as the disease free survivals ðRR1:52; p ¼ 0:25Þ: Overall, the prognostic value of HER-2 over-expression in NSCLC currently remains uncertain. 3.3. Hepatocyte growth factor and its receptor Met/HGFR Met/hepatocyte growth factor receptor (HGFR) is a unique RTK with close homology to the insulin receptor [41]. Interaction of Met with its ligand HGF results in pleotropic effects on cell proliferation, differentiation and motility [42]. Both Met and HGF are commonly expressed in various types of human cancers [43]. HGF is synonymous with the scatter factor (SF) named for its ability to stimulate cell movement, especially in epithelial cells [44]. This lead to the hypothesis that HGF–Met loop plays an important role in invasion and metastasis. Until recently, the HGF–Met interaction has primarily been considered a paracrine one with the ligand mainly produced by the stromal cells, while the receptor is expressed mainly on the epithelial cells [45]. Using in situ hybridization and IHC, Tsao et al. [46] have demonstrated that the HGF mRNA expressed in NSCLC is predominantly by the tumor cells, suggesting that the HGF–Met interaction in lung cancer involves both autocrine and paracrine functions, the latter through the potent angiogenic effect of HGF. Siegfried et al. [47] reported that the levels or concentration of HGF in resected NSCLC tissue is an independent poor prognostic factor. Takanami et al. [48] also reported that lung ADC patients with tumors that stained positively for Met had a significantly worse prognosis

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in multivariate analysis than those patients whose tumors demonstrated negative staining. 3.4. Retinoblastoma (Rb) and p16INK4A The cell cycle checkpoints are governed by families of genes including cyclins, cyclin dependent kinases (cdks), retinoblastoma (Rb), p53 and E2F (Fig. 3) [49]. Rb is a potent inhibitor of G1-S progression through its binding and sequestration of the E2F transcription factor. In order for the G1 phase to proceed, Rb protein has to be phosphorylated resulting in its release from E2F, which then activates the transcription of genes necessary for S phase progression. Rb phosphorylation is regulated by cdk4 and cdk6 whose activities in turn are inhibited by the INK family of cdk inhibitors p21cip1=waf 1 and p16INK4A [50,51]. The p16 competes with cyclin D for binding to cdk4/6. Cancer cells can deregulate the G1 phase of the cell cycle checkpoint by inactivation of the Rb gene, amplification or overexpression of cyclin D or cdk4, or inactivation of the p16 gene. Rb gene inactivation occurs in almost all (90%) SCLC but in only 10–30% of NSCLC. In contrast, the loss of p16 expression occurs in 50–60% of NSCLC [51]. Inactivation may be caused by homozygous deletion (20%), point mutations (5–10%), and promoter methylation (20%). Rb and p16 inactivation appear to be mutually exclusive, consistent with their shared function. Overall, approximately 75% of NSCLC are expected to show aberrant (loss of) Rb/p16-regulated cell cycle checkpoint. A few reports have correlated the abnormalities in Rb and p16 gene expression with patient outcome. Reissmann et al. [52] studied 219 primary NSCLC tumors of patients enrolled into the LCSG (Lung Cancer Study Group) protocol 871. A lack of Rb immunostaining in at least 80% tumor cells was found in 32% of the cases studied. However, there was no correlation between the loss of Rb staining in tumor cells with the relapse-free or overall survival. In contrast,

Fig. 3. A simplified diagram of the G1 cell cycle checkpoint regulations.

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Kratzke et al. [53] studied 100 NSCLC and found that loss of p16 rather than Rb expression correlated with poorer survival. A recent report indicated that the other member of the Rb family genes, Rb2/p130 may also play an important role in the biology of NSCLC [54]. 3.5. Cyclin E and p27kip1 Aside from the Cdk4/6-cyclinD complex, the G1/S cell cycle transition is also regulated by the Cdk2cyclinE/A complexes (Fig. 3). Cyclin E activation of Cdk2 also inhibits Rb function. The expression of cyclin E has recently been correlated with poor prognosis in early stage (I and/or II) NSCLC patients [55–58]. On the other hand, Cdk2 activity is potently inhibited by p27kip1 ; and the level of p27kip1 is modulated through activation of the p27-directed ubiquitin degradation pathway [59] and by the transforming growth factor (TGF)-b1 [60]. Reduced p27kip1 expression in both resected early and non-resected advanced NSCLC have been correlated with poor prognosis [61–64]. The potential interaction of cyclin E and p27kip1 as prognostic factors remains to be investigated. 3.6. p53 The normal p53 (wild-type) protein has major functions in regulating apoptosis and G1 -S cell cycle checkpoint, in sensing stress or apoptotic signals, and in instituting cellular defense mechanisms against mutagenic DNA damages (Fig. 3) [65,66]. Recent evidence also suggests a role for p53 in regulating angiogenesis [67]. Thus, it is not difficult to imagine the critical role of p53 mutation in all stages of carcinogenesis, including tumor progression and response to therapeutic interventions [68,69]. Perhaps no other molecular/genetic marker has been studied more extensively than p53 for its prognostic value in NSCLC. Meta-analyses have been used to assess the overall outcome of these studies [70,71]. The overall frequency of p53 mutations in NSCLC is 48% as detected by positive immunohistochemical staining of the nuclei, and 37% by single strand conformation polymorphism (SSCP) detection of gene mutations. It is worth noting that most SSCP studies have been limited to exons 5–8, which account for only 75–80% of p53 mutations. There is a consensus that both p53 protein overexpression and gene mutations are less common among ADC (36% and 29% respectively) than SQCC (54% and 49% respectively). The meta-analysis result indicates that p53 alterations have negative prognostic effect in patients with ADC but not SQCC [70]. Most studies that involved a large number of early stage (I–II) NSCLC patients have also showed good correlation between positive p53 staining with poorer prognosis [72–77]. Nevertheless, there is as yet no plan to

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incorporate p53 mutational analysis into the clinical management of NSCLC patients. This is mainly due to the lack of standardized methods and criteria for routine scoring of the p53 status in clinical samples. It is commonly regarded that positive nuclear immuno-staining is equivalent to p53 gene mutation identified by molecular techniques, but extensive data have shown that the overall concordance rate between positive staining and p53 mutation is only 66% [78]. However, it is also clear that there is a better correlation between positive nuclear p53 staining and the presence of mis-sense mutation or in-frame deletion that produce more stable mutant proteins; the latter accumulate and become detectable by IHC. There is controversy whether IHC and mutational analyses (PCR-SSCP) provide similar or different biological meanings to the abnormalities detected. There is evidence to suggest that only p53 mis-sense mutations but not the null mutations have negative impact on prognosis when compared to patients with wild type p53 gene [79]. 3.7. Bcl-2 family genes The bcl-2 family gene includes both negative regulators of apoptosis such as bcl-2, and enhancers of apoptosis such as BAX. These protein family members are capable of forming homodimers, heterodimers, or both and these complexes are tightly controlled [80]. Under homeostatic conditions, bcl-2 and BAX form heterodimers. Under conditions such as radiationinduced injury, there is increased transcription of BAX but decreased transcription of bcl-2. This tips the equilibrium towards the formation of BAX homodimers that eventually leads to apoptosis [80]. The basal cells of normal bronchial epithelium stain positively by immunohistochemistry for bcl-2 [81]. The expression of bcl-2 protein by IHC has been detected in approximately 20–40% of various subtypes of NSCLC. Despite the initial reports by Pezzella et al. [82] and Fontanini et al. [83] that indicated a correlation of positive bcl-2 staining in NSCLC with better survival, several subsequent reports have failed to confirm bcl-2 as an independent prognostic factor for NSCLC [84–86].

3.9. Marker interactions While studies on single genes/factors as independent prognostic markers remain valid, there is an emerging concept that a comprehensive multivariate analysis taking into account the interactions and synergism of many critical genes appears more likely to yield a clinically useful molecular classification and subgrouping. Kwiatkowski et al. [77] performed a multiparametric analysis of the impact of three demographic characteristics, surgical extent, 11 pathologic features, and seven molecular factors on the overall and diseasefree survivals of 244 stage I NSCLC patients. Multivariate analysis using the Cox proportional hazards regression model identified nine markers that independently conferred increased risk for recurrence for all patients, and six markers for patients that were treated by complete resection. Among patients treated by complete resection and taking into account the six risk factors (adenocarcinoma solid tumor with mucin subtype, lymphatic invasion, TX4 cm; K-ras mutation, positive p53 staining and lack of H-ras expression), those with 2 or less risk factors had a 5-year cancer free survival (CFS) rate of 87%, while those with 4 or more risk factors had a CFS rate of only 21%. D’Amico et al. [97] performed a similar study using 10 molecular markers representing 5 groups of oncogenic pathways on 408 stage I NSCLC treated by complete resection and. Five markers (HER-2, Rb, p53, Factor VIII, CD44) were significantly associated with elevated risk for cancer recurrence and death, but survival was strongly associated with the cumulative number of molecular risk factors.

4. Predictive markers Predictive factors are defined as markers, both clinical and molecular that predict response to adjuvant treatments such as chemotherapy or radiotherapy. While prognostic factors define the effects of tumor characteristics on the patient, predictive factors define the effect of treatment on the tumor. In lung cancer, few molecular predictive factors have been studied and most of them are related to response to chemotherapy.

3.8. Other marker genes 4.1. Ras The number of genes that have been studied for their potential prognostic value in lung cancer is quite vast, and this limited review does not allow their exhaustive descriptions. Among these genes include the FHIT (fragile histidine triad) gene [87], vascular endothelial growth factor (VEGF) [88–90], cyclo-oxygenase 2 (COX-2) [91–93] and MMPs and TIMPs [94–96]. Some of these candidate prognostic marker genes are ‘‘hot’’ targets for development of novel therapeutic agents.

There have been few studies that have assessed whether ras mutations predict response to chemotherapy. Most studies have been undertaken either in cell lines or in other in vitro systems. Koo et al. [98] reported that activated ras enhanced the sensitivity of human tumor cells to some chemotherapy agents by potentiating an apoptotic response. However, this theory was not confirmed in US Intergroup Trial 0115 (EST3590) in

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which completely resected patients with stage II and IIIA NSCLC treated with post-operative thoracic radiation were randomized to receive chemotherapy with etoposide (an epipodophyllin) and cisplatin or no further therapy [99]. Additional studies are needed to confirm interaction (or the lack of) between K-ras mutations and chemotherapy. 4.2. p53 P53 gene alteration has been evaluated as a predictor of response to chemotherapy in many tumor types [100,101] (see Table 1). In NSCLC, conflicting results were reported from two trials of adjuvant chemotherapy after surgical resection. Schiller et al. [99] found no differential effect from chemotherapy with etoposide and cisplatin in patients who had either wild type or mutated p53 (evaluated by SSCP). However Tanaka et al. [102] reported that normal p53 gene as assessed by IHC was associated with significantly longer survival for stage I NSCLC patients treated with adjuvant UFT (tegafur and uracil), while the same chemotherapy conferred no survival advantage when p53 was overexpressed.

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4.4. Beta-tubulin gene mutation Tubulin is a heterodimer consisting of alpha and beta sub-units that form the microtubule. The alpha-tubulin subunit is primarily a structural role, whereas the betasubunit is critical in microtubule assembly, hence cell division and replication. Monzo et al. [106] reported that 16 of 49 (33%) patients with NSCLC had beta-tubulin gene mutations in exons 1 or 4. More interestingly, however, none of the patients with mutations responded to paclitaxel, compared to 13/33 (39.4%) patients with normal tubulin genes ðp ¼ 0:001Þ: Similar studies have not been done for the other commercially available taxane, docetaxel, nor for any of the vinca alkaloids which also act at the level of tubulin. Clearly, betatubulin gene mutation has the potential to become one of the most important predictive tools for NSCLC since almost all patients with this cancer are treated with either a taxane or a vinca alkaloid at some time during the course of their disease. However, it should be cautioned that two recent reports have questioned the existence of beta-tubulin mutations in primary NSCLC [107,108].

4.3. HER-2/c-erbB2/neu

5. Early detection markers

Using a panel of 20 NSCLC cell lines, Tsai et al. [103] showed that high HER-2 expression was correlated with chemoresistance, low S-phase fraction and long doubling times. Multivariate analysis revealed that HER-2 level was the only independent predictor for chemoresistance to doxorubicin, etoposide and cisplatin. In two small studies of induction chemotherapy for stage III NSCLC, high HER-2 expression was not predictive of response to chemotherapy although it was associated with poor overall survival [104,105]. These trials involved only 28 and 30 patients each and the patients were not randomized to receive or not to receive chemotherapy, thus other clinical factors may have influenced response to chemotherapy.

Since lung cancer staging is the most important determinant of survival, detection of cancers at an earlier stage would result in a significant improvement in the outcome of this disease. Major efforts during the last two decades have been devoted to better recognition of pre-malignant cells in sputum that might indicate high risk of lung cancer development. Combined with technical advances in bronchoscopy and imaging, molecular diagnostics could also contribute significantly to increasing the sensitivity and specificity of lung cancer early detection programs. The concept that ongoing genetic alterations in the bronchial epithelium will eventually lead to cancer is based on the frequent occurrence of multifocal

Table 1 The role of p53 aberrations in response to chemotherapy p53 Status

Schiller et al. [99] p53 Wildtype p53 Mutant Tanaka et al. [102] p53 Normal p53 Overexpressed ns: non-significant

Chemotherapy

P value

Survival No chemotherapy

Chemotherapy

51.7 months 38.2 months

41.8 months 40.3 months

ns ns

74.3% 5-year 62.8% 5-year

95.2% 5-year 55.8% 5-year

0.022 ns

Etoposide/Cisplatin

UFT

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synchronous or metachronous tumors and dysplasias in the airways of patients with lung cancer. Broad areas of the tracheobronchial tree in high-risk patients, especially smokers, are prone to premalignant or multifocal malignant change. This concept of ‘‘field carcinogenesis’’ leads to two mechanistic hypotheses [109]. It is possible that carcinogens in tobacco smoke can induce genetic mutations at dispersed sites that are exposed to high concentrations of tobacco smoke; this would lead to the development of multiple primary lung tumors in which each tumor had a separate genetic profile. Alternatively, it is also possible that a single clone of mutant progenitor epithelial cells expands over time to populate widespread areas of the respiratory tract, which would result in common mutations found at different sites. In the lung, identical abnormalities have been found in lung carcinoma and adjacent nonmalignant epithelium [110]. These mutations include partial loss of genomic materials resulting in loss of heterozygosity (LOH) or point mutations. Extensive studies on cancerous and pre-cancerous lesions of the lung have indicated high frequency of genetic changes involving the 3p, 9p and 17p chromosomal regions where many candidate tumor suppressor genes are putatively located. 5.1. hnRNP In the 1980s, The NCI-Navy group studied the efficacy of a series of monoclonal antibodies raised against small cell and non-small cell lung cancer cell lines to improve the detection rate of lung cancer cells in the sputum [111,112]. Immunohistochemical staining was performed using these antibodies on sputum specimens of subjects with moderate to severe dysplasia while being followed up longitudinally in the John Hopkins Lung Cancer Detection project. The results indicated that these monoclonal antibodies could detect lung cancer on average 2 years prior to the development of clinical cancer with a sensitivity of 91%, and a specificity of 88%. One of the antibodies detects the heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 that was recently characterized as an onco-developmental protein. A study on bronchial lavage specimens stained immunohistochemically by this hnRNP A2/B1 (monoclonal antibody 703D4) also yielded 96% sensitivity and 82% specificity in detecting the presence of cancer cells [113]. A prospective study was carried out to evaluate hnRNPA2/B1 as an early detection biomaker in postresection stage I lung cancer patients with high risks of developing second or primary lung cancer. The results indicated that hnRNPA2/B1 overexpression in sputum specimens may accurately predict 67% and 69% of lung cancer development during the first year of follow-up, as compared to 2.2% and 0.9% of lung cancer risks in this study population [114].

5.2. p16 promoter methylation Inactivation of p16 may occur through homozygous deletion or hemizygous deletion coupled with inactivation of the second p16 allele by point mutation or hypermethylation of the p16 promoter CpG islands [51]. The frequency of p16 methylation increases during disease progression from basal cell hyperplasia (17%) to squamous metaplasia (24%) to carcinoma in situ (50%) [115]. Using a polymerase chain reaction (PCR) based technique to detect specifically the presence of methylated and unmethylated p16 promoter sequences, methylation can be detected in greater than 50% of exfoliated bronchial or bronchial lavage specimens of lung cancer patients. In one study, aberrant methylation of the p16 and/or 06 -methyl-guanine-DNA methyltransferase gene promoters in DNA isolated from sputum specimens was detected in 100% of squamous cell carcinoma patients at the time of or up to 3 years before clinical diagnosis [116]. It should be noted, however, that methylation of p16 promoter has also been detected in 20% of cancer-free individuals who are at high risk for lung cancer development [117]. 5.3. FIHT gene Alternative strategies that have recently been reported for early detection of lung cancer include assay of loss of heterozygosity (LOH) in the plasma DNA of NSCLC patients. Such plasma DNA is presumably derived from tumor cell DNA. Sozzi et al. reported the results of assays for microsatellite instability (D21S1245) and LOH of FHIT locus in plasma DNA of 87 stage I–III NSCLC patients [118]. The two markers were detected in 56% and 40% of tumor tissues and plasma samples, respectively, with 61% of patients with tumor alteration also showing plasma changes. Plasma changes were not detected in 14 control subjects. Of interest was the detection of plasma changes in 43% of stage I patients and in 45% of patients with tumors o2 cm: However, plasma changes were not able to predict risk of recurrence. The results of this study further suggest the possibility of using blood or plasma biomarker assays for early detection strategies. 5.4. Ras gene and p53 mutations Mutations on the Ki-ras and p53 genes have also been detected in sputum and bronchial alveolar lavage specimens of lung cancer patients. In one study, these mutations were detected up to 1 year before the diagnosis of clinical lung cancers [119]. Circulating p53 antibody has also been detected in 30% of lung cancer patients [120], especially in patients whose tumor demonstrate mis-sense mutations in the p53 gene [121].

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5.5. Retinoic acid receptors Vitamin A and its related analogues retinoids, are important in lung development and maturation, and they have been tested as potential chemoprevention agents in lung cancer [122]. The cellular effects of retinoids are mediated by the various retinoic acid receptor isoforms RARs and RXRs. RARb is located on 3p24.2, a chromosomal area that is commonly deleted in lung cancers [123]. Subsequent studies have shown that RARb inactivation or loss of expression by deletions or promoter hypermethylation occur commonly in both SCLC and NSCLC, and the frequency is correlated with increasing exposure to tobacco smoking and to increasing grade of bronchial dysplasia [124]. A recent placebo-controlled study has demonstrated that 6-month treatment of smokers whose bronchial brushing samples demonstrated depressed RARb expression by 13-cis-retinoic acid, is significantly associated with upregulation of the RARb expression levels, thus providing a candidate biomarker for future clinical trials involving the retinoids [125]. 5.6. Other circulating biomarkers Circulating fragments of cytokeratins have also been detected in the blood samples of lung cancer patients [126]. These include tissue polypeptide antigen (TPA), tissue polypeptide-specific antigen (TPS) and cytokeratin-19-fragments (Cyfra 21-1); the latter is measured by ELISA assay. The sensitivity of Cyfra 21-1 in detecting lung cancer ranged from 61% for squamous cell carcinoma to 36% for small cell carcinoma, and the plasma levels were correlated with almost all parameters of disease extent. Other studies have also reported elevated levels of matrix metalloproteinases (MMP), the tissue inhibitors metalloproteinases (TIMP) and type I collagen degradation marker ICTP [127]. These results suggest that serum/plasma of lung cancer patients most likely will reveal many more proteins that may be used as early detection biomarkers for lung cancer patients. A recent report that showed serum proteomic patterns capable of identifying ovarian cancer patients strongly suggest that novel techniques in proteomics will have a major impact on lung cancer early detection effort and strategy [128].

6. Discussion and future consideration During the last 10–15 years, significant advances have been made in our knowledge on the molecular biology and pathology of lung cancer. Despite many studies, the impact of molecular pathology at the clinical level remains invisible. The value of many candidate prognostic, predictive and early detection biomarkers remain

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unconfirmed and controversial. Numerous reasons may be argued for the tardiness of this achievement, foremost among them include: 1. the use of diverse techniques to assay a specific molecular aberration; 2. the imprecise/subjective nature of current method to score expression changes and the lack of standardized scoring criteria; 3. the paucity of studies performed on carefully defined or controlled cohorts of patients or their respective tumor samples enrolled in large randomized phase III clinical trials. Without significant efforts made to correct these shortcomings, the prospect of making important advances in bringing molecular pathology to the bedside appears pessimistic. It is quite apparent, however, that conventional histopathology including the classification system and technologies have reached the limits of their roles in providing additional novel markers to improve the management of lung cancer patients. Although the completion of the human genome sequencing program is still 1–2 years away, the impact of this project has already become apparent in the development of new global approaches and high throughput technologies for studying the genetic basis of diseases. Preliminary results from DNA microarray studies have confirmed the existence of large-scale gene expression profiles that correlate with lung cancer biology including histopathology and clinical outcome [129–131]. The next few years will likely involve fresh approaches to define molecular signatures of specific cancer type of their cells. The parallel development of new high throughput and quantitative technologies to analyse genomic or expression aberrations will undoubtedly provide additional means to translate our newly found knowledge into overall better care of cancer patients. In the future, the care of cancer patients will undoubtedly be defined by specific molecular pathology profiles of their particular tumor.

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