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Journal of Thoracic Oncology:
doi: 10.1097/JTO.0b013e3181a4b8fb
State of the Art: Concise Review

Prognostic and Predictive Markers of Benefit from Adjuvant Chemotherapy in Early-Stage Non-small Cell Lung Cancer

Custodio, Ana Belén MD; González-Larriba, José Luis MD, PhD; Bobokova, Jana MD; Calles, Antonio MD; Álvarez, Rafael MD; Cuadrado, Eugenio MD; Manzano, Aranzazu MD; Díaz-Rubio, Eduardo MD, PhD

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Author Information

Medical Oncology Department, Hospital Universitario Clínico San Carlos, Center affiliate to the “Red Temática de Investigación Cooperativa (RD06/0020/0021)” and Programme “Acción Transversal del Cáncer,” Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Science and Innovation, Madrid, Spain.

Disclosure: The authors declare no conflicts of interest.

Ana Belén Custodio, MD, C/Professor Martín Lagos, s/n. Ciudad Universitaria, Madrid 28040, Spain. E-mail: anabelencustodio@hotmail.com

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Abstract

Lung cancer—predominantly non-small cell lung cancer (NSCLC)—is the leading cause of death from cancer in most industrialized countries. Patients with early-stage NSCLC are at substantial risk for recurrence and death even after potentially curative surgery. Multiple large randomized trials have demonstrated that adjuvant chemotherapy using modern cisplatin-based regimens can significantly improve 5-year survival in carefully selected patients with NSCLC.

The current staging system is inadequate for predicting the outcome of treatment and the prognosis in an individual patient. Molecular markers may provide additional information about the likelihood of relapse beyond that obtained from pathologic staging. They may also have value in determining which patients will benefit from adjuvant platinum-based chemotherapy.

This is a review focused on approaches and specific markers under study, including gene expression profiles, DNA repair pathways, class III β-tubulin expression, abnormalities in the k-ras oncogene and p53 tumor suppressor gene, and DNA methylation markers. Additional studies will be required to determine whether these markers are useful in selecting patients for adjuvant platinum-based chemotherapy.

Lung cancer is the most common cause of death from cancer worldwide, and non-small cell lung cancer (NSCLC) accounts for almost 80% of such deaths.1 Patients with early-stage NSCLC are at substantial risk for recurrence even after potentially curative surgical resection and 5-year survival ranges from only 30 to 60%. As many as 40% of patients with stage I, 66% of stage II and 75% of stage IIIA patients will develop recurrence and die as a result of their disease within 5 years of resection.2–4

Several recent trials using cisplatin-based doublets have demonstrated that adjuvant chemotherapy improves 5-year overall survival (OS) by 8 to 15% in carefully selected patients with completely resected stages II and IIIA NSCLC.5–7 Its role in stage IB disease is less established, and it is not currently recommended for routine use. Neither the Lung Adjuvant Cisplatin Evaluation meta-analysis8 nor any of the recently published large randomized clinical trials (NCIC-CTG JBR.10,7 ANITA,6 and International Adjuvant Lung Cancer Trial (IALT)5) have shown a significant OS benefit for cisplatin therapy in stage IB subgroup. Nevertheless, the Cancer and Leukemia Group B (CALGB) 9633 study9,10 has found a nonsignificant survival benefit for carboplatin-based therapy in stage IB patients (harzard ratio [HR], 0.80; 90% two-sided confidence interval [CI], 0.60–1.07; p = 0.10) and a significant benefit in terms of relapse-free survival (RFS) (HR, 0.74; 90% two-sided CI, 0.57–0.96; p = 0.027). Moreover, the trial reported significant OS and RFS advantages in an unplanned subset analysis of patients with tumors ≥4 cm (HR, 0.66; 90% CI, 0.045–0.97, and HR = 0.62; 90% CI, 0.44–0.89, respectively). To date, very few patients with stage IA NSCLC have been enrolled onto randomized clinical trials, and adjuvant chemotherapy is not recommended in these cases.

Although the current standard of treatment for patients with stage I NSCLC is surgical resection, nearly 30 to 35% of them will relapse after initial surgery and thus have a poor prognosis, indicating that a subgroup of these patients might benefit from adjuvant chemotherapy.11 Similarly, as a population, patients with stage II or stage IIIA NSCLC receive adjuvant chemotherapy, but some may receive potentially toxic treatment unnecessarily. Thus, we can consider NSCLC as a heterogeneous disease. Even in patients with similar clinical and pathologic features, the outcome varies: some are cured, whereas in others, the cancer recurs.

Prognostic markers are patient or tumors factors that, independent of treatment, predict patient survival outcome. Predictive markers are factors that may influence and predict the outcome of treatment in terms of either response or survival benefit.

Although other clinical and pathologic markers have prognostic significance,12–15 the clinic-pathologic staging system has been the standard for determining NSCLC prognosis.13 But this classification scheme is probably an imprecise predictor of the prognosis of an individual patient. Thus, main investigational studies nowadays are focused in identifying molecular markers of recurrence, beyond pathologic stage, after surgical treatment and factors that can predict a benefit from platinum adjuvant chemotherapy in poor prognosis subgroups, to individualize treatments. This ability to identify subgroups of patients more accurately may improve health outcomes across the spectrum of disease. The study of molecular factors that influence drug responsiveness is also a potentially promising approach to decrease treatment toxicity and costs by avoiding the administration of ineffective therapy to patients destined not to benefit.

Many molecular markers that predict patient survival independent of the tumor-node-metastasis staging system have been reported.14,16–19 These include oncogenes (K-ras, BclII, Her2/neu, EGFR), tumor suppressor genes (p53, RB, p16, p27), cell cycle modulators (cyclins), molecules related to tumor invasion and metastasis (CD 44, cathepsin B, matrix metalloproteinase), telomerase, molecules involved in tumor angiogenesis (vascular endothelial growth factor, vascular endothelial growth factor receptor), and cyclo-oxygenase 2.

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GENE EXPRESSION PROFILES

Gene expression profiling may identify patient groups with significantly different prognosis. They may also have value in determining, more selectively than stage, which patients will benefit from adjuvant chemotherapy. A number of prognostic gene expression signatures have been reported to predict survival in NSCLC.

Gene-expression profiling by means of microarrays20,21 and reverse-transcriptase polymerase chain reaction (RT-PCR)16 is useful for classifying tumors and formulating a prognosis for patients with various types of cancer, including lung cancer. The use of microarrays in clinical practice is limited, however, by the need for complicated methods, the large number of genes used in genes profiling, the need for fresh-frozen tissue and the lack of both reproducibility and independent validation of the results.22 The genes selected for profiling in studies of lung cancer have varied considerably and only a few genes have been consistently included.23,24 Moreover, gene-expression profiles can vary according to the microarray platform and the analytic strategy used.

The RT-PCR method can be applied to paraffin-embedded pathologic specimens and is more useful in clinical practice. It allows for accurate and reproducible quantification of results for RNA obtained from histologic specimens. However, RT-PCR can be used to analyze only a small number of genes.22

The potential value of gene expression profiling in NSCLC was illustrated by a Duke University study.25 Authors identified a gene-expression profile—the lung metagene model—that predicted the risk of recurrence in a cohort of 89 patients with early-stage NSCLC enrolled through the Duke Lung Cancer Prognostic Laboratory. Pathologic stages in the study cohort were as follow: 39 patients (44%) had stage IA, 30 (34%) stage IB, four (5%) stage IIA, 10 (11%) stage IIB, and six (7%) stage IIIA. This genetic strategy was then validated in two separate cohorts from multicenter cooperative group trials: 25 patients from the American College of Surgeons Oncology Group Z0030 study and 84 from the prospective CALGB 9761 trial. Neither adjuvant chemotherapy nor radiotherapy was allowed in any patient.

The lung metagene model represents the dominant average pattern of expression of the gene cluster across the tumor samples. The confirmation that it represents the biology of the tumor was provided by the finding that the metagenes with the greatest discriminatory capability in the model included genes that have previously been shown to have clinical relevance in NSCLC. In some instances, a metagene represented a single molecular process such as angiogenesis (metagene 19), which is a proven target for therapy in NSCLC. Other key metagenes, such as metagene 41, represented a combination of biological processes—for example, the BRAF, phosphatidylinositol 3′ quinase, TP 53, and MYC signaling pathways.

The lung metagene model for the prediction of recurrence was superior to a predictive model generated with the same methods but that included clinical data alone (including age, sex, tumor diameter, stage of disease, histologic subtype, and smoking history). In the Duke cohort, the lung metagene model predicted disease recurrence with an overall accuracy of 93%. The model built with clinical data had an accuracy of only 64%. Inclusion of the clinical data with the genomic data did not further improve the accuracy of the prediction of recurrence over that of the genomic data alone. This model was consistently accurate across all the early stages of NSCLC and between the major histologic subtypes, not only in the estimated risk of recurrence but also in the results of the Kaplan-Meier survival analysis for each stage or subtype. Applied to the cohorts from the American College of Surgeons Oncology Group Z0030 trial and the CALGB 9761 trial, this genomic strategy had an overall predictive accuracy of 72 and 79%, respectively.

This gene expression profile also was applied to 68 patients with stage IA disease, who are not usually candidates for adjuvant chemotherapy. Kaplan-Meier survival curves were generated for the group as a whole and for the subgroups predicted to be at high or low risk for recurrence by the lung metagene model. Although the survival rate for the group was approximately 70% at 4 years, the survival rate for those predicted to be at low risk was 90% and less than 10% for those predicted to be at high risk, thus identifying the subgroup of patients with stage IA NSCLC at high risk of recurrence, who might benefit from adjuvant chemotherapy (Figure 1).

Figure 1
Figure 1
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In another important study from Taiwan University,26 authors examined the expression of multiple genes associated with invasive activity in frozen specimens of lung-cancer tissue from 125 randomly selected patients who underwent surgical resection of NSCLC and not received adjuvant chemotherapy, to identify a gene signature that is correlated with clinical outcome.

Sixteen genes were initially identified by analyzing microarray data and then confirmed by RT-PCR. From these, the authors further identified five genes that were significantly associated with survival. The levels of expression of these five genes were used to construct a decision tree to classify patients as having a high-risk gene signature or a low-risk gene signature. The five selected genes were: dual-specificity phosphatase 6 (DUSP6), monocyte-to-macrophage differentiation-associated protein (MMD), signal transducer and activator of transcription 1 (STAT1), v-erb b2 avian erythroblastic leukemia viral oncogene homolog 3 (ERBB3), and lymphocyte-specific protein tyrosine kinase (LCK).

The authors identified 59 patients with high-risk gene signatures and 42 with low-risk gene signatures, according to gene expression as measured with RT-PCR and decision-tree analysis. The five-gene signature was strongly associated with OS (sensitivity 98%; specificity 93%; positive predictive value 95%; negative predictive value 98%; and overall accuracy 96%). The presence of a high-risk five-gene signature in the NSCLC tumors was associated with an increased risk of recurrence and decreased OS. With a median follow-up of 20 months, the patients with a high-risk gene signature had a shorter median OS than the patients with a low-risk gene signature (20 months versus 40 months, p < 0.001). The high-risk gene signature was associated with a median RFS of 13 months, whereas the low-risk gene signature was associated with a median RFS of 29 months (p = 0.002) (Figure 2).

Figure 2
Figure 2
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According to multivariate regression analysis, the high-risk five-gene signature, tumor stage III and older age were significantly associated with death from any cause among the 101 patients, and the high-risk five-gene signature and tumor stage III were significantly associated with recurrence of cancer as well (HR for the high-risk signature versus the low-risk signature, 1.92; 95% CI, 1.06–3.46; p = 0.03).

In a subgroup analysis of 59 patients with stage I or II disease, those with a high-risk gene signature had a shorter OS and a shorter RFS than those with a low-risk gene signature (Figure 2).

These results were validated in an independent cohort of 60 patients with NSCLC and with the use of a set of published microarray data from 86 patients from a Western population with NSCLC.

The identification of five genes that are closely associated with the outcomes in patients with NSCLC has clinical implications. Patients who have tumors with a high-risk gene signature could benefit from a cisplatin-based adjuvant chemotherapy, whereas those with a low-risk gene signature could be spared what may be unnecessary treatment. Prospective, large scale, multicenter studies are necessary to test this idea.

These five genes that can predict the clinical outcome in patients with NSCLC may also reveal targets for the development of therapy for lung cancer. STAT1 causes arrested growth and apoptosis in many types of cancer cells by inducing the expression of p21WAF1 and caspase.27,28 MMD is preferentially expressed in mature macrophages.29 Some studies have shown that macrophage activation promotes cancer metastasis,30 although the function of the MMD protein is unknown. DUSP6 inactivates extracellular signal-regulated kinase 2 (also known as mitogen-activated protein kinase 1), resulting in tumor suppression and apoptosis.31 ERBB3, a member of the epidermal growth factor receptor family of tyrosine kinases, can shorten cell survival.32 LCK, a member of the Src family of protein tyrosine kinases, is expressed mainly in T cells and is one of the first signaling molecules downstream of the T-cell receptor. It plays a key role not only in the differentiation and activation of T cells but also in the induction of apoptosis.33 In addition, LCK is expressed in many cancers and regulates the mobility of cancer cells.34,35

Skrzypski et al.36 examined the expression pattern of 29 genes selected by cDNA studies to test their clinical prognostic value in early-stage squamous cell carcinoma (SCC) of the lung. From 2000 to 2004, freshly frozen primary tumor specimens were obtained at the time of the surgery from 66 SCC patients and gene expression of the 29 genes was assessed by quantitative RT-PCR using low-density arrays. Expression values were dichotomized using the median value as the cutoff. The univariate analysis showed 10 genes with prognosis value: PH4 (p = 0.01); macrophage-colony stimulating factor (CSF1), which attracts macrophages and induce them to express EGF (p = 0.002); EGFR (p = 0.05); KIAA0974 (p = 0.02); ANLN (p = 0.02); carbonic anhydrase IX (CA IX), which is regulated by hypoxia and plays a role in chemoresistance (p = 0.007); VEGFC (p = 0.03); neurotrophic tyrosine receptor kinase 1 (p = 0.04); fibronectin (p = 0.002); insulin receptor (p = 0.03). In the multivariate analysis of survival, CSF1, EGFR and CA IX, and tumor size emerged as significant variables.

The validation of these five-gene and three-gene signatures was performed in tumors from an independent cohort.37 From 2000 to 2005, freshly frozen primary tumor specimens were obtained from 142 patients. Eighty percent of them had stage I and stage II; 66% had SCC and 33% adenocarcinoma. Expression of genes included in the five-gene signature (MMD, ERBB3, LCK, STAT1, and DUSP6) and the three-gene signature (CSF1, EGFR, and CA IX) was assessed by quantitative RT-PCR. Risk scores were used to classify patients into high or low risk according to the expression of the genes in the two signatures. All genes included in the five-gene signature were more highly expressed in adenocarcinoma than in SCC. According to the original risk score for the five-gene signature, median survival (MS) for low-risk patients was not reached, whereas it was 29.7 months for high risk (p = 0.22). However, using the adjusted risk score for SCC, MS for low-risk patients was 63 months versus 24 months for high-risk patients (p = 0.003). Using the adjusted risk score for adenocarcinoma, MS for low-risk patients was not reached, whereas it was 46.2 months for high-risk patients (p = 0.006). According to the risk score for the three-gene signature, MS for high-risk patients was 41 months versus 62.4 months for low-risk patients (p = 0.009). For stage I to II SCC patients, MS in low-risk patients was not reached, whereas it was 34 months for high risk (p = 0.05). For adenocarcinoma patients, MS was not reached for low-risk patients, whereas it was 61.2 months for high-risk ones (p = 0.04). The authors concluded that the five-gene signature is highly predictive of survival when adjusted for histology, whereas the three-gene signature is predictive of survival without adjustment for histology. These gene signatures can be useful for selecting high-risk patients for adjuvant chemotherapy.

Finally, in another study presented at 2008 ASCO Annual Meeting, gene expression profiling was performed on RNA isolated from tumor tissues of 133 (62 on observation and 71 on chemotherapy) JBR.10 patients.38 JBR.107 is a North American phase III Intergroup trial led by the National Cancer Institute of Canada Clinical Trials Group (NCIC CTG), in which 482 patients with completely resected stages IB and II—excluding T3N0-NSCLC were randomly assigned to receive four cycles of adjuvant cisplatin plus vinorelbine or observation alone. Chemotherapy-treated patients enjoyed a significant survival advantage (HR, 0.70; p = 0.03), although a significant interaction with stage was seen, with benefit limited to stage II patients.

Authors identified a prognostic 15-gene expression signature that can classify surgery only patients into risk groups with significantly different survival outcomes. The prognostic signature separated the 62 observation patients into groups with high (n = 33) and low risk (n = 29) for death (HR for high/low risk, 15.02; 95% CI, 5.12–44.04; p > 0.0001). It had prognostic value both in stage IB (n = 34) (HR, 13.32; 95% CI, 2.86–62.11; p < 0.0001) and stage II observation patients (n = 28) (HR, 13.47; 95% CI, 3.00–60.43; p < 0.0001). But it had not prognostic value in chemotherapy-treated patients (n = 71) (HR for high/low risk, 1.15; 95% CI, 0.56–2.37; p = 0.6942). The validation of the genetic profile in five independent public gene expression datasets (stage I–II patients, total n = 372) showed that it had prognostic value in observation patients (HR for high/low risk, 3.21; 95% CI, 1.69–6.11; p = 0.0002), but not in treated ones (HR, 1.10; 95% CI, 0.47–2.53; p = 0.8294). The predictive value of the 15-gene expression signature for benefit from adjuvant chemotherapy was also tested; chemotherapy significantly reduces the risk of death in JBR.10 high-risk patients (n = 67) (HR, 0.33; 95% CI, 0.17–0.63; p = 0.0005) but patients with the low-risk profile (n = 66) have a worse outcome from this adjuvant treatment (HR, 3.67; 95% CI, 1.22–11.06; p = 0.0133). Interaction of chemotherapy and expression signature was highly significant (p = 0.0001). If the 15-gene signature is validated by further testing, it may improve the current method for deciding which patients should receive adjuvant chemotherapy.

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EXCISION REPAIR CROSSCOMPLEMENTATION GROUP 1 EXPRESSION

Clinical trials have tested the ability of adjuvant chemotherapy to improve survival after complete resection of NSCLC. The IALT5 demonstrated with a 4.7-year follow-up an absolute benefit of 4.1% in 5-years OS among 1867 patients with completely resected NSCLC stages I through III who were randomly assigned to receive either cisplatin plus an additional drug (etoposide or a vinca alkaloid) or to be observed only. An updated interim analysis was presented at the 2008 ASCO Annual Meeting.39 At a median follow-up of 7.5 months, there was a beneficial effect on disease-free survival (DFS) (HR, 0.88; 95% CI, 0.78–0.98; p = 0.02) and a nonstatistically significant trend toward improved OS (HR, 0.91; 95% CI, 0.81–1.02; p = 0.10) in patients who had received adjuvant chemotherapy. However, there was a significant difference between the results of OS before and after 5 years (HR, 0.86; 95% CI, 0.76–0.97; p = 0.01 versus HR, 1.45; 95% CI, 1.02–2.07; p = 0.04; p value for interaction 0.006). DFS benefit was also different according to the follow-up duration (first 5 years: HR, 0.85; p = 0.006; after 5 years: HR, 1.33; p = 0.16; p value for interaction 0.04).

However, no validated clinical or biological predictor of benefit from chemotherapy was identified in this trial. The IALT Biology (IALT Bio) study40 was subsequently designed to examine whether tumor markers could be used to predict a survival benefit from adjuvant cisplatin-based chemotherapy between patients who had enrolled in the IALT study.

DNA repair mechanisms are important in the resistance to cisplatin. The destruction of cells by cisplatin requires the binding of the drug to DNA and the creation of platinum-DNA adducts. Some of these adducts establish covalent crosslinking between DNA strands, thereby inhibiting DNA replication. Nucleotide excision repair has a central role in DNA repair and is associated with resistance to platinum-based chemotherapy.41 The excision repair crosscomplementation group 1 (ERCC1) enzyme plays a rate-limiting role in the nucleotide excision repair pathway that recognizes and removes cisplatin-induced DNA adducts.42 ERCC1 is also important in the repair of interstrand crosslinks in DNA and in recombination processes.43,44

For more than a decade, small, retrospective clinical studies have repeatedly reported an association between low levels of expression of ERCC1 mRNA in several solid tumors and improved clinical outcome among patients treated with platinum-containing regimens.45–47 In particular, it has been reported that the expression of ERCC1 mRNA predicts a response to chemotherapy in advanced NSCLC.48 Furthermore, two common polymorphisms of the ERCC1 gene (codon 118 C/T and C8092A) have been correlated with the response to platinum-based chemotherapy in colorectal cancer49 and NSCLC.45 These polymorphisms are mainly associated with lower rates of translation of the ERCC1 gene, which results in low levels of the protein in nuclei. Previous studies50 have also suggested that, in the absence of adjuvant chemotherapy, ERCC-1 is a prognostic factor of better outcome.

These data led authors of the IALT-Bio study to hypothesize that ERCC1 expression could predict a survival benefit from cisplatin-based adjuvant chemotherapy in completely resected NSCLC. They used immunohistochemical analysis to determine the expression of this protein in 761 paraffin-embedded tumor samples from patients enrolled in the IALT-study, 389 (51%) of whom were assigned to the chemotherapy group and 372 (49%) to the control group.

ERCC1 expression was positive in 335 (44%) and negative in 426 (56%) patients. A multivariate logistic model showed that the expression of this enzyme was significantly correlated with age (p = 0.03; less common in patients younger than 55 years of age than in patients 55–64 years of age), histologic type (p < 0.001; less common in adenocarcinomas than in SCC), and pleural invasion (p = 0.01; less common in the absence than in the presence of pleural invasion). A benefit from cisplatin-based adjuvant chemotherapy was associated with the absence of ERCC1 (test for interaction, p = 0.009). Adjuvant treatment, when compared with observation, significantly prolonged OS among patients with ERCC1-negative tumors (HR for death, 0.65; 95% CI, 0.50–0.86; p = 0.002) but not among patients with ERCC-1 positive tumors (adjusted HR for death, 1.14; 95% CI, 0.84–1.55; p = 0.40). The 5-year OS rates among patients with ERCC1-negative tumors were 47% (95% CI, 40–55%) in the chemotherapy group and 39% (95% CI, 32–47%) in the control group. Median OS was 14 months longer in the adjuvant chemotherapy group (56 months) than in the control group (42 months) (Table 1). DFS among patients with ERCC1-negative tumors was also longer in the chemotherapy group than in the control one (adjusted HR for recurrence and death, 0.65; 95% CI, 0.50–0.85; p = 0.001).

Table 1
Table 1
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However, in the control group, the 5-year OS rate was significantly higher among patients with ERCC1-positive tumors than among patients with ERCC1-negative tumors (HR, 0.66; 95% CI, 0.49–0.90; p = 0.009). This finding is in contrast to the results observed in patients who received adjuvant chemotherapy and favors the interpretation that the presence or absence of ERCC1 is a determinant of the sensitivity of NSCLC cells to platinum.

In conclusion, if these results are confirmed by large, independent, and prospective studies, determination of ERCC1 expression in NSCLC cells before chemotherapy could be widely applicable as an independent predictor of the effect of adjuvant chemotherapy; patients with ERCC1-negative tumors derived a substantial benefit from adjuvant cisplatin-based chemotherapy, when compared with ERCC1-positive tumors.

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RIBONUCLEOTIDE REDUCTASE SUBUNIT M1 EXPRESSION

Ribonucleotide reductase subunit M1 (RRM1), the gene that encodes the regulatory subunit of ribonucleotide reductase is located on chromosome segment 11p15.5, a region with a frequent loss of heterozygosity in NSCLC.51 RRM1 is involved in tumor invasiveness and metastasis.52,53 Phospathase and tensin homologue (PTEN), a bifunctional phospathase that regulates cellular signaling, survival, and migration,54 is thought to mediate these effects of RRM1. The increased expression of RRM1 decreases the formation of metastases, inhibits the development of carcinogen-induced lung tumors, and prolongs survival in tumor-bearing mice.52,53,55 In previous studies, an association between high expression of RRM1, as determined by quantitative RT-PCR, and prolonged survival has been reported in patients with NSCLC.56 RRM1 is also the predominant cellular determinant of the efficacy of the nucleoside analogue gemcitabine.57,58

Dr. Bepler’s group performed a study to validate RRM1 as a marker of the clinical outcome in a large cohort of patients with NSCLC.59 Authors describe an immunohistochemical method for the determination of RRM1 expression in histologic specimens of 187 patients with early-stage NSCLC who had received only surgical treatment. Patients were eligible for inclusion in the study if they had an adenocarcinoma, SCC, or large-cell carcinoma; had undergone a complete resection of the tumor; and had stage I disease by pathologic staging. None of them received any form of preoperative or adjuvant therapy. The primary objective was to determine the association between RRM1 expression in the tumor and survival. Secondary objectives were to assess the associations between the expression of RRM1 and ERCC1, between RRM1 and PTEN, and between mRNA and protein levels of RRM1.

This study showed a significant correlation between the RRM1 protein and its mRNA (p = 0.004). High expression of RRM1 protein is associated with better outcome. The median DFS exceeded 120 months in the group of patients with tumors that had high expression of RRM1 and was 54.5 months in the group with low expression of RRM1 (HR for disease progression in the high-expression group, 0.46; p = 0.004). The OS was more than 120 months for patients with tumors with high expression of RRM1 and 60.2 months for those with low expression of RRM1 (HR for death, 0.61; p = 0.02). In a multivariate analysis that included RRM1 expression, tumor stage, ECOG performance status, sex, and smoking status, RRM1 was the only variable that was significantly associated with DFS (p = 0.03); the association with OS, however, was not statistically significant (p = 0.11). There was neither significant association between RRM1 expression and tumor stage, histologic type, age, sex, ECOG performance status, absence or presence of weight loss, and smoking status.

On the other hand, the scores for RRM1 were not correlated with those for PTEN (p > 0.07) and PTEN expression was not significantly associated with survival. However, RRM1 scores were significantly correlated with ERCC1 (p < 0.001), and ERCC1 expression was associated with survival (p = 0.11 for DFS and p = 0.01 for OS). So, authors grouped the 184 patients with scores for both proteins into four categories: 55 patients had tumors with high expression of both proteins (high/high), 54 had low expression of both (low/low), 38 had high RRM1 expression and low ERCC1 expression (high/low), and 37 had low RRM1 and high ERCC1 (low/high). The coordinate high expression of RRM1 and ERCC1 defines a subgroup of patients with an excellent outcome, with a median DFS and a median OS of more than 120 months, which were significantly longer than those for the patients in the other groups (p = 0.01 for DFS, and p = 0.02 for OS) (Figure 3).

Figure 3
Figure 3
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The apparent lack of an association between RRM1 and PTEN contrasts with the previously reported positive correlation between these genes at the RNA level.55 This discrepancy may be due to differential, posttranslational processing or compartmentalization for PTEN and RRM1 or to technical issues.60,61

In conclusion, given that high levels of expression of both ERCC1 and RRM1 are associated with long survival among patients with completely resected lung cancer and are also associated with a poor response to chemotherapy containing gemcitabine and platinum, a trial comparing the current standard of care with adjuvant treatment selected on the basis of RRM1 and ERCC1 expression seems to be warranted.

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BREAST CANCER 1 EXPRESSION

Breast cancer 1 (BRCA1) plays a crucial role in DNA repair and it is implicated in transcription-coupled nucleotide excision repair (TC-NER), and modulation of its expression leads to modification of TC-NER and hence to radio- and chemoresistance.62 BRCA1 is also involved in homologous recombination repair and nonhomologous end joining, in response to DNA damage.63 In addition, it is a component of a large DNA repair complex termed the BRCA1-associated genome surveillance complex, which contains a number of mismatch repair proteins, indicating a potential role for BRCA1 in mismatch repair.62,63 BRCA1 may also be a regulator of mitotic spindle assembly, as BRCA1 and β-tubulin colocalize to the microtubules of the mitotic spindle and to the centrosomes.64 Finally, enhanced BRCA1 expression has been linked to apoptosis through the c-Jun N-terminal kinase pathway,65 which is activated by cisplatin-induced DNA damage; inhibition of this pathway increased cisplatin sensitivity in cell lines.66 Decreased BRCA1 mRNA expression in a breast cancer cell line led to greater sensitivity to cisplatin and etoposide, but to a greater resistance to the microtubule-interfering agents paclitaxel and vincristine.67

On the basis of the evidence for the role of BRCA1 in breast and ovarian cancers, Taron et al.68 examined the potential role of BRCA1 mRNA expression in predicting differential chemotherapy sensitivity in NSCLC. They used quantitative RT-PCR to determine BRCA1 mRNA levels in 55 surgically resected tumors of NSCLC patients who had received neoadjuvant gemcitabine/cisplatin chemotherapy, and divided the gene expression values into quartiles. When results were correlated with outcome, two cutoffs were observed: patients with levels <0.61 had better outcome, and those >2.45 had poorer outcome. MS was not reached for the 15 patients in the bottom quartile, whereas for the 28 in the two middle quartiles, it was 37.8 months (95% CI, 10.6–65), and for the 12 patients in the top quartile, it was 12.7 months (95% CI, 0.28–28.8) (p = 0.01). Moreover, when patients were stratified by pathologic stage, those in the bottom quartile had a decreased risk of death (HR, 0.206; 95% CI, 0.05–0.83; p = 0.026) compared with those in the top quartile, and those in the two middle quartiles also had a decreased risk of death (HR, 0.294; 95% CI, 0.10–0.83; p = 0.020) compared with those in the top quartile.

In a recent study Rosell et al.69,70 hypothesized that altered mRNA expression in nine genes could help to identify patients with a higher risk of recurrence within each stage of the disease. They chose to examine the following genes, based on previous reports of their predictive value: ERCC1, which is involved in keeping genetic alterations to a minimum45; myeloid zinc finger 1 (MZF1),71–73 which mediates ERCC1 expression; Twist1,74,75 which regulates N-cadherin expression and contributes to metastasis by promoting epithelial-mesenchymal transition; thioredoxin 1 (TRX1),76,77 a redox protein overexpressed in NSCLC that is correlated with poor prognosis; the RRM1,59,78,79 regulated by TRX1, which induces HIF-1 and VEGF; tyrosyl-DNA-phosphodiesterasa (Tdp1),80–82 which is overexpressed in NSCLC; nuclear factor of activated T cells,83,84 which promotes carcinoma invasion in vitro; the human homolog of yeast budding uninhibited by benzimidazole 1,85–87 which confers poor survival in colorectal cancer and BRCA1,68 which regulates the expression of budding uninhibited by benzimidazole 1.

To shed light on the prognostic value of these genes, authors examined their expression by quantitative RT-PCR in frozen lung cancer tissue specimens from 126 chemonaive NSCLC patients who had undergone complete surgical resection between 2000 and 2004 and correlated the results with survival.

The patients were 98 men and 28 women, with age at diagnosis ranging from 37 to 77 years (median age, 64 years). Ninety-three patients (73.8%) had SCC and 20 (22.2%) adenocarcinomas. In relation with pathologic stage, 18 patients (14.3%) had stage IA disease, 53 (42.1%) stage IB, 35 (26.2%) stage II, and 22 (17.5%) stage IIIA. One hundred and twenty-two patients underwent formal pulmonary lobectomy or more, with systematic ipsilateral mediastinal lymph node dissection; the four remaining patients underwent segmentectomy due to poor pulmonary reserve. None of the patients received adjuvant chemotherapy. The BCRA1 prognostic value was validated in an independent cohort of 58 stage IB–IIB NSCLC patients.

There were significant differences in expression according to histology for all genes except nuclear factor of activated T cells, with higher levels observed in SCC than in adenocarcinomas. There were no differences in gene expression according to stage or tumor size (<4 versus >4 cm). A strong correlation was observed between expression levels of different genes, for example, between levels of TRX and RRM1 (ρ = 0.52; p = 0.0003) and between ERCC1 and BRCA1 (ρ = 0.62; p = 0.0001).

With a median follow-up of 29.7 months, overall event-free survival (EFS) and MS have not been reached. When EFS and MS was analyzed according to expression levels of the nine genes, TRX and BRCA1 showed significant differences (Figure 4). EFS for 21 patients with low TRX levels has not been reached, whereas it was 32 months (95% CI) for the remaining 93 patients with high levels (p = 0.02). For 77 patients with low levels of BRCA1, EFS has not been reached, whereas it was 22 months (95% CI, 14.9–29 months) for those with high levels (p = 0.04). MS for 24 patients with low TRX levels has not been reached, whereas it was 39 months for the remaining 101 patients with high levels (p = 0.03). For 83 patients with low levels of BRCA1, MS has not been reached, whereas it was 29 months (95% CI, 22.2–35.7 months) for those with high levels (p = 0.04). MS according to expression of the other seven genes are shown in Table 2. Expression levels of these genes did not have prognostic value for the entire study population, although low-MZF1 levels showed a trend toward better outcome. For 56 patients with low levels of MZF1, MS has not been reached, whereas it was 33 months (95% CI, 21.9–44.1 months) for the 66 patients with high levels (p = 0.05). Further study is warranted in this area to elucidate the predictive role of these NER-related genes and to correctly customize treatments.

Figure 4
Figure 4
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Table 2
Table 2
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When only stage I patients were examined, EFS was significantly different according to expression levels of MZF1 and BRCA1, and MS was significantly different according to expression levels of ERCC1, MZF1, Twist, and BRCA1. These findings highlight the potential role of ERCC1 and MZF1, which are highly correlated with BRCA1, as strong prognostic markers in stage I NSCLC. Not unexpectedly, however, considering the high correlation between the expression levels of these three genes, when they were combined, no further improvement over the prognostic value of BRCA1 alone was observed.

In a multivariate Cox model, pathologic stage IIIA (HR, 7.91; 95% CI, 2.27–27.54; p = 0.001) and BRCA1 mRNA expression (HR, 1.98; 95% CI, 1.11–6; p = 0.02) were the only independent prognostic factors for OS. In the validation cohort, the HR for patients with high levels of BRCA1 was 2.4 (95% CI, 1.01–5.92; p = 0.04) according to the Cox proportional hazards model. There were no stage IIIA patients in this cohort.

In conclusion, overexpression of BRCA1 mRNA was strongly associated with poor survival in NSCLC patients, and the validation of this finding in an independent data set further strengthened this association. Patients whose tumors had high BRCA1 expression had significantly worse survival and should be candidates for adjuvant chemotherapy.

Results of previous studies of ERCC140 (stage I–IIIA) and RRM159 (stage I) protein expression are in contrast to the findings reported here. It is not clear why low ERCC1 or RRM1 expression was found to confer poor survival in these studies, whereas in the present study high expression of ERCC1—and specially MZF1—had a detrimental effect in stage I and low expression was beneficial. These conflicting results highlight the need for further research. Patients should be stratified according to the expression of these genes in prospective clinical trials of adjuvant chemotherapy to clarify their role.

On the other hand, in vitro studies have shown that BRCA1 can regulate differential sensitivity to different classes of chemotherapy agents.88 The absence of BRCA1 results in high sensitivity to cisplatin, whereas its presence increases sensitivity to antimicrotubule agents. Moreover, some reports have demonstrated that patients with high-BRCA1 levels attained longer survival when treated with taxane-based therapy.88,89 Based on this results, the Spanish Lung Cancer Group performed an adjuvant trial90 in which adjuvant chemotherapy was customized based on BRCA1 mRNA levels in 84 completely resected N1 and N2 NSCLC patients. Eleven patients with high-BRCA1 levels received docetaxel, 29 patients with intermediate BRCA1 levels received docetaxel/cisplatin and 44 patients with low-BRCA1 levels received cisplatin/gemcitabine. At the time of the analysis, MS has not been reached in patients with high or intermediate BRCA1 levels, whereas it is 25.6 months in patients with low levels (p = 0.04). In a multivariate analysis for survival in all 84 patients, the HR were 5.23 for patients with high-BRCA1 levels (p = 0.07) and 3.57 for patients with tumor size >4 cm (p = 0.07).

Forty-two N2 patients were included in the study, 18 of which were treated with postoperative radiotherapy. In relation with BRCA1 expression, 24 patients had low-BRCA1 mRNA levels and 15 intermediate or high levels. In the multivariate analysis, postoperative radiotherapy was a statistically significant good-prognostic factor (HR, 0.12; 95% CI, 0.02–0.89; p = 0.04), whereas intermediate or high-BRCA1 levels were associated with worse outcome (HR, 22.43; 95% CI, 2.02–248.62; p = 0.01).

In conclusion, single-agent docetaxel has no detrimental effect on survival in comparison with docetaxel/cisplatin. Moreover, high-BRCA1 mRNA expression could be a poor prognostic marker. These results are not definitive and require confirmation. If this hypothesis is validated in prospective clinical trials, patients with the highest BRCA1 expression levels should receive antimicrotubule, nonplatinum-based adjuvant chemotherapy.

The influence of XPG mRNA levels on the effect of BRCA1 mRNA levels in prognosis of early NSCLC has been shown in another study presented at the 2008 American Society of Clinical Oncology Annual Meeting.91 Xeroderma pigmentosum complementation group G (XPG) belongs to the flap endonuclease 1 family of nucleases. XPG expression is correlated with cellular nucleotide excision repair activity and may be a useful marker to predict sensitivity to platinum compounds. In this study BRCA1, ERCC1, RRM1, and XPG transcripts were examined by quantitative PCR in tumors of 54 resected stage I and stage II NSCLC patients. Median DFS was 40 months for stage IA, not reached for stage IB, and 25 months for stage II. DFS was not reached in 18 patients with low-BRCA1 mRNA levels, whereas 37 patients with high-BRCA1 mRNA levels had a DFS of 22.4 months (p = 0.02). In contrast, patients with high XPG had a 51-months DFS, whereas patients with low XPG had a 26-months DFS. When BRCA1 and XPG levels were considered jointly, DFS was not reached for patients with low levels of BRCA1 regardless of whether XPG was high (10 patients) or low (17 patients). However, for patients with high-BRCA1 levels, DFS was shorter for 10 patients with low XPG (16.4 months) than for 17 patients with high XPG (26 months). Authors concluded that high-BRCA1 mRNA expression confers poor prognosis in early NSCLC and the combination of high BRCA1 plus low XPG expression still further increases the risk of shorter DFS.

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CLASS III β-TUBULIN EXPRESSION

Among the described mechanisms of resistance to antitubulin agents, class III β-tubulin (bTubIII) overexpression is of particular interest. Many preclinical studies have shown high levels of expression of bTubIII are associated with paclitaxel resistance in human cancer cells lines (lung,92 ovarian,93 prostate,94 and breast92) and with docetaxel resistance in human pancreatic cancer cell lines.95 Similar results have been obtained for vinca alkaloids.96,97 The mechanistic involvement of bTubIII overexpression in the determination of resistance remains open to debate. Current hypotheses are that these alterations could alter drug binding to the tubulin dimer,98 or, alternatively, that the microtubule contained in the tumor cells could have different intrinsic dynamic properties and thus might be less sensitive to antitubulin agents.99,100

Several studies have shown that levels of expression of bTubIII could possess predictive value for response and outcome in patients with advanced NSCLC receiving tubulin-binding agents.78,101–103 Rosell et al.78 showed that patients with low levels of bTubIII mRNA had better response rates when treated with carboplatin plus paclitaxel and longer time to progression when given vinorelbine plus gemcitabine compared with individuals with high levels of such mRNA. By contrast, levels of bTubIII mRNA were not found to be predictive in patients receiving a gemcitabine/cisplatin regimen. It has also been shown that high level of expression of bTubIII in tumor cells, assessed by a semiquantitative immunohistochemical assay, was associated with a lower response rate and a poor clinical outcome in advanced NSCLC patients receiving vinorelbine-based chemotherapy.101 In another study of 19 patients102 receiving taxane-based regimens, progression-free survival was shorter in patients whose tumors expressed high levels of bTubIII. Using a similar approach, expression of bTubIII was examined in 91 tumor samples obtained from patients with stage III and stage IV NSCLC, including 47 who received paclitaxel-based regimens and 44 who received regimens without tubulin-binding agents.103 This retrospective study showed high levels of bTubIII were associated with a lower response rate and shorter survival in patients with advanced NSCLC receiving paclitaxel, but not in patients receiving regimens without tubulin-binding agents.

To determine whether bTubIII might be a useful marker in early NSCLC patients undergoing adjuvant chemotherapy with a vinorelbine-based regimen, Sève et al.104 assessed the levels of bTubIII in tumor samples from patients treated on the NCIC-CTG JBR.10 study7 and correlated them with outcome in both treated and control patients groups.

A semiquantitative immunohistochemical assay for class bTubIII was done on primary tumor tissues available for 265 (55%) of the 482 patients in the trial. High-bTubIII expressors (n = 133) included more women (42% versus 30%; p = 0.04), fewer with squamous histology (25% versus 48%; p < 0.001), more with RAS mutations (31% versus 20%; p = 0.05), more patients ≤60 years old (53% versus 42%; p = 0.09), and more PS1 patients (58% versus 47%; p = 0.09) compared with the low-tubulin expressors (n = 132). Gender, stage, type of lung resection, and chemotherapy treatment assignment were not related to bTubIII expression.

High versus low-bTubIII expression was associated with inferior outcome for the entire study population. The result was statistically significant for RFS (HR, 1.52; 95% CI, 1.05–2.22; p = 0.03) and a similar trend was seen for OS (HR, 1.39; 95% CI, 0.96–2.01; p = 0.08). But the value of tubulin expression in predicting RFS and OS seemed to be largely confined to those patients assigned to the observation arm of JBR.10. High-bTubIII expression was associated with poorer survival in patients treated with surgery alone (RFS: HR, 1.92; 95% CI, 1.16–3.18; p = 0.01; OS: HR, 1.72; 95% CI, 1.02–2.88; p = 0.04). Tubulin expression was not a statistically significant predictor of outcome in the patients assigned to receive chemotherapy (RFS: HR, 1.10; 95% CI, 0.62–1.95; p = 0.75; OS: HR, 1.11; 95% CI, 0.65–1.88; p = 0.7). Cox regression stratified by treatment arm was used to examine the relationship between bTubIII expression and RFS or OS after adjusting for other prognostic factors. In this type of model, high-bTubIII expression remained as a significant adverse prognostic factor for RFS (HR, 1.78; 95% CI, 1.06–3.00; p = 0.03). Similar results were seen in a model of OS (HR, 1.42; 95% CI, 0.97–2.09; p = 0.07).

This study also compared the benefits of adjuvant chemotherapy in high versus low-bTubIII expressors (Figure 5). In the low-bTubIII group (n = 132), no significant difference in RFS (HR, 0.78; 95% CI, 0.44–1.37; p = 0.4) or OS (HR, 1.00; 95% CI, 0.57–1.75; p = 0.99) was seen in between patients assigned to chemotherapy (n = 72) and observation (n = 60). However, in the high-bTubIII group (n = 133), patients receiving chemotherapy (n = 68) had significantly improved RFS (HR, 0.45; 95% CI, 0.27–0.75; p = 0.002) and a trend toward improved OS (HR, 0.64; 95% CI, 0.39–1.04; p = 0.07) compared with patients in the observation arm (n = 65). But in a Cox regression analysis, the interaction between levels of bTubIII and chemotherapy treatment predicting RFS and OS did not reach statistical significance (p = 0.15 for RFS; p = 0.25 for OS).

Figure 5
Figure 5
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In conclusion, the results of this study suggest that high-bTubIII expression in resected NSCLC is associated with poorer survival in the absence of adjuvant chemotherapy treatment but not in patients who receive adjuvant chemotherapy. These findings suggest that adjuvant cisplatin/vinorelbine chemotherapy can overcome the adverse biology of cancers that express higher amounts of bTubIII. Furthermore, adjuvant chemotherapy significantly prolonged the RFS and OS in the high-bTubIII expressors, but its effect was not clear for the low-bTubIII expressors in this study. High-bTubIII expression is associated not only with a higher risk of relapse after surgery alone but also with a higher probability of benefit from adjuvant cisplatin plus vinorelbine chemotherapy.

The adverse prognostic significance of high-bTubIII expression observed in this study is consistent with prior published reports in the setting of advanced NSCLC. However, this report is contrary to the data from advanced NSCLC about the value of bTubIII expression in predicting benefit from chemotherapy. In the setting of advanced disease, low-bTubIII expression is associated with a higher objective response rate to chemotherapy containing the antimicrotubule agents but does not seem to predict response to regimens that do not target microtubules.78,101–103 On the other hand, the results of the present study suggest that in early lung cancer, it is the patients with high-bTubIII expression that are most likely to benefit from adjuvant cisplatin and vinorelbine. It is possible that all the studies are correct and that bTubIII has differential predictive implications in early versus advanced NSCLC. This discrepancy between the metastatic and adjuvant setting is not without precedent. In colorectal cancer, the relationship between thymidylate synthase and benefit from chemotherapy differs between operable and advanced disease.105,106 The reason for the current discrepancy is as yet unexplained.

On the basis of previous results, Reiman et al.107 tried to verify that high-bTubIII expression might correlate with greater benefit from adjuvant chemotherapy in an analysis of tumor tissues from 737 patients in the IALT trial. Because some chemotherapy regimens in this trial did not include an antitubulin agent, authors also tested the hypothesis that the value of bTubIII expression in predicting benefit from chemotherapy was specific to antitubulin containing regimens. High-bTubIII expression significantly correlated with adenocarcinoma histology (p = 0.0001), lymphatic (p = 0.03) and vascular invasion (p = 0.01), and DFS (HR, 1.26; 95% CI, 1.01–1.58; p = 0.04), with a similar trend in OS (p = 0.12). There was no significant interaction between bTubIII status and treatment assignment in predicting DFS (HR for chemotherapy effect in high versus low-bTubIII groups, 0.92; p = 0.71) or OS (HR, 1.01; p = 0.98), when the analysis was restricted to antitubulin containing regimens (DFS: HR, 0.90; p = 0.71; OS: HR, 0.93; p = 0.82). In conclusion, high-bTubIII expression is also an adverse prognostic factor in IALT, in concordance with prior observations in JBR.10. However, unlike what was suggested in JBR.10, in IALT bTubIII expression did not correlate with benefit from adjuvant chemotherapy.

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CELL CYCLE REGULATORS

P53 and RAS are multifunctional proteins that play key roles in regulating cell cycle progression, apoptosis, gene transcription, response to stress, and DNA repair.108–110 They are the most extensively investigated prognostic markers in NSCLC, with each having more than 50 reported studies. Although meta-analyses generally have indicated that aberrations of these genes are weak prognostic markers of poorer outcome in NSCLC, results from individual studies have been inconsistent.110–113 Meta-analysis cannot eliminate potential biases that may exist in published data,114 making it imperative that promising markers identified in meta-analyses be confirmed prospectively or retrospectively in large phase III randomized trials.115,116 Moreover, because p53 is an important factor in the regulation and initiation of DNA repair, aberrations in p53 expression may also affect response to chemotherapy.117

In a recently published study,118 authors evaluated the prognostic and predictive value of p53 and RAS gene mutations and p53 protein overexpression using tumor samples from NCIC-CTG JBR.10 trial.7 P53 protein expression was evaluated by immunohistory (IHC) in paraffin-embedded tissue sections of 253 patients. The prevalence of p53 overexpression was 52% (132 patients). P53 positive tumors were more frequent in men, SCC, and tumors with wild-type RAS. In the observation arm, patients with p53-positive tumors had significantly shorter survival than did those with p53-negative tumors (p = 0.03; HR, 1.89; 95% CI, 1.07–3.34) indicating that p53 protein overexpression is a significant marker of poor prognosis, even after multivariate adjustment for other potential prognostic factors (p = 0.02).

However, patients with p53-positive tumors derived significant benefit from adjuvant chemotherapy (HR, 0.54; 95% CI, 0.32–0.92; p = 0.02), whereas patients with p53 negative tumors had not (HR, 1.40; 95% CI, 0.78–2.52; p = 0.26) (Figure 6). In the multivariate model the interaction of chemotherapy and p53 overexpression remained significant (p = 0.05). Furthermore, this significant interaction was maintained even when multivariate regression modeling was applied to all 482 patients in JBR.10, including those who did not have p53 IHC (p = 0.01).

Figure 6
Figure 6
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Authors also investigated exons 5 to 9 for the presence of mutations because these constitute more than 90% of all p53 mutations in NSCLC.110,119 They were determined by denaturing high performance liquid chromatography and confirmed by sequencing. It was found that 124 (31%) of 397 patients had functional mutations, including six patients whose tumors revealed multiple mutations. P53 mutation was not prognostic for survival in the observation arm (HR for mutant versus wild type, 1.15; 95% CI, 0.75–1.77; p = 0.45). Patients with p53 mutations did not derived significant survival benefit from adjuvant chemotherapy (HR, 0.78; 95% CI, 0.46–1.32; p = 0.35). For 273 patients with wild-type p53, chemotherapy significantly prolonged survival compared with observation (HR, 0.67; 95% CI, 0.46–0.98; p = 0.04), but the interaction p value in the multivariate analysis was insignificant at 0.65.

Finally, RAS mutations were identified by allelic-specific oligonucleotide hybridation. Because JBR.10 stratified patients according to their RAS mutation status, it was known in more than 90% of randomly assigned patients. Authors identified 119 mutations in 117 of 450 patients (26%), which were significantly higher in large-cell and adenocarcinoma and in women. RAS mutation was not a significant prognostic marker for survival in univariate or multivariate analyses. In 333 patients with wild-type RAS, survival was significantly prolonged with adjuvant chemotherapy compared with observation (HR, 0.69; 95% CI, 0.49–0.97; p = 0.03). In contrast, there was no apparent benefit from chemotherapy in 117 patients with RAS mutant tumors (HR, 0.95; 95% CI, 0.53–1.71; p = 0.87). However, in the multivariate model, significant interaction between chemotherapy and RAS mutation was not detected (p = 0.29).

P53 nuclear immunoreactivity in tumors had been regarded as a surrogate marker for the presence of p53 gene mutation, because missense mutant p53 protein demonstrates a longer half life than does wild-type protein.120 However, Greenblatt et al.121 reported than in 84 studies evaluating p53 mutation and p53 protein by IHC simultaneously, the overall sensitivity of IHC to predict p53 mutation status was 75%, whereas the positive predictive value was only 63%. In this study, 56 (75%) of 75 mutant p53 tumors were positive for p53 staining, but 68 (43%) of 158 of wild-type p53 tumors were also positive for IHC. Although a majority of IHC-negative mutant cases could usually be accounted for by deletion/nonsense mutations, the mechanistic basis, and biological significance of wild-type p53 protein overexpression in tumors is less clearly understood.

Recent discoveries place increasing importance on MDM2 and p14ARF as regulators of cellular p53 protein levels.122 The degradation of p53 protein by the ubiquitin pathway is mediated by its binding to MDM2, an E2 ligase, and the expression of MDM2 mRNA and protein is negatively regulated by p14ARF. The stability of mutant p53 protein in tumor cell seems more dependent on its inability to bind MDM2,123 and the level of wild-type p53 protein is also significantly regulated by the MDM2 expression level. Wang et al.124 studied 94 NSCLC and identified 16 p53 mutant tumors (17%). Although 45 tumors were p53 IHC positive, 37 overexpressed the wild-type p53 protein. Among the latter, 35 (95%) and 34 (92%) had low expression of MDM2 and high expression of p14ARF, respectively. They reported that overexpression of p53 and low expression of MDM2 are poor prognostic markers. Their study provides compelling evidence that the biological effects of p53 mutation and p53 protein overexpression may not be identical, and that the regulation of p53-MDM2 pathway may influence the outcome of NSCLC patients.

The regulation of expression, signaling pathways, and biological activity of p53 is complex.108,125 It remains speculative why p53 protein expression rather than p53 mutation imposes more aggressive clinical behavior in NSCLC. One hypothesis could be that high levels of p53 protein, regardless of mutation status, are reflective of significant oncogenic (e.g., myc, β-catenin) activation pathways, leading to p14 overexpression and stabilized p53 protein.125,126 The role of p53 mutation and/or aberrant protein expression (positive IHC staining) in DNA repair and response to chemotherapy is also complex and remains controversial.126 There is contradictory evidence as to whether or not p53 mutation/aberrant protein expression could affect the sensitivity of solid tumors to anticancer agents.117,125 Results of this study indicate that adjuvant chemotherapy seems not to be very effective in p53 mutant patients, but p53 IHC-positive tumors remain sensitive to treatment. On the other hand, there is some evidence to suggest that the disruption of p53 function could sensitize tumor cells to the effect of chemotherapeutic drugs such as cisplatin, whose DNA damage is repaired by nucleotide excision pathways.127 Sensitization could possibly be caused by an inability of p53 aberrant tumor cells to transactivate p21 and allow DNA repair to occur, or by an interference of tumor cellular ability to sense DNA damage or initiate/effect DNA repair.115 The discrepancy between the role of p53 mutation and aberrant protein expression suggest that the biological effects of these 2 p53 abnormalities are not equivalent and their roles warrant further mechanistic studies.

In conclusion, of the markers assessed in this study, p53 protein overexpression is both prognostic for poorer survival and predictive of a differentially greater survival benefit from adjuvant chemotherapy. Although p53 and RAS wild-type patients seem to derive greater benefit from adjuvant chemotherapy than do patients with p53 or RAS mutant tumors, the differences in this study was not statistically significant. These observations, together with those demonstrating a differential benefit from adjuvant chemotherapy in patients with low ERCC1 protein expression,40 suggest that the greatest benefit from platinum-based adjuvant chemotherapy should be in NSCLC patients with low ERCC1 but high p53 protein expression. An international collaborative BIO-Lung Adjuvant Cisplatin Evaluation study is planned to test this hypothesis in a large cohort of patients’ samples that should have the statistical power to test multiple markers. It seems that we are on the threshold of molecular selection of NSCLC patients for postoperative adjuvant chemotherapy.

Authors from the Medical University of Vienna performed an study to determine whether cell cycle regulators are of prognostic and/or predictive value in patients who were enrolled onto the IALT trial.128 Expression of p27Kip1, p16INK4A, cyclin D1, cyclin D3, cyclin E, and Ki-67 was immunohistochemically assessed in tumors specimens obtained from 778 IALT patients. A relationship between p27kip1 status and benefit of cisplatin-based chemotherapy was found (test for interaction, p = 0.02). Among patients with p27Kip1-negative tumors, cisplatin-based chemotherapy resulted in longer OS compared with controls (HR for death 0.66, 95% CI, 0.50–0.88; p = 0.006). In patients with p27Kip1-positive tumors, OS was not different between patients treated with cisplatin-based chemotherapy and controls (HR for death, 1.09; 95% CI, 0.82–1.45; p = 0.54). The other cell cycle regulators and Ki-67 did not predict benefit of adjuvant cisplatin-based chemotherapy. None of these biomarkers was significantly associated with OS of the patients in the total study population. In conclusion, NSCLC patients with p27Kip1-negative tumors benefit from adjuvant cisplatin-based chemotherapy after complete tumor resection. But before establishing p27Kip1 as a routine marker for selection of patients for adjuvant chemotherapy, the predictive value of p27Kip1 has to be confirmed in patients from others trials.

In another study presented at 2008 ASCO Annual Meeting, patterns of coexpression of ERCC1 and p27 were analyzed by IHC in surgical resection specimens from 18 patients with NSCLC.129 Of 18 tumors, seven (39%) were ERCC1 positive and three (17%) were p27Kip1 positive. Coexpression of ERCC1 and p27Kip1 was as follows: one case (6%) double positive, nine cases (50%) double negative, six cases (33%) ERCC1 positive/p27Kip1 negative, and two cases (11%) ERCC1 negative/p27Kip1 positive. P27Kip1 expression correlated with better differentiated adenocarcinoma, but there was no correlation of ERCC1 or p27Kip1 with other clinic-pathologic parameters. The authors concluded that the most common pattern of ERCC1 and p27Kip1 expression is double negativity, characteristic of 50% of NSCLC, predicting the patient is more likely to benefit from cisplatin-based adjuvant chemotherapy. Only 6% of patients show coexpression of these markers, which would predict the lowest likelihood of benefit.

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DNA METHYLATION MARKERS

Epigenetic gene silencing is a molecular mechanism of silencing a gene by methylating its promoter region. Epigenetic silencing is involved in the initiation and progression of several types of cancer, including lung cancer.130,131 The detection of epigenetic alterations with the use of a method like the methylation-specific PCR assay may allow for the molecular staging of cancer.132 With this method, relatively few genes are required to analyze each type of cancer.

The value of the methylation-specific PCR assay for predicting the recurrence of early stage, resected NSCLC has been examined in a recently published study.133 The authors designed a nested case-control study of T1-2N0 NSCLC to test the association between clinical outcome and the DNA methylation status of tumor, ipsilateral peribronchial and/or hilar lymph nodes, and mediastinal lymph nodes. Seven genes thought to be important in the biological development of lung cancer and frequently methylated in this tumors were studied134–138: the cyclin-dependent kinase inhibitor 2A gene p16, the H-cadherin gene CDH13, the adenomatous polyposis coli gene APC, the Ras association domain family one gene RASSF1A, the O6-methylguanine-DNA methyl-transferase gene MGMT, the PYD and CARD domain-containing gene ASC, and the death-associated protein kinase one gene DAPK. The authors hypothesized that the methylation-specific PCR assay could be used to define patterns of DNA methylation that can delineate the behavior of the primary tumor and to detect micrometastases in histologically negative lymph nodes.

Evidence of recurrent disease was evaluated in 715 patients with pathologically verified stage I cancer who underwent lobectomy or greater resection at the Johns Hopkins Hospital between January 1, 1986, and July 31, 2002. Fifty-one patients who underwent curative resection but who had a recurrence within 40 months after surgery (case patients) were matched on the basis of age, NSCLC stage, sex, and date of surgery (±5 years) to 116 patients who did not have a recurrence within 40 month after resection (controls). Neither case patients nor controls received adjuvant chemotherapy. The authors also evaluated 162 paraffin-embedded tissue blocks from 20 patients in a validation cohort (11 case patients and nine matched controls).

Clinical and demographic variables were similar in case patients and controls. The most frequent site of recurrence was the ipsilateral lung (in 45.1% of patients), followed by metastasis to bone (13.7%), brain (11.7%), and mediastinum (11.7%).

The covariates of pathologic stage, age, sex, histologic characteristics of the tumor, smoking status, and race were not associated with the risk of recurrence in patients with histologically negative lymph nodes.

However, when compared with controls, the largest differences in the univariate distribution among case patients of the frequency of methylation in any type of tissue were found in four genes—p16, CDH13, RASSF1A, and APC—especially in tumors or mediastinal lymph nodes. When p16 or CDH13 was methylated in the primary tumor, the adjusted odds ratio (OR) for recurrence was 3.50 (95% CI, 1.65–7.41; p = 0.001) and 2.12 (95% CI, 0.98–4.59; p = 0.06), respectively. When these same genes were methylated in peribronchial and/or hilar lymph nodes, the OR was 3.62 (95% CI, 1.41—9.32; p = 0.008) and 1.99 (95% CI, 0.81–4.88; p = 0.13), respectively. If methylation of p16 or CDH13 was found in mediastinal lymph nodes, the OR for recurrence was 4.67 (95% CI, 1.53–14.42; p = 0.007) and 3.98 (95% CI, 1.22–13.01; p = 0.02), respectively. Methylation of RASSF1A or APC in the tumor or mediastinal nodes was not significantly associated with recurrence.

The six possible pairs of these four genes were examined for an association with recurrence. Among these pairs, four had a significant association with recurrence in at least one type of tissue: p16 and CDH13, CDH13 and APC, APC and p16, and RASSF1A and p16. Methylation of the gene pair p16 and CDH13 in the primary tumor alone was associated with and OR for recurrence of 8.00 (95% CI, 2.50–25.51; p < 0.001) for the case patients compared with the controls; when methylation of the two genes was found in both the tumor and the mediastinal lymph nodes, the estimated OR for recurrence was 15.50 (95% CI, 1.61–185.02; p = 0.03) in the original cohort and 25.25(95% CI, 2.53–252.35; p = 0.006) when the original cohort was combined with the independent validation cohort (Figure 7). Methylation of p16 and CDH13, RASSF1A, or APC in paired tumor and mediastinal lymph-node samples from the 51 case patients was associated with early recurrence (median, 9 months; range, 5–30), whereas in the absence of methylation of these markers, the median time to recurrence was 25 month after surgery (range, 6–40; p = 0.04).

Figure 7
Figure 7
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In addition, Kaplan-Meier plots indicated that methylation of one or more of four genes—p16, CDH13, RASSF1A, and APC—in any sample from the patient was related to the duration of RFS. For example, the 5-year RFS rate in the group of patients with two or more of the four methylated genes in the primary tumor and mediastinal lymph nodes was 27.3% (95% CI, 6.5–53.9), when compared with 65.3% (95% CI, 53.1–75.0) in the group with fewer than two methylated genes at those sites (p < 0.001). Methylation of both p16 and CDH13 in tumor and mediastinal lymph nodes was associated with a 5-year RFS rate of 14.3% (95% CI, 0.7–46.5), when compared with 63.1% (95% CI, 50.2–73.5) in the absence of methylation of these genes (p < 0.001).

In conclusion, this study indicates that the methylation of the promoter regions of certain genes (p16, CDH13, APC, and RASSF1A) in a resected NSCLC specimen is associated with a significantly increased risk of early recurrences. These genes are involved in cell-cycle control (p16), invasion and metastasis (CDH13, APC), and RAS signaling (RASSF1A). Other studies of p16 expression or promoter-region methylation in lung cancer have focused mostly on the primary tumors,138–143 but this one found that molecular examination of lymph nodes improves the assessment of risk of recurrence. The methylation of these genes in histologically normal regional lymph nodes probably indicates the presence of microscopically undetectable micrometastases. Immunohistochemical analyses, for example, may miss a rare cell in the background of normal tissue, whereas the methylation-specific PCR assay is sufficiently sensitive to detect a signal of DNA methylation. These results also suggest that the detection of promoter methylation of certain genes may identify cells with a potential for metastatic spread not only within NSCLC but also in lymph nodes. The correlation between short survival and the number of methylated genes in the regional and mediastinal lymph nodes supports the presence of micrometastases in those sites.

Recent promising results for predicting the risk of lung cancer144 or its recurrence137 have been obtained by examining changes in gene methylation in sputum.144

As with other molecular markers, these results require confirmation in a larger, prospectively studied cohort, before the-four gene panel can be used to select patients for adjuvant therapy in clinical practice.

Table 3 summarizes the role of the molecular markers discussed in the review as prognostic factors for survival and predictive markers of benefit from adjuvant chemotherapy.

Table 3
Table 3
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As we previously mentioned, gene expression profiles are useful in defining favorable and unfavorable prognostic subsets and in selecting high-risk patients for adjuvant treatment. These genomic strategies are consistently accurate in estimating the risk of recurrence since they have been validated in independent cohorts of patients from multicenter cooperative groups trials.

Among the molecular markers used as potential predictors of a survival benefit from adjuvant chemotherapy, ERCC1, RRM1, and BRCA1 are the most well-established ones. For more than a decade, a number of clinical studies have reported the association between levels of expression of ERCC1 in several solid tumors and clinical outcome or response to platinum-based chemotherapy. Data of BRCA1 are also fairly consistent, because several studies have examined its prognostic value and its potential role in predicting differential chemotherapy sensitivity in NSCLC, showing similar results.

Nevertheless, the prognostic significance of bTubIII expression is less established and published reports in early lung cancer are contrary to the data from advanced disease about the value of bTubIII expression in predicting benefit from antitubulin agents. So, further studies are needed to shed light to these conflicting findings.

In relation with cell cycle regulators, p53, Ras, and p27 have also been extensively investigated as prognostic markers in NSCLC, but results from individual studies have been inconsistent, making it imperative that promising published data be confirmed prospectively or retrospectively in large phase III randomized trials.

Finally, the mechanism of aberrant gene silencing in cancer by hypermethylation of DNA in promoter regions and the clinical potential of this knowledge for risk assessment, early diagnosis, prognostic monitoring, treatment, and the prevention of cancer is a promising area of biological research. Nowadays, published results are preliminary and larger studies must be performed to determine the clinical applicability of such findings.

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Keywords:

Non-small cell lung cancer; Early stages; Platinum-based adjuvant chemotherapy; Molecular prognostic and predictive markers

© 2009International Association for the Study of Lung Cancer

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