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Prognostic and Predictive Importance of p53 and RAS for Adjuvant Chemotherapy in Non–Small-Cell Lung Cancer

Ming-Sound Tsao, Sarit Aviel-Ronen, Keyue Ding, Davina Lau, Ni Liu, Akira Sakurada, Marlo Whitehead, Chang-Qi Zhu, Robert Livingston, David H. Johnson, James Rigas, Lesley Seymour, Timothy Winton, Frances A. Shepherd

From the University Health Network, Princess Margaret Site and Ontario Cancer Institute, University of Toronto, Toronto; National Cancer Institute of Canada Clinical Trials Group and Queen's University, Kingston, Ontario; University of Alberta Hospital, Edmonton, Alberta, Canada; Southwest Oncology Group, San Antonio, TX; Eastern Cooperative Oncology Group, Boston, MA; and Cancer and Leukemia Group B, Chicago, IL

Address reprint requests to Frances Shepherd, MD, Department of Hematology and Oncology, Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada; e-mail: frances.shepherd{at}uhn.on.ca

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS Appendix REFERENCES

 
Purpose p53 and RAS are multifunctional proteins that are critical to cell cycle regulation, apoptosis, cell survival, gene transcription, response to stress, and DNA repair. We have evaluated the prognostic and predictive value of p53 gene/protein aberrations using tumor samples from JBR.10, a North American phase III intergroup trial that randomly assigned 482 patients with completely resected stage IB and II non–small-cell lung cancer (NSCLC) to receive four cycles of adjuvant cisplatin plus vinorelbine or observation alone.

Methods p53 protein expression was evaluated by immunohistochemistry. Mutations in exons 5 to 9 of the p53 gene were determined by denaturing high-performance liquid chromatography and confirmed by sequencing. RAS mutations were identified by allelic specific oligonucleotide hybridization.

Results Of 253 patients, 132 (52%) were positive for p53 protein overexpression. Untreated p53-positive patients had significantly shorter overall survival than did patients with p53-negative tumors (hazard ratio [HR] = 1.89; 95% CI, 1.07 to 3.34; P = .03). However, these p53-positive patients also had a significantly greater survival benefit from adjuvant chemotherapy (HR = 0.54; P = .02) compared with patients with p53-negative tumors (HR = 1.40; P = .26; interaction P = .02). Mutations of p53 and RAS genes were found in 124 (31%) of 397 and 117 (26%) of 450 patients, respectively. Mutations in these genes were neither prognostic for survival nor predictive of a differential benefit from adjuvant chemotherapy.

Conclusion p53 protein overexpression is a significant prognostic marker of shortened survival, and also a significant predictive marker for a differentially greater benefit from adjuvant chemotherapy in completely resected NSCLC patients.

	INTRODUCTION

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Non–small-cell lung carcinoma (NSCLC) represents approximately 80% of all lung cancers. Although early-stage (I and II) NSCLC patients are treated surgically with curative intent, 30% to 60% will develop recurrence and die as a result of their disease.^1,2 Three recent trials using cisplatin-based doublet chemotherapy have demonstrated significant survival benefits with postoperative chemotherapy.^3-5 JBR.10, a North American intergroup trial led by the National Cancer Institute of Canada Clinical Trials Group (NCIC CTG) randomly assigned patients with completely resected stage IB and II NSCLC to receive adjuvant cisplatin/vinorelbine or observation alone.⁴ Chemotherapy-treated patients enjoyed a significant survival advantage (hazard ratio [HR] = 0.70; P = .03).

It has long been recognized that differences in clinical factors such as stage and sex, and tumor factors such as cellular differentiation, vascularity, and vascular invasion are prognostic of outcome.⁶ More recently, molecular alterations in key pathways have also been found to be prognostic, and some of these markers have also been shown to predict a differential effect of adjuvant chemotherapy on survival. An analysis of tumor samples from the International Adjuvant Lung Trial (IALT) showed that the expression of DNA excision repair cross complementing-1 (ERCC1) protein is a favorable prognostic marker in untreated patients (adjusted HR for death = 0.66; 95% CI, 0.49 to 0.90; P = .009). However, the benefit of platinum-based chemotherapy was significant only in patients whose tumors did not express ERCC1 (HR = 0.65; P = .002, interaction P = .009).⁷

Prognostic markers are patient or tumor factors that, independent of treatment, predict patient survival outcome.⁸ Predictive markers are factors that may influence and predict the outcome of treatment in terms of either response or survival benefit. Currently, apart from stage, neither prognostic nor predictive markers are used to select NSCLC patients for adjuvant chemotherapy. Patients with a poor prognosis have the greatest potential to benefit from adjuvant therapy; however, only through the evaluation of predictive markers will it be known whether adjuvant chemotherapy can, in fact, improve their survival.

p53 and RAS are multifunctional proteins that play key roles in regulating cell cycle progression, apoptosis, gene transcription, response to stress, and DNA repair.^9-11 Oncogenic activation of RAS, p53 protein overexpression, and p53 gene mutations have been reported as prognostic markers of poor outcome in NSCLC patients.^11-14 Because p53 is an important factor in the regulation and initiation of DNA repair, aberrations in p53 expression may also affect response to chemotherapy.¹⁵ We report herein our evaluation of these three markers and their ability to predict prognosis and differential benefit from adjuvant chemotherapy for patients in JBR.10.

	METHODS

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Patients and Tissue
These studies were approved by the University Health Network Research Ethics Board. JBR.10 compared the effect of four cycles of adjuvant chemotherapy (vinorelbine/cisplatin) with observation alone in 482 patients with completely resected T2N0, T1-2N1 NSCLC.⁴ All patients provided written informed consent for study participation and RAS mutational analyses, for which samples from 452 patients were collected (Fig A1, online only). Among these, 445 patients consented to tumor banking for future studies. These included snap-frozen tissue collected within 30 minutes of resection from 169 patients, paraffin blocks only from 121 patients, and 10 unstained slides only from 155 patients. Paraffin blocks from 280 consented patients were available to construct tissue microarrays (TMAs), using the Manual Tissue Arrayer (Beecher Instruments, Silver Spring, MD). Guided by hematoxylin and eosin (H&E)-stained slides, 0.6-mm cores were taken from three areas of high tumor cellularity, and one from non-neoplastic lung tissue. These cores were arrayed into eight TMA blocks. Serial 4-µ sections were mounted on silane-coated slides for H&E and immunohistochemical (IHC) staining.

p53 Immunohistochemistry and Scoring
Because of the limited number of unstained slides remaining after DNA extraction and mutation analyses, p53 protein expression was evaluated using only TMA sections and using the DO7 p53 antibody (NovoCastra Laboratories, Newcastle upon Tyne, United Kingdom) that recognizes wild-type and mutant p53 proteins and the Ultra-Streptavidin detection system (Signet Pathology System, Dedham, MA) to demonstrate immunoreactivity. After microwave antigen retrieval using Milestone T/T Mega (Shelton, CT) for 10 minutes in 10 mmol/L citrate buffer (pH 6.0) and endogenous biotin blocking with Vector's biotin blocking kit (Burlingame, CA), sections were incubated in primary antibody (1:200 dilution) for 16 hours at room temperature in a moist chamber. The antibody dilution was optimized a priori to show absent p53 staining in more than 99% of cells in non-neoplastic lung tissue. The slides were evaluated independently by the two study pathologists (S.A.R. and M.S.T.). For each core, staining intensity was qualitatively scored from 0 (absent) to 3 (strong), and the percentage of tumor cells with nuclear staining was estimated (Fig A2, online only). Only cases with two or more assessable cores were included in the analyses, because this has been validated to reflect results obtained from evaluation of whole sections.¹⁶ For each case, the final score represented the mean score of all cores provided by the two evaluators. A final staining score of 15% or greater was chosen as the cutoff to designate p53 positivity, with all positive tumors showing intensity scores of 1 or more. The cutoff was defined on the basis of a graphic representation, which showed that dichotomization at 15% gives differential benefit from adjuvant therapy (K. Ding, manuscript in preparation).

p53 and RAS Gene Mutation Assay
Mutation analyses were conducted using genomic DNA isolated from 170 snap-frozen tumors and 280 paraffin-embedded tissue sections. For samples with less than 40% tumor cellularity, tumor DNA was enriched by microdissection. DNA was isolated by standard phenol-chloroform methods and each exon 5 to 9 was amplified by polymerase chain reaction (PCR) using their respective primer sequences (Table A1, online only) and the AmpliTaq Gold PCR kit (Applied Biosystems, Foster City, CA).¹⁷ The PCR conditions were identical for all exons aside from the annealing temperatures (Table A1). PCR reaction included 35 cycles of denaturation at 94°C for 45 seconds at the exon-specific annealing temperature for 45 seconds, extension at 72°C for 1 minute, final extension at 72°C for 7 minutes, and cool down to 4°C. PCR products were analyzed by denaturing high-performance liquid chromatography on the Transgenomic WAVE Nucleic Acid High Sensitivity Fragment Analysis System (Omaha, NE). PCR products with aberrant heteroduplex formation were reanalyzed by independent PCR and sequencing to confirm the presence and nature of mutations. Allele-specific oligonucleotide hybridization was used to detect mutations on codons 12, 13, and 61 of HRAS, KRAS and NRAS, as described previously.¹⁸

Statistical Analyses
Exploratory analyses were performed to characterize relationships between marker levels, baseline clinical characteristics, and survival.^{4 2} or Fisher's exact test was used to compare relationships between marker levels and baseline factors; Kaplan-Meier product-limit methods and the log-rank test were used to estimate and test overall survival distributions and their difference between markers and treatment arms, and multivariable Cox regression models were used to validate the prognostic and predictive effects of markers on survival while adjusting for baseline factors. All reported P values are two sided, and a level of 5% (P = .05) was considered statistically significant.

	RESULTS

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IHC scores for p53 protein expression were obtained from 253 patients; disqualified cases included those with exhausted tumor tissue, those with loss of two or more cores during staining, or cores lacking tumor. Assay for p53 gene mutation was successful for all exons in 397 patients who consented to future studies. RAS mutation status was a trial stratification factor, and successful assay was accomplished for 450 patients.

Comparisons of baseline stratification and potential prognostic factors are shown in Table 1. For 253 patients with p53 IHC/protein results, more patients with marker data (compared with those without) were stage IB and smokers; their survival benefit from adjuvant chemotherapy was also slightly less than that in JBR.10⁴ (HR = 0.83; 95% CI, 0.55 to 1.20; P = .33; Fig 1A). For 397 patients with p53 and 450 with RAS mutation results, there were no significant imbalances in any factors between those with and without biomarker data. With both markers, patients treated with chemotherapy demonstrated significant survival benefits compared with observed patients. (Figs 2A and 3A).

View larger version (17K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Overall survival curves of patients with p53 protein expression (immunohistochemistry) results. (A) p53 IHC results known; (B) p53 IHC results, observation; (C) p53 IHC positive; and (D) p53 IHC negative. IHC, immunohistochemistry; HR, hazard ratio.

View larger version (17K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. Overall survival curves of patients with p53 mutation results. (A) p53 mutation results known; (B) p53 mutation results, observation; (C) mutant p53; and (D) wild-type p53. HR, hazard ratio.

View larger version (17K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 3. Overall survival curves of patients with RAS mutation results. (A) RAS mutation data available; (B) RAS mutation results, observation; (C) wild-type RAS; (D) mutant RAS. HR, hazard ratio.

RAS Mutation
We identified 119 mutations in 117 of 450 patients (26%; Table A4, online only). Mutations were significantly higher in large-cell and adenocarcinoma and in females (Table 2). RAS mutation was not a significant prognostic marker for survival in univariate or multivariate analyses (Fig 3B). In 333 patients with wild-type RAS, survival was significantly prolonged with adjuvant chemotherapy compared with observation (HR = 0.69; 95% CI, 0.49 to 0.97; P = .03; Table 3; Fig 3C). In contrast, there was no apparent benefit from chemotherapy in 117 patients with RAS mutant tumors (HR = 0.95; 95% CI, 0.53 to 1.71; P = .87; Fig 3D). However, in the Cox model, significant interaction between chemotherapy and RAS mutation was not detected (P = .29; Table 3).

	DISCUSSION

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p53 and RAS are the most extensively investigated prognostic markers in NSCLC, with each having more than 50 reported studies. Although meta-analyses generally have indicated that p53 and RAS gene mutations and p53 protein overexpression are weak prognostic markers of poorer outcome in NSCLC, results from individual studies have been inconsistent.^11-14 Meta-analysis cannot eliminate potential biases that may exist in published data,¹⁹ making it imperative that promising markers identified in meta-analyses be confirmed prospectively or retrospectively in large phase III randomized trials.^20,21 Because JBR.10 stratified patients according to their RAS mutation status, RAS mutation status was known in more than 90% of randomly assigned patients. Using one of the most sensitive techniques available, our results failed to confirm RAS oncogenic activation as a significant marker of poor prognosis after surgery for NSCLC. Our p53 mutation and protein expression studies were conducted retrospectively. As was the case with RAS mutation, our results show that p53 mutation is not a significant marker of poor prognosis. In contrast, p53 protein overexpression was associated with significantly shorter survival for patients in the observation arm in both univariate and multivariate analyses.

The prevalence rates of p53 and RAS mutations and p53 protein expression by IHC are comparable to previous reports (Table A5, online only). Tumors with p53 mutation and p53-positive immunohistochemistry are slightly more common among males and squamous histology, whereas RAS mutations are mainly associated with adenocarcinoma and current or former smokers. Although p53 protein overexpression is more frequent in males, this alone cannot explain the association of overexpression with poor outcome since the difference in survival between p53-positive and -negative patients remained significant in multivariate analysis.

In contrast to the numerous studies on prognosis, data on the predictive value of p53 or RAS in early stage NSCLC patients treated by adjuvant chemotherapy are lacking. In Eastern Cooperative Oncology Group (ECOG) study 3590, a study that randomly assigned completely resected stage II to IIIA NSCLC patients to receive adjuvant radiotherapy with or without etoposide/cisplatin, p53 mutation, p53 overexpression, and KRAS mutation were neither prognostic of poorer survival nor predictive of a differential benefit from chemotherapy.²²

In our study, patients with p53 or RAS wild-type tumors demonstrated a significant survival benefit from adjuvant chemotherapy, whereas those with functionally aberrant p53 or oncogenic RAS mutations did not. However, in univariate and multivariate analyses, significant interactions with chemotherapy could not be confirmed for either gene. In contrast, although untreated patients whose tumors demonstrated p53 overexpression by IHC had a significantly poorer prognosis compared with patients with negative p53 staining, their survival benefit from adjuvant chemotherapy was significantly greater than that of p53-negative patients. Furthermore, the Cox regression model with chemotherapy and p53 protein expression showed significant interaction in both the univariate and multivariate models, and the significant interaction was maintained even when multivariate Cox regression modeling was applied to all 482 patients in JBR.10, including those who did not have p53 IHC results available.

p53 nuclear immunoreactivity in tumors has 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.²³ However, Greenblatt et al²⁴ reported that in 84 studies evaluating p53 mutation and p53 protein by IHC simultaneously, the overall sensitivity of IHC to predict p53 mutation status was 75% (range, 36% to 100%), whereas the positive predictive value was only 63% (range, 8% to 100%). In our study, 56 (75%) of 75 nonsilent 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 biologic significance of wild-type p53 protein overexpression in tumors is less clearly understood.

Recent discoveries place increasing importance on MDM2 and p14^ARF as regulators of cellular p53 protein levels.²⁵ 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 p14^ARF. The stability of mutant p53 protein in tumor cells appears more dependent on its inability to bind MDM2,²⁶ and the level of wild type p53 protein is also significantly regulated by the MDM2 expression level. Wang et al²⁷ 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 P14^ARF, respectively. They reported that overexpression of p53 and low expression of MDM2 are poor prognostic markers. Their study provides compelling evidence that the biologic effects of p53 mutation and p53 protein overexpression may not be identical, and that the regulation of the p53-MDM2 pathway may influence the outcome of NSCLC patients.

The regulation of expression, signaling pathways and biologic activity of p53 is complex.^9,28 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 (eg, myc, ß-catenin, and so on) activation pathways, leading to p14 overexpression and stabilized p53 protein.^28,29 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.²⁹ There is contradictory evidence as to whether or how p53 mutation/aberrant protein expression could affect the sensitivity of solid tumors to anticancer agents.^15,28 Our results indicate that adjuvant chemotherapy appears 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.³⁰ Sensitization could possibly be caused by an inability of p53 aberrant tumor cells to transactivate p21^waf1 and allow DNA repair to occur, or by an interference of tumor cellular ability to sense DNA damage or initiate/effect DNA repair.¹⁵ Efficient DNA repair capacity is thought to account for the lack of survival benefit from platinum-based chemotherapy in NSCLC patients whose tumors have high ERCC1.⁷ The discrepancy between the role of p53 mutation and aberrant protein expression suggests that the biologic effects of these two p53 abnormalities are not equivalent and their roles warrant further mechanistic studies.

In conclusion, we have demonstrated that, 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 appear to derive greater benefit from adjuvant chemotherapy than do patients with p53 or RAS mutant tumors, the differences in our study was not statistically significant. These observations, together with those demonstrating a differential benefit from adjuvant chemotherapy in patients with low ERCC1 protein expression, 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-LACE study is being planned to test this hypothesis in a large cohort of patient samples that should have the statistical power to test multiple markers. Notwithstanding, it appears that we are on the threshold of molecular selection of NSCLC patients for postoperative adjuvant chemotherapy.

	AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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The author(s) indicated no potential conflicts of interest.

	AUTHOR CONTRIBUTIONS

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Conception and design: Ming-Sound Tsao, Sarit Aviel-Ronen, Keyue Ding, Robert Livingston, David H. Johnson, James Rigas, Lesley Seymour, Timothy Winton, Frances A. Shepherd

Financial support: Ming-Sound Tsao, Frances A. Shepherd

Administrative support: Ming-Sound Tsao, Lesley Seymour, Frances A. Shepherd

Provision of study materials or patients: Ming-Sound Tsao, Robert Livingston, David H. Johnson, James Rigas, Timothy Winton, Frances A. Shepherd

Collection and assembly of data: Ming-Sound Tsao, Sarit Aviel-Ronen, Davina Lau, Ni Liu, Akira Sakurada, Marlo Whitehead, Chang-Qi Zhu

Data analysis and interpretation: Ming-Sound Tsao, Sarit Aviel-Ronen, Keyue Ding, Davina Lau, Ni Liu, Marlo Whitehead, Chang-Qi Zhu, Lesley Seymour, Frances A. Shepherd

Manuscript writing: Ming-Sound Tsao, Sarit Aviel-Ronen, Keyue Ding, Davina Lau, Ni Liu, Lesley Seymour, Frances A. Shepherd

Final approval of manuscript: Ming-Sound Tsao, Sarit Aviel-Ronen, Keyue Ding, Davina Lau, Ni Liu, Akira Sakurada, Marlo Whitehead, Chang-Qi Zhu, Robert Livingston, David H. Johnson, James Rigas, Lesley Seymour, Timothy Winton, Frances A. Shepherd

	Appendix

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View larger version (33K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig A1. Consort chart for JBR.10 patient samples used in this study showing the number of patients available for various marker studies. FFPE, formalin fixed, paraffin embedded; TMA, tissue microarray; IHC, immunohistochemistry.

View larger version (137K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig A2. Representative images of p53 nuclear immunostaining and intensity scoring. (A) Lack of staining scored as 0; (B) intensity 1 in 20% of tumor cells; (C) intensity 2 in 40% of cells; and (D) intensity 3 in 100% of cells. Tumors with nuclear staining intensity 1 to 3 were considered positive for p53 protein overexpression when > 15% of the tumor cells were stained.

View larger version (9K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig A3. Distribution of tumors according to (A) percentage of p53 immunohistochemistry (IHC)-positive tumor cells or (B) staining intensity. No cases had percentage scores between 15% and 20% (A) and only 13% (17 of 132) of IHC-positive tumors had staining intensity of 1.

	ACKNOWLEDGMENTS

 
We thank Jean Viallet, MD, for his contribution and effort to the establishment of JBR.10 tumor bank.

	NOTES

 
Supported by grants from the Ontario Cancer Research Network (02-MAY-0132), and the Canadian Cancer Society. The JBR.10 trial was supported by the Canadian Cancer Society, the National Cancer Institute of the United States, and GlaxoSmithKline. S.A.-R. is a Fellow of the CIHR Training Program for Clinician Scientists in Molecular Oncologic Pathology (STP-53912) and is also supported by Knudson Research Fellowship (Ontario Cancer Institute) and NCIC Terry Fox Foundation Clinical Research Fellowship.

Presented at the 43rd Annual Meeting of the American Society of Clinical Oncology, June 1-5, 2007, Chicago, IL.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted May 18, 2007; accepted August 6, 2007.

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