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RRM1 and PTEN As Prognostic Parameters for Overall and Disease-Free Survival in Patients With Non–Small-Cell Lung Cancer

From the Thoracic Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL.

Address reprint requests to Gerold Bepler, MD, PhD, Moffitt Cancer Center and Research Institute, Thoracic Oncology Program, MRC-4 West, 12902 Magnolia Dr, Tampa, FL 33612-9497; e-mail: beplerg{at}moffitt.usf.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
PURPOSE: RRM1 has important functions in the determination of the malignant phenotype. It controls cell proliferation through deoxynucleotide production and metastatic propensity through PTEN induction. It is located in a region of loss of heterozygosity in non–small-cell lung cancer (NSCLC), which is a predictor of poor survival. We hypothesized that RRM1 expression would be a significant predictor of outcome in NSCLC.

PATIENTS AND METHODS: A retrospective data set of 49 patients and a prospective data set of 77 patients with resectable NSCLC were studied. RNA was extracted from tumor and normal lung tissue, and expression of the genes RRM1, PTEN, and RRM2 was determined by real-time quantitative polymerase chain reaction.

RESULTS: RRM1 expression was significantly correlated with PTEN and RRM2 expression in tumor tissue. RRM1 and PTEN expression in tumor tissue was highly predictive of overall (P = .011 and .018, respectively) and disease-free survival (P = .002 and .026, respectively). Patients with high levels of expression lived longer and had disease recurrence later than patients with low levels of RRM1 and PTEN. In a multivariate analysis, high RRM1 expression was predictive of long survival independent of tumor stage, performance status, and weight loss.

CONCLUSION: RRM1 is a biologically and clinically important determinant of malignant behavior in NSCLC. Knowing the level of expression of this gene adds significant information to management decisions independent of the currently used outcome predictors of tumor stage, performance status, and weight loss. Future clinical trials should stratify patients based on expression of this gene to avoid unwanted biases.

	INTRODUCTION

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Cancer morbidity and mortality are among the most challenging issues in health care in the industrialized nations. The hallmarks of cancer are invasion and metastasis formation, which are the cause of its morbidity and mortality. Among cancers, lung cancer is the leading cause of death. It is responsible for 28% of cancer mortality and causes more deaths than colorectal cancer, breast cancer, and prostate cancer combined.¹

RRM1 is a gene important in the determination of the malignant phenotype. It is a component of ribonucleotide reductase, the key enzyme in production of the deoxynucleotides required for DNA synthesis and repair.^2–4 Another function of RRM1 is suppression of cell migration and metastasis formation,^5,6 which is at least partially mediated through induction of PTEN.⁶ RRM1 is in a region of frequent loss of heterozygosity (LOH),^7–9 and LOH at this locus was found to be a significant adverse prognostic factor of survival in patients with non–small-cell lung cancer (NSCLC).¹⁰

PTEN, a lipid and protein phosphatase, is a tumor suppressor gene that was discovered through LOH and functional studies.^11–13 Among its many downstream targets is focal adhesion kinase (FAK), which becomes inactivated on PTEN-induced dephosphorylation.^14,15 FAK is important in regulation of cell migration and adhesion,^16,17 and its overexpression has been associated with poor outcome in patients with cancer.¹⁸

In this study, we show that RRM1 and PTEN expression are closely associated in human NSCLC, and both are significant clinical predicators of overall and disease-free survival.

	PATIENTS AND METHODS

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Exploratory Data Set
Randomly available fresh-frozen tumor specimens from patients with resected NSCLC from 1991 to 1994 and 2000 to 2001 were obtained from the tissue procurement facility. Normal lung tissue from the same resection specimens was also obtained. The two time periods were chosen based on availability of the longest stored specimens to assess RNA stability and target gene expression over a decade. Formalin-fixed and paraffin-embedded specimens were reviewed to verify diagnosis and to determine tumor cell content. Normal lung specimens were microscopically reviewed to ensure absence of premalignant or malignant cells. Specimens were uniquely identified by laboratory numbers that allowed cross-referencing with clinical data from the tumor registry without disclosure of patient identity. Use of specimens and data was approved by the institutional review board.

Validation Data Set
An institutional review board–approved prospective tissue and clinical data collection protocol was used to obtain and store freshly resected lung cancer specimens from 1993 to 2001. Specimens were macrodissected to enrich the tissue of interest. They were bisected, and one half was snap frozen in liquid nitrogen, and the other half was fixed in neutral-buffered formalin. Specimens were microscopically examined to assess quality and to verify the histopathology. Collection on this protocol required documentation of time elapsed from resection to freezing, and it was 20 minutes or less for all specimens. There was no overlap between patients in the exploratory and validation datasets. Specimens of adequate quality were available on 77 patients comprising three from 1993, two from 1994, 11 from 1995, 18 from 1996, 21 from 1997, two from 1998, one from 1999, 14 from 2000, and five from 2001. They were kept frozen at –80°C in aliquots of approximately 0.1 g.

Demographic, tobacco use (lifetime never smoker is a person that smoked 100 cigarettes, and former smoker is a person that smoked > 100 cigarettes and quit 12 months before first encounter), and clinical information was obtained on a questionnaire that patients were asked to complete before their first encounter. This information was reviewed and verified in a face-to-face interview by the treating surgeon. It was used to score the performance status (Eastern Cooperative Oncology Group scale) and absence or presence of weight loss (> 5% in the 3 months before diagnosis).

Tumor staging was based on computed tomography of the chest and upper abdomen, mediastinal lymph node dissection, and gross and microscopic evaluation of the resected lung tissue. Other staging studies were performed at the discretion of the surgeon. The date of first pathologic verification of malignancy was used as date of diagnosis.

After surgery, patients were followed for 3 months by the surgeon. They were then given a choice of follow-up at regular intervals (every 3 to 12 months) by the referring physician or at our institution with standard radiographs and/or computed tomography of the chest. The first date of unequivocal clinical evidence of disease or histologic confirmation, in questionable cases, was recorded as recurrence date. The date of death was obtained through family or care provider contact, and it was verified by review of public records. Overall survival was defined as the time elapsed from histologic diagnosis to death. Disease-free survival was defined as the time elapsed from surgery to recurrence.

Only one of these patients had induction chemotherapy and adjuvant radiation and chemotherapy. This was the only patient with pathologically proven mediastinal lymph node metastases (pN2) in the data set. All other patients did not receive any neoadjuvant or adjuvant therapy.

Biochemical Analysis
Specimens (100 mg) were pulverized with separate, sterilized, and frozen mortar and pestles. Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA) and dissolved in water. For the exploratory data set, RNA extraction had been performed at different time points, and RNA specimens were aliquoted and kept frozen at –80°C. For the validation data set, RNA extraction was performed within a 2-week time period for all specimens. Five micrograms of RNA were reverse transcribed with Superscript II and oligo-dT (Invitrogen) for generation of cDNA. For each real-time quantitative polymerase chain reaction gene analysis, 5 ng of sample cDNA was used in triplicate and amplified in 96-well plates. Each plate contained a serial dilution of reference cDNA for standard curve determination and negative controls without template. The reference cDNA was a mixture of cDNAs from various cell lines, and the identical preparation batch was used throughout this study. Intron-spanning primers were designed to avoid amplification of traces of genomic DNA (Table 1). Probes were labeled with 6-FAM on the 5' end and TAMRA on the 3' end. Fluorescent emission as a result of cleavage of the probe through exonuclease activity of Taq polymerase was recorded in real time (ABI prism 7700; Perkin-Elmer, Foster City, CA). The relative amount of target RNA in a sample was determined by comparing the threshold cycle with the standard curve, and the standardized amount was then determined by dividing the target amount by the 18SrRNA amount (Perkin-Elmer; #4310893E-0203015, VIC-TAMRA–labeled reporter probe). We had determined that 18SrRNA was the gene with the best correlation coefficient and steepest slope in a series of experiments with a variety of housekeeping genes. The slopes and correlation coefficients for the standard curves were (–)4.6 and 0.99 for RRM1, (–)4.5 and 0.99 for PTEN, (–)4.0 and 0.99 for RRM2, and (–)3.9 and 0.99 for 18SrRNA, respectively.

Statistical Analysis
Correlation coefficients between the expression of two genes and between gene expression and age were calculated using the Spearman rank correlation coefficient. The Wilcoxon rank sum test was used to test for significant associations between dichotomous variables and gene expression, and the Kruskal-Wallis test was used for variables with more than two categories. Overall and disease-free survival probabilities were estimated using the Kaplan-Meier method, and the log-rank test was used to determine the level of significance between survival curves. Cox regression analysis was used to assess the association between gene expression and survival while controlling for pathologic tumor stage, performance status, and weight loss as potential confounders. Each variable was entered singly into the model to obtain unadjusted hazard ratios.

	RESULTS

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Exploratory Data Set
Freshly frozen tumor and normal lung specimens from 1991 to 1994 (n = 28) and 2000 to 2001 (n = 21) were collected from pathologically staged patients undergoing complete resection for NSCLC. Specimen diagnosis and the content of viable tumor cells compared with all cellular elements was determined by a pathologist. Only specimens with 60% of cells consisting of tumor were processed. They included 25 adenocarcinomas, 21 squamous carcinomas, and three large-cell carcinomas. Tumor stage, survival status, and overall survival based on last contact date or date of death were obtained from the tumor registry. Thirty patients had stage I disease (single tumor lesion located within one lobe of the lung only), 12 had stage II disease (tumor within hilar lymph nodes), six had stage III disease (tumor within mediastinal lymph nodes, n = 4; two tumor lesions within one lobe of the lung, n = 2), and one had stage IV disease (two lesions in separate lobes of the lung).

Total RNA was extracted, reverse transcribed, and amplified for the genes RRM1, PTEN, and RRM2. 18SrRNA was used as reference for normalization of expression. Summary data are listed in Table 2. Normalized gene expression was not significantly different between specimens collected in the early 1990s and early 2000s or among adenocarcinomas, squamous carcinomas, and large-cell carcinomas.

There was no significant correlation between gene expression in tumor and normal lung from 44 patients for RRM1 (r = 0.08, P = .60) and PTEN (r = 0.09, P = .28); however, RRM2 expression was significantly correlated (r = 0.57, P < .0001) between tumor and normal lung.

The expression of these three genes in tumors was investigated for overall survival associations using Kaplan-Meier estimates and the log-rank test for significance. Patients were dichotomized into two groups, those with expression equal to or higher than the median and those with expression below the median. RRM1 and PTEN were significantly associated with survival (P = .013 and .011, respectively), whereas expression of RRM2 was not. Survival was longer for patients whose tumors expressed high levels of the respective gene compared with low levels (median survival time of 52 months v 24 months for RRM1, and 62 months v 23 months for PTEN).

Validation Data Set
For 77 patients, tumor specimens and clinical variables were collected prospectively, and pertinent information is listed in Table 3. This data set was similar to the exploratory data set regarding tumor histology (38 of 77 v 25 of 49 adenocarcinomas, 31 of 77 v 13 of 49 squamous carcinomas, and eight of 77 v three of 49 large-cell carcinomas); however, there was a higher rate of patients with stage I tumors (84.4%) and a lower rate of stage II (7.8%) and stages III and IV tumors (7.8%) than in the exploratory data set (stage I, 61.2%; stage II, 24.5%; and stages III and IV, 16.3%). Actual overall median survival was 39.7 months (range, 2.0 to 106.1 months) in the validation data set compared with 28.6 months (range, 3.5 to 132.1 months) in the exploratory data set. Gene expression levels were comparable in both datasets (median in validation and exploratory datasets: RRM1, 12.24 v 12.57; RRM2, 4.23 v 6.02; and PTEN, 39.56 v 37.46, respectively). Normalized gene expression was not significantly different for specimens collected by calendar year between 1993 and 2001 (P = .6783; Spearman correlation coefficient, r = 0.04803). As in the exploratory data set, the expression of RRM1 was significantly correlated with the expression of PTEN (r = 0.348, P = .002) and RRM2 (r = 0.714, P < .0001).

View larger version (18K): [in this window] [in a new window]   Fig 1. Kaplan-Meier overall survival estimates from 77 patients (validation data set) for (A) RRM1 and (B) PTEN expression. Black lines depict patients with marker expression greater than or equal to the respective median value, and red lines depict expression less than the median value. RRM1 and PTEN expression: P = .011 and .018, respectively.

View larger version (18K): [in this window] [in a new window]   Fig 2. Kaplan-Meier disease-free survival estimates from 77 patients (validation data set) for (A) RRM1 and (B) PTEN expression. Black lines depict patients with marker expression greater than or equal to the respective median value, and red lines depict expression less than the median value. RRM1 and PTEN expression: P = .002 and .026, respectively.

	DISCUSSION

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Genomic and correlative human tissue studies have strongly suggested a role for a gene on chromosome segment 11p15.5 in the malignant phenotype of multiple epithelial malignancies including gastric carcinoma,¹⁹ esophageal carcinoma,²⁰ and breast cancer.^21,22 In addition, reduced PTEN expression was found to be associated with poor clinical outcome in cancer of the esophagus,²³ stomach,²⁴ prostate,²⁵ ovary,²⁶ endometrium,²⁷ breast,²⁸ and glia.^29–31 Our results, generated through quantitative measurement of RRM1 and PTEN in fresh tumor specimens from patients with NSCLC, are consistent with these observations. The gene expression data were generated in an objectively measurable, robust, and reproducible way, and corrected expression values remained stable over a decade in tissues stored in liquid nitrogen.

A role for PTEN in lung cancer has not been convincingly demonstrated. This gene is rarely mutated or deleted in lung cancer,³² and its expression may be influenced by epigenetic DNA modification.³³ Homozygous deletions of PTEN have not been observed in NSCLC,³² and whether homozygous loss-of-function coding region mutations exist is controversial.⁶ Our results clearly show a strong association between PTEN expression and the clinical behavior of lung cancer. This together with the established function of PTEN in controlling the phosphorylation status of multiple proteins with crucial roles in cell motility, proliferation, and survival, strongly supports a key role for this gene in the pathogenesis of lung cancer.

In our previous in vitro investigations, we found that RRM1 expression was strongly associated with PTEN expression and that reduction in PTEN expression through antisense oligonucleotides did not result in a concomitant reduction of RRM1 expression.⁶ In this study, we confirmed this strong association between RRM1 and PTEN expression in two separate datasets and show that RRM1 expression seems to be marginally better in predicting clinical outcome than PTEN expression. Taken together, these results favor the assumption that RRM1 is upstream of PTEN and a key component in a network of genes that controls malignant behavior. Other effectors of the RRM1-controlled phenotype have not yet been identified.

The strong positive prognostic significance of RRM1 in lung cancer is surprising given the role of this gene in ribonucleotide reductase holoenzyme formation. Ribonucleotide reductase is a heterotetramer, and activity requires two 90-kd large subunits and two 45-kd small subunits. The enzyme is crucial for the supply of deoxynucleotides for de novo synthesis of DNA and, thus, cell proliferation. Its function is rate limited by the small RRM2 component. In mammalian cells, it is the expression of the RRM2 gene that is tightly controlled during cell cycle progression, with a sharp induction at the end of G1 phase. In contrast, the RRM1 gene is expressed constitutively, with higher levels during chief demand for ribonucleotide reductase formation.^2–4 This discrepancy in the control of expression for both genes supports the hypothesis that the RRM1 gene has alternate functions in the control of the cellular phenotype aside from its role in ribonucleotide reductase formation.

The role of RRM1 in ribonucleotide reductase formation has also made it an attractive molecular target for the development of chemotherapeutic agents, such as gemcitabine.³⁴ In fact, gemcitabine has become an important agent used in the treatment of NSCLC.³⁵ It has recently been reported that high RRM1 levels are associated with poor outcome in patients with metastatic NSCLC who received combination chemotherapy with gemcitabine and cisplatin.^36,37 The mechanism for this finding is presumably a decreased cytotoxicity of gemcitabine in tumors with high levels of expression. However, alternate explanations are that high levels of RRM1 may lead to global desensitization to cytotoxic agents through increased DNA repair efficiency from increased activity of the RRM1/p53R2 DNA damage repair complex³⁸ or through dormancy from disruption of extracellular signaling pathways such as FAK-mediated extracellular matrix signaling.³⁹ These preliminary results and hypotheses await confirmation in ongoing clinical and biochemical investigations.

Given the multiple roles of RRM1 in suppression of motility and production of deoxynucleotides for proliferation and DNA damage repair, it is plausible that patients whose tumors express high levels of RRM1 have intrinsically a better outcome resulting from a decreased tumor propensity for dissemination; however, once dissemination has occurred, such tumors may prove to be resistant to therapy.

We conclude that RRM1 and PTEN expression are highly correlated in NSCLC, and both are strong predictors of outcome in patients with resectable disease. Future randomized trials in NSCLC should stratify patients based on RRM1 and/or PTEN expression because tumors with high levels of expression have an intrinsically less malignant phenotype, as shown here, that is potentially less responsive to cytotoxic therapy.

	Authors' Disclosures of Potential Conflicts of Interest

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The authors indicated no potential conflicts of interest.

	NOTES

 
Supported in part by grant No. R01-CA102726 from the National Cancer Institute, Bethesda, MD.

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

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Submitted December 1, 2003; accepted February 25, 2004.

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bepler, G. Articles by Robinson, L. Search for Related Content PubMed PubMed Citation Articles by Bepler, G. Articles by Robinson, L.