Originally published as JCO Early Release 10.1200/JCO.2006.06.1101 on September 11 2006

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 Rights & Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bepler, G. Articles by Simon, G. Search for Related Content PubMed PubMed Citation Articles by Bepler, G. Articles by Simon, G.

RRM1 Modulated In Vitro and In Vivo Efficacy of Gemcitabine and Platinum in Non–Small-Cell Lung Cancer

Gerold Bepler, Irina Kusmartseva, Swati Sharma, Ashish Gautam, Alan Cantor, Anupama Sharma, George Simon

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

Address reprint requests to Gerold Bepler, MD, PhD, H. Lee Moffitt Cancer Center and Research Institute, MRC-4W, Room 4046, 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... Author Contributions GLOSSARY REFERENCES

 
PURPOSE: RRM1 encodes the regulatory subunit of ribonucleotide reductase and is a molecular target of gemcitabine. Previous studies showed increased RRM1 expression on continuous exposure of cell lines to gemcitabine and suggested improved survival for patients with low as opposed to high tumoral RRM1 expression when treated with gemcitabine-containing chemotherapy. However, the principal hypothesis that intratumoral levels of gene expression are associated with disease response has not been addressed.

PATIENTS AND METHODS: We constructed genetically modified lung cancer cell lines with increased and decreased RRM1 expression to investigate the in vitro 50% inhibitory concentration (IC₅₀) for gemcitabine, cisplatin, and carboplatin. A prospective phase II clinical trial in patients with locally advanced non–small-cell lung cancer was conducted with pretreatment tumor collection for determination of RRM1 and ERCC1 expression by real-time reverse transcriptase polymerase chain reaction. The levels of gene expression were correlated with tumor response after two cycles of gemcitabine and carboplatin.

RESULTS: In cell lines with a genetically engineered 15-fold RRM1 expression range, the gemcitabine IC₅₀ had a 100-fold range, and the cisplatin and carboplatin IC₅₀ had a two-fold range. They were highest in constructs with high RRM1 expression. In the prospective clinical trial, RRM1 expression was significantly (P = .002) and inversely correlated (r = –0.498) with disease response. ERCC1 expression showed a similar trend (P = .099).

CONCLUSION: The results strongly suggest that tumoral RRM1 expression is a major predictor of disease response to gemcitabine/platinum chemotherapy. ERCC1 expression is predictive of response albeit to a lesser degree.

	INTRODUCTION

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

 
The principal chemotherapeutic agents used for treatment of patients with non–small-cell lung cancer (NSCLC) are platinum analogs, taxanes, gemcitabine, and vinorelbine. Response rates in patients with advanced stages of the disease to dual agent therapy, the current standard of care, are 17% to 37%; the median overall survival is 6.7 to 11.3 months, the 1-year survival rate is 31% to 46%, and the 2-year survival rate is 9% to 21%.^1-7 After patients experienced treatment failure of initial therapy, response to further systemic treatment is approximately 10% for single agents.^8-10 Thus, resistance to systemic therapy does not appear to be an all-or-none phenomenon, but rather a function of molecular characteristics of individual tumors. In fact, specific molecular characteristics highly associated with tumor response to a class of therapeutic agents used in NSCLC, epidermal growth factor receptor tyrosine kinase inhibitors, has recently been reported.^11-13

The RRM1 gene encodes the regulatory subunit of ribonucleotide reductase, an essential enzyme that catalyzes the reduction of ribonucleoside diphosphates to the corresponding deoxyribonucleotides.^14-17 It is the molecular target of gemcitabine (2',2'-difluorodeoxycytidine), an antimetabolite with activity in several malignancies including NSCLC.^18-20 Earlier work had suggested that patients with low as compared with high levels of tumoral RRM1 expression had improved survival when treated with gemcitabine-based therapy.²¹ In addition, continuous exposure of lung cancer cell lines to increasing amounts of gemcitabine resulted in increased RRM1 expression.²⁰ However, the principal hypothesis for these investigations (ie, intratumoral levels of RRM1 expression are associated with disease response) has not been addressed.

	PATIENTS AND METHODS

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

 
Generation of RRM1-Modified Stable Lung Cancer Cell Lines
H23siR1 was generated by stable transfection of NCI-H23 with a pSUPER-siRRM1 construct. To generate this construct, the pSUPER-GFP/neo vector (OligoEngine, Seattle, WA) was digested with BgIII and HindII and the annealed oligonucleotides (5'-GATCCCCGACGCTAGAGCGGTCTTATTTCAAGAGAATAAGACCGCTCTAGCGTCTTTTTGGAAA-3'and 5'-AGCTTTTCCAAAAAGACGCTAGAGCGGTCTTATTCTCTTGAAATAAGACCGCTCTAGCGTCGGG-3') were ligated into the vector. The 19-nucleotide RRM1 target sequences are underlined. The H23siCt cell line was generated with the targeting sequence GCTAATAGCGCGGAGTCTT, which has no similarity to any known gene. H23-R1 is a stable RRM1 overexpressing cell line, and H23-Ct is its corresponding control. Both were generated by transfection with full-length RRM1 cDNA cloned into the expression plasmid pCMV-Tag2 (Stratagene, La Jolla, CA) as previously described.²² The level of RRM1 expression in these cell lines was assessed at the RNA level by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) using custom-designed and validated primers and probes and at the protein level by Western blot analysis.²³

In Vitro Drug Testing
A modified MTT assay with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium as the reagent was used, MTS assay (Promega, Madison, WI). It was performed by seeding 4,000 cells/well in 96-well flat bottom plates. H23-Ct, H23-R1, H23siR1, and H23siCt cells were continuously exposed to drug for 72 or 96 hours. After 3 hours of incubation at 37°C, the absorbance was measured at 490 nm in a microplate reader (Benchmark Plus; Bio-Rad, Hercules, CA). All experiments were repeated three times. The in vitro drug concentration that inhibited cell growth by 50% (IC₅₀) was calculated as ([mean absorbance in triplicate wells containing the drug – mean absorbance in triplicate blank wells]/[mean absorbance in triplicate drug-free wells – mean absorbance in triplicate blank wells])x100.

To determine drug-induced in vitro apoptosis, 3x10⁵ cells/well were seeded in six well plates and allowed to attach overnight. Chemotherapeutic agents were added to the culture at the indicated concentrations. After 2, 4, and 6 hours of treatment, annexin V labeling of the cells was performed as recommended by the manufacturer (BD Pharmingen, San Diego, CA). The percentage of labeled cells was determined by flow cytometry (FACScalibur, Becton Dickinson, Franklin Lakes, NJ).

Clinical Trial
A trial was designed to prospectively assess the impact of tumoral RRM1 expression on efficacy of gemcitabine-based chemotherapy in previously untreated patients with NSCLC. A secondary trial end point was to determine the impact of tumoral ERCC1 (excision repair cross-complementing gene) expression on treatment efficacy. For this, we determined the response to gemcitabine and carboplatin as induction therapy (IndGC) in patients with locally advanced and inoperable NSCLC. Double agent chemotherapy was chosen because single-agent therapy is deemed inadequate for previously untreated patients with any stage of NSCLC and a performance status of 0 to 1.²⁴ Trial eligibility included histologically confirmed NSCLC, inoperable stage IIIA or IIIB disease (patients with cytologically positive pleural effusion were excluded; and exemption was made for one patient with stage IIB disease), measurable disease by Response Evaluation Criteria in Solid Tumors (RECIST),²⁵ no prior systemic chemotherapy or thoracic radiation, Eastern Cooperative Oncology Group performance status 0 or 1, absence of weight loss ( 5% in the 3 months preceding diagnosis), and adequate bone marrow, liver, and kidney function. All patients were staged with computed tomography (CT) of the chest and upper abdomen, whole body [¹⁸F]fluorodeoxyglucose positron emission tomography (PET), and magnetic resonance imaging of the brain. IndGC consisted of two 28-day cycles of gemcitabine, 1,000 mg/m², given on days 1 and 8 and carboplatin, area under the curve 5, given on day 1. CT and PET were repeated in week 8, and disease response was assessed. Thereafter, patients received concurrent radiation and chemotherapy for definitive treatment of their disease. The study required the collection of tumor specimens specifically for molecular analyses before therapy, which was performed by core needle biopsy that produced a tissue specimen of 0.8 mm diameter. Specimens were immediately frozen in liquid nitrogen, embedded in OCT, and processed as frozen sections. Tumor cells were collected by laser capture microdissection (LCM), and RNA was extracted. The institutional review board approved the clinical trial and all patients provided written informed consent.

Disease Response Assessment
For disease response determination all measurable cancer lesions were assessed in greatest diameter using images obtained with intravenous contrast on a multichannel helical CT scanner at 5 mm intervals. Measurements were performed on a picture-archive communication system workstation (Siemens MagicView 1000, Munich, Germany), and they were repeated in week 8. The percentage of change of the sum of tumor diameters comparing the post-treatment with the pretreatment measurements was calculated using the formula 1–(SumCTpost/SumCTpre). A positive value indicated tumor shrinkage and a negative value tumor growth (ie, disease response was assessed as a continuous variable). In addition, disease response was assessed as a discrete variable using RECIST (ie, complete remission [CR], partial remission [PR], stable disease [SD], and progressive disease). The appearance of a new and previously not observed tumor lesion on imaging studies, which included a repeat PET scan in week 8, or by physical exam was coded as disease progression.

Gene Expression Analysis
Frozen tissue samples, embedded in OCT, were cut in 5 to 7 µm sections. Tumor cells were collected by LCM using the Arcturus system (60 mW, 1.5 milliseconds, intensity 100, spot size 20 µm), and total RNA was extracted using a commercially available method (PicoPure RNA isolation kit, #KIT0204; Arcturus, Mountain View, CA). Complementary DNA was generated with Superscript II and oligo-dT (Invitrogen, Carlsbad, CA). Real-time quantitative RT-PCR gene analysis was performed in triplicate per sample and gene in 96-well plates (ABI prism 7700; Perkin-Elmer, Foster City, CA). Each plate contained a serial dilution of reference cDNA for standard curve determination and negative controls without template. We had designed and validated the primers and probes as previously described.^23,26 Commercially available primers and probes were used for expression analysis of the housekeeping gene 18SrRNA (Perkin-Elmer, #4310893E-0203015), which was used as internal reference standard. 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.

Statistical Methods
Correlation coefficients between gene expression and the continuous variables tumor response, number of cells collected by LCM, and patients' age were calculated according to Spearman. The Wilcoxon rank sum test was used to test for significance between dichotomous variables and gene expression. The Kuskal Wallis test was used to test for significance between noncontinuous variables with more than two values and gene expression.

	RESULTS

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

 
We used a modified MTT assay to assess the impact of RRM1 expression on therapeutic efficacy of gemcitabine and platinum analogs in vitro. H23, originating from a lung adenocarcinoma, was used as the parent cell line.²⁷ H23-R1 had a 2.5- to 3.5-fold increased RRM1 expression compared with H23.²² H23siR1 had a five-fold reduced RRM1 expression. The expression levels of RRM1 and control genes were determined by real-time quantitative RT-PCR and Western blotting. The expression of RRM2, the catalytic ribonucleotide reductase subunit, was unaffected. The sensitivity of these cell lines to gemcitabine, cisplatin, and carboplatin was compared with transfected control cell lines (H23-Ct and H23siCt). The results are summarized in Table 1. Increased RRM1 expression resulted in resistance to gemcitabine (Fig 1). The gemcitabine IC₅₀ of H23-R1 was eight-fold higher than the IC₅₀ of H23-Ct. Reduced RRM1 expression increased sensitivity to gemcitabine (Fig 1). The gemcitabine IC₅₀ of H23siR1 was 10-fold lower than the IC₅₀ of H23siCt. The response of the parental cell line (H23) to gemcitabine was similar to the response of H23-Ct and H23siCt. There was a similar relationship between RRM1 expression and cytotoxicity response to cisplatin (Fig 1 and Table 1) and carboplatin albeit to a lesser degree. H23-R1 was 1.2- to 1.5-fold more resistant and H23siR1 was 1.1- to 1.3-fold more sensitive to platinum analogs than the corresponding control cell lines.

View larger version (16K): [in this window] [in a new window]   Fig 1. In vitro cytotoxicity of gemcitabine and cisplatin in H23-R1, H23-Ct, H23siR1, and H23siCt. Cells were exposed to various drug concentrations for 72 hours, and viability was subsequently determined by MTS assays (Promega, Madison, WI). Data are means with or without SE from three independent experiments.

View larger version (17K): [in this window] [in a new window]   Fig 2. In vitro apoptosis induction by gemcitabine and cisplatin in H23-R1, H23-Ct, H23siR1, and H23siCt. Cells were exposed to 250 nmol/L gemcitabine or 200 nmol/L cisplatin for 6 hours, and the proportion of cells labeled with annexin V was determined. Data are means with or without SE from three independent experiments.

Between November 2003 and January 2006, 40 eligible patients were enrolled, and the required tumor biopsies were obtained from all. Three patients withdrew consent before initiation of therapy because they desired treatment closer to home. In 28 patients, the biopsies were done to obtain tissue for molecular studies only; in nine patients, they were also used to establish a diagnosis. In one patient, a pneumothorax developed that required chest tube placement. In two patients tumor collection did not yield sufficient material for gene expression analysis. From the remaining 35 patients, between 30 and 4,500 tumor cells were collected by LCM. All 35 patients completed IndGC, and disease response ranged from a 9% increase to a 100% decrease in tumor diameters. Twenty patients had SD, fourteen had PR, and one had CR. The patients' ages ranged from 47 to 87 years; 17 were women; 18 had stage IIIA, 16 had stage IIIB, and one had stage IIB disease; 11 had squamous cell carcinoma, 11 had adenocarcinoma, and 13 had large cell carcinoma or unspecified NSCLC. Two patients had been life-time never smokers, 20 had quit smoking for 1 to 46 years, and 13 were current smokers.

RRM1 expression ranged from 0.18 to 129.3. We found a significant (P = .002) inverse correlation (r = –0.498, Spearman correlation coefficient) between RRM1 expression and the magnitude of disease response (Fig 3). When grouping patients into those with response (CR/PR) and without response (SD), RRM1 expression was significantly associated with response (P = .03, ² test), where patients with high tumoral expression of RRM1 were less likely to respond to IndGC than those with low levels of expression. ERCC1 expression was determined as a secondary trial end point, and it was also inversely correlated (r = –0.283; P = .099) with the magnitude of response but not with the response classification (P = .32) to IndGC. Results are summarized in Table 2. There was no significant association between RRM1 or ERCC1 expression and the number of tumor cells collected, patients' ages or sex, tumor histopathology, tumor stage, and smoking status.

View larger version (13K): [in this window] [in a new window]   Fig 3. Scatter plot of (A) RRM1 and (B) ERCC1 expression in relation to the percent change in tumor size after two cycles of gemcitabine and carboplatin chemotherapy in 35 patients. Red dots indicate patients with less than 30% tumor shrinkage (stable disease [SD]), and green dots indicate patients with greater than 30% tumor shrinkage (partial remission/complete remission [PR/CR]). The Spearman correlation coefficient was r = –0.498 (P = .002) for RRM1 and r = –0.283 (P = .099) for ERCC1.

	DISCUSSION

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

Gemcitabine efficacy is thought to be a function of proteins involved in activation, metabolism, and transport of the drug. Deoxycytidine kinase is required for phosphorylation of gemcitabine to its active metabolite, and low levels have been associated with drug resistance.²⁸ High levels of cytidine deaminase inactivate gemcitabine and confer resistance to the drug.²⁹ Equilibration-sensitive nucleoside transporter 1 facilitates intracellular gemcitabine update, and deficiency has been associated with drug resistance.³⁰ Finally 5' nucleotidase dephosphorylates dFdCMP thus antagonizing the production of active dFdCTP. High levels of this enzyme have been found in gemcitabine resistant cell lines; and in patients with acute myelogenous leukemia receiving gemcitabine, high 5NT expression was associated with poor survival.

A recent report suggested that gemcitabine resistance, generated in vitro through exposure of two NSCLC cell lines (H358 and H460) to increasing concentrations of the drug, was primarily a function of increased expression of RRM1.²⁰ These data were confirmed in a subcutaneous murine colon tumor model (Colon 26) where gemcitabine resistance had been generated through prolonged gemcitabine exposure and serial transplantation.³¹ However, induction of drug resistance through continuous exposure results in alterations in multiple genes as demonstrated by these authors. Thus, our results in genetically modified lung cancer cell lines are the first to demonstrate directly that RRM1 is a major cellular determinant of cytotoxic efficacy of gemcitabine. In addition our data demonstrate that RRM1 is a minor determinant of platinum efficacy.

We confirmed these in vitro data by demonstrating a statistically significant inverse correlation between the magnitude of change in tumor diameter and RRM1 expression in patients with NSCLC that received gemcitabine and carboplatin as first-line therapy. In addition, we found a similar but less striking relationship between ERCC1 expression and tumor response. Although previous retrospective studies had suggested this relationship for both molecules by demonstrating better patient survival in those having low RRM1 or ERCC1 expression as compared with high levels of expression if treated with gemcitabine and platinum,^21,32 a direct and prospective demonstration had not been performed. Thus, our results strongly support the primary study hypothesis that RRM1 expression is predictive of response to treatment with gemcitabine.

In summary, the cumulative body of indirect and direct in vitro data and murine and human investigations provide convincing support for RRM1 as the major determinant of gemcitabine efficacy. Several issues should be addressed to facilitate implementation of RRM1 gene expression analysis into patient management. First, it is important to verify our prospective clinical results in a second prospective treatment trial and to assess if RRM1 and ERCC1 expression are similar among a wider spectrum of disease stages. Second, for clinical management of patients a categorization with defined cutoff levels for RRM1 and perhaps ERCC1 would be desirable to obtain clear positive and negative predictive values. For instance, our data suggest that none of the patients with RRM1 expression above 10 achieved a PR (0 of 11; positive predictive value for resistance, 1.0), while 15 of 24 patients with levels below 10 had an objective disease response (positive predictive value for sensitivity, 0.6). Third, the general feasibility of performing core needle biopsies of patients' tumors with immediate freezing, LCM, and subsequent sophisticated gene expression analysis appears limited given the required infrastructure. Thus, the development of a more generally applicable methodology based on technology familiar to clinical laboratories and pathologists, such as immunocytochemistry, is desirable. Fourth, it is likely that other molecular characteristics of tumor cells and the host significantly impact the RRM1-affected treatment response. It is thus important to further elucidate specific molecular features and pathways that modulate therapeutic efficacy to the agents used for lung cancer therapy.

	Authors' Disclosures of Potential Conflicts of Interest

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

 
Although all authors completed the disclosure declaration, the following authors or their immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.

Authors Employment Leadership Consultant Stock Honoraria Research Funds Testimony Other Gerold Bepler Eli Lilly (A) Eli Lilly (A); Astra Zeneca (A); Genentech (A) George Simon Eli Lilly (A); Sanofi Aventis (A); Genentech (A); Astra Zeneca (A)

Dollar Amount Codes (A) <$10,000 (B) $10,000-99,999 (C) $100,000 (N/R) Not Required

	Author Contributions

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

 

Conception and design: Gerold Bepler, Irina Kusmartseva, Alan Cantor, Anupama Sharma, George Simon Financial support: Gerold Bepler Administrative support: Gerold Bepler Provision of study materials or patients: Gerold Bepler, Irina Kusmartseva, Swati Sharma, Ashish Gautam, Anupama Sharma, George Simon Collection and assembly of data: Gerold Bepler, Irina Kusmartseva, Swati Sharma Data analysis and interpretation: Gerold Bepler, Irina Kusmartseva, Swati Sharma, Alan Cantor, Anupama Sharma Manuscript writing: Gerold Bepler, Irina Kusmartseva, Alan Cantor Final approval of manuscript: Gerold Bepler, Irina Kusmartseva, Swati Sharma, Ashish Gautam, Alan Cantor, Anupama Sharma, George Simon

 

	GLOSSARY

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

 

Annexin V labeling:

Annexin V is used as a probe in an affinity assay to detect cells that have expressed phosphatidylserine on the cell surface.

ERCC1 (excision repair cross-complementing gene):

Encodes a nucleotide excision repair protein that repairs a range of lesions, including UV-induced thymine dimers and other photoproducts, and also lesions caused by a variety of chemical agents.

MTT assay:

A laboratory test that measures cell growth.

RRM1 (ribonucleotide reductase):

A gene that encodes the regulatory subunit of ribonucleotide reductase and is a molecular target of gemcitabine.

RT-PCR (reverse-transcriptase polymerase chain reaction):

PCR is a method that allows logarithmic amplification of short DNA sequences within a longer, double-stranded DNA molecule. Gene expression can be measured after extraction of total RNA and preparation of cDNA by a reverse-transcription step. Thus, RT-PCR enables the detection of PCR products on a real-time basis, making it a sensitive technique for quantitating changes in gene expression.

	NOTES

 
published online ahead of print at www.jco.org on September 11, 2006.

Supported by Grants No. R01 CA102726 and R21 CA106616 from the National Cancer Institute.

Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org.

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

	REFERENCES

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

 
1. Kelly K, Crowley J, Bunn PA Jr, et al: Randomized phase III trial of paclitaxel plus carboplatin versus vinorelbine plus cisplatin in the treatment of patients with advanced non-small-cell lung cancer: A Southwest Oncology Group trial. J Clin Oncol 19:3210-3218, 2001[Abstract/Free Full Text]

2. Schiller JH, Harrington D, Belani CP, et al: Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med 346:92-98, 2002[Abstract/Free Full Text]

3. Kosmidis P, Mylonakis N, Nicolaides C, et al: Paclitaxel plus carboplatin versus gemcitabine plus paclitaxel in advanced non-small-cell lung cancer: A phase III randomized trial. J Clin Oncol 20:3578-3585, 2002[Abstract/Free Full Text]

4. Rosell R, Gatzemeier U, Betticher DC, et al: Phase III randomized trial comparing paclitaxel/carboplatin with paclitaxel/sicplatin in patients with advanced non-small-cell lung cancer: A cooperative multinational trial. Ann Oncol 13:1539-1549, 2002[Abstract/Free Full Text]

5. Fossella F, Pereira JR, von Pawel J, et al: Randomized, multinational, phase III study of docetaxel plus platinum combinations versus vinorelbine plus cisplatin for advanced non-small-cell lung cancer: The TAX 326 study group. J Clin Oncol 21:1-9, 2003[Free Full Text]

6. Gridelli C, Gallo C, Shepherd FA, et al: Gemcitabine plus vinorelbine compared with cisplatin plus vinorelbine or cisplatin plus gemcitabine for advanced non-small-cell lung cancer: A phase III trial of the Italian GEMVIN Investigators and the National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol 21:3025-3034, 2003[Abstract/Free Full Text]

7. Smit EF, van Meerbeeck JPAM, Lianes P, et al: Three-arm randomized study of two cisplatin-based regimens and paclitaxel plus gemcitabine in advanced non-small-cell lung cancer: A phase III trial of the European Organization for Reserach and Treatment of Cancer Lung Cancer Group - EORTC 08975. J Clin Oncol 21:3909-3917, 2003[Abstract/Free Full Text]

8. Shepherd FA, Dancey J, Ramlau R, et al: Prospective randomized trial of docetaxel versus best supportive care in patients with non-small-cell lung cancer previously treated with platinum-based chemotherapy. J Clin Oncol 18:2095-2103, 2000[Abstract/Free Full Text]

9. Fossella FV, DeVore R, Kerr RN, et al: Randomized phase III trial of docetaxel versus vinorelbine or ifosfamide in patients with advanced non-small-cell lung cancer previously treated with platinum-containing chemotherapy regimens: The TAX 320 Non-Small Cell Lung Cancer Study Group. J Clin Oncol 18:2354-2362, 2000[Abstract/Free Full Text]

10. Hanna N, Shepherd F, Fossella FV, et al: Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small-cell lung cancer previously treated with chemotherapy. J Clin Oncol 22:1589-1597, 2004[Abstract/Free Full Text]

11. Lynch TJ, Bell DW, Sordella R, et al: Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350:2129-2139, 2004[Abstract/Free Full Text]

12. Paez JG, Janne PA, Lee JC, et al: EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science 304:1497-1500, 2004[Abstract/Free Full Text]

13. Tsao MS, Sakurada A, Cutz JC, et al: Erlotinib in lung cancer: Molecular and clinical predictors of outcome. N Engl J Med 353:133-144, 2005[Abstract/Free Full Text]

14. Elledge SJ, Zhou Z, Allen JB: Ribonucleotide reductase: Regulation, regulation, regulation. Trends Biochem Sci 17:119-123, 1992[CrossRef][Medline]

15. Reichard P: From RNA to DNA, why so many ribonucleotide reductases? Science 260:1773-1777, 1993[Abstract/Free Full Text]

16. Filatov D, Ingemarson R, Johansson E, et al: Mouse ribonucleotide reducatse: From genes to proteins. Biochem Soc Trans 23:903-905, 1995[Medline]

17. Stubbe J: Ribonucleotide reductases in the twenty-first century. Proc Natl Acad Sci U S A 95:2723-2724, 1998[Free Full Text]

18. Johnson DH: Gemcitabine for the treatment of non-small-cell lung cancer. Oncology 3:33-39, 2001 (suppl)

19. Bergman AM, Pinedo HM, Peters GJ: Determinants of resistance to 2',2'-difluorodeoxycytidine (gemcitabine). Drug Resist Updat 5:19-33, 2002[CrossRef][Medline]

20. Davidson JD, Ma L, Flagella M, et al: An increase in the expression of ribonucleotide reductase large subunit 1 is associated with gemcitabine resistance in non-small cell lung cancer cell lines. Cancer Res 64:3761-3766, 2004[Abstract/Free Full Text]

21. Rosell R, Danenberg K, Alberola V, et al: Ribonucleotide reductase mRNA expression and survival in gemcitabine/cisplatin-treated advanced non-small-cell lung cancer patients. Clin Cancer Res 10:1318-1325, 2004[Abstract/Free Full Text]

22. Gautam A, Li ZR, Bepler G: RRM1-induced metastasis suppression through PTEN-regulated pathways. Oncogene 22:2135-2142, 2003[CrossRef][Medline]

23. Bepler G, Sharma S, Cantor A, et al: RRM1 and PTEN as prognostic parameters for overall and disease-free survival in patients with non-small-cell lung cancer. J Clin Oncol 22:1878-1885, 2004[Abstract/Free Full Text]

24. National Comprehensive Cancer Network: Guidelines for NSCLC treatment. http://www.nccn.org/professionals/physician_gls/PDF/nscl.pdf

25. Therasse P, Arbuck SG, Eisenhauer EA, et al: New guidelines to evaluate the response to treatment in solid tumors: European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst 92:205-216, 2000[Abstract/Free Full Text]

26. Simon GR, Sharma S, Cantor A, et al: ERCC1 expression is a predictor of survival in resected patients with non-small cell lung cancer. Chest 127:978-983, 2005[Abstract/Free Full Text]

27. Carney DN, Gazdar AF, Bepler G, et al: Establishment and identification of small cell lung cancer cell lines having classic and variant features. Cancer Res 45:2913-2923, 1985[Abstract/Free Full Text]

28. Plunkett W, Huang P, Xu YZ, et al: Gemcitabine: Metabolism, mechanism of action, and self potentiation. Semin Oncol 22:3-10, 1995 (suppl 11)[Medline]

29. Eliopoulos N, Cournoyer D, Momparler RL: Drug resistance to 5-aza-2'deoxycytidine and 2,2'-difluorodeoxycytidine conferred by retroviral mediated transfer of human cytidine deaminase cDNA into murine cells. Cancer Chemotherapy Pharmacol 42:373-378, 1998

30. Rauchwerger DR, Firby PS, Hedley DW, et al: Equilibrative-sensitive nucleoside transporter and its role in gemcitabine sensitivity. Cancer Res 60:6075-6079, 2000[Abstract/Free Full Text]

31. Bergman A, Eijk P, van Haperen V, et al: In vivo induction of resistance to gemcitabine results in increased expression of ribonucleotide reductase subunit M1 as a major determinant. Cancer Res 65:9510-9516, 2005[Abstract/Free Full Text]

32. Lord RVN, Brabender J, Gandara D, et al: Low ERCC1 expression correlates with prolonged survival after cisplatin plus gemcitabine chemotherapy in non-small cell lung cancer. Clin Cancer Res 8:2286-2291, 2002[Abstract/Free Full Text]

Submitted February 8, 2006; accepted June 23, 2006.

This article has been cited by other articles:

				S.-O. Kim, J.-Y. Jeong, M.-R. Kim, H.-J. Cho, J.-Y. Ju, Y.-S. Kwon, I.-J. Oh, K.-S. Kim, Y.-I. Kim, S.-C. Lim, et al. Efficacy of Gemcitabine in Patients with Non-Small Cell Lung Cancer According to Promoter Polymorphisms of the Ribonucleotide Reductase M1 Gene Clin. Cancer Res., May 15, 2008; 14(10): 3083 - 3088. [Abstract] [Full Text] [PDF]

				D. Breen and F. Barlesi The place of excision repair cross complementation 1 (ERCC1) in surgically treated non-small cell lung cancer Eur. J. Cardiothorac. Surg., May 1, 2008; 33(5): 805 - 811. [Abstract] [Full Text] [PDF]

				G. Bepler Genes Predictive of Chemotherapeutic Efficacy in Non-small Cell Lung Cancer ASCO Educational Book, January 1, 2008; 2008(1): 350 - 352. [Abstract] [Full Text] [PDF]

				J.-C. Soria ERCC1-Tailored Chemotherapy in Lung Cancer: The First Prospective Randomized Trial J. Clin. Oncol., July 1, 2007; 25(19): 2648 - 2649. [Full Text] [PDF]

				G. Simon, A. Sharma, X. Li, T. Hazelton, F. Walsh, C. Williams, A. Chiappori, E. Haura, T. Tanvetyanon, S. Antonia, et al. Feasibility and Efficacy of Molecular Analysis-Directed Individualized Therapy in Advanced Non-Small-Cell Lung Cancer J. Clin. Oncol., July 1, 2007; 25(19): 2741 - 2746. [Abstract] [Full Text] [PDF]

				M. Cobo, D. Isla, B. Massuti, A. Montes, J. M. Sanchez, M. Provencio, N. Vinolas, L. Paz-Ares, G. Lopez-Vivanco, M. A. Munoz, et al. Customizing Cisplatin Based on Quantitative Excision Repair Cross-Complementing 1 mRNA Expression: A Phase III Trial in Non-Small-Cell Lung Cancer J. Clin. Oncol., July 1, 2007; 25(19): 2747 - 2754. [Abstract] [Full Text] [PDF]

				Z. Zheng, T. Chen, X. Li, E. Haura, A. Sharma, and G. Bepler DNA Synthesis and Repair Genes RRM1 and ERCC1 in Lung Cancer N. Engl. J. Med., February 22, 2007; 356(8): 800 - 808. [Abstract] [Full Text] [PDF]

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 Rights & Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bepler, G. Articles by Simon, G. Search for Related Content PubMed PubMed Citation Articles by Bepler, G. Articles by Simon, G.