1318 Vol. 10, 1318–1325, February 15, 2004 Clinical Cancer Research Ribonucleotide Reductase Messenger RNA Expression and Survival in Gemcitabine/Cisplatin-Treated Advanced Non-Small Cell Lung Cancer Patients Rafael Rosell,1 Kathleen D. Danenberg,2 from bronchoscopy by real-time quantitative reverse tran- Vincente Alberola,3 Gerold Bepler,4 scription-PCR. Results were correlated with survival using Jose Javier Sanchez,5 Carlos Camps,6 the Kaplan-Meier method. 7 8 Results: A total of 100 patients were assessed. There was Mariano Provencio, Dolores Isla, a strong correlation between RRM1 and ERCC1 mRNA 1 9 P < 0.001). In the ;0.410 ؍ Miquel Taron, Pilar Diz, and expression levels (Spearman r Angel Artal10 on behalf of the Spanish Lung gemcitabine/cisplatin arm, patients with low RRM1 mRNA Cancer Group expression levels had significantly longer median survival 1Medical Oncology Service, Institut Catala`d’Oncologia, Hospital than those with high levels [13.7 versus 3.6 months; 95% -Me .[0.009 ؍ Germans Trias i Pujol, Barcelona, Spain; 2Response Genetics Inc, confidence interval (CI), 9.6–17.8 months; P Los Angeles, California; 3Medical Oncology Service, Hospital Arnau dian survival was also significantly longer among patients 4 de Vilanova, Valencia, Spain; Thoracic Oncology Program, H. Lee with low mRNA expression levels of both RRM1 and ERCC1 Moffitt Cancer Center and Research Institute, Tampa, Florida; 5 (not reached), than among those with high levels of both ؍ ,Facultat de Medicina, Autonomous University of Madrid, Madrid Spain; 6Medical Oncology Service, Hospital General de Valencia, genes (6.8 months; 95% CI, 2.6–11.1 months; P 0.016). Valencia, Spain; 7Medical Oncology Service, Puerta de Hierro Conclusions: RRM1 mRNA expression is a crucial pre- Hospital, Madrid, Spain; 8Medical Oncology Service, Hospital dictive marker of survival in gemcitabine/cisplatin-treated 9 Clinico de Zaragoza, Zaragoza, Spain; Medical Oncology Service, patients. Genetic testing of RRM1 mRNA expression levels Hospital de Leon, Leon, Spain; and 10Medical Oncology Service, Hospital Miguel Servet, Zaragoza, Spain can and should be used to personalize chemotherapy. INTRODUCTION ABSTRACT The prognosis of advanced non-small cell lung cancer Purpose: No chemotherapy regimen, including the (NSCLC) is dismal. A recent Eastern Cooperative Oncology widely used combination of gemcitabine/cisplatin, confers Group trial of 1155 patients showed no differences among significantly improved survival over any other in metastatic cisplatin/paclitaxel, cisplatin/gemcitabine, cisplatin/docetaxel, non-small cell lung cancer (NSCLC); however, the selection and carboplatin/paclitaxel. Overall median time to progression of patients according to key genetic characteristics can help was 3.6 months, and median survival was 7.9 months (1). to tailor chemotherapy. Ribonucleotide reductase subunit Although clear pharmacogenetic guidelines for personalized M1 (RRM1) is involved in DNA synthesis and repair and chemotherapy have not yet been exactly defined, the assessment in gemcitabine metabolism, and the excision repair cross- of abnormalities in DNA repair pathways and enzymes involved complementing group 1 (ERCC1) gene has been related to in gemcitabine metabolism can help to distinguish between cisplatin activity. patients who are sensitive or resistant to gemcitabine or cis- Experimental Design: Patients were part of a large ran- platin. domized trial carried out from September 1998 to July 2000, Ribonucleotide reduction is the reaction whereby ribo- comparing gemcitabine/cisplatin versus gemcitabine/cispla- nucleotides, the precursors of RNA synthesis, are reduced to tin/vinorelbine versus gemcitabine/vinorelbine followed by form deoxyribonucleotides, the precursors of DNA synthesis vinorelbine/ifosfamide. We analyzed RRM1 and ERCC1 (2). Ribonucleotide reductase converts ribonucleotide 5Ј- mRNA expression in paraffin-embedded samples obtained diphosphate to deoxyribonucleotide 5Ј-diphosphate, which is essential for DNA synthesis (Fig. 1). Gemcitabine, an analogue of deoxycytidine (2Ј,2Ј-difluorodeoxycytidine), is phosphoryl- ated to the 5Ј-monophosphate form by deoxycytidine kinase. Received 9/4/03; revised 10/14/03; accepted 10/17/03. Subsequent phosphorylation by uridylate-cytidylate monophos- Grant support: This research was funded in part by a grant from Eli phate kinase generates difluorodeoxycytidine 5Ј-diphosphate, Lilly and Company (master agreement 2001–2003) and in part by grants which interferes with the function of ribonucleotide reductase from Red Tema´tica de Investigacio´n Cooperativa de Centros de Ca´ncer and reduces the pool of deoxyribonucleotide 5Ј-diphosphate and from Red Respira. The costs of publication of this article were defrayed in part by the available for DNA synthesis (Ref. 3; fig. 1). Although RRM1 is payment of page charges. This article must therefore be hereby marked a tumor suppressor gene (4), up-regulation of human RRM1 has advertisement in accordance with 18 U.S.C. Section 1734 solely to been observed during DNA repair after chemotherapy damage indicate this fact. (5). Overexpression of ribonucleotide reductase was observed in Requests for reprints: Rafael Rosell, Chief, Medical Oncology Ser- vice, Hospital Germans Trias i Pujol, Institut Catala`d’Oncologia, Ctra a gemcitabine-resistant human oropharyngeal carcinoma KB Canyet, s/n, 08916 Badalona (Barcelona), Spain. Phone: 34-93-497-89- clone (6) and in a gemcitabine-resistant human leukemic cell 25; Fax: 34-93-497-89-50; E-mail: [email protected]. line K562 (7). Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2004 American Association for Cancer Research. Clinical Cancer Research 1319 Fig. 1 Gemcitabine and cisplatin DNA damage. Gemcitabine metabolism competes with ribonucleotide 5Ј-diphosphate for incorporation into DNA. Ribonucleotide reductase converts ribonucleotide 5Ј-diphosphate to deoxyribonucleotide 5Ј-diphosphate, which is essential for DNA synthesis. Gemcitabine, an analogue of deoxycytidine (2Ј,2Ј-difluorodeoxycytidine), is phosphorylated to the 5Ј-monophosphate form by deoxycytidine kinase. Subsequent phosphorylation by uridylate-cytidylate monophosphate kinase generates difluorideoxycytidine 5Ј-diphosphate, which interferes with the function of ribonucleotide reductase and reduces the pool of deoxyribonucleotide diphosphate available for DNA synthesis. Overexpression of ribonucleotide reductase abrogates gemcitabine depletion of deoxyribonucleotide diphosphate, leading to efficient DNA synthesis and repair. Table 1 Clinical and genetic characteristicsa Gemcitabine plus Gemcitabine plus Gemcitabine cisplatin plus vinorelbine–vinorelbine Characteristic All patients plus cisplatin vinorelbine plus ifosfamide No. of patients 81 21 (25.9) 31 (38.3) 29 (35.8) Age, yr (range) 59 (37–75) 61 (45–75) 57.4 (37–75) 60.3 (48–73) Sex Male 77 (95.1) 21 (100) 30 (96.8) 26 (89.7) Female 4 (4.9) 1 (3.2) 3 (10.3) Performance status 0–1 63 (77.8) 16 (76.2) 24 (77.4) 23 (79.3) 2 18 (22.2) 5 (23.8) 7 (22.6) 6 (20.7) Weight loss Ͼ5% 34 (42) 8 (38.1) 15 (48.1) 11 (37.9) Histology Adenocarcinoma 39 (48.1) 10 (47.6) 13 (41.9) 16 (55.2) Squamous cell carcinoma 39 (48.1) 10 (47.6) 16 (51.6) 13 (44.8) Large cell carcinoma 3 (3.7) 1 (4.8) 2 (6.5) — Stage IIIB 12 (14.8) 5 (23.8) 5 (16.1) 2 (6.9) IV 69 (85.2) 16 (76.2) 26 (83.9) 27 (93.1) Metastatic site Liver 15 (18.5) 5 (23.8) 5 (16.1) 5 (17.2) Lung 35 (43.2) 9 (42.9) 14 (45.2) 12 (41.4) Bone 22 (27.2) 3 (14.3) 8 (25.8) 11 (37.9) Brain 4 (4.9) 4 (13.8) Adrenal 20 (24.7) 3 (14.3) 9 (45) 8 (27.6) Skin 6 (7.4) 3 (14.3) 2 (6.5) 1 (3.4) Lymph node 9 (11.1) 3 (14.3) 3 (9.7) 3 (10.3) Others 8 (9.9) 3 (14.3) 3 (9.7) 2 (6.9) ERCC1b mRNA, median (range) 1.6 (0.6–10.7) 1.5 (0.6–3.9) 1.7 (0.6–10.7) 1.6 (0.7–4.3) RRM1 mRNA, median (range) 1.1 (0.3–6.9) 1.3 (0.3–3.5) 1.1 (0.3–6.9) 0.9 (0.3–4.9) ERCC1 mRNAb Յ1.7 45 (55.6) 13 (61.9) 15 (48.4) 17 (58.6) Ͼ1.7 36 (44.4) 8 (38.1) 16 (51.6) 12 (41.4) RRM1 mRNAc Յ1.4 46 (63) 12 (60) 17 (63) 17 (65.4) Ͼ1.4 27 (37) 8 (40) 10 (37) 9 (34.6) No. of chemotherapy cycles, median 5(1–7) 5 (1–6) 4 (1–7) 5 (1–7) (range) a Numbers in parentheses indicate percentages except when specified as a range. b ERCC1, excision repair cross-complementing group 1; RRM1, ribonucleotide reductase subunit M1. c Possible discrepancies between numbers of patients may occur because PCR was not amplified in some instances. Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2004 American Association for Cancer Research. 1320 RRM1 and Outcome in Advanced NSCLC The nucleotide excision repair (NER) pathway is involved Table 2 Survival and time to progression according to in eliminating both cisplatin DNA adducts and nucleotides biological markers damaged by UV-irradiation, and the resulting gaps are filled Median time to with precursors for DNA synthesis provided by RRM1 (8). Median survival progression ERCC1 is a NER component that participates in DNA damage Mo 95% CIa P Mo 95% CI P recognition and DNA strand incision. The inhibition of ERCC1 Gemcitabine plus cisplatin expression by antisense approaches has been reported to reduce RRM1 cisplatin-DNA adduct repair (9). In human cell lines and in Յ1.4 13.7 9.6–17.8 0.009 8.4 5.4–11.3 0.020 mouse organs, ERCC1 has been reported to be transcribed at Ͼ1.4 3.6 0–8.1 2.7 2.5–2.9 low constitutive levels (10), and in human ovarian and gastric ERCC1 Յ1.7 13.7 0.3–27 0.190 8.4 4.5–12.2 0.070 cancer, elevated ERCC1 mRNA expression in tumor tissue has Ͼ1.7 9.5 6.3–12.8 5.1 1–9.3 been related to cisplatin resistance (11, 12).