RRM1 gene expression in peripheral blood is predictive of shorter survival in Chinese patients with advanced non-small-cell lung cancer treated by gemcitabine and platinum

RRM1 gene expression in peripheral blood is predictive of shorter survival in Chinese patients with advanced non-small-cell lung cancer treated by gemcitabine and platinum 1 first-author 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:myathena100@yahoo.com.cnstjz@zju.edu.cn †E-mail:myathena100@yahoo.com.cnstjz@zju.edu.cn myathena100@yahoo.com.cn 1 1 2 3 1 4 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:myathena100@yahoo.com.cnstjz@zju.edu.cn †E-mail:myathena100@yahoo.com.cnstjz@zju.edu.cn myathena100@yahoo.com.cn Department of Pharmacy, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Department of Pharmacy, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Department of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Department of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Department of Medical Oncology, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Department of Medical Oncology, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Clinical Laboratory, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Clinical Laboratory, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Objective: 2 To evaluate the predictive values of gene expressions of ribonucleotide reductase M1 ( RRM1 ) and breast cancer susceptibility gene 1 ( BRCA1 ) in peripheral blood from Chinese patients with non-small-cell lung cancer (NSCLC) treated with gemcitabine plus platinum. Methods: 2 Forty Chinese patients with advanced NSCLC were recruited and received gemcitabine 1 200 mg/m 2 on Days 1 and 8 plus carboplatin AUC 5 on Day 1. RRM1 and BRCA1 expression levels in peripheral blood were detected by quantitative reverse transcription-polymerase chain reaction (RT-PCR). Kaplan-Meier survival curve and log-rank test were performed to evaluate the correlation between gene expression and overall survival for these subjects. Results: 2 No correlation was observed between gene expression of RRM1 and that of BRCA1 ( P > 0.05), but there was a strong correlation between the expression of RRM1 and the response to chemotherapy ( P =0.003). Subjects with low RRM1 expression levels in peripheral blood had longer survival time than those with high RRM1 expression levels (16.95 vs. 12.76 months, log-rank 3.989, P =0.046). However, no significant association between BRCA1 expression levels and survival time was found (16.80 vs. 13.77 months, log-rank 0.830, P =0.362). Conclusions: 2 Patients with low RRM1 expression levels in peripheral blood have a greater response to chemotherapy and longer survival time. Advanced NSCLC patients with low RRM1 expression levels may benefit from gemcitabine plus platinum therapy. RRM1 mRNA expression in peripheral blood could be used to predict the prognosis of NSCLC treated by gemcitabine and platinum. Gemcitabine Ribonucleotide reductase M1 ( RRM1 ) Breast cancer susceptibility gene 1 ( BRCA1 ) Non-small-cell lung cancer (NSCLC) Gene expression Introduction 1 Lung cancer remains the leading cause of cancer death (Jemal et al., 2007 2007 ), and its overall five-year survival rate is still unsatisfactory. More than 75% of lung cancers are non-small-cell lung cancers (NSCLCs) at diagnosis. Recently, the combinations of third-generation cytotoxic agents (such as gemcitabine, vinorelbine, and taxane) and platinum have emerged as a new chemotherapy standard for NSCLC. In our previous phase II clinical trials in advanced NSCLC (Wang et al., 2007b 2007b ), the median survival time and one-year survival for the combination chemotherapy of platinum and gemcitabine were 8.2–14.8 months and 38.1%–41.2%, respectively. Retrospective studies of stage IV NSCLC have reported that patients with low ribonucleotide reductase M1 ( RRM1 ) or excision repair cross completion gene 1 ( ERCC1 ) messenger RNA (mRNA) levels had a median survival up to 15 months when treated with gemcitabine plus cisplatin, with more significant differences in survival according to RRM1 levels (Rosell et al., 2003 2003 ; 2004a 2004a ; Ceppi et al., 2006 2006 ). Resistance to gemcitabine has been associated with RRM1 overexpression (Goan et al., 1999 1999 ; Davidson et al., 2004 2004 ). RRM1 is a key enzyme involved in DNA synthesis, which could catalyze the biosynthesis of deoxyribonucleotide, and previous data showed that higher levels of RRM1 are associated with chemoresistance to gemcitabine-based therapies. A close correlation has also been observed between expression levels of RRM1 and breast cancer susceptibility gene 1 ( BRCA1 ) (Taron et al., 2004 2004 ; Rosell et al., 2004b 2004b ; 2007 2007 ). BRCA1 expression confers differential chemosensitivity in cancer cell lines (Quinn et al., 2003 2003 ; 2007 2007 ). Low levels of BRCA1 also correlated with increased survival in NSCLC patients treated with gemcitabine plus cisplatin (Rosell et al., 2004b 2004b ). Hopefully, the search for genetic markers for predicting a better response may lead to customized chemotherapy. In order to further investigate the relationship between the RRM1 / BRCA1 expression in peripheral blood and the sensitivity to platinum plus gemcitabine, we collected the peripheral blood samples from 40 Chinese patients with advanced NSCLC treated by gemcitabine plus platinum, and analyzed the expressions of RRM1 and BRCA1 , as well as their clinical predictive significance. Materials and methods 1 Patients and samples 2 Forty Chinese patients with histologically-confirmed stages III and IV NSCLC were recruited and the peripheral blood samples were collected. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the expression levels of RRM1 and BRCA1 . Subjects received gemcitabine (Gemzar; Eli Lilly, USA) 1 200 mg/m 2 on Days 1 and 8 and carboplatin AUC 5 on Day 1. Patient evaluations were carried out at baseline and after every two cycles of chemotherapy (Wang et al., 2007a 2007a ; 2007b 2007b ). The study protocol was approved by the Ethical Committee of the First Affiliated Hospital of Zhejiang University, China. All subjects signed an informed consent before entry into the study. Gene expression analysis 2 Total RNA was purified from peripheral blood by column chromatography method (E.Z.N.A® Blood RNA Mini Kit; Omega, Berkeley, USA). Complementary DNA (cDNA) synthesis was performed using reverse transcription system (Promega, Madison, WI, USA) and the products were stored at −80 °C. Relative quantifications of gene expressions for RRM1 , BRCA1 , and β-actin (internal reference gene) were performed using quantitative RT-PCR [RealMasterMix (SYBR Green) Kit; TIANGEN, Beijing, China]. The PCR was carried out as follows: 50 °C for 2 min, an initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 20 s, and annealing at 60 °C for 1 min. The sequences of the primers used were as follows: RRM1 forward 5′-ACTAAGCACCCTGACTATGCTATCC-3′, reverse 5′-CTTCCATCACATCACTGAACACTTT-3′; BRCA1 forward 5′-CCCATTTTCCTCCCGCA-3′, reverse 5′-GGACCTTGGTGGTTTCT-3′; β-actin forward 5′-TGAGCGCGGCTACAGCTT-3′, reverse 5′-TCCTTAATGTCACGCACGATTT-3′. Statistical analysis 2 Statistical analyses were performed with SPSS software (Version 15.0; SPSS Inc., Chicago, IL, USA). For statistical analyses, the responses to the therapy were simply split into “responders” (partial responders) and “non-responders” (stable and progressive diseases). Mann-Whitney t -test was used to evaluate associations between the gene expression and clinical and pathological factors (including gender, tumor stage, smoker, and performance status; Kruskal-Wallis test for histology). Correlation between RRM1 and BRCA1 mRNA levels was evaluated by Spearman correlation coefficients. Log-rank test and Kaplan-Meier survival curves were used to analyze the potential association between each clinical factor and the survival rate. Factors with significant influence on survival in univariate analysis were further analyzed by multivariate Cox regression analysis. Results 1 Patient characteristics 2 Three subjects dropped out of treatment due to drug side effects, and a total of 37 patients with a median age of 64 (range 24–77) years were included. The baseline characteristics of the 37 subjects are shown in Table 1 1 . All of these subjects received gemcitabine plus platinum as first-line chemotherapy, including 4 subjects treated with gefitinib (an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor) as further therapy and 2 subjects with a concomitant radiotherapy treatment. Thirteen subjects were diagnosed with stage III NSCLC (including 5 of IIIA and 8 of IIIB) and 24 with stage IV. Overall mean survival time (MST) was 15.23 months (stage III 15.79 months, stage IV 14.55 months). Characteristics Number Percentage (%) Smoker  Yes 15 40.5  No 22 59.5 Gender  Male 27 73.0  Female 10 27.0 Performance status  0 9 24.3  1 26 70.3  2 2 5.4 Stage  III 13 35.1  IV 24 64.9 Histology  Squamous cell carcinoma 12 32.4  Adenocarcinoma 25 67.6 Response to therapy  Partial 12 32.4  Stable 16 43.3  Progressive 9 24.3 Metastasis sites  Lung 7 18.9  Liver 3 8.1  Bone 13 35.1  Nodes 11 29.7  Others 8 21.6 Number of metastasis sites  1 13 35.1  2 8 21.6  ≥3 4 10.8 RRM1 and BRCA1 mRNA expression levels 2 RRM1 and BRCA1 mRNA expression levels were analyzed by quantitative RT-PCR. Median mRNA expression levels were 1.14×10 −2 (range 0.03×10 −2 –20.15×10 −2 , mean 3.46×10 −2 , standard deviation 4.59×10 −2 ) for RRM1 , and 7.87×10 −2 (range 0.09×10 −2 –98.47×10 −2 , mean 17.68×10 −2 , standard deviation 24.53×10 −2 ) for BRCA1 . By adopting cut-off values according to median expression levels, no correlation was found between RRM1 expression and the clinical factors including gender ( P =0.122), stage ( P =0.405), smoker ( P =0.092), performance status ( P =0.596), and histology ( P =0.412). Similar results were observed between BRCA1 expression and the clinical factors. However, a strong correlation between gene expression levels of RRM1 and the response to chemotherapy was observed ( P =0.003). Ten of nineteen subjects with low RRM1 mRNA levels got partial response to chemotherapy (52.6%), and only two of eighteen patients with high RRM1 mRNA levels got partial response (11.1%). Also, the percentages of responses in subjects with low and high BRCA1 mRNA levels were 33.3% (6/18) and 31.6% (6/19), respectively, and no statistical significance was found ( P > 0.05). Correlation between RRM1 and BRCA1 mRNA levels 2 No correlation was found between RRM1 and BRCA1 expression levels ( P > 0.05) by means of the Spearman’s rank correlation method. Overall survival time associated with clinical and pathological factors and gene expression 2 In the univariate analysis model of survival, the median value of RRM1 / BRCA1 gene expression was used as the cut-off value. It was found that MST in subjects with low RRM1 mRNA levels was significantly longer than that in subjects with high RRM1 mRNA levels (16.95 vs. 12.76 months, log-rank 3.989, P =0.046; Fig. 1a 1a ). However, no significant association between BRCA1 expression levels and survival was found (16.80 vs. 13.77 months, log-rank 0.830, P =0.362; Fig. 1b 1b ), and no significant difference was observed on the overall survival time between patients with low RRM1 and BRCA1 expression levels and the others (18.01 vs. 13.92 months, log-rank 1.801, P =0.180; Fig. 1c 1c ). Other selected factors including gender, stage, smoker, performance status, and histology were also not statistically correlated with the overall survival time (Table 2 2 ). In multivariate Cox regression analysis, mRNA expression levels of RRM1 in peripheral blood appeared to be a possible predictive factor for response to chemotherapy with gemcitabine and cisplatin, and possibly for survival (Hazard ratio 2.586, 95% confidence interval (CI) 0.967–6.916, P= 0.058; Table 2 2 ). 2 Factor and gene expression 2 Number of patients 2 Mean survival time (month) 2 Univariate analysis 3 Multivariate analysis Log-rank P Hazard ratio 95% CI P Gender  Male 27 15.11 0.044 0.833  Female 10 15.10 Stage  III 13 15.79 0.005 0.943  IV 24 14.55 Smoker  Yes 15 15.29 0.008 0.927  No 22 14.36 Performance status  0 9 15.20 0.062 0.803  1 or 2 28 14.88 Histology  Squamous cell carcinoma 12 13.95 0.484 0.487  Adenocarcinoma 25 15.78 Response  Partial 12 19.50 8.392 0.004 10.55 1.40–79.41 0.022  Stable/progressive 25 12.88 RRM1  Low 19 16.95 3.989 0.046 2.586 0.967–6.916 0.058  High 18 12.76 BRCA1  Low 18 16.80 0.830 0.362  High 19 13.77 RRM1 / BRCA1  Both low 11 18.01 1.801 0.180  Others 26 13.92 Discussion 1 Many researchers have proven that DNA repair capacity is associated with the pathogenesis and drug-resistance of lung cancer. As one of the targets of gemcitabine, ribonucleotide reductase (RR) is a rate-limiting enzyme in the DNA synthesis pathway and is responsible for transforming bisphosphate nucleotide to bisphosphate deoxynucleotide. The latter is the necessary material for DNA synthesis and repair. Thus, RR is key for cell survival. It was presumed that RRM1 expression may be related to the resistance to gemcitabine that could interfere with the function of ribonucleotide reductase. Clinical studies have also shown that high expression level of RRM1 was correlated with a poor prognosis in NSCLC patients. Gautam et al. ( 2003 2003 ) detected the RRM1 mRNA expression in tumor tissues of 22 NSCLC patients receiving the combination chemotherapy of cisplatin/gemcitabine by means of quantitative RT-PCR, and found that low expression level of RRM1 mRNA of patients was associated with better responses to chemotherapy, higher impact on the time to progression ( P =0.05), and higher survival rate ( P =0.0028). In another retrospective study (Rosell et al., 2004a 2004a ), the survival time of NSCLC patients receiving cisplatin/gemcitabine chemotherapy with low expression levels of RRM1 mRNA was found to be significantly longer than that for those with higher RRM1 mRNA levels (13.7 vs. 3.6 months, P =0.009). BRCA1 may also play an important role in NSCLC not only as a prognostic factor, but also as a valuable tool for choosing the optimal treatment regimen. Patients with negative or low expression of BRCA1 were more sensitive to cisplatin combined with gemcitabine or etoposide, while those with positive expression of BRCA1 were resistant to antimicrotubular drugs. Several clinical studies have already shown that quantitative analyses of RRM1 and BRCA1 mRNA expression levels were mainly applied to tumor tissue from an operative resection and with sufficient amount. Given that the definite diagnosis of advanced NSCLC was mainly by cellular pathology, the main obstacle for detection was the difficulty of tumor tissue extraction from bronchoscopic biopsy and insufficient tissue samples. Therefore, clinical practice may need a simpler and more convenient detection method to realize individualized treatment for patients with NSCLC. A simple, convenient, and non-invasive method, such as assessment of RRM1 and BRCA1 mRNA expression levels in peripheral blood lymphocytes from NSCLC patients, could provide guidance for the treatment of NSCLC especially in advanced stage. Isla et al. ( 2004 2004 ) detected ERCC1 expression in peripheral blood by means of fluorescence quantitative PCR, but did not find a correlation between ERCC1 expression and prognosis of advanced NSCLC. Dong et al. ( 2010 2010 ) detected RRM1 mRNA expression of peripheral blood mononuclear cells by fluorescence quantitative PCR method, and found that it showed a higher sensitivity and specificity and was suitable for clinical practice. Whether the mRNA expression levels of RRM1 and/or BRCA1 reflect the tolerance status of tumor cells to gemcitabine plus platinum is unclear. In the present study, we recruited patients with advanced NSCLC and not suitable for operative treatment. All subjects received gemcitabine plus platinum as follows: intravenous gemcitabine 1 200 mg/m 2 on Days 1 and 8; intravenous carboplatin AUC 5 on Day 1. The therapeutic effects were evaluated after every two cycles of chemotherapy, including gene expression levels of RRM1 and BRCA1 in peripheral blood and their prognostic significance. RRM1 and BRCA1 mRNA expression levels relative to housekeeping gene expression in peripheral blood of NSCLC subjects were assessed by fluorescence quantitative RT-PCR. Results showed that there was a significant difference in patients’ survival due to the variable sensitivities to chemotherapy, in which low RRM1 expression conferred longer survival. Furthermore, the results of multivariate Cox regression analysis also indicated that mRNA expression levels of RRM1 in peripheral blood appear to be a possible predictive factor for response to chemotherapy with gemcitabine and cisplatin, and possibly for survival. Though it failed to reach statistical significance due to the small number of subjects, the trend could still be observed. In summary, this study preliminarily validated the mRNA level of RRM1 in peripheral blood as a determinant factor for the prognosis of NSCLC treated by gemcitabine and platinum. 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Phase II study of gemcitabine and carboplatin in patients with advanced non-small-cell lung cancer 10.1631/jzus.B1000197 Project supported by the Science and Technology Planning Project of Zhejiang Province (No. 2009C33165) and the Medical Science Research Fund Scheme of Zhejiang Province (No. 2008A060), China 2010-06-01 2011-02-15 2010-10-18 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2011 2011 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 03 12