Response to First-Line Chemotherapy in Patients with Non-Small Cell Lung Cancer According to RRM1 Expression

Abstract

Background

The response to cytotoxic chemotherapy varies greatly in patients with advanced non-small cell lung cancer (NSCLC), and molecular markers may be useful in determining a preferable therapeutic approach for individual patients. This retrospective study was performed to evaluate the predictive value of ribonucleotide reductase regulatory subunit M1 (RRM1) on the therapeutic efficacy of platinum-based chemotherapy in patients with NSCLC.

Methods

Patients with advanced NSCLC who received platinum doublet chemotherapy (n = 229) were included in this retrospective study, and their clinical outcomes were analyzed according to RRM1 expression.

Results

In patients receiving gemcitabine-based therapy, the disease control rate (DCR) and progression-free survival (PFS) of patients with RRM1-negative tumors were significantly higher than in patients with RRMI-positive tumors (P = 0.041 and P = 0.01, respectively), and multivariate analysis showed that RRM1 expression was an independent prognostic factor (P = 0.013). No similar differences were found in patients receiving docetaxel- or vinorelbine-based therapy. In RRM1-positive patients, the DCRs for docetaxel and vinorelbine were higher than for gemcitabine (P = 0.047 and P = 0.047, respectively), and docetaxel and vinorelbine showed a longer PFS than gemcitabine-based chemotherapy (P = 0.012 and P = 0.007). No similar differences were found among patients with RRM1-negative tumors.

Conclusions

Negative RRM1 expression in advanced NSCLC is associated with a higher response rate to gemcitabine-based chemotherapy. In patients with RRM1-positive tumors, docetaxel and vinorelbine showed a higher therapeutic efficacy than gemcitabine-based therapy. Additional prospective studies are needed to investigate the predictive meaning of RRM1 in the response to chemotherapy.

About the Authors:

Xiaopeng Dong

Affiliation: Department of Thoracic Surgery, Second Hospital of Shandong University, Jinan, China

Yingtao Hao

Affiliation: Department of Thoracic Surgery, Second Hospital of Shandong University, Jinan, China

Yucheng Wei

Affiliation: Department of Thoracic Surgery, Affiliated Hospital of Qingdao University Medical College, Qingdao, China

Qiuwei Yin

Affiliation: Department of Thoracic Surgery, Qilu Hospital of Shandong University, Jinan, China

Jiajun Du

Affiliation: Department of Thoracic Surgery, Shandong Provincial Hospital, Jinan, China

Xiaogang Zhao

* E-mail: [email protected]

Affiliation: Department of Thoracic Surgery, Second Hospital of Shandong University, Jinan, China

Introduction

Platinum-based chemotherapy is considered the main therapeutic approach for advanced non-small cell lung cancer (NSCLC) [1], 2. However, the selection of chemotherapeutic agents is primarily based on the clinician’s experience and preference, and studies have shown a great deal of variability with respect to their therapeutic efficacy and toxicity. Even with newly developed chemotherapy regimens, the prognosis of patients with advanced NSCLC remains dismal [3]–[5].

At present, promising results on the utility of molecular markers in predicting efficacy of cytotoxic therapy in NSCLC have been reported. Excision repair cross-complementation group 1 (ERCC1) was shown to be associated with the response to platinum-based chemotherapy [6]–[8], and in another recent study, taxane-based therapies showed a higher disease control rate (DCR) and longer progression-free survival (PFS) than gemcitabine in patients with epidermal growth factor receptor (EGFR) mutations [9]. These studies suggest that the tumor biology and the response to cytotoxic chemotherapy vary greatly among NSCLC patients, and individualized therapies may help reduce the resistance to chemotherapeutic agents.

Ribonucleotide reductase regulatory subunit M1 (RRM1) is a molecule involved in DNA synthesis and damage repair. Preclinical studies have shown that RRM1 is involved in sensitivity to gemcitabine in NSCLC [10], [11]. Lower RRM1 expression was associated with a high response rate to platinum agents and gemcitabine, and patients with high expression of RRM1 showed a decreased response to gemcitabine therapy [12]–[15]. However, in other reports, RRM1 was either not associated or was inversely associated with the survival of NSCLC patients receiving gemcitabine-containing regimens [16], [17]. Therefore, the correlation between RRM1 expression and the response to chemotherapy is still uncertain.

In the present study, we reviewed 229 patients with advanced NSCLC who had received platinum-based doublet chemotherapy as a first-line therapy, and evaluated their clinical outcomes according to RRM1 expression.

Patients and Methods

Ethics Statement

This retrospective study was approved by the ethics committee of second hospital of Shandong university. And all patient records were anonymized and de-identified prior to analysis.

Patients

In this retrospective analysis, 680 patients diagnosed with advanced NSCLC between 2007 and 2010 were screened, 325 of whom had received carboplatin-based doublet chemotherapy as a first-line treatment. A cohort of 229 patients, for whom clinical records and computed tomography (CT) scans were complete and tumor specimens were available to screen for RRM1 expression, was selected. Histological type was determined according to the World Health Organization criteria. During the treatment period, a chest CT scan was taken every 6–8 weeks, and independent reviews of these CT scans were performed in this retrospective study to confirm the response to therapy and to assess disease progression. The treatment response was classified as progressive disease (PD), stable disease (SD), partial response (PR), or complete response (CR), according to RECIST (Response Evaluation Criteria in Solid Tumors). Patients showing a CR or PR were regarded as responders. The DCR included patients with CR, PR, and SD lasting longer than three months. PFS was the time between the first day of treatment and the first sign of disease progression or death.

RRM1 Expression Analysis

Immunohistochemistry was performed using 5 μm-thick sections from paraffin-embedded tissue blocks and a Bond Polymer Intense Detection System (VisionBioSystems, Vic, Australia), according to the manufacturer’s instructions. As a negative control, the same immunohistochemical staining protocol was used except the specific primary antibody (ProteinTech Group, Chicago, USA) was replaced with distilled water. Formalin-fixed, paraffin-embedded human colonic adenocarcinoma tissue was used as a positive control.

Five fields at 400× magnification were selected for each section to assess immunoreactivity. RRM1 immunoreactivity was evaluated semi-quantitatively based on the staining intensity and the proportion of positively staining cells by two independent observers blinded to patient status. The proportion of staining was scored from 0 to 3 as follows: diffuse, ≥50% positive (score 3); regional, 10–49% positive (score 2); focal, 1–9% positive (score 1); and negative, <1% positive (score 0). The intensity of staining was also scored from 0 to 3 (0, absent; 1, weak; 2, moderate; 3, intense). The immunoreactive score for each sample was determined by multiplying the two individual scores. A score of ≥9 was defined as a positive/high expression, and a score of <9 was considered a negative/low expression.

Statistical Analysis

Statistical analyses of categorical variables, including response rate (RR) and DCR, were performed using Fisher’s exact test. Comparisons of the mean between different groups were calculated using the Student’s t-test. The median duration of PFS was calculated using the Kaplan-Meier method. Multivariate analyses were performed using Cox regression analysis for PFS to identify independent factors. Two-sided P-values of less than 0.05 were considered significant. All analyses were performed using SPSS 17.0 for Windows.

Results

Patient Characteristics and RRM1 Expression

A total of 229 NSCLC patients were included in the study. The ages ranged from 39 to 75 years (median age, 61 years), and 127 patients (55.5%) were male. The majority of the tumors were adenocarcinoma (112 patients, 48.9%) and 123 patients had stage IV disease (53.7%). All patients received carboplatin-based doublet chemotherapy as a first-line treatment. Gemcitabine, docetaxel, and vinorelbine regimens were administered in 81 (35.4%), 77 (33.6%) and 71 (31.0%) cases, respectively, and the choice of regimen was made by the responsible clinician (Table 1). Of the 229 tumors, 146 (63.8%) were negative for RRM1 expression, and 83 (36.2%) were positive for RRM1 (Table 1).

[Figure omitted. See PDF.]

Table 1. Basic characteristics of NSCLC patients.

https://doi.org/10.1371/journal.pone.0092320.t001

The relationship between patient characteristics and chemotherapy regimens according to RRM1 expression was analyzed. The patient characteristics were similar among patients receiving gemcitabine-, docetaxel-, and vinorelbine-based therapies (Table 2).

[Figure omitted. See PDF.]

Table 2. Characteristics of patients receiving chemotherapeutic regimens according to RRM1 expression.

https://doi.org/10.1371/journal.pone.0092320.t002

Tumor Response and PFS According to RRM1 Expression

In the 229 patients, 3 CRs, 77 PRs, 101 SDs, and 48 PDs were observed, for an overall RR and DCR of 34.9% and 79.0%, respectively. There were no differences in the RR and DCR between patients with RRM1-negative tumors and those with RRM1-positive tumors. However, in patients receiving gemcitabine-based therapy, the DCR of RRM1-negative patients was significantly higher than that of RRM1-positive cases (78.8% vs. 55.2%, P = 0.041). No similar difference was found in patients receiving docetaxel- or vinorelbine-based therapy (Table 3).

[Figure omitted. See PDF.]

Table 3. Response to chemotherapy according to RRM1 expression.

https://doi.org/10.1371/journal.pone.0092320.t003

The median PFS was 8.7 months (95% confidence interval (CI): 8.5–9.0 months) in all patients. No difference in PFS was found between patients with RRM1-negative tumors and those with RRM1-positive tumors (8.9 months vs. 8.5 months, P = 0.316) (Fig. 1A). However, in patients receiving gemcitabine-based therapy, the PFS of RRM1-negative patients was significantly higher than that of RRM1-positive patients (8.8 months vs. 7.6 months, P = 0.01) (Fig. 1B). No similar difference was observed in patients receiving docetaxel- or vinorelbine-based therapy (Figs. 1C and 1D).

[Figure omitted. See PDF.]

Figure 1. Kaplan-Meier curve of progression-free survival (PFS) according to ribonucleotide reductase M1 (RRM1) expression.

(A) PFS for all patients with negative or positive RRM1 expression. (B) PFS for patients receiving gemcitabine-based therapy. (C) PFS for patients receiving docetaxel-based therapy. (D) PFS for patients receiving vinorelbine-based therapy.

https://doi.org/10.1371/journal.pone.0092320.g001

In multivariate analysis adjusted for gender, smoking history, and stage of disease, RRM1 expression emerged as an independent predictive factor for PFS in patients receiving gemcitabine-based therapy (95% CI: 1.135–2.907, P = 0.013).

Tumor Response and PFS According to Chemotherapy Regimen

In patients with RRM1-negative tumors, no differences were observed in terms of RR, DCR, or PFS among patients that received gemcitabine-, docetaxel-, or vinorelbine-based therapies. However, in patients with RRM1-positive tumors, the DCR of patients receiving docetaxel or vinorelbine was higher than that of patients receiving gemcitabine (81.5% and 81.5% vs. 55.2%, respectively; P = 0.047 and P = 0.047) (Table 3). In addition, docetaxel and vinorelbine showed a longer PFS than gemcitabine-based chemotherapy (8.9 months and 9.1 months vs. 7.6 months, respectively; P = 0.012 and P = 0.007) (Figs. 2A and 2B).

[Figure omitted. See PDF.]

Figure 2. Kaplan-Meier curve of progression-free survival (PFS) according to chemotherapy regimen.

(A) PFS for patients with RRM1-negative tumors. (B) PFS for patients with RRM1-positive tumors.

https://doi.org/10.1371/journal.pone.0092320.g002

Discussion

In the present study, we analyzed 229 patients with NSCLC who had received carboplatin-based doublet chemotherapy. In patients receiving gemcitabine-based therapy, the DCR and PFS in patients with RRM1-negative tumors was significantly higher than in RRM1-positive cases, and multivariate analysis showed that RRM1 expression was an independent predictive factor for outcome. RRM1 overexpression in tumor tissue may induce resistance to gemcitabine-based therapy. Ribonucleotide reductase (RR) is an essential enzyme for DNA synthesis, and is inhibited by the active metabolite of gemcitabine, difluorideosycytidine 5-diphosphate. RRM1 depletes difluorideosycytidine 5-diphosphate and promotes DNA synthesis, thereby enabling tumor survival. In studies with lung cancer cell lines, RRM1 overexpression is associated with resistance to gemcitabine therapy [13], [18]. Consistently, clinical studies have also suggested that overexpression of RRM1 correlates with resistance to gemcitabine-based therapy [19], [20]. Conversely, low RRM1 mRNA expression was associated with a high response rate [21]. These studies demonstrate that RRM1 could be a predictive marker of the response to gemcitabine-based chemotherapy in patients with NSCLC [22].

The present study also showed that the DCR was higher in RRM1-positive patients that received docetaxel or vinorelbine, rather than gemcitabine-based therapy. In addition, docetaxel and vinorelbine each showed a longer PFS than gemcitabine-based therapy. Simon et al. used RRM1 and ERCC1 as molecular determinants, and found that RRM1- and ERCC1-tailored selection of first-line therapy could improve response, overall survival (OS), and PFS over standard treatments in patients with NSCLC [23]. These studies suggest that responses to cytotoxic chemotherapy vary greatly in patients with NSCLC, and individualized therapy based on RRM1 expression may help improve the efficacy of chemotherapeutic agents [24]. Our research was performed retrospectively, and this is the major limitation of the study. However, the current results provide new information and further insight that can assist clinicians in selecting appropriate and individualized chemotherapy for patients with NSCLC based on RRM1 expression.

Several molecular markers have been used as predictive markers of the response to chemotherapy in NSCLC patients. ERCC1 has been used for the prediction of platinum sensitivity in the treatment of NSCLC [6]–[8]. Park et al. analyzed 217 patients with NSCLC who had received gemcitabine- or taxane-based chemotherapy, and found that taxane was associated with a higher response than gemcitabine treatment in patients with EGFR mutations [9]. Another study found that low thymidylate synthase (TS) expression is significantly associated with better clinical outcomes in non-squamous NSCLC patients who were treated with pemetrexed-based chemotherapy [25]. Therefore, more prospectively designed studies with combined detection of these markers (RRM1, ERCC1, EGFR, and TS) will provide valuable information that will ultimately be used to determine preferable therapeutic approaches for individual patients with NSCLC.

In conclusion, the results of this study suggest that negative RRM1 expression in advanced NSCLC is associated with a higher response rate to gemcitabine-based chemotherapy. Moreover, RRM1 may be used as a predictive marker for conventional chemotherapy regimens involving gemcitabine, docetaxel, and vinorelbine. Additional prospective studies are needed to evaluate the effect of RRM1 expression on the response to various chemotherapeutic regimens in patients with NSCLC.

Author Contributions

Conceived and designed the experiments: XZ XD. Performed the experiments: YH YW QY JD. Analyzed the data: XZ XD. Contributed reagents/materials/analysis tools: YW QY JD. Wrote the paper: XD.

Citation: Dong X, Hao Y, Wei Y, Yin Q, Du J, Zhao X (2014) Response to First-Line Chemotherapy in Patients with Non-Small Cell Lung Cancer According to RRM1 Expression. PLoS ONE 9(3): e92320. https://doi.org/10.1371/journal.pone.0092320

References

1. Schiller JH, Harrington D, Belani CP, Langer C, Sandler A, et al. (2002) Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med 346: 92–98.

2. Pfister DG, Johnson DH, Azzoli CG, Sause W, Smith TJ, et al. (2003) American society of clinical oncology treatment of unresectable non-small-cell lung cancer guideline: update 2003. J Clin Oncol 22: 330–353.

3. Kelly K, Crowley J, Bunn PA Jr, Presant CA, Grevstad PK, et al. (2001) 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.

4. Scagliotti GV, De Marinis F, Rinaldi M, Crinò L, Gridelli C, et al. (2002) Phase III randomized trial comparing three platinum-based doublets in advanced non small cell lung cancer. J Clin Oncol 20: 4285–4291.

5. Schiller JH, Harrington D, Belani CP, Langer C, Sandler A, et al. (2002) Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med 346: 92–98.

6. Roth JA, Carlson JJ (2011) Prognostic role of ERCC1 in advanced non-small-cell lung cancer: a systematic review and meta-analysis. Clin Lung Cancer 6: 393–401.

7. Papadaki C, Sfakianaki M, Ioannidis G, Lagoudaki E, Trypaki M, et al. (2012) ERCC1 and BRAC1 mRNA expression levels in the primary tumor could predict the effectiveness of the second-line cisplatin-based chemotherapy in pretreated patients with metastatic non-small cell lung cancer. J Thorac Oncol 7: 663–671.

8. Olaussen KA, Dunant A, Fouret P, Brambilla E, André F, et al. (2006) DNA repair by ERCC1 in non-smallcell lung cancer and cisplatin-based adjuvant chemotherapy. N Engl J Med 355: 983–991.

9. Park JH, Lee SH, Keam B, Kim TM, Kim DW, et al. (2012) EGFR mutations as a predictive marker of cytotoxic chemotherapy. Lung Cancer 77: 433–437.

10. Bergman AM, Eijk PP, Ruiz VHV, Smid K, Veerman G, et al. (2005) In vivo induction of resistance to gemcitabine results in increased expression of ribonucleotide reductase subunit M1 as the major determinant. Cancer Res 65: 9510–9516.

11. Oguri T, Achiwa H, Sato S, Bessho Y, Takano Y, et al. (2006) The determinants of sensitivity and acquired resistance to gemcitabine differ in non-small cell lung cancer: a role of ABCC5 in gemcitabine sensitivity. Mol Cancer Ther 5: 1800–1806.

12. Gong W, Zhang X, Wu J, Chen L, Li L, et al. (2012) RRM1 expression and clinical outcome of gemcitabine-containing chemotherapy for advanced non-small-cell lung cancer: a meta-analysis. Lung Cancer 75: 374–380.

13. Bepler G, Kusmartseva I, Sharma S, Gautam A, Cantor A, et al. (2006) RRM1 modulated in vitro and in vivo efficacy of gemcitabine and platinum in nonsmall-cell lung cancer. J Clin Oncol 24: 4731–4737.

14. Ceppi P, Volante M, Novello S, Rapa I, Danenberg KD, et al. (2006) ERCC1 and RRM1 gene expressions but not EGFR are predictive of shorter survival in advanced non-small-cell lung cancer treated with cisplatin and gemcitabine. Ann Oncol 17: 1818–1825.

15. Rosell R, Danenberg KD, Alberola V, Bepler G, Sanchez JJ, et al. (2004) Ribonucleotide reductase messenger RNA expression and survival in gemcitabine/cisplatin-treated advanced non-small cell lung cancer patients. Clin Cancer Res 10: 1318–1325.

16. Bepler G, Sommers KE, Cantor A, Li X, Sharma A, et al. (2008) Clinical efficacy and predictive molecular markers of neoadjuvant gemcitabine and pemetrexed in resectable non-small cell lung cancer. J Thorac Oncol 3: 1112–1118.

17. Reynolds C, Obasaju C, Schell MJ, Li X, Zheng Z, et al. (2009) Randomized phase III trial of gemcitabine-based chemotherapy with in situ RRM1 and ERCC1 protein levels for response prediction in non-small-cell lung cancer. J Clin Oncol 27: 5808–5815.

18. Davidson JD, Ma L, Flagella M, Geeganage S, Gelbert LM, et al. (2004) 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.

19. Simon GR, Begum M, Bepler G (2008) Setting the stage for tailored chemotherapy in the management of non-small cell lung cancer. Future Oncol 4: 51–59.

20. Simon GR, Ismail-Khan R, Bepler G (2007) Nuclear excision repair-based personalized therapy for non-small cell lung cancer: from hypothesis to reality. Int J Biochem Cell Biol 39: 1318–1328.

21. Souglakos J, Boukovinas I, Taron M, Mendez P, Mavroudis D, et al. (2008) Ribonucleotide reductase subunits M1 and M2 mRNA expression levels and clinical outcome of lung adenocarcinoma patients treated with docetaxel/gemcitabine. Br J Cancer 98: 1710–1715.

22. Reynolds C, Obasaju C, Schell MJ, Li X, Zheng Z, et al. (2009) Randomized phase III trial of gemcitabine-based chemotherapy with in situ RRM1 and ERCC1 protein levels for response prediction in non-small-cell lung cancer. J Clin Oncol 27: 5808–5815.

23. Simon GR, Schell MJ, Begum M, Kim J, Chiappori A, et al. (2012) Preliminary indication of survival benefit from ERCC1 and RRM1-tailored chemotherapy in patients with advanced non small cell lung cancer: evidence from an individual patient analysis. Cancer 118: 2525–2531.

24. Bepler G, Williams C, Schell MJ, Chen W, Zheng Z, et al. (2013) Randomized International Phase III Trial of ERCC1 and RRM1 Expression-Based Chemotherapy Versus Gemcitabine/Carboplatin in Advanced Non-Small-Cell Lung Cancer. J Clin Oncol 31: 2404–2412.

25. Sun JM, Han J, Ahn JS, Park K, Ahn MJ (2011) Significance of thymidylate synthase and thyroid transcription factor 1 expression in patients with nonsquamous non-small cell lung cancer treated with pemetrexed-based chemotherapy. J Thorac Oncol 6: 1392–1399.