Abstract

Background

Platinum-based doublets (PBDs) remain the cornerstone of treatment in non-small-cell lung cancer (NSCLC) and may include gemcitabine. A biomarker predicting sensitivity to this antimetabolite would represent a major step forward. Accordingly, we explored the predictive role of ribonucleotide reductase subunit M (RRM1) in advanced NSCLC.

Patients and methods

A total of 443 patients were randomly assigned to regimen A [paclitaxel (Taxol) and cisplatin with gemcitabine] or regimen B (cisplatin and vinorelbine). Immunohistochemical evaluation of RRM1 was correlated to clinical end-points.

Results

A total of 261 (58.9%) patients had representative tissue samples for RRM1 evaluation. Disease control rate, progression-free survival (PFS) and overall survival (OS) were substantially improved in patients with RRM-negative (neg) tumors receiving regimen B when compared with patients with RRM-positive (pos) tumors (68.8% versus 31.2%, P = 0.046, 6.90 months versus 3.93 months, P = 0.000 and 11.57 months versus 7.4 months, P = 0.002, respectively). Interaction analysis between RRM1-neg status and adenocarcinomas yielded a hazard ratio (HR) of 0.36 for death (P = 0.000).

Conclusions

RRM1 protein expression was without any predictive impact in patients treated with cisplatin, paclitaxel and gemcitabine. Surprisingly, the predictive power was demonstrated in the cisplatin and vinorelbine arm and may suggest that RRM1 is involved in vinorelbine sensitivity warranting further research.

introduction

Advanced non-small-cell lung cancer (NSCLC) constitutes the bulk of new cases in this devastating disease and represents a heterogeneous group of patients. An individualized patient approach, based on patient characteristics and tumor molecular-biological features, may be of significant benefit in specific subgroups. These may constitute entirely new NSCLC entities and should accordingly receive antineoplastic treatment customized to target the genetic lesions responsible for the malignant phenotype.

This paradigm switch has already improved prognosis for a small number of patients carrying EGFR mutation [1, 2] or EML4–ALK gene fusion [3], while the majority of NSCLC patients receive the standard of care with platinum-based doublets (PBDs). Overall median survival remains <1 year [4], while patients carrying the above-mentioned driver mutations transcend this therapeutic plateau. Impressive response rates (RR) (60%–80%) [1, 2, 5] has been reported in the patients harboring mutation-positive (pos) NSCLC treated with targeted agents in large randomized trials, while the large group of patients given PBDs only achieve RR of 25%–30% [4].

This emphasizes the need for biomarkers in this huge population combined with histology-based stratification before treatment. It has previously been demonstrated that histological subtype is of great impact when evaluating chemotherapy efficacy [6]. The PBDs may often contain gemcitabine, especially in patients having squamous cell carcinoma subtype, and a biomarker predicting resistance to this compound would represent a great step forward.

Ribonucleotide reductase subunit M1 (RRM1) is a regulatory component of ribonucleoside-diphosphate reductase, a key enzyme in DNA synthesis that catalyzes the formation of deoxyribonucleotides, by reducing ribonucleotides. The reaction requires generation of a radical allowing the 2′-hydroxyl of ribose to be reduced, which is carried out by the RRM1 enzyme [7]. The antimetabolite gemcitabine interferes with the function of RRM1 by reducing the pool of deoxyribonucleotide-5′-diphosphate available for DNA synthesis [8]. A combined in vitro and in vivo study demonstrated that RRM1 mRNA overexpression in cell lines leads to gemcitabine resistance and that advanced NSCLC patients with low levels respond better to gemcitabine-based chemotherapy [9], which was subsequently confirmed clinically by Ceppi et al. in a retrospective study [10]. More recently, a prospective phase II feasibility study has been published supporting the predictive role of RRM1 in gemcitabine efficacy [11]. Furthermore, similar results have been reported by using immunohistochemistry (IHC) [12, 13].

Looking at other important compounds, the vinca alkaloid vinorelbine is frequently used in combination with platinum. Vinorelbine seems to work by suppressing spindle-microtubule dynamics, which induces apoptosis by slowing or blocking the transition from metaphase to anaphase in mitosis [14]. The microtubules are complex polymers consisting of tubulin dimers (one α-tubulin and one β-tubulin) and a variety of microtubule-associated proteins [15].

Up-regulation of the spindle-microtubule dynamics during carcinogenesis may lead to a cellular state resistant to vinorelbine-induced apoptosis. Previous studies in lung cancer have suggested that high expression levels of the brain-specific class III β-tubulin (TUBB3) is associated with vinorelbine resistance in patients with advanced NSCLC [16, 17]. However, other potential targets for vinorelbine such as chromatin-associated proteins, DNA and histones have been suggested [18] and could pave the way for novel biomarkers involved in the DNA synthesis pathways predicting sensitivity to vinorelbine.

The prognostic and predictive role of RRM1 in NSCLC has been extensively investigated; however, the evidence is limited by small populations receiving a variety of different chemotherapy regimens. Furthermore, the biomarker in question is analyzed by the use of different methodologies compromising comparability.

Accordingly, we explored the predictive value of RRM1 expression analyzed by IHC in a large, homogeneous, population of advanced NSCLC patients participating in a randomized chemotherapy trial.

patients and methods

patient population

A total of 443 chemotherapy-naïve patients aged 18–75 years with histologically verified, unresectable NSCLC, performance status 0–2 and normal organ function were included in the study (LU2007) between January 2003 and February 2006 and randomly assigned to regimen A [paclitaxel (Bristol-Myers Squibb, Lyngby, Denmark) (Taxol) 180 mg/m2 and cisplatin 100 mg/m2 day 1 with gemcitabine 1000 mg/m2 day 1 and 8 every 3 weeks] or regimen B [cisplatin (Hospira Nordic AB, Stockholm, Sweden) 100 mg/m2 day 1 every 3 weeks and weekly i.v vinorelbine for a maximum of six cycles]. Patients with brain metastasis were excluded. Patients gave informed written consent. The retrospective biomarker study and LU2007 were approved by the ‘Danish National Committee on Biomedical Research Ethics and the Danish Data Protection Agency’. Characteristics are summarized in Table 1. Clinical end-points in the RRM1 tumor-marker study were RR (according to RECIST criteria), disease-free survival (DFS) and median overall survival (OS). Patients were followed up through December 2008 or until death before. Patients still alive were censored at the end of December 2008.

Table 1.

Characteristics of the patients in the RRM1 tumor-marker study according to regimen

 Number of patients
 
 Regime A (Tax + Gem + Cis) (n = 140)
 
Regime B (Na + Cis) (n = 121)
 
 RRM1-negative (n = 93)
 
RRM1-positive (n = 47)
 
RRM1-negative (n = 75)
 
RRM1-positive (n = 46)
 
 n n P n n P 
Gender 
 Male 50 53.8 33 70.2 0.061 43 57.3 32 69.6 0.178 
 Female 43 46.2 14 29.8  32 42.7 14 30.4  
Performance status (WHO) 
 PS = 0 34 36.6 20 42.6 0.622 27 36.0 17.8 0.054 
 PS = 1 49 52.7 24 51.1  43 57.3 30 66.7  
 PS = 2 10 10.8 6.4  6.7 15.6  
Stage 
 IIIA/N2 (unresectable) 1.1 4.3 0.099 4.0 0.637 
 IIIA/T3 (unresectable) 1.1 8.5  4.0 4.3  
 IIIB (dry) 21 22.6 13 27.7  20 26.7 10 21.7  
 IIIB (wet) 11 11.8 10.6  8.0 10.9  
 IV 59 63.4 23 48.9  43 57.3 29 63.0  
Histological subtype 
 Adenocarcinoma 45 48.4 16 34.0 0.196 39 52.0 19 41.3 0.040 
 Squamous cell carcinoma 22 23.7 18 38.3  16 21.3 19 41.3  
 Large-cell carcinoma 3.2  8.0  
 NOS 22 23.7 13 27.7  14 18.7 17.4  
 Adenosquamous carcinoma 1.1   
 Number of patients
 
 Regime A (Tax + Gem + Cis) (n = 140)
 
Regime B (Na + Cis) (n = 121)
 
 RRM1-negative (n = 93)
 
RRM1-positive (n = 47)
 
RRM1-negative (n = 75)
 
RRM1-positive (n = 46)
 
 n n P n n P 
Gender 
 Male 50 53.8 33 70.2 0.061 43 57.3 32 69.6 0.178 
 Female 43 46.2 14 29.8  32 42.7 14 30.4  
Performance status (WHO) 
 PS = 0 34 36.6 20 42.6 0.622 27 36.0 17.8 0.054 
 PS = 1 49 52.7 24 51.1  43 57.3 30 66.7  
 PS = 2 10 10.8 6.4  6.7 15.6  
Stage 
 IIIA/N2 (unresectable) 1.1 4.3 0.099 4.0 0.637 
 IIIA/T3 (unresectable) 1.1 8.5  4.0 4.3  
 IIIB (dry) 21 22.6 13 27.7  20 26.7 10 21.7  
 IIIB (wet) 11 11.8 10.6  8.0 10.9  
 IV 59 63.4 23 48.9  43 57.3 29 63.0  
Histological subtype 
 Adenocarcinoma 45 48.4 16 34.0 0.196 39 52.0 19 41.3 0.040 
 Squamous cell carcinoma 22 23.7 18 38.3  16 21.3 19 41.3  
 Large-cell carcinoma 3.2  8.0  
 NOS 22 23.7 13 27.7  14 18.7 17.4  
 Adenosquamous carcinoma 1.1   

NOS, the histological subtype could not be classified on the basis of the available bioptic material.

tissue samples

Archival paraffin blocks containing formalin-fixed NSCLC tissue from the 443 patients enrolled in LU2007 were mainly obtained from the Departments of Pathology at the University Hospitals of Copenhagen, Odense and Aalborg. 261 patients (58.9%) had sufficient biopsy material for RRM1 evaluation. The histological samples consisted of 37 surgical resections, 195 biopsies (117 endoscopical, 57 mediastinoscopical and 21 transthoracic biopsies) and 27 miscellaneous [local biopsies from metastatic lesions including 6 clot-preparations of cytological specimens (pleural/pericardial effusions, fine needle aspirations)]. Information on sample type was unavailable for two patients. Tissue samples were obtained from the primary lesion in 158 patients, from pulmonal-, bronchial- or mediastinal lymph nodes in 20 patients and from distant metastatic lesions in 35 patients. The remaining 48 patients had combinations of these three categories.

immunohistochemical preparation of tissue samples

four micrometer-thick formalin-fixed paraffin-embedded sections were cut and mounted on coated glass slides. From each tissue specimen, sections stained with hematoxylin–eosin were histologically evaluated for verification of diagnosis and eligibility for IHC analysis.

Tissue sections for RRM1 immunostaining were antigen-retrieved with DAKO target retrieval solution (code S1700) of high pH for 20 min at 97°C using a DAKO Pt. link machine according to manufacturer's instructions. The tissue sections were then processed with the Envision Flex + kit (DAKO K 8002, DAKO, Denmark) blocking endogenous peroxidase activity for 5 min and then incubating for 20 min with the rabbit anti-human RRM1 polyclonal antibody (Protein Tech Group cat. 10526-1-AP, Protein Tech Group Inc) (diluted 1:100). The reactions were visualized by incubation with Envision Linker (Rabbit) for 15 min followed by Envision Flex + horseradish peroxidase for 20 min and finally diaminobenzidine for 10 min. The sections were counterstained with Mayer's hematoxylin for 1 min.

immunohistochemical evaluation for biomarker- status

The expression of RRM1 (cytoplasmaic and nuclear) was analyzed as previously described [19, 20]. Briefly, two observers (AV and ES-R) blinded to the clinical data, independently evaluated the immunostaining of the eligible tissue samples under a light microscope at a magnification of ×400. A semi-quantitative H-score for each tissue sample was calculated multiplying the staining intensity of tumor cells (0, no expression; 1, weak expression; 2, moderate expression; 3, strong expression) by a proportion score based on the percentage of positive tumor cells (0 if 0%, 0.1 if 1% to 9%, 0.5 if 10% to 49%, and 1.0 if ≥50%). As positive control, placental tissue (metabolically active tissue, corresponding to intensity 2) was used. Omission and substitution of the RRM1 antibody with unspecific IgG were used as negative control. In the event of discordance between the observers, the tissue section was re-evaluated with a two-head microscope to reach consensus.

The cut-off point was chosen a priori as the median value of the H-scores to separate patients with biomarker-pos (H-score > median) tumors from biomarker-neg (H-score ≤ median) ones.

statistical analyses

All statistical analyses were carried out with the use of SPSS-software (SPSS version 17.0). Proportions were compared by the chi-square test or Fisher's exact test. Survival curves are shown as Kaplan–Meier plots and compared by log-rank analyses. Examination for independent prognostic variables was analyzed by Cox regression that yielded hazard ratios (HR). The P values <0.05 were considered statistically significant.

results

characteristics of the population

A total of 443 patients were randomly assigned in the chemotherapy trial (LU2007). Two-hundred and sixty-one patients (58.9%) of the 443 patients originally randomly assigned to the two treatment arms could be immunohistochemically evaluated for RRM1 expression. The remaining 41.1% of patients could not be evaluated due to unavailable tissue samples or no tumor tissue left in the paraffin blocks (Figure 1).

Figure 1

Flow chart of the patients' tissue samples from the RRM1 tumor-marker study. TS, tissue samples; QOL, quality of life.

Figure 1

Flow chart of the patients' tissue samples from the RRM1 tumor-marker study. TS, tissue samples; QOL, quality of life.

As shown in Table 1, more males had RRM1-pos tumors (∼70%) in both the treatment arms. Patients with RRM1-pos tumors receiving regimen B had a poorer performance status (PS) (P = 0.054) and had a higher frequency of squamous cell carcinomas (P = 0.040) in contrast to their RRM1-neg counterparts.

immunohistochemical evaluation for RRM1- expression

A median H-score value of 1 in both the treatment arms was observed that separated the population into 93 (66.4%) patients with RRM1-neg tumors (H-score ≤1) and 47 (33.6%) patients with RRM1-pos tumors (H-score >1) in regimen A. The corresponding numbers in regimen B were 75 (62%) and 46 (38%), respectively. Considerable variation of the intratumoral immunostaining intensity and frequency of positive cells was observed.

outcome according to RRM1 tumor marker status

Disease control rate (complete remission + partial response + stable disease), PFS and OS were significantly improved in patients with RRM1-neg tumors receiving regimen B when compared with patients with RRM1-pos tumors (68.8% versus 31.2%, P = 0.046, 6.90 months (95% confidence interval (CI) 5.83–7.96) versus 3.93 months (95% CI 3.11–4.76), P = 0.000 and 11.57 months (95% CI 9.65–13.48) versus 7.4 months (95% CI 5.37–9.43), P = 0.002, respectively) (Figure 2), while this was not the case for patients receiving regimen A (73% versus 35%, P = 0.366, 6.73 months versus 7.6 months, P = 0.207 and 11.07 months versus 12.37 months and 0.103, respectively).

Figure 2

Progression-free survival (PFS) and overall survival (OS) in months for patients with advanced non-small-cell lung cancer (NSCLC) treated with regimen B stratified according to the RRM1 tumor-marker status.

Figure 2

Progression-free survival (PFS) and overall survival (OS) in months for patients with advanced non-small-cell lung cancer (NSCLC) treated with regimen B stratified according to the RRM1 tumor-marker status.

significance of histopathology

The favorable outcome in regimen B could mainly be ascribed to the patients with RRM1-neg adenocarcinomas with an OS of 18.77 months versus 9.0 months for the RRM1-pos counterparts (P = 0.000).

multivariate analyses of survival

A Cox proportional hazards model for the patients receiving regimen B was fitted to test specific variables in multivariate analyses including relevant interactions. Apart from disease stage, an interaction analysis of patients with RRM1-neg status and adenocarcinomas emerged as the only other significant prognostic variable with a HR of 0.36 (95% CI 0.24–0.56, P = 0.000).

discussion

Improving prognosis in advanced NSCLC through customized treatment has a huge potential, but also significant obstacles. The TKIs have been implemented as first-line treatment in patients harboring EGFR mutations, while the choice of methodology for mutation analysis continues to be an area of debate. However, this individualised approach has dramatically improved outcome in small subgroups, whereas reliable biomarkers for patients receiving classic platinum-based chemotherapy are still greatly needed. While evidence concerning biomarkers predicting response to platinum, such as ERCC1, seems promising [19, 21, 22], the predictive value of RRM1 remains controversial.

Surprisingly, our findings showed no predictive impact of RRM1 protein expression in a subgroup of NSCLC patients randomly assigned to cisplatin-based chemotherapy including gemcitabine. This lack of predictive impact is not without precedents. Reynolds et al. [23] used a fluorescent-based IHC method (AQUA) and failed to demonstrate substantial difference in survival according to RRM1 levels in patients treated with gemcitabine and carboplatin or gemcitabine monotherapy. These results were further supported by Wang et al. [24] using classical IHC. However, only 54 of 124 patients received cisplatin and gemcitabine, while the remaining population was treated with other chemotherapy combinations. In contrast, several groups have demonstrated significant differences in univariate survival analysis in patients treated mainly with cisplatin and gemcitabine according to mRNA-expression in favor of patients with RRM1-neg tumors [10, 25]. Similar results have been shown by RRM1 protein expression receiving comparable gemicitabine-containing regimens as mentioned above [12, 26, 27]. None of these results, however, remained significant in multivariate analyses. Furthermore, it should be noted that the patient populations were small and chemotherapy regimens heterogeneous, thus limiting reproducibility.

Alternative methodological reasons could explain our negative results in the gemcitabine-containing regimen A, such as poor specificity and/or sensitivity of the antibody, the choice of cut-off values applied and the scoring method of the stained tissue sections. Our group decided a priori to use the H-score due to our experience with this system [15, 19] and the fact that it has been the scoring method of choice in larger biomarker studies [19–21, 28].

It is somewhat surprising that RRM1 protein expression predicted outcome in patients treated with cisplatin and vinorelbine in our biomarker study. In this treatment arm, patients with RRM1-neg tumors showed improved disease control rates, PFS and OS. The favorable prognosis was mainly carried by the adenocarcinoma subgroup and multivariate analysis revealed an interaction meaning that both factors contribute. These results are counterintuitive but demand attention and further research. Such findings, to our knowledge, have not previously been described in the literature. The Spanish Lung Cancer Group observed no survival difference in their NSCLC subgroups stratified according to RRM1 gene expression levels receiving vinorelbine-containing chemotherapy regimens. However, their patient cohort was limited to 60 patients [25].

Prognostic and predictive immunohistochemical biomarkers are an area of controversy and greatly depend on the characteristics of the antibody (Ab) employed. This issue is illustrated by an ongoing debate on specificity of the ERCC1 Ab (Clone 8F1) used in well-conducted ERCC1 protein biomarker studies in lung cancer [18–21, 27]. Obviously, the Abs used are of great importance to the outcome of the study and its reproducibility. Abs can be monoclonal or polyclonal. The monoclonal ones are more specific to the target antigen since they show specificity for a single epitope, while the polyclonal Abs are a heterogenous mix and thus recognize several epitopes [29]. The RRM1 Ab used by our group and by others [12, 24] is polyclonal which could explain the recognition of alternative epitopes that may be involved in vinorelbine sensitivity. A less theoretical explanation could be simple cross reactivity with an epitope unrelated to RRM1 and raises an important question our group is currently investigating: how is this epitope involved in vinorelbine efficacy?

As suggested by Rabbani-Chadegani et al. [18], potential targets for vinorelbine may exist such as DNA and to a greater extent other chromatin components apart from microtubuli. Since the antibody is designed to target a specific peptide region in the RRM1 enzyme, it seems plausible that this region may share molecular similarities with other epitopes involved in nucleotide synthesis. These epitopes could serve as targets for vinorelbine and accordingly represent a novel, predictive biomarker. However, this hypothesis is speculative and should be further tested.

Among the limitations of our project count the relatively high number of unavailable tissue samples (41.1%) and the fact that the biomarker study was not preplanned in the randomized treatment trial. The prognostic value of RRM1 as proposed by Zheng et al. [30] could influence our results. Furthermore, the tissue samples were obtained not only from the primary tumor but also, in some cases, from metastatic lesions and it is known that a biomarker may differ in expression between lesions [31]. The retrospective nature of this study opens up for the possibility of random observations. Finally, the cut-off value applied skewed our populations characteristics meaning that the RRM1-pos patients receiving the vinorelbine-containing regimen B had higher frequencies of poor PS and squamous cell carcinomas.

In conclusion, we observed that the predictive value of RRM1 protein expression was without impact in the subpopulation treated with cisplatin, paclitaxel and gemcitabine. However, in the cisplatin and vinorelbine arm, the predictive power was demonstrated across classic end-points. These findings warrant further research.

funding

The Harboe foundation, Augustinus foundation and the Research Council of Rigshospitalet supported this study.

disclosure

The authors have declared no conflicts of interest.

acknowledgements

We thank Mia Romero-Karlsen, Lone Svendstrup, Michelle S. Lage, Camilla C. Mortensen and Maria Anderberg for expert technical assistance and Claus B. Andersen for excellent assistance in design and conception.

references

1
Maemondo
M
Inoue
A
Kobayashi
K
, et al.  . 
A randomized phase III study comparing gefitinib with carboplatin (CBDCA) plus paclitaxel (TXL) for the first-line treatment of non-small cell lung cancer (NSCLC) with sensitive EGFR mutations: NEJ002 study
Eur J Cancer Suppl
 , 
2009
, vol. 
7
 
3
 
6. 9-10-0009
2
Mok
TS
Wu
YL
Thongprasert
S
, et al.  . 
Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma
N Engl J Med
 , 
2009
, vol. 
361
 (pg. 
947
-
957
)
3
Kwak
EL
Bang
YJ
Camidge
DR
, et al.  . 
Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer
N Engl J Med
 , 
2010
, vol. 
363
 (pg. 
1693
-
1703
)
4
Ardizzoni
A
Boni
L
Tiseo
M
, et al.  . 
Cisplatin- versus carboplatin-based chemotherapy in first-line treatment of advanced non-small-cell lung cancer: an individual patient data meta-analysis
J Natl Cancer Inst
 , 
2007
, vol. 
99
 (pg. 
847
-
857
)
5
Mitsudomi
T
Morita
S
Yatabe
Y
, et al.  . 
Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial
Lancet Oncol
 , 
2010
, vol. 
11
 (pg. 
121
-
128
)
6
Scagliotti
GV
Parikh
P
von
PJ
, et al.  . 
Phase III study comparing cisplatin plus gemcitabine with cisplatin plus pemetrexed in chemotherapy-naive patients with advanced-stage non-small-cell lung cancer
J Clin Oncol
 , 
2008
, vol. 
26
 (pg. 
3543
-
3551
)
7
Poole
AM
Logan
DT
Sjoberg
BM
The evolution of the ribonucleotide reductases: much ado about oxygen
J Mol Evol
 , 
2002
, vol. 
55
 (pg. 
180
-
196
)
8
Gazdar
AF
DNA repair and survival in lung cancer––the two faces of Janus
N Engl J Med
 , 
2007
, vol. 
356
 (pg. 
771
-
773
)
9
Bepler
G
Kusmartseva
I
Sharma
S
, et al.  . 
RRM1 modulated in vitro and in vivo efficacy of gemcitabine and platinum in non-small-cell lung cancer
J Clin Oncol
 , 
2006
, vol. 
24
 (pg. 
4731
-
4737
)
10
Ceppi
P
Volante
M
Novello
S
, et al.  . 
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
 , 
2006
, vol. 
17
 (pg. 
1818
-
1825
)
11
Simon
G
Sharma
A
Li
X
, et al.  . 
Feasibility and efficacy of molecular analysis-directed individualized therapy in advanced non-small-cell lung cancer
J Clin Oncol
 , 
2007
, vol. 
25
 (pg. 
2741
-
2746
)
12
Lee
JJ
Maeng
CH
Baek
SK
, et al.  . 
The immunohistochemical overexpression of ribonucleotide reductase regulatory subunit M1 (RRM1) protein is a predictor of shorter survival to gemcitabine-based chemotherapy in advanced non-small cell lung cancer (NSCLC)
Lung Cancer
 , 
2010
, vol. 
70
 (pg. 
205
-
210
)
13
Wang
X
Zhao
J
Yang
L
, et al.  . 
Positive expression of ERCC1 predicts a poorer platinum-based treatment outcome in Chinese patients with advanced non-small-cell lung cancer
Med Oncol
 , 
2010
, vol. 
27
 
2
(pg. 
484
-
490
)
14
Jordan
MA
Toso
RJ
Thrower
D
, et al.  . 
Mechanism of mitotic block and inhibition of cell proliferation by taxol at low concentrations
Proc Natl Acad Sci USA
 , 
1993
, vol. 
90
 (pg. 
9552
-
9556
)
15
Morris
PG
Fornier
MN
Microtubule active agents: beyond the taxane frontier
Clin Cancer Res
 , 
2008
, vol. 
14
 (pg. 
7167
-
7172
)
16
Vilmar
AC
Santoni-Rugiu
E
Sorensen
JB
Class III beta-tubulin in advanced NSCLC of adenocarcinoma subtype predicts superior outcome in a randomized trial
Clin Cancer Res
 , 
2011
, vol. 
17
 (pg. 
5205
-
5214
)
17
Seve
P
Mackey
J
Isaac
S
, et al.  . 
Class III beta-tubulin expression in tumor cells predicts response and outcome in patients with non-small cell lung cancer receiving paclitaxel
Mol Cancer Ther
 , 
2005
, vol. 
4
 (pg. 
2001
-
2007
)
18
Rabbani-Chadegani
A
Chamani
E
Hajihassan
Z
The effect of vinca alkaloid anticancer drug, vinorelbine, on chromatin and histone proteins in solution
Eur J Pharmacol
 , 
2009
, vol. 
613
 (pg. 
34
-
38
)
19
Olaussen
KA
Dunant
A
Fouret
P
, et al.  . 
DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy
N Engl J Med
 , 
2006
, vol. 
355
 (pg. 
983
-
991
)
20
Vilmar
AC
Santoni-Rugiu
E
Sorensen
JB
ERCC1 and histopathology in advanced NSCLC patients randomized in a large multicenter phase III trial
Ann Oncol
 , 
2010
, vol. 
21
 
9
(pg. 
1817
-
1824
)
21
Azuma
K
Komohara
Y
Sasada
T
, et al.  . 
Excision repair cross-complementation group 1 predicts progression-free and overall survival in non-small cell lung cancer patients treated with platinum-based chemotherapy
Cancer Sci
 , 
2007
, vol. 
98
 (pg. 
1336
-
1343
)
22
Holm
B
Mellemgaard
A
Skov
T
, et al.  . 
Different impact of excision repair cross-complementation group 1 on survival in male and female patients with inoperable non-small-cell lung cancer treated with carboplatin and gemcitabine
J Clin Oncol
 , 
2009
, vol. 
27
 (pg. 
4254
-
4259
)
23
Reynolds
C
Obasaju
C
Schell
MJ
, et al.  . 
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
 , 
2009
, vol. 
27
 (pg. 
5808
-
5815
)
24
Wang
X
Zhao
J
Yang
L
, et al.  . 
Positive expression of ERCC1 predicts a poorer platinum-based treatment outcome in Chinese patients with advanced non-small-cell lung cancer
Med Oncol
 , 
2010
, vol. 
27
 (pg. 
484
-
490
)
25
Rosell
R
Danenberg
KD
Alberola
V
, et al.  . 
Ribonucleotide reductase messenger RNA expression and survival in gemcitabine/cisplatin-treated advanced non-small cell lung cancer patients
Clin Cancer Res
 , 
2004
, vol. 
10
 (pg. 
1318
-
1325
)
26
Gao
Z
Han
B
Shen
J
, et al.  . 
Relations between RRM1 protein expression levels and effects of gemcitabine and cisplatin chemotherapy in advanced non-small cell lung cancer patients
Zhongguo Fei Ai Za Zhi
 , 
2011
, vol. 
14
 (pg. 
340
-
344
)
27
Liang
W
Hu
C
Gu
Q
, et al.  . 
Effects of expression of ERCC1, RRM1 on survival trend of lung cancer with cisplatin combine gemcitabine chemotherapy after surgical resection.
Zhongguo Fei Ai Za Zhi
 , 
2009
, vol. 
12
 (pg. 
403
-
407
)
28
Lee
HW
Choi
YW
Han
JH
, et al.  . 
Expression of excision repair cross-complementation group 1 protein predicts poor outcome in advanced non-small cell lung cancer patients treated with platinum-based doublet chemotherapy
Lung Cancer
 , 
2009
, vol. 
65
 (pg. 
377
-
382
)
29
Ramos-Vara
JA
Technical aspects of immunohistochemistry
Vet Pathol
 , 
2005
, vol. 
42
 (pg. 
405
-
426
)
30
Zheng
Z
Chen
T
Li
X
, et al.  . 
DNA synthesis and repair genes RRM1 and ERCC1 in lung cancer
N Engl J Med
 , 
2007
, vol. 
356
 (pg. 
800
-
808
)
31
Gomez-Roca
C
Raynaud
CM
Penault-Llorca
F
, et al.  . 
Differential expression of biomarkers in primary non-small cell lung cancer and metastatic sites
J Thorac Oncol
 , 
2009
, vol. 
4
 
10
(pg. 
1212
-
1220
)