1 Hospital Clinico Lozano Blesa, Zaragoza; 2 Institut Catala d'Oncologia, Hospital Germans Trias i Pujol, Barcelona; 3 Hospital Juan Canalejo, La Coruña; 4 Fundación Jimenez Diaz, Madrid; 5 Hospital de Cruces, Bilbao; 6 Hospital General de Valencia, Valencia; 7 Facultad de Medicina, Autonomous University of Madrid, Madrid; 8 Hospital Valdecilla, Santander; 9 Hospital Reina Sofia, Cordoba; 10 Hospital de Donostia, San Sebastian; 11 Hospital Ciudad de Jaen, Jaen; 12 Hospital Miguel Servet, Zaragoza; 13 Hospital de Mataro, Mataro, Spain
* Correspondence to: Dr R. Rosell, Institut Català d'Oncologia, Hospital Germans Trias i Pujol, Ctra Canyet, s/n, 08916 Barcelona, Spain. Tel: +34-93-497-89-25; Fax: +34-93-497-89-50; Email: rrosell{at}ns.hugtip.scs.es
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patients and methods: Using the TaqMan 5' nuclease assay, we examined ERCC1 118, XPD 751 and 312, RRM1 37C/A, and MDR1 C3435T SNPs in peripheral blood lymphocytes (PBLs) obtained from 62 docetaxelcisplatin-treated advanced NSCLC patients. ERCC1 expression was measured in RNA isolated from PBLs using real-time reverse transcriptase PCR.
Results: Overall median survival was 10.26 months. Median survival was 9.67 months for 34 patients with ERCC1 118 C/T, 9.74 months for 17 patients with T/T, and not reached for 11 patients with C/C (P=0.04). Similar significant differences in time to progression were observed according to ERCC1 118 genotype (P=0.03). No other significant differences were observed.
Conclusions: Patients homozygous for the ERCC1 118 C allele demonstrated a significantly better survival. ERCC1 SNP assessment could be an important component of tailored chemotherapy trials.
Key words: cisplatin, docetaxel, ERCC1, non-small-cell lung cancer, single nucleotide polymorphisms
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Loss of heterozygosity of NER genes has been observed in lung carcinomas [4], and reduced mRNA expression levels of NER genes have been observed in peripheral blood lymphocytes (PBLs) from lung cancer patients [5
]. It has also been demonstrated that the expression of NER genes in PBLs may be used as a surrogate for estimating expression levels of these genes in proliferating tissues [6
]. In a host-cell reactivation assay, ERCC1 and XPD mRNA levels in PBLs correlated both with each other and with DNA repair capacity (DRC) [7
]. Reduced DRC has been shown to be related to lung oncogenesis [8
], while elevated DRC has been related to chemoresistance in NSCLC [9
, 10
]. High ERCC1 expression in tumor tissue has been related to cisplatin resistance in ovarian [11
, 12
] and gastric [13
] cancer. Colorectal cancer patients whose tumors showed low levels of ERCC1 gene expression had superior overall survival when treated with second-line fluorouraciloxaliplatin [14
], and low levels of ERCC1 expression also correlated with significantly longer survival in gemcitabinecisplatin-treated advanced NSCLC patients [15
].
Single nucleotide polymorphisms (SNPs) in any of the NER genes may modulate DRC and contribute to individual variations in chemotherapy response. About 2032% of non-synonymous SNPs in coding regions of NER genes [16] may alter protein structure, function, stability or folding [17
]. At codon 118 of ERCC1, 42 bases prior to the beginning of the helixturnhelix sites at exon 4, a nucleotide alteration AAC to AAT resulted in the same amino acid (asparagine). ERCC1 118C/T SNP has been associated with diminished mRNA and protein levels [18
], which can lead to differential cisplatin sensitivity [19
]. SNPs at the XPD locus have also been associated with suboptimal DRC in lymphocytes. In a case-control study of 316 lung cancer cases and 316 healthy controls, a greater reduction in DRC was observed in patients who were homozygous for two wild-type XPD SNPs, Asp312Asn at exon 10 and Lys751Gln at exon 23 [20
]. Other studies have indicated that cell lines homozygous for the XPD Asn allele at codon 312 showed higher DRC [21
]. A significant association has also been observed between homozygosity for the wild-type XPD 751 allele (Lys/Lys) and better response to fluorouraciloxaliplatin in metastatic colorectal cancer [22
].
Ribonucleotide reductase is a highly regulated enzyme in the deoxyribonucleotide synthesis pathway, and alterations in ribonucleotide reductase levels can affect tumor progression [23, 24
]. An adenine
cytosine substitution in the 5' non-coding region of the ribonucleotide reductase subunit M1 (RRM1) gene, located 37 nucleotides upstream of the start codon has been associated with higher RRM1 expression levels [25
]. The multidrug resistance (MDR1) gene product P-glycoprotein is a membrane protein that functions as an ATP-dependent exporter of drugs from cells. Subjects homozygous for the C in position 3435 (SNP C3435T at exon 26) of the MDR1 gene had higher MDR1 mRNA expression in leukocytes than subjects with the TT genotype [26
]. This MDR1 SNP has been associated with outcome in acute myeloid leukemia [27
] and with drug-responsive epilepsy [28
].
In order to elucidate the discriminant role of SNPs and gene expression in cisplatin and docetaxel sensitivity, we used the TaqMan 5' nuclease assay to test ERCC1, XPD, RRM1 and MDR1 SNPs in genomic DNA from PBLs. The TaqMan 5' nuclease assay combines target DNA amplification with allele discrimination in a single reaction, relying on the hybridization of two doubly labeled allele-specific fluorescence probes to the target SNPs. We also examined ERCC1 mRNA expression in PBLs by real-time reverse transcription PCR (RTPCR). All analyses were performed at baseline in 62 advanced NSCLC patients treated with docetaxelcisplatin.
![]() |
Patients and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patient evaluation
All patients underwent a complete history and physical examination, including routine hematology and biochemistry analyses, and staging with chest radiographs and computed tomography of the thorax and abdomen. Bone scan or computed tomography of the brain was required only for patients with suspected bone or brain metastases. Hematology and biochemistry analyses were repeated at the end of each cycle. Toxicity was classified according to WHO criteria at each cycle for each patient. Response was assessed after two cycles of chemotherapy and every two cycles thereafter, using Response Evaluation Criteria in Solid Tumor Group (RECIST) guidelines [29].
Treatment plan
Patients received intravenous doses of cisplatin 75 mg/m2 plus docetaxel 75 mg/m2 on day 1, every 3 weeks, for a maximum of eight cycles, unless disease progression or unacceptable toxicity occurred. Chemotherapy was given only if the patient had a minimum neutrophil count of 1.5 x 109/l, a minimum platelet count of 100 x 109/l, a hemoglobin level of 8 g/dl and no sign of organ toxicity. Antiemetics and mannitol diuresis were undertaken according to individual institutional protocols. Second-line treatment was not planned.
Sample collection and SNP genotyping
Venous blood (10 ml) was collected from each subject into tubes containing 50 mmol of EDTA per liter, and genomic DNA was isolated with the QIAmp® DNA blood Mini kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. SNPs in ERCC1 (C/T in codon 118), XPD (T/G in codon 751 and G/A in codon 312), RRM1 (37C/A; the minus sign refers to the 5' upstream region relative to the transcription-initiation site) and MDR1 (C3435T) were assessed using the 5' nuclease allelic discrimination assay. In this method, the region flanking the polymorphism is amplified by PCR in the presence of two probes, each specific for one or the other allele. Each oligonucleotide probe is 5'-labeled with a different fluorescent reporter dye (typically FAM or VIC) to differentiate the amplification of each allele, and with a quencher dye. During PCR, each probe anneals specifically to complementary sequences between the forward and reverse primer sites. DNA polymerase can cleave only probes that fully hybridize to the allele. Cleavage separates the reporter dye from the quencher dye, which results in increased fluorescence by the reporter dye. The PCR-generated fluorescent signal(s) indicates which alleles are present in the sample. The PrimerExpress program (Applied Biosystems) was used to design probes and primers for the analysis of each SNP. All oligonucleotide sequences were checked in the BLAST search program (http://www.ncbi.nlm.nih.gov/LocusLink/BLAST/) (primer and probe sequences are available as supplementary data at http://annonc.oupjournals.org). Each PCR reaction mixture (25 µl) contained 50 ng of DNA, 900 nM of each forward and reverse primer, 300 nM of each allele specific probe, and 12.5 µl of Taqman Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Amplification was carried out under the following conditions: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 92°C for 15 s and 60°C for 1 min. Fluorescence in each sample well was measured before and after PCR using the ABI Prism 7000 Sequence Detection System (Applied Biosystems). Data were analyzed using the Allelic Discrimination Program (Applied Biosystems). For each SNP, a minimum of 20 randomly selected DNA samples were genotyped at least twice to confirm the results. In addition, to ensure the accuracy of this method, 20 randomly selected DNA samples were subjected to PCR and direct DNA sequencing in an ABI Prism 310 Sequence Detection System (Applied Biosystems). In all cases, the genotype determined by the 5' nuclease allelic discrimination assay was identical to that determined by DNA sequencing.
RNA, cDNA and RTPCR
Blood samples for RNA expression analysis were collected in K3/EDTA-containing tubes. Since participating hospitals were located throughout Spain, special containers were used for permanent sample refrigeration at 4°C, and a special express courier delivered the samples to the central laboratory in <24 h. After standard erythrocyte lyses, RNA was isolated from PBLs using the RNeasy Blood Mini Kit (Qiagen) according to manufacturer's instructions. Between 50 and 500 ng of total RNA was reverse transcribed into cDNA using regular M-MLV Reverse Transcriptase (Gibco, Cambridge, UK) following the manufacturer's instructions.
ERCC1 cDNA and reference cDNA (ß-actin) were amplified separately with the Taqman using an oligonucleotide probe with 5' fluorescent reporter dye (6FAM) and 3' quencher dye (TAMRA). Template cDNA was added to Universal Master Mix (Applied Biosystems) in a 25 µl reaction with 900 nM primers and 300 nM probe for each gene. The primer and probe sets were designed using Primer Express 2.0 Software (Applied Biosystems) and are described in the Supplementary Appendix.
Relative quantitation was carried out using the comparative CT method. The CT is defined as the fractional cycle number at which the fluorescence generated by cleavage of the probe exceeds a fixed threshold above the baseline; at a given threshold, a higher CT value indicates a lower starting copy number. Since the precise amount of total RNA added to each reaction mix is difficult to assess, transcripts of the target genes and of the endogenous control ß-actin gene were quantified in the same plate. The relative target gene expression level was also normalized to the expression in RNA controls (UPE, Applied Biosystems; human normal liver, Stratagene, La Jolla, CA, USA) used as calibrators.
Final results, expressed as N-fold differences in target gene expression relative to the ß-actin gene and the calibrators, were determined as follows: 2(CT sample
CT calibrator), where
CT values of the calibrator and sample are determined by subtracting the CT value of the target gene from the value of the ß-actin gene.
Statistical analyses
Survival curves were drawn with the KaplanMeier product limit method for each of the different genotypes. Comparisons were made with the log-rank or TaroneWare test. Hazard ratios of both death and progression with 95% confidence intervals (CIs) were estimated by using the Cox model, with genotype, sex, age, stage of disease, histological type and WHO PS as covariates. Student's t-test and the 2-test were used to compare response rates according to genotype. The Spearman correlation coefficient analysis was used to determine the correlation between different genotypes. All analyses were performed with the SPSS software package, version 11.5 (SPSS Inc., Chicago, IL, USA).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To date, few studies have addressed the issue of the impact of SNPs on chemotherapy efficacy. The 5' tandem repeat polymorphism on the thymidylate synthase (TS) gene has been identified as a predictor of clinical outcome for 5-fluorouracil-based chemotherapy in both adjuvant and metastatic settings in colorectal cancer [30], and in acute lymphoblastic leukemia [31
]. However, the molecular mechanisms to explain why differences in nucleotide sequences of SNPs or tandem repeats enhance transcription of genes such as ERCC1 remain largely unknown. It is known that a novel SNP within the 5' tandem repeat polymorphism of the TS gene alters the enhancer function of an extra repeat. A single G
C base transition found at the 12th nucleotide of the second repeat in the 3-tandem genotype changes a critical residue in the upstream stimulatory factor (USF) E-box consensus element (5'-CACGTG-3') [32
, 33
]. The same sequence at a different exon is found in ERCC1, but at present there is no evidence that USF stimulates ERCC1 gene expression.
ERCC1 and XPD are separated by <250 kbp on chromosome 19q 13.2-13.3, suggesting a close link in DNA repair function [34]. A significant relationship between survival and XPD Lys751Gln was found in colorectal cancer patients treated with fluorouraciloxaliplatin. Patients with the Lys751Lys genotype had the longest median survival (17.4 months), followed by those with Lys751Gln (12.8 months) and those with Gln751Gln (3.3 months) [22
]. Significant differences according to the XPD 312 genotype have also been found in a retrospective study of 100 NSCLC patients treated with cisplatin-based chemotherapy [35
]. In the present study, we found the same frequencies of XPD SNPs as in other studies [20
, 36
, 37
]. However, no significant differences in survival were observed either for XPD, RRM1 or MDR1 SNPs, perhaps due to the small number of patients included.
Upregulation of ERCC1 mRNA expression has been observed in malignant and non-malignant tissue after exposure to platinum compounds [34]. A positive correlation between ERCC1 mRNA levels in PBLs and DRC has been reported, indicating that ERCC1 mRNA levels can be used as a proxy when tissue is not available [7
].
In a large, randomized study, the median survival of 406 patients treated with docetaxelcisplatin was 11.3 months [1]. If the ERCC1 118 SNP had been analyzed in this study,
73 patients may have been identified as carrying wild-type ERCC1 and having a substantially better potential for survival. In the present study, ERCC1 118 C/T SNP correlated with time to progression and survival in docetaxelcisplatin-treated NSCLC patients. Patients harboring one or two copies of the T allele may attain better survival when treated with non-cisplatin combinations. An ongoing prospective study, which has so far included 320 patients of a planned total of 480, will further examine the role of the ERCC1 118 SNP in docetaxelcisplatin-treated NSCLC, and may validate its predictive value.
![]() |
Acknowledgements |
---|
![]() |
Notes |
---|
Received for publication March 25, 2004. Accepted for publication April 21, 2004.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Furuta T, Ueda T, Aune G et al. Transcription-coupled nucleotide excision repair as a determinant of cisplatin sensitivity of human cells. Cancer Res 2002; 62: 48994902.
3. Reed E, Yu JJ, Davies A et al. Clear cell tumors have higher mRNA levels of ERCC1 and XPB than other histological types of epithelial ovarian cancer. Clin Cancer Res 2003; 9: 52995305.
4. Takebayashi Y, Nakayama K, Kanzaki A et al. Loss of heterozygosity of nucleotide excision repair factors in sporadic ovarian, colon and lung carcinomas: implication for their roles of carcinogenesis in human solid tumors. Cancer Lett 2001; 174: 115125.[CrossRef][ISI][Medline]
5. Cheng L, Spitz MR, Hong WK et al. Reduced expression levels of nucleotide excision repair genes in lung cancer: a case-control analysis. Carcinogenesis 2000; 21: 15271530.
6. Cheng L, Guan Y, Li L et al. Expression in normal human tisssues of five nucleotide excision repair genes measured simultaneously by multiplex reverse transcription-polymerase chain reaction. Cancer Epidemiol Biomarkers Prev 1999; 8: 801807.
7. Vogel U, Dybdahl M, Frentz G et al. DNA repair capacity: inconsistency between effect of over-expression of five NER genes and the correlation to mRNA levels in primary lymphocytes. Mutat Res 2000; 461: 197210.[ISI][Medline]
8. Wei Q, Cheng L, Hong WK et al. Reduced DNA repair capacity in lung cancer patients. Cancer Res 1996; 56: 41034107.[Abstract]
9. Zeng-Rong N, Paterson J, Alpert L et al. Elevated DNA repair capacity is associated with intrinsic resistance of lung cancer to chemotherapy. Cancer Res 1995; 55: 47604764.[Abstract]
10. Bosken CH, Wei Q, Amos CI et al. An analysis of DNA repair as a determinant of survival in patients with non-small-cell lung cancer. J Natl Cancer Inst 2002; 94: 10911099.
11. Dabholkar M, Vionnet J, Bostick-Bruton F et al. Messenger RNA levels of XPAC and ERCC1 in ovarian cancer tissue correlate with response to platinum-based chemotherapy. J Clin Invest 1994; 94: 703708.[ISI][Medline]
12. Li Q, Yu JJ, Mu C et al. Association between the level of ERCC1 expression and the repair of cisplatin-induced DNA damage in human ovarian cancer cells. Anticancer Res 2000; 20: 645652.[ISI][Medline]
13. Metzger R, Leichman G, Danenberg KD et al. ERCC1 mRNA levels complement thymidylate synthase mRNA levels in predicting response and survival for gastric cancer patients receiving combination cisplatin and fluorouracil chemotherapy. J Clin Oncol 1998; 16: 309316.[Abstract]
14. Shirota Y, Stoehlmacher J, Brabender J et al. ERCC1 and thymidylate synthase mRNA levels predict survival for colorectal cancer patients receiving combination oxaliplatin and fluorouracil. J Clin Oncol 2001; 19: 42984304.
15. 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 2002; 8: 22862291.
16. Shen MR, Jones IM, Mohrenweiser H. Nonconservative amino acid substitution variants exist at polymorphic frequency in DNA repair genes in healthy humans. Cancer Res 1998; 58: 604608.[Abstract]
17. Hu G, Modrek B, Stensland HMFR et al. Efficient discovery of single-nuceotide polymorphisms in coding regions of human genes. Pharmacogenomics J 2002; 2: 236242.[CrossRef][Medline]
18. Yu JJ, Mu C, Lee KB et al. A nucleotide polymorphism in ERCC1 in human ovarian cancer cell lines and tumor tissues. Mutat Res 1997; 382: 1320.[ISI][Medline]
19. Ford BN, Ruttan CC, Kyle VL et al. Identification of single nucleotide polymorphisms in human DNA repair genes. Carcinogenesis 2000; 21: 19771981.
20. Spitz MR, Wu X, Wang Y et al. Modulation of nucleotide excision repair capacity by XPD polymorphisms in lung cancer patients. Cancer Res 2001; 61: 13541357.
21. Seker H, Butkiewicz D, Bowman ED et al. Functional significance of XPD polymorphic variants: attenuated apoptosis in human lymphoblastoid cells with the XPD 312 Asp/Asp genotype. Cancer Res 2001; 61: 74307434.
22. Park DJ, Stoehlmacher J, Zhang W et al. A xeroderma pigmentosum group D gene polymorphism predicts clinical outcome to platinum-based chemotherapy in patients with advanced colorectal cancer. Cancer Res 2001; 61: 86548658.
23. Xue L, Zhou B, Liu X et al. Wild-type p53 regulates human ribonucleotide reductase by protein-protein interaction with p3R2 as well as hRRM2 subunits. Cancer Res 2003; 63: 980986.
24. Pitterle DM, Kim Y-C, Jolicoeur EMC et al. Lung cancer and the human gene for ribonucleotide reductase subunit M1 (RRM1). Mamm Genome 1999; 10: 916922.[CrossRef][ISI][Medline]
25. Bepler G, Sharma S, Gautam A et al. Tumor genotype, RRM1 expression and outcome of patients with lung cancer. Eur J Cancer 2003; 38 (Suppl 7): S82 (Abstr 265).
26. Hitzl M, Drescher S, Van Der Kuip H et al. The C3435T mutation in the human MDR1 gene is associated with altered efflux of the P-glycoprotein substrate rhodamine 123 from CD56+ natural killer cells. Pharmacogenetics 2001; 11: 293298.[CrossRef][ISI][Medline]
27. Illmer T, Schuler UL, Thiede C et al. MDR1 gene polymorphisms affect therapy outcome in acute myeloid leukemia patients. Cancer Res 2002; 62: 49554962.
28. Siddiqui A, Kerb R, Weale M et al. Association of multidrug resistance in epilepsy with a polymorphism in the drug-transporter gene ABCB1. N Engl J Med 2003; 348: 14421448.
29. Therasse P, Arbuck SG, Eisenhauer EA et al. New guidelines to evaluate the response to treatment in solid tumor. J Natl Cancer Inst 2000; 92: 205216.
30. Stoehlmacher J, Lenz HJ. Implications of genetic testing in the management of colorectal cancer. Am J Pharmacogenomics 2003; 3: 7388.[Medline]
31. Krajinovic M, Costea I, Chiasson S. Polymorphism of the thymidilate synthase gene and outcome of acute lymphoblastic leukaemia. Lancet 2002; 359: 10331035.[CrossRef][ISI][Medline]
32. Goueli BS, Janknecht R. Regulation of telomerase reverse transcriptase gene activity by upstream stimulatory factor. Oncogene 2003; 22: 80428047.[CrossRef][ISI][Medline]
33. Mandola MV, Stoehlmacher J, Muller-Weeks S et al. A novel single nucleotide polymorphism within the 5' tandem repeat polymorphism of the thymidylate synthase gene abolishes USF-1 binding and alters transcriptional actvity. Cancer Res 2003; 63: 28982904.
34. Dabholkar MD, Berger MS, Vionnet JA et al. Malignant and nonmalignant brain tissues differ in their messenger RNA expression patterns for ERCC1 and ERCC2. Cancer Res 1995; 55: 12611266.[Abstract]
35. Gurubhagavatula S, Liu G, Park S et al. XPD and XRCC1 genetic polymorphisms are associated with overall survival in advanced NCLC patients treated with platinum chemotherapy. Proc Am Soc Clin Oncol 2003; 22: 123 (Abstr).
36. Zhou W, Liu G, Miller DP et al. Gene-environment interaction for the ERCC2 polymorphisms and cumulative cigarette smoking exposure in lung cancer. Cancer Res 2002; 62: 13771381.
37. Booton R, Ward TH, Ashcroft L et al. Randomised phase III trial of docetaxel/carboplatin vs. MIC/MVP chemotherapy in inoperable advanced non-small cell lung cancer. Do XPD polymorphisms predict for sensitivity or resistance to platinum based chemotherapy. Lung Cancer 2003; 41 (Suppl 2): S101S102.