Purpose: Platinum agents cause DNA cross-linking and adducts. Xeroderma pigmentosum group D (XPD) plays a key role in the nucleotide excision repair pathway of DNA repair. Genetic polymorphisms of XPD may affect the capacity to remove the deleterious DNA lesions in normal tissues and lead to greater treatment-related toxicity. This study aimed to investigate the association of three polymorphisms of XPD at codons 156, 312, and 711, with the occurrence of grade 3 or 4 toxicity in advanced non–small cell lung cancer patients.

Experimental Design: We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to genotype the three polymorphisms in 209 stage III and IV non–small cell lung cancer patients treated with platinum-based chemotherapy.

Results: The variant homozygotes of XPD p.Arg156Arg (rs238406) polymorphism were associated with a significantly increased risk of grade 3 or 4 hematologic toxicity (adjusted odds ratios, 3.24; 95% confidence interval, 1.35-7.78; P for trend = 0.009), and, more specifically, severe leukopenia toxicity (P for trend = 0.005). No statistically significant association was found for the three polymorphisms and grade 3 or 4 gastrointestinal toxicity. Consistent with these results of single-locus analysis, both the haplotype and the diplotype analyses revealed a protective effect of the haplotype “CG” (in the order of p.Arg156Arg-p.Asp312Asn) on the risk of grade 3 or 4 hematologic toxicity.

Conclusions: This investigation, for the first time, provides suggestive evidence of an effect of XPD p.Arg156Arg polymorphism on severe toxicity variability among platinum-treated non–small cell lung cancer patients.

Translational Relevance

Chemotherapy with a platinum-based regimen is the standard care for advanced non–small cell lung cancer (NSCLC) patients. Nonetheless, the success of chemotherapy is still far from perfect, as tumor response is at the expense of toxicities, which in most subjects are mild to moderate but for other patients may be unpredictable and severe. Our study indicates, for the first time, that XPD p.Arg156Arg polymorphism might be important for prediction of severe toxicity after platinum-based chemotherapy. It can potentially be applied in advanced NSCLC patients to identify genetically high-risk subgroup that might suffer disproportionately from chemotherapy, which would limit the incidence and severity of toxicities and improve quality of life. Even in the context of prognostic marker, a simple blood test has much appeal, especially in the advanced cancer setting. It can help us understand the functional consequences of chemotherapy and construct genetic profiles directing the choice of optimal therapy.

Lung cancer continues to be a serious global health problem and is the leading cause of cancer death worldwide (1), and non–small cell lung cancer (NSCLC) accounts for ∼80% of such deaths. Most NSCLC patients are diagnosed in the advanced stages, with the majority of patients presenting with stage III or IV disease. Five-year survival rates for NSCLC remain less than 15% (2, 3).

Standard treatment for NSCLC involves chemotherapy with a platinum agent and another cytotoxic agent (4, 5). Nonetheless, one limitation of platinum-based chemotherapy is the unpredictable and occasionally significant side effects, including gastrointestinal and hematologic toxicity, which often complicate the clinical situation as it may impair the functional status of patients or their ability to tolerate further therapies. Incidence and severity of toxicities vary greatly between individuals (6, 7), which in most subjects are mild to moderate but for other patients may be unpredictable and severe. The variability of treatment-related toxicity for NSCLC highlights the need for the identification of pharmacogenetic markers for optimal individualized therapy.

Platinum compounds form both intrastrand and interstrand DNA adducts that result in bulky distortion of DNA and destabilization of the double helix. Unless these adducts are repaired before the DNA replicates, they may lead to nucleotide substitutions, deletions, and chromosome rearrangements (mutagenesis) or to activation of cell signaling pathways that result in cell death (apoptosis; refs. 8, 9). These adducts are responsible for the cytotoxicity of the platinum agents, and clinical outcome seems to be related with the level of platinum-DNA adducts in the circulation (10). In mammalian cells, nucleotide excision repair is the major pathway for removing damaged bases from DNA. It suggests that suboptimal DNA repair actually may lead to the decreased removal of deleterious DNA lesions in normal bystander cells and therefore increase toxicity to platinum therapy. As such, constitutive variation in nucleotide excision repair (NER) activity may be a prognostic factor for treatment-related toxicities in advanced NSCLC (11, 12).

XPD protein possesses both single-strand DNA-dependent ATPase and 5′-3′ DNA helicase activities and mediates DNA unwinding required for initiation of both NER and basal transcription. Rare constitutive mutations in XPD diminish nucleotide excision repair, resulting in hypersensitivity to UV light and increased risk of three distinct human disorders: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy (13). Although associations between genetic polymorphisms of XPD and the risk of developing certain cancers (14, 15) and survival outcome have been reported (12, 16), a similar study with toxicity has been rarely reported.

In this study, three putative functional single nucleotide polymorphisms (SNP) in the XPD gene (in codons 156, 312, and 711) were investigated. The XPD codon 312 polymorphism (rs1799793) is characterized by a G to A substitution causing an Asp (D) to Asn (N) amino acid exchange at codon 312, which is associated with DNA repair capacity in lymphocytes (17). The p.Arg156Arg (rs238406) and p.Asp711Asp (rs1052555) polymorphisms both result in common silent substitution that may influence the rate of translation by altering codon usage or reduce XPD protein levels through an effect on mRNA stability. The genetic variation of p.Arg156Arg has been reported as risk factor for lung cancer, glioma, melanoma, and basal cell carcinoma in previous studies (15).

Using DNA samples obtained from a series of consecutive patients with advanced NSCLC treated with chemotherapy, we assessed the association between the XPD polymorphisms at codons 156, 312, and 711 and grade 3 or 4 toxicities.

Patient recruitment and follow-up. All patients with newly diagnosed advanced lung cancer were enrolled for the study at the Shanghai Chest Hospital in Shanghai, China, between March, 2005, and September, 2007. Patients with histologically confirmed NSCLC were eligible for the study if they fulfilled all of the following criteria: aged 18 to 70 y; inoperable stage IIIA-IV presence of a measurable or assessable lesion; no prior history of malignancy except nonmelanoma skin cancer, in situ carcinoma of the cervix or “cured” malignant tumor (>5-y disease-free survival); no prior chemotherapy; Eastern Cooperative Oncology Group 0-2; required laboratory values for inclusion of neutrophil count ≥1.5 × 109/L, platelet count ≥100 × 109/L, serum creatinine ≤1.5 × upper limit normal, estimated creatinine clearance ≥60 mL/min, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤1.5 × upper limit normal; no recent (<3 mo before the date of treatment) myocardial infarction and no active congestive heart failure or cardiac arrhythmia requiring medical treatment; no uncontrolled infectious disease; no other serious medical or psychological factors that might prevent adherence to the treatment schedule. Patients had to be available for follow-up and informed consent had to be provided. The study protocol was approved by the ethical review committee of the hospital.

Clinical data were systematically recorded at entry (including age at diagnosis, sex, smoking history, family history of cancer, clinical stage, and tumor histology). Before starting any treatment, all patients underwent a complete medical history interview, physical examination and laboratory testing, including routine hematology and biochemistry analyses, staging with chest radiographs and computed tomography of the thorax and abdomen, and magnetic resonance imaging of brain and bone scan.

The incidence of grade 3 or 4 toxicity was assessed twice a week during first-line chemotherapy, according to the National Cancer Institute Common Toxicity Criteria version 3.0.4

Toxicities included leukocytopenia, anemia, thrombocytopenia, nausea, vomiting. Severe hematologic toxicity consisted of grade 3 or 4 leukocytopenia, anemia, or thrombocytopenia. Patient charts were reviewed to extract data on toxicities experienced during chemotherapy. The complete medical record, including progress notes of the treating oncologist and treating nurses, chemotherapy infusion orders, and infusion flow sheets, was reviewed to collect these data. The investigators were blinded to the polymorphism status of the patients.

Chemotherapy regimens. All the 209 patients enrolled in the study were inoperable and were given first-line platinum-based chemotherapy (navelbine: 25 mg/m2, day 1 and day 8 every 3 wk in combination with cisplatin 75 mg/m2 or carboplatin AUC 5, both administered on day 1, every 3 wk; gemcitabine 1,250 mg/m2, days 1 and 8 every 3 wk in combination with cisplatin 75 mg/m2 or carboplatin AUC 5, both administered on day 1, every 3 wk; Taxol 175 mg/m2, day 1 every 3 wk in combination with cisplatin 75 mg/m2 or carboplatin AUC 5, both administered on day 1, every 3 wk; docetaxel 75 mg/m2, day 1 every 3 wk in combination with cisplatin 75 mg/m2, also administered on day 1, every 3 wk). Few patients were treated with other platinum-based combinations. All chemotherapeutic drugs were administered i.v. and patients were treated for two to six cycles.

XPD genotyping. Blood samples were collected from all study subjects at the time of recruitment. Then, the genomic DNA was prepared from peripheral leukocytes using QIAamp DNA Maxi Kit (Qiagen GmbH). SNPs were typed using iPLEX chemistry on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (Sequenom, Inc.). PCR reactions were carried out in standard 384-well plates in 5 μL per reaction with 10 ng of genomic DNA, 0.5 units of Taq polymerase (HotStarTaq, Qiagen), 500 μmol of each deoxynucleotide triphosphate, and 100 nmol of each PCR primer. PCR thermal cycling was carried out in an ABI-9700 instrument for 15 min at 94°C, followed by 45 cycles of 20 s at 94°C, 30 s at 56°C, and 60 s at 72°C. After PCR reaction, 2 μL containing 0.3 units of Shrimp Alkaline Phosphatase was added, and the reaction was incubated at 37°C for 20 min followed by inactivation for 5 min at 85°C. After adjusting the concentrations of extension primers to equilibrate signal-to-noise ratios, the post-PCR primer extension reaction of the iPLEX assay was done in a final 9 μL volume extension reaction containing 0.2 μL of termination mix, 0.04 μL of DNA polymerase (Sequenom, Inc.), and 625 to 1,250 nmol/L extension primers. A 200-short-cycle program was used for the iPLEX reaction: initial denaturation was for 30 s at 94°C followed by 5 s at 94°C and five cycles of 5 s at 52°C and 5 s at 80°C. An additional 40 annealing and extension cycles were then looped back to 5 s at 94°C, five cycles of 5 s at 52°C and 5 s at 80°C. The final extension was carried out at 72°C for 3 min and the sample was cooled to 4°C. The samples were then manually desalted by using 6 mg of clean resin and a dimple plate and subsequently transferred to a 384-well SpectroCHIP (Sequenom, Inc.) using a nanodispenser. Mass spectrum was acquired by Compact Mass Spectrometer and analyzed by MassARRAY Typer 3.4 Software. The PCR assay was arrayed with two no-template controls and four duplicated samples in each 384-well format as quality controls. One hundred percent of genotypes were successfully determined for all SNPs. All genotyping results were generated and checked by laboratory staff unaware of patient status.

Statistical analysis. Toxicity outcomes were grouped into (a) any grade 3 or 4 toxicity, (b) any grade 3 or 4 hematologic toxicity, and (c) any grade 3 or 4 gastrointestinal toxicity. Toxicity outcome in each of these groups was dichotomized by the presence or absence of grade 3 or 4 toxicity during the first-line treatment. The associations between each genetic polymorphism/haplotype and grade 3 or 4 toxicity were estimated by odds ratios (OR) and their 95% confidence intervals (95% CI), which were calculated by unconditional logistic regression. Adjusting covariates were performance status, type of treatment regimen, and gender. Tests for trend were done by including genotypes as an ordinal variable in regression models. For pairwise linkage disequilibrium analysis, D' and r2 for each pair of SNPs were calculated by the Haploview Software.5

Haplotype blocks were defined by the four-gamete rule (15) using standard Haploview parameters (minor allele frequency [MAF]). Individual haplotype frequencies were estimated from genotype data using the PHASE 2.0 program (version 2.0.2), which implemented a Bayesian statistical method for reconstructing haplotypes from population genotype data (18). The HAPLO.STATS package6 in the software language R was used for the haplotype analysis (19). This method, based on the generalized linear model framework, allows adjustment for possible confounding variables and provides both global and haplotype-specific tests. All P values reported were two-sided, and a level of 0.05 was considered statistically significant. All statistical analyses used SPSS, version 15.0. For each gene, the Bonferroni correction was made for P value for the results of any SNP by multiplying the number of SNPs tested for the gene.

Patient characteristics and toxicity outcomes. All study patients had advanced (stage III or IV) NSCLC diagnosed histologically and had received platinum-based chemotherapy as first-line treatment. The median age at diagnosis was 57 years (range, 32-80 years). Of the subjects, 158 (75.6%) were male. All of the patients had advanced inoperable tumors, and there were 22 (10.5%) with stage IIIA, 58 (27.8%) with stage IIIB, and 129 (61.7%) with stage IV disease. Adenocarcinoma was the most common histology (n = 121, 57.9%), with 38 (18.2%) squamous cell carcinoma, 10 (4.8%) adenosquamocarcinoma, and 40 (19.2%) other carcinoma. Table 1 shows the clinical and pathologic characteristics of patients along with their SNP genotypes.

Table 1.

Clinical characteristics of NSCLC patients

Patient characteristicsn (%)
Total no. patients = 209  
Median age (range)  
    57 (32-80)  
Gender  
    Male 158 (75.6) 
    Female 51 (24.4) 
PS*  
    0-1 203 (97.1) 
    2 6 (2.9) 
TNM stage  
    IIIA 22 (10.5) 
    IIIB 58 (27.8) 
    IV 129 (61.7) 
Histologic type  
    Adenocarcinoma 121 (57.9) 
    Squamous cell 38 (18.2) 
    Adenosquamocarcinoma 10 (4.8) 
    Others* 40 (19.2) 
Chemotherapy regimens  
    Platinum-navelbine 124 (59.3) 
    Platinum-gemcitabine 34 (16.3) 
    Platinum-paclitaxel 26 (12.4) 
    Platinum-docetaxel 13 (6.2) 
    Other platinum combinations 12 (5.8) 
XPD Arg156Arg  
    C/C 55 (26.3) 
    C/A 11 (52.6) 
    A/A 44 (21.1) 
XPD Asp312Asn  
    G/G 184 (88.0) 
    G/A 22 (10.5) 
    A/A 3 (1.4) 
XPD Asp711Asp  
    C/C 184 (88.0) 
    C/T 25 (12.0) 
    T/T 0 (0) 
Patient characteristicsn (%)
Total no. patients = 209  
Median age (range)  
    57 (32-80)  
Gender  
    Male 158 (75.6) 
    Female 51 (24.4) 
PS*  
    0-1 203 (97.1) 
    2 6 (2.9) 
TNM stage  
    IIIA 22 (10.5) 
    IIIB 58 (27.8) 
    IV 129 (61.7) 
Histologic type  
    Adenocarcinoma 121 (57.9) 
    Squamous cell 38 (18.2) 
    Adenosquamocarcinoma 10 (4.8) 
    Others* 40 (19.2) 
Chemotherapy regimens  
    Platinum-navelbine 124 (59.3) 
    Platinum-gemcitabine 34 (16.3) 
    Platinum-paclitaxel 26 (12.4) 
    Platinum-docetaxel 13 (6.2) 
    Other platinum combinations 12 (5.8) 
XPD Arg156Arg  
    C/C 55 (26.3) 
    C/A 11 (52.6) 
    A/A 44 (21.1) 
XPD Asp312Asn  
    G/G 184 (88.0) 
    G/A 22 (10.5) 
    A/A 3 (1.4) 
XPD Asp711Asp  
    C/C 184 (88.0) 
    C/T 25 (12.0) 
    T/T 0 (0) 

Abbreviations: PS, performance status; TNM, tumor-node-metastasis.

*

Other carcinomas include mixed cell, neuroendocrine carcinoma, or undifferentiated carcinoma.

For the XPD C/A polymorphism at codon 156, 55 (26.3%) patients were homozygous for the C/C genotype, whereas 11 (52.6%) were heterozygous C/A and 44 (21.1%) were variant homozygotes A/A. For the XPD p.Asp312Asn polymorphism, 184 (88.0%) patients were homozygous for the G/G genotype, whereas 22 (10.5%) were heterozygous G/A and 3 (1.4%) were variant homozygotes A/A. For the codon 711 polymorphism, 184 (88%) patients had the reference C/C genotype, whereas 25 (12%) were C/T. All the genotype distributions were in Hardy-Weinberg equilibrium (P > 0.05).

All chemotherapy-related toxicities were recorded for each treatment cycle. Of the 209 patients enrolled, hematologic toxicity was evaluated in 199 patients, and gastrointestinal toxicity was obtained for 201 patients. Table 2 shows incidences of all grades 3 and 4 toxicities. Eighty-five (42.7%) patients suffered from grade 3 or 4 toxicity. Seventy-seven patients (38.7%) experienced grade 3 or 4 hematologic toxicity, of whom 64 (31.9%) had grade 3 or 4 leukocytopenia, 10 (5.0%) grade 3 or 4 anemia, and 16 (7.9%) grade 3 or 4 thrombocytopenia. Fifteen (7.5%) patients experienced grade 3 or 4 gastrointestinal toxicity. There were patients who experienced more than one of the toxicities.

Table 2.

Toxicity outcomes

n (%)
Any grade 3 or 4 toxicity 85 (42.7) 
Any grade 3 or 4 hematologic toxicity 77 (38.7) 
Leukocytopenia 64 (31.9) 
Anemia 10 (5.0) 
Thrombocytopenia 16 (7.9) 
Any grade 3 or 4 gastrointestinal toxicity  
Nausea/vomiting 15 (7.5) 
n (%)
Any grade 3 or 4 toxicity 85 (42.7) 
Any grade 3 or 4 hematologic toxicity 77 (38.7) 
Leukocytopenia 64 (31.9) 
Anemia 10 (5.0) 
Thrombocytopenia 16 (7.9) 
Any grade 3 or 4 gastrointestinal toxicity  
Nausea/vomiting 15 (7.5) 

Association between XPD polymorphisms and grade 3 or 4 toxicity. The incidence of grade 3 or 4 hematologic toxicity was significantly higher in variant homozygotes A/A of XPD p.Arg156Arg polymorphism (adjusted OR, 3.24; 95% CI, 1.35-7.78), compared with their wild-type homozygotes. The significance levels of the increasing trend were apparent for an indication of a dose-response relationship between the polymorphism and grade 3 or 4 hematologic toxicity risk (P for trend = 0.009; the significance remained after the Bonferroni correction, P = 0.027). There was no significant association between the risk of grade 3 or 4 hematologic toxicity and p.Asp312Asn (adjusted OR, 0.51; 95% CI, 0.18-1.39) and p.Asp711Asp (adjusted OR, 0.85; 95% CI, 0.36-2.00) polymorphisms (Table 3).

Table 3.

Association between XPD polymorphisms and grade 3 or 4 hematologic toxicity

XPD genotypeTotal, nAny grade 3 or 4 hematologic toxicity, n (%)OR (95% CI)*P for trend
Arg156Arg     
C/C 49 9 (11.7) 1.00 (reference) 0.009 
C/A 108 43 (55.8) 2.21 (0.99-4.92)  
A/A 42 25 (32.5) 3.24 (1.35-7.78)  
Asp312Asn    0.251 
G/G 175 72 (93.5) 1.00 (reference)  
A/G+A/A 24 5 (6.5) 0.51 (0.18-1.39)  
Asp711Asp    0.716 
C/C 175 69 (89.6) 1.00 (reference)  
C/T+T/T 24 8 (10.4) 0.85 (0.36-2.00)  
XPD genotypeTotal, nAny grade 3 or 4 hematologic toxicity, n (%)OR (95% CI)*P for trend
Arg156Arg     
C/C 49 9 (11.7) 1.00 (reference) 0.009 
C/A 108 43 (55.8) 2.21 (0.99-4.92)  
A/A 42 25 (32.5) 3.24 (1.35-7.78)  
Asp312Asn    0.251 
G/G 175 72 (93.5) 1.00 (reference)  
A/G+A/A 24 5 (6.5) 0.51 (0.18-1.39)  
Asp711Asp    0.716 
C/C 175 69 (89.6) 1.00 (reference)  
C/T+T/T 24 8 (10.4) 0.85 (0.36-2.00)  
*

Data were calculated by unconditional logistic regression and adjusted for performance status and type of treatment regimen.

Data were adjusted for performance status and type of treatment regimen.

Significance remained after the Bonferroni correction.

When only severe leukopenia toxicity was considered, more evident statistically significant difference was found for p.Arg156Arg polymorphism, in the frequency of the different genotypes versus occurrence of grade 3 or 4 leukopenia [P for trend = 0.005, the significance remained after the Bonferroni correction (P = 0.015); Table 4].

Table 4.

Association between XPD polymorphisms and grade 3 or 4 leukopenia toxicity

XPD genotypeTotal, nGrade 3 or 4 leukopenia toxicity, n (%)OR (95% CI)*P for trend
Arg156Arg     
    C/C 50 5 (7.8) 1.00 (reference) 0.005 
    A/A 42 20 (31.2) 4.88 (1.67-14.26)  
Asp312Asn     
    G/G 177 61 (95.3) 1.00 (reference) 0.104 
    A/G+A/A 24 3 (4.7) 0.36 (0.10- 1.24)  
Asp711Asp     
    C/C 177 57 (89.1) 1.00 (reference) 0.830 
    C/T+T/T 24 7 (10.9) 0.91 (0.37-2.23)  
XPD genotypeTotal, nGrade 3 or 4 leukopenia toxicity, n (%)OR (95% CI)*P for trend
Arg156Arg     
    C/C 50 5 (7.8) 1.00 (reference) 0.005 
    A/A 42 20 (31.2) 4.88 (1.67-14.26)  
Asp312Asn     
    G/G 177 61 (95.3) 1.00 (reference) 0.104 
    A/G+A/A 24 3 (4.7) 0.36 (0.10- 1.24)  
Asp711Asp     
    C/C 177 57 (89.1) 1.00 (reference) 0.830 
    C/T+T/T 24 7 (10.9) 0.91 (0.37-2.23)  
*

Data were calculated by unconditional logistic regression and adjusted for performance status and type of treatment regimen.

Data were adjusted for performance status and type of treatment regimen.

Significance remained after the Bonferroni correction.

No significant association between genotype and gastrointestinal toxicity was observed for the p.Arg156Arg, p.Asp312Asn, or p.Asp711Asp polymorphisms (Supplementary Table S1).

Analysis of grade 3 or 4 overall toxicity revealed statistically significant association with the p.Arg156Arg polymorphism [adjusted OR, 2.31; 95% CI, 1.05-5.11; P for trend = 0.037, no more significance after the Bonferroni correction (P = 0.111)]. However, there was no significant association between the grade 3 or 4 overall toxicity and the p.Asp312Asn or the p.Asp711Asp polymorphisms (Supplementary Table S2).

Haplotype analysis. Pairwise linkage disequilibriums for the three SNPs are presented, respectively, in Supplementary Table S3. p.Arg156Arg and p.Asp312Asn polymorphisms were in strong linkage disequilibrium with each other and therefore formed a haplotype block. The two most common haplotypes, AG and CG (in the order of p.Arg156Arg-p.Asp312Asn), were found to account for 93% of the study populations. Global score test showed statistically significant differences in haplotype frequency distribution versus occurrence of grade 3 or 4 hematologic toxicity [global statistics = 7.3541, df = 2, P = 0.025, the significance remained after the 10,000 time permutation tests (P sim = 0.027)]. The haplotype CG was associated with a reduced risk of grade 3 or 4 hematologic toxicity (adjusted OR, 0.64; 95% CI, 0.43-0.95) compared with the most common haplotype AG (Table 5). No significant association between haplotypes and gastrointestinal toxicity/overall toxicity was observed.

Table 5.

XPD haplotypes and grade 3 or 4 hematologic toxicity

Haplotypes*Frequencies
PP simLogistic regression, OR (95% CI)Global score test
nAny grade 3 or 4 hematologic toxicity, n (%)
AG 192 93 (60.4) 0.007 0.011 1.00 (reference) χ2 = 7.3541, df = 2, P = 0.025, P sim* = 0.027 
CG 179 55 (35.7) 0.035 0.039 0.64 (0.43-0.95)  
CA 27 6 (3.9) 0.240 0.224 0.46 (0.18-1.16)  
Haplotypes*Frequencies
PP simLogistic regression, OR (95% CI)Global score test
nAny grade 3 or 4 hematologic toxicity, n (%)
AG 192 93 (60.4) 0.007 0.011 1.00 (reference) χ2 = 7.3541, df = 2, P = 0.025, P sim* = 0.027 
CG 179 55 (35.7) 0.035 0.039 0.64 (0.43-0.95)  
CA 27 6 (3.9) 0.240 0.224 0.46 (0.18-1.16)  
*

Haplotypes were composed in order of p.Arg156Arg-p.Asp312Asn.

Generated by permutation test with 10,000 times simulation.

Data were calculated by unconditional logistic regression and adjusted for performance status and type of treatment regimen.

A total of six diplotypes were found. The three most common diplotypes, AG/CG, AG/AG, and CG/CG (in the order of p.Arg156Arg-p.Asp312Asn), accounted for 88.0% of all the diplotypes. Results showed that diplotypes CG/CG were significantly associated with reduced risk of grade 3 or 4 hematologic toxicity, with adjusted ORs of 0.32 (95% CI, 0.12-0.87), compared with the most common diplotype AG/CG (Table 6). However, gastrointestinal toxicity/overall toxicity risks were not modified by any diplotypes.

Table 6.

XPD diplotypes (combined haplotypes) and grade 3 or 4 hematologic toxicity

Diplotype*nAny grade 3 or 4 hematologic toxicity, n (%)OR (95% CI)P
AG/CG 97 42 (54.5) 1.00 (reference)  
AG/AG 42 25 (32.5) 1.35 (0.73-2.52) 0.338 
CG/CG 36 5 (6.5) 0.32 (0.12-0.87) 0.026 
AG/CA 11 1 (1.3) 0.21 (0.03-1.68) 0.141 
CG/CA 10 3 (3.9) 0.67 (0.17-2.63) 0.564 
CA/CA 1 (1.3) 0.76 (0.08-7.57) 0.818 
Diplotype*nAny grade 3 or 4 hematologic toxicity, n (%)OR (95% CI)P
AG/CG 97 42 (54.5) 1.00 (reference)  
AG/AG 42 25 (32.5) 1.35 (0.73-2.52) 0.338 
CG/CG 36 5 (6.5) 0.32 (0.12-0.87) 0.026 
AG/CA 11 1 (1.3) 0.21 (0.03-1.68) 0.141 
CG/CA 10 3 (3.9) 0.67 (0.17-2.63) 0.564 
CA/CA 1 (1.3) 0.76 (0.08-7.57) 0.818 
*

Diplotypes were composed in order of p.Arg156Arg-p.Asp312Asn.

Data were calculated from logistic regression models, adjusting for performance status and type of treatment regimen.

In this study, we investigated whether polymorphisms in the DNA repair gene XPD were associated with increased toxicity among platinum-based chemotherapy-treated advanced NSCLC patients. We found that variant homozygotes of XPD p.Arg156Arg polymorphism were significantly associated with an increased risk of grade 3 or 4 hematologic toxicity and, more specifically, severe leukopenia toxicity. Besides, a statistically significant association was found with the occurrence of severe hematologic toxicity in both the haplotype and diplotype analysis.

Gene polymorphisms in the DNA repair system may be regarded as a double-edged sword for the determination of efficacy of chemotherapy. Proficient nucleotide excision repair would protect tissues from cancer relevant with favorable prognosis but may yield the tumor resistant to chemotherapy (20). Conversely, sufficient DNA repair capacity may result in increased damage response to the mutagenesis in normal tissue by platinum agents and hence reduced toxicity. NER activity may be crucial to repair damage in normal host tissues during the course of cytotoxic treatment and prevent overwhelming treatment-related toxicity (11, 21).

Suk et al. reported that variant genotypes of ERCC1 8092A allele turned out to be a risk factor for grade 3 or 4 gastrointestinal toxicity among platinum-treated NSCLC patients (11). In a recent work that included 139 patients with advanced NSCLC and extensive SCLC, a significantly increased risk of grade 3 or 4 gastrointestinal/overall toxicity was observed with variant genotypes of XRCC1 Arg399Gln (Arg/Gln or Gln/Gln; ref. 22).

XPD is the leading enzyme in the NER process. At least eight core NER genes, including XPD, are involved in the NER pathway. One thousand ninety-eight SNPs have been identified in these eight core NER genes, but only five are common nonsynonymous SNPs, of which two are in the XPD gene (23). Epidemiologic studies have indicated that sequence variations within XPD may influence cancer risk, along with clinical outcome of platinum-based cancer therapy, whereas association study with toxicity has been scarce. In a preliminary study with 62 patients treated with docetaxel-cisplatin, Isla et al. observed a higher rate of ≥2 grade neutropenia toxicity in patients with Lys/Lys genotype of XPD at codon 751 versus those with Lys/Gln or Gln/Gln genotypes. However, the difference was not statistically significant for XPD p.Asp312Asn polymorphism, which is in accordance with our study (24).

The genetic variation at codon 156 is a common silent substitution and has been risk factors for various cancers. The variant A allele of XPD p.Arg156Arg was associated with increased risk of lung cancer in adenocarcinoma cases (P = 0.02) in a Chinese population (15). Another previous study reported the association between XPD p.Arg156Arg and age at onset of skin cancer in psoriasis patients (14). A study in Caucasian American patients suggested that the A/A genotype of p.Arg156Arg polymorphism was associated with an increased risk of glioma (25). Our results confirmed these findings that the adverse A/A genotype might confer less efficient DNA repair capacity that favors the carcinogenetic process and leads to greater damage in normal tissues when exposed to platinum or other cytotoxic agents. Whereas the exact functional consequence of this polymorphism has not been elucidated yet, it could possibly affect the stability of the mRNA or disturb protein synthesis by converting a high-usage codon to a low-usage codon. Alternatively, this polymorphism might be in linkage disequilibrium with some functional polymorphisms of the gene. Finally, because of the close proximity of XPD and ERCC1 on chromosome region 19q13.2-13.3, along with DNA ligase and XRCC1, these genes may cosegregate with the polymorphisms. All of the four genes play critical roles in DNA repair system (14, 26, 27). It has been reported that ERCC1 and XPD mRNA quantities were highly correlated in primary lymphocytes and both correlated moderately with DNA repair capacity, which suggests that transcription of these two genes may be controlled by the same regulatory elements and XPD mRNA levels would be used as a predictive marker as well with ERCC1 for DNA repair capacity, thus influencing treatment-related toxicity (28).

In this study, we found that the XPD p.Arg156Arg variant A-allele frequency was 0.48, similar to those published in Chinese population (15). Compared with around 0.39 and 0.48, respectively, among Caucasians from the United States and Denmark, respectively (14, 25), the A-allele frequencies in Caucasian and Asian populations are quite similar. It would also be interesting to evaluate this association in other ethic populations.

For the p.Asp312Asn polymorphism, the incidence of the A allele in this study is similar to that Chinese population (29), but considerably lower than that for Caucasians and Non-Hispanic White populations, with the A-allele frequency of 0.36 and 0.33, respectively (30, 31). The allele frequency for the T allele of p.Asp711Asp in our population occurs similarly to that in reported in Chinese populations, but is lower than that in the European population according to the dbSNPs databases. The allele and genotype frequencies of p.Asp312Asn and p.Asp711Asp vary with ethnicity, which warrants additional comparative studies.

Our study has several limitations. First, the sample size of our study may not be large enough to detect a small effect from SNPs of low penetrance. We performed standard power calculations, assuming α = 0.05. Minimum detectable OR was calculated for each sequence variant based on its genotype frequency, our study sample size, and a statistical power of 80%. In terms of grade 3 or 4 hematologic toxicity, our study had an 80% power to detect a minimum OR of 1.82 for common polymorphisms p.Arg156Arg and a minimum OR of 2.33 and 2.70 for the rare polymorphisms p.Asp312Asn and p.Asp711Asp, respectively. There may be moderate or weak associations between the polymorphisms at the 312 and 711 codons and platinum- related toxicities that would have a low probability of being detected (32). Second, treatment heterogeneity is another concern. Different combination regimens may modify the association. However, all the ORs and their 95% CIs were estimated by adjusting for types of treatment regimens in logistic regression model; the potential confounding factors were minimized (33). Third, we did not include other DNA repair genes or platinum-metabolizing genes. Given that the basis for treatment of advanced NSCLC involves platinum-based doublets combinations, careful consideration of other pathways involved in the metabolism of commonly used cytotoxic agents will be important (12). Finally, prospective validation studies in an independent patient population should be carried out to replicate the findings.

Our study has several strengths. A relatively large number of patients with advanced NSCLC receiving platinum-based chemotherapy were enrolled in this study, compared with published studies. Both patient recruitment and clinical outcome data collection were carried out independently without knowledge of polymorphism status. All patients were treated at the same hospital. The relatively homogeneous therapeutic standard limits the potential confounding effect of difference among various hospitals. In addition, we evaluated multiple XPD variants individually as an allele and collectively as haplotypes. A statistically significant difference for p.Arg156Arg polymorphism was also observed in more specific phenotype, a higher grade of leukopenia. The consistency of these results confirmed the findings. A potential concern includes its retrospective nature and possible evaluations bias inherent in toxicity outcomes. However, we assessed the toxicity twice a week during the treatment, and the documentation was based on multiple sources. Grading of toxicities was standardized according to the National Cancer Institute Common Toxicity Criteria version 3.0 among clinicians. Our study chose to focus mainly on hematologic toxicity, which is the more objective outcome. It seems that our finding is unlikely to have been obtained by chance.

In lung cancer, the choice of the cytotoxic chemotherapy is currently based on tumor (histology and disease extent) and patient features (age and performance status). However, chemotherapeutic treatment is widely associated with dose-limiting toxicity, which may reduce the life quality of patients. Toxicity has presented a challenge in the care of cancer patients. Better predictive methods are necessary for patients who tend to experience severe toxicity during the therapy course to make their choice. Assessing germ-line genetic polymorphisms as either prognostic or predictive markers has much appeal especially in the setting of advanced NSCLC where tumor tissues are rarely available (9, 34).

To the best of our knowledge, this is the first study demonstrating that the XPD p.Arg156Arg polymorphism is associated with grade 3 or 4 hematologic toxicity and, more specifically, severe leukopenia toxicity in advanced NSCLC patients treated with platinum-based chemotherapy. Although it is still hypothetical, our results suggest that XPD genotypes may play an important role in the pharmacogenetic basis for variability in severe toxicity following platinating agent treatment in advanced NSCLC patients. As little is known about the underlying physiology, further in vivo functional studies should be conducted with an emphasis on the biological basis of these findings.

No potential conflicts of interest were disclosed.

Grant support: China National Key Basic Research Program Grants 2002CB512902 and Shanghai Science and Technology Research Program 06DZ19501 and Shanghai Municipal Health Board Program 08GWD07.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

W. Wu and W. Zhang contributed equally to this work.

We thank Hongliang Liu and Yu Zhong for their help with sample collection, Professor Naiqing Zhao, Dr. Xueyan Yang, and Dr. Shuhua Xu for their helpful comments and discussion, our volunteers for donating their blood, and our collaborators for collection of blood sample and information.

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Supplementary data