1
Genetic variation predicting cisplatin cytotoxicity associated with overall
survival in lung cancer patients receiving platinum-based chemotherapy †, ‡
Xiang-Lin Tan1,2, Ann M. Moyer1,4, Brooke L. Fridley3, Daniel J. Schaid3, Nifang Niu1, Anthony
J. Batzler3, Gregory D. Jenkins3, Ryan P. Abo1, Liang Li1, Julie M. Cunningham4, Zhifu Sun2,
Ping Yang2, Liewei Wang1,*
1Division of Clinical Pharmacology, Department of Molecular Pharmacology and Experimental
Therapeutics, 2Division of Epidemiology, Department of Health Sciences Research, 3Division of
Biomedical Statistics and Informatics, Department of Health Sciences Research, 4Department of
Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN, 55905,
United States of America
Running title: Genetic variation and platinum response in lung cancer
Key words: Lung cancer, cisplatin, pharmacogenomics, lymphoblastoid cell lines, GWAS
† Sources of Support: This study was supported by U.S. National Institutes of Health research
grants, R01 CA80127 (to PY), R01 CA84354 (to PY), K22 CA130828 (to LW), R01 CA138461
(to LW), R25T CA92049 (to XT) and U19 GM61388 (The Pharmacogenomics Research
Network), Mayo Foundation funds (to PY), ASPET-Astellas Award (to LW) and a PhRMA
Foundation “Center of Excellence in Clinical Pharmacology” Award (to LW)
‡ Author disclosures: no conflicts of interest.
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
2
* To whom correspondence and requests for reprints should be addressed: Liewei Wang M.D.,
Ph.D., Division of Clinical Pharmacology, Department of Molecular Pharmacology and
Experimental Therapeutics, Mayo Clinic, 200 First Street SW, Rochester, MN, U.S.A. 55905
Email: [email protected] or [email protected] ; Telephone: 507-284-5264; Fax:
507-284-4455
5Abbreviations used: GWAS, genome-wide association study; LCLs, lymphoblastoid cell lines;
SCLC, small cell lung cancer, NSCLC, non-small cell lung cancer; OS, overall survival; SNPs,
single-nucleotide polymorphisms; AA, African-American; CA, Caucasian-American; HCA, Han
Chinese-American; DMSO, dimethyl sulfoxide; HWE, Hardy-Weinberg Equilibrium; MAF,
minor allele frequency; HR, hazard ratio
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
3
Translational Relevance
Chemotherapy with platinum-based regimens is the standard of care for both small cell and non-
small cell lung cancer, but large inter-individual variations are observed in platinum efficacy and
toxicity. However, reliable genetic biomarkers for the prediction of response to platinum-based
therapy are still not well established. Our study identified several genetic variants that are
potentially associated with the efficacy of platinum-based chemotherapy in patients with lung
cancer by using genome-wide analysis of data generated with lymphoblastoid cell lines followed
by genotyping studies using DNA samples from lung cancer patients, and functional validation
in lung cancer cells. These results provide important insight into mechanisms that might
contribute to variation in response to platinum-based therapy of lung cancer.
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
4
Abstract
Purpose: Inherited variability in the prognosis of lung cancer patients treated with platinum-
based chemotherapy has been widely investigated. However, the overall contribution of genetic
variation to platinum response is not well established. To identify novel candidate SNPs/genes,
we performed a genome-wide association study (GWAS) for cisplatin cytotoxicity using
lymphoblastoid cell lines (LCLs), followed by an association study of selected SNPs from the
GWAS with overall survival (OS) in lung cancer patients.
Experimental Design: GWAS for cisplatin were performed with 283 ethnically diverse LCLs.
168 top SNPs were genotyped in 222 small cell and 961 non-small cell lung cancer (SCLC,
NSCLC) patients treated with platinum-based therapy. Association of the SNPs with OS was
determined using the Cox regression model. Selected candidate genes were functionally
validated by siRNA knockdown in human lung cancer cells.
Results: Among 157 successfully genotyped SNPs, 9 and 10 SNPs were top SNPs associated
with OS for patients with NSCLC and SCLC, respectively, although they were not significant
after adjusting for multiple testing. Fifteen genes, including 7 located within 200 kb up or
downstream of the four top SNPs and 8 genes for which expression was correlated with three
SNPs in LCLs were selected for siRNA screening. Knockdown of DAPK3 and METTL6, for
which expression levels were correlated with the rs11169748 and rs2440915 SNPs, significantly
decreased cisplatin sensitivity in lung cancer cells.
Conclusions: This series of clinical and complementary laboratory-based functional studies
identified several candidate genes/SNPs that might help predict treatment outcomes for
platinum-based therapy of lung cancer.
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
5
Introduction
Platinum compounds are the major chemotherapeutic agents used to treat both small cell
and non-small cell lung cancer (SCLC and NSCLC), with platinum compounds as the base for
combination chemotherapy. Meta-analyses from randomized trials of cisplatin-based
chemotherapy versus best supportive care have shown that cisplatin-based chemotherapy was
associated with a modest improvement in overall survival (OS) (1). Unfortunately, although
these agents have shown success in treating lung cancer, the use of platinum-based
chemotherapy is limited by the development of chemoresistance and toxicity. In addition,
response rates vary greatly among individuals for any given cisplatin-based chemotherapy
regimen. Factors such as disease stage, tumor histology, sex, tobacco exposure and patient age
may influence platinum-based chemotherapy outcomes. Although tumor DNA is important for
drug response, previous studies have shown clearly that germline genetic variations also play an
important role in determining treatment outcome (2-4). Therefore, identification and
characterization of the role of genetic variation in differential response to platinum may help
optimize individual treatment plans and improve platinum treatment outcomes.
Chemoresistance and toxicity associated with platinum-based therapy are complex
phenotypes that may involve many processes (5). These processes include alterations in drug
influx or efflux, detoxification through glutathione conjugation, DNA repair capacity and other
cellular pathways required for proper response to DNA damage (6). The development of
chemoresistance to platinum agents can be due to germline genetic variation or acquired
mutation through their effects on mRNA and protein levels in key pharmacokinetic or
pharmacodynamic pathways. Candidate gene and/or candidate pathway approaches indicated
that single nucleotide polymorphisms (SNPs) in genes responsible for drug influx and efflux (7),
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
6
metabolism and detoxification (8-10), and DNA damage repair pathways (2-4, 11-17), were
associated with treatment outcome of lung cancer patients. However, the overall contribution of
these genetic biomarkers in the prediction of response to platinum-based therapy is still not well
established, suggesting that additional genes might be involved.
Therefore, in the current study, we took a genome-wide approach using 283
lymphoblastoid cell lines (LCLs) to identify SNPs/genes that might contribute to variation in
cisplatin cytotoxicity as represented by IC50 values. This step was followed by genotyping top
candidate SNPs from the LCL GWAS studies using 1183 DNA samples from lung cancer
patients treated with platinum-based therapy to identify SNPs that might be associated with OS.
Finally, functional studies of candidate genes identified during the association studies were
performed using siRNA knockdown in human lung cancer cells.
Materials and Methods
Cell lines
This study was reviewed and approved by the Mayo Clinic Institutional Review Board.
Human Variation Panel LCLs from sample sets HD100AA, HD100CAU, HD100CHI,
corresponding to 100 African-American (AA), 100 Caucasian-American (CA), and 100 Han
Chinese-American (HCA) unrelated subjects, were obtained from the Coriell Cell Repository
(Camden, NJ). The National Institute of General Medical Sciences had obtained and
anonymized these cell lines before deposit, and all subjects had provided written informed
consent for the use of their samples for research purposes. Human NSCLC cell lines H1437,
H1299 and the SCLC cell line H196 were obtained from the American Type Culture Collection
(Manassas, VA). LCLs were cultured in RPMI 1640 medium (Mediatech, Manassas, VA)
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
7
supplemented with 15% FBS (Mediatech). H1437, H1299 and H196 cell lines were cultured in
RPMI 1640 medium containing 10% FBS.
Patient samples
The study group included 1183 Caucasian patients with pathologically confirmed primary
lung cancer, 222 SCLC and 961 NSCLC, who were treated with platinum-based chemotherapy
at the Mayo Clinic (Rochester, MN) between 1997 and 2008. Details with regard to clinical
characteristics of patients, patient enrollment, diagnosis, and data collection procedures were
described previously (18-20). Briefly, each patient was identified through the Mayo Clinic
pathologic database. After written informed consent had been obtained, a blood sample was
collected. The characteristics of patients, including demographics, lung cancer pathology,
anatomic site, and types and timing of treatment and chemotherapeutic agents, were abstracted
from patient medical records by a trained nurse. Vital status and cause of death were determined
by reviewing the Mayo Clinic registration database and medical records, correspondence from
patients' next-of-kin, death certificates, obituary documents, the Mayo Clinic Tumor Registry,
and the Social Security Death Index website. Clinical staging and recurrence or progression
information was determined by results from available chest radiography, computerized
tomography, bone scans, positron emission tomography scans, and magnetic response imaging.
All patients were actively followed up during the initial six months after diagnosis, with
subsequent annual follow-up by mailed questionnaires and annual verification of the patients’
vital status.
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
8
Cisplatin cytotoxicity assay
Cisplatin was obtained from Sigma-Aldrich (St. Louis, MO) and was dissolved in dimethyl
sulfoxide (DMSO) immediately before use. Cells (4 ×104 cells/well) were plated into 96-well
plates, and incubated with cisplatin for 72 h in eight concentrations ranging from 0.1 to 80
μmol/L. DMSO alone was used as a control. Cisplatin cytotoxicity was evaluated by
determining the concentration of cisplatin required to inhibit growth and/or survival by 50%
(IC50) using the CellTiter 96@ Aqueous Non-Radioactive Cell Proliferation Assay (Promega,
Madison, WI). Experiments were performed successfully for 283 LCLs (91 AA, 96 CA, and 96
HCA).
Genome-wide SNP and expression arrays for LCLs
The genotyping and expression array data for all LCLs were described previously (21-23)
and are publically available from NCBI Gene Expression Omnibus
(http://www.ncbi.nlm.nih.gov/geo) under SuperSeries accession No. GSE24277 and accession
No. GSE23120. Briefly, Illumina HumanHap 550K and 510S BeadChips were used for
genotyping of DNA samples from all of the LCLs in the Genotype Shared Resource at Mayo
Clinic. Publicly available Affymetrix SNP Array 6.0 Chip SNP data were also obtained for the
same cell lines. SNPs that deviated from Hardy-Weinberg Equilibrium (HWE) (24) based on
minimum P value from an exact test for HWE (25) and the stratified test for HWE (26) (P <
0.001); SNPs with call rates < 95%; or SNPs with minor allele frequency (MAF) < 5% were
removed from the analysis (27, 28). As a result, 1,348,798 SNPs that passed these quality
control measures were included in the GWA analyses of SNP vs. IC50 and SNP vs. expression.
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
9
SNP selection and genotyping in lung cancer patients
SNP selection was based on P values obtained during the analysis of genome-wide SNP vs.
IC50, and SNP vs. expression, as well as expression vs. IC50 using LCL data. We selected all
SNPs with P < 10-5 from the SNP vs. IC50 analysis. Additionally, we defined a SNP peak as a
locus that contained at least two SNPs associated with cisplatin IC50 with P <10-4. SNPs within
the SNP peaks were used to perform an association study with 54,613 expression probesets to
identify SNPs that were associated with gene expression (P < 10-4). Finally an association study
was performed with gene expression and cisplatin IC50 to identify SNPs that might be associated
with cisplatin IC50 through an influence on gene expression. Therefore, we also selected SNPs
with SNP vs. expression: P value < 10-4 and expression vs. IC50: P value < 10-4 to genotype the
patient samples (Supplementary Table 1).
A total of 168 top hit SNPs were selected and genotyped in 1183 lung cancer patients who
received platinum-based chemotherapy. Genotyping was performed in the Mayo Clinic
Genomics Shared Resource using a custom-designed Illumina GoldenGate panel. Quality
control tests of the genotyping results were performed by assessing concordance among three
control DNA samples that were present in duplicate, SNP call rates, sample call rates, MAF of
SNPs or departure of SNP genotypes from HWE. SNPs were excluded if they failed genotyping,
or displayed ambiguous clustering monomorphic genotyping, MAFs of <0.01, or significant
departures from HWE (P < 0.001). SNPs having call rates > 95% but which passed all other
quality control tests were included in the analysis. Of the SNPs with genotyping data, 157 SNPs
passed the quality control tests and were included in the analyses.
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
10
Functional validation by siRNA knockdown
Human NSCLC cell lines, H1437, H1299 and the SCLC cell line, H196, were used in
siRNA screening studies. siRNAs for the candidate genes and non-targeting negative control
siRNA pool were purchased from Dharmacon (Hiden, Germany). Specifically, approximately
3,000-4,000 cells were seeded into 96-well plates, mixed with an siRNA-mixture consisting of
25 nmol/L of specific or negative control siRNAs and the LipofectamineTM RNAiMAX reagent
(Invitrogen, Carlsbad, CA). Twenty-four hours after transfection, the cells were treated with
vehicle or increasing concentrations of cisplatin for an additional 72 h, followed by MTS assay
using the CellTiter 96@ Aqueous Non-Radioactive Cell Proliferation Assay.
Knockdown efficiency determination by real-time RT-PCR and Western blot analysis
Total RNA was isolated from cultured cells transfected with controls or specific siRNAs
with the Mini RNA isolation kit (ZYMO Research, Orange, CA), followed by qRT-PCR
performed with the 1-step, Brilliant SYBR Green qRT-PCR master mix kit (Stratagene, La Jolla,
CA). Specifically, primers purchased from QIAGEN were used to perform qRT-PCR using the
ABI StepOne™ Real-Time PCR System (Applied Biosystems). Western Blots were performed
for DAPK3, METTL6 and RUFY1 using goat polyclonal antibodies purchased from Santa Cruz
biotechnology, Inc. All experiments were performed in triplicate with β-actin as an internal
control.
Statistical analysis
A detailed description for GWAS of LCLs has been described elsewhere (21-23). Briefly,
the cisplatin cytotoxicity IC50 phenotype was calculated for each cell line based on a logistic
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
11
dose-response model using the R package ‘drc’ (http://cran.r-project.org/doc/packages/ drc.pdf).
Logarithm transformed IC50 values were then compared between genders, batches of samples
based on time since purchase, and between the CA race and all other samples using independent
samples t-tests. An overall comparison of transformed IC50 values among ethnic groups was
performed using an F-test based on ANOVA. Because we used LCLs from multiple races/ethnic
groups, population stratification was adjusted using the method developed by Price et al. (29),
which uses an eigen analysis for detecting and adjusting. SNPs. The log transformed IC50
values and GCRMA normalized expression data were adjusted for race using the five
eigenvectors. The GWA analysis of the association of SNP vs. IC50 or SNP vs. expression was
performed with Pearson correlations using adjusted SNP, IC50 and expression values for each
individual. For the siRNA knockdown experiments, group mean values of IC50 were compared
using Student's t test.
The overall survival time was used as the primary endpoint, defined as the time from lung
cancer diagnosis to either death or the last known date alive. Patients known to be alive were
censored at the time of last contact. All the patients included in the analysis were Caucasians.
To test for the effect of SNP on OS, we used the Cox regression model that included the effects
of a SNP genotype dosage (count of minor alleles). A total of 157 SNPs were included in this
analysis, and the association was performed for NSCLC and SCLC separately because of
significant differences between the two diseases. To correct for multiple testing of the 157 SNPs
assayed in the initial experiment, the Bonferroni corrected P value threshold of 0.0003 was used
to determine statistically significant associations. To determine whether associations with SNPs
should be adjusted for the clinical covariates of age at diagnosis, gender, smoking status, disease
stage, and treatment, backward selection was done. The disease stage was included in the final
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
12
multivariate Cox-regression model since it was significantly associated with OS of the lung
cancer patients. The disease stage was divided into five categories: small cell lung cancer with
limited versus extensive stages; NSCLC with stages I and II, versus III, versus IV. We used 0.05
as a cutoff for P values (not adjusted for multiple testing) to select SNPs/genes for further
functional validation.
Results
Correlation analysis of genome-wide SNP vs cisplatin IC50 in LCLs
Cytotoxicity assays were performed for cisplatin to determine the range of variation in drug
response, and IC50 was used as a phenotype to indicate the drug sensitivity for each cell line.
The unadjusted average IC50 value for cisplatin in these 283 cell lines was 1.79 ± 1.64 (mean ±
SD) µmol/L. Gender had no significant effect on IC50 values (P = 0.15). Caucasian Americans
(CAs) were more sensitive to cisplatin compared to the other two races (AAs and HCAs) (P =
0.0001). Time since the Coriell Institute acquired the cell lines had no significant effect on
cisplatin cytotoxicity (P = 0.64).
A total of 1,348,789 SNPS were used in the genome-wide SNP analysis for 283 cell lines.
Figure 1A depicts the association results graphically. 364 SNPs were associated with cisplatin
IC50 with P values < 10-4, and 50 SNPs had P values < 10-5. None of the top 50 SNPs were
within the coding region of a gene. Twenty-three, 16 and 11 SNPs were in introns, 5’-upstream
or 3’-downstream flanking regions of genes, respectively. The top three SNPs from the GWAS
were rs6633130 (P = 1.66 × 10-7), rs6946197 (P = 1.99 × 10-7) and rs12304656 (P = 3.64 × 10-7)
at loci containing the PPEF1 (intron), LOC100128030 (3’downstream) and STYK1 (intron)
genes, respectively.
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
13
Survival analysis for the selected SNPs and lung cancer patient samples
A total of 168 SNPs were selected and genotyped to determine whether any of those SNPs
might be associated with patient OS. Table 1 lists basic demographic and clinic data with regard
to the patients studied. Of these patients, 656 (55.5%) were male and 527 (44.5%) were female,
with a median age of 63.0 years. Most patients had a history of smoking, with 47.3% (n = 560)
being former smokers and 36.5% (n = 431) current smokers. 550 (46.5%) tumors were classified
as adenocarcinoma, 194 (16.4%) as squamous cell carcinoma and 222 (18.8%) as small cell
carcinoma. 881 (74.5%) patients received platinum drugs in combination of surgery and/or
radiation, and 302 (25.5%) patients received only platinum drugs.
Only disease stage was included in the analysis as a confounder among all the others tested
including age at diagnosis, gender and smoking status. Nine and 10 different SNPs were
associated with OS for NSCLC and SCLC patients, respectively (P < 0.05) (Table 2 and
Supplemental Fig. 1). However none of the SNPs were statistically significant after correcting
for multiple testing. The most significant SNPs for NSCLC and SCLC, rs1287276 (located
within an intron of UGT3A2 on chromosome 5) and rs12669805 (located 3’-downstream of the
LOC100128030 pseudogene on chromosome 7) were associated with OS in NSCLC (P =
0.0004) and SCLC (P = 0.0021), respectively. A hazard ratio > 1 indicated that patients carrying
the minor allele had worse survival. These SNPs would also be expected to be associated with
higher IC50 values in LCLs, and the cells carrying these SNPs would be predicted to be more
resistant to cisplatin. Therefore, 12 out of the 19 SNPs showed concordant association directions
between the two results (Table 2), when we compared the association direction of SNPs with
clinical OS with that of the same SNP with cisplatin IC50 values in LCLs.
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
14
Functional characterization of candidate gene
Although none of the SNPs studied in the patient samples reached statistical significance
after multiple testing, to further pursue the possibility that the locus harboring these SNPs might
include functional genes, we took advantage of the expression data we had for the cell lines (21-
23) to help interpret SNP function and guide the selection of genes for siRNA screening. The
overall selection strategy is depicted graphically in Figure 1B. First, we focused on genes
located within 200 kb up or downstream of the 12 SNPs which showed a concordant association
with the IC50 phenotype in LCLs and the OS phenotype in patients. 39 genes were found to be
located within 200 kb up or downstream of these SNPs, and 7 out of the 39 genes close to 4
SNPs had the expression levels > 50 in LCLs after GCRMA normalization (Fig. 1B). Next,
since SNPs might influence cisplatin response through the regulation of gene expression by
either cis- or trans-regulation, we also performed association analysis using the genotyping data
for these 12 SNPs and basal expression array data (54,613 expression probesets) available for all
of the LCLs. Eight of 12 SNPs were associated with the expression of 212 probesets (154
unique genes) using a cutoff P value <10-4, and 4 of 12 SNPs were associated with mRNA
expression levels of 54 genes with P value <10-5 (Fig. 1B). No cis-regulation was identified.
We selected these 54 genes plus 2 genes with multiple probesets associated with the SNPs to
check expression levels in LCLs. However, only 8 of these genes had the expression levels > 50
after GCRMA normalization. Finally, 15 genes, including 7 genes located within 200 kb up or
downstream of 4 SNPs (rs11169748, rs2440915, rs5952066 and rs7620841) and 8 genes for
which expression was correlated with three SNPs (rs11169748, rs2440915 and rs5952066) were
selected for siRNA screening (Fig. 1B).
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
15
We performed knockdown experiments for these 15 genes in the NSCLC cell lines H1437
and H1299 and in the SCLC cell line H196. Knockdown of DAPK3 and METTL6 significantly
decreased cisplatin sensitivity in all three lung cancer cell lines, while knockdown of RUFY1 did
not significantly alter cisplatin cytotoxicity (Fig. 2). The rs11169748 and rs2440915 SNPs that
were associated with lung cancer OS were also correlated with cisplatin IC50 values in LCLs. In
addition, these two SNPs were also associated with expression of DAPK3 and METTL6 in LCLs
(Fig. 3). However only DAPK3 expression level was associated with cisplatin IC50 (P = 0.03) in
LCLs, while METTL6 was not (P = 0.32). To obtain ungenotyped SNPs, we imputed SNPs that
were not on our genotyping platform and that were located within 200 kb up or downstream of
these two SNPs (rs11169748 and rs2440915), using MACH 1.0 (30), with 1000 Genome Project
data (31) as the reference panel. We observed no stronger association with cisplatin IC50 for the
imputed SNPs, compared to the observed SNPs (Fig. 3E and F).
Discussion
In the current study, we took advantage of the extensive genomic data that we have already
obtained for 283 human LCLs, together with DNA samples obtained from lung cancer patients
treated with platinum-based therapy to determine whether SNPs identified during a GWAS
performed with LCLs might contribute to our understanding of the drug actions of platinum
compounds and the response to platinum compounds in the treatment of lung cancer. Therefore,
we first conducted a GWAS for cisplatin in 283 LCLs to identify top candidate SNPs that were
associated with platinum cytotoxicity in vitro (Fig. 1 and Supplemental Table 1). We then
performed an association study with selected top candidate SNPs using DNA samples obtained
from NSCLC and SCLC patients (Table 2), followed by functional validation of candidate genes
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
16
using human lung cancer cell lines (Fig. 2 and Table 3). We found that 19 SNPs were associated
with OS with P values <0.05 of either NSCLC or SCLC patients (Table 2), and that knockdown
of DAPK3 and METTL6, for which expression levels were correlated with the rs11169748 and
rs2440915 SNPs (Fig. 3), significantly decreased cisplatin sensitivity in the lung cancer cell lines
H1437, H1299 and H196 (Fig. 2).
Previous candidate gene and candidate pathway-based pharmacogenomic analyses have
shown that genetic variation may play a role in determining response to platinum-based
chemotherapy. Much of the focus has been on SNPs within the DNA damage and repair
pathway (2-4, 11-17, 32). However, those results remain controversial. For example, Zhou et al.
reported that the ERCC1 8092C>A variant allele was associated with a 50% increased risk of
death in Stage III (A+B) and IV NSCLC patients (32), while Wu et al. observed that the variant
A allele was significantly associated with a shorter OS (HR = 0.68, 95%CI. 0.48-0.95) (3).
These previous studies indicate that the effects of each individual SNP are modest and suggest
that additional SNPs/genes and/or other possible genetic mechanisms such as CpG methylation
or copy number variation might influence response to platinum-based therapy.
With advances in genotyping and gene expression technologies, several studies have taken
an unbiased approach towards understanding genetic factors influencing response to platinum-
based chemotherapy. In LCLs obtained from pedigrees of Centr d’Etudes du polymorphism
Human (CEPH) individuals of European background, it was estimated that 30-40% of the
variation in cisplatin-induced cytotoxicity was due to heritable factors (33, 34). Using this cell
line model system, those investigators subsequently identified several genetic variants that
contributed to cisplatin and carboplatin cytotoxicity (24, 34-36). However, the results of those
studies have not been replicated in cancer patients treated with platinum-based
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
17
chemotherapeutics. Our genome-wide pharmacogenomics analysis did not confirm those
variants associated with response to cisplatin (24). The difference could be due to differences
between populations from which these LCLs are derived. In Huang’s study, the LCLs derived
from CEU, European descent and YRI, African descent while our LCLs derived from CA, AA
and HCA. It could also be due to confounding factors associated with the use of LCLs, such as
cell growth rate and ATP levels (37). Therefore, we performed siRNA screening for selected
candidate genes to pursue the underlying biology. Several previous studies have suggested an
important contribution of trans-acting variants to individual variation in human gene expression
(38, 39). Among the genes tested, knockdown of DAPK3 and METTL6, for which expression
levels correlated with the rs11169748 and rs2440915 SNPs, significantly decreased cisplatin
sensitivity in three lung cancer cell lines, although METTL6 expression levels were not
significantly associated with cisplatin IC50 in LCLs, suggesting a potential difference between
LCLs and lung cancer cell lines.
DAPK3, also known as zipper interacting protein kinase (ZIPK) or DAPK like kinase
(DLK), is a member of the DAPK family. DAPK was initially identified as being encoded by a
gene whose reduced expression, mediated by antisense cDNA transfection, protected HeLa cells
from gamma interferon-induced cell death. DAPK3 contains several putative nuclear localization
signal sequences and a leucine zipper domain, required for homo-oligomerization, interaction
with other leucine zipper-containing proteins and for its death-promoting effects (40, 41).
DAPK3 has been shown to play distinct roles in tumor suppression and the regulation of
apoptosis (42-44). We have shown that knockdown of DAPK3 significantly decreased cisplatin
sensitivity in lung cancer cell lines and might be related to OS of NSCLC patients treated with
platinum-based therapy.
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
18
METTL6 belongs to the methyltransferase superfamily. It has been suggested that
chemoresistance and toxicity associated with platinum compound treatment might be mediated
through methylation-dependent gene silencing. For example, methylation of FANCF, a gene
associated with Fanconi anemia, confers cisplatin sensitivity in ovarian cancer cell lines (45).
Additionally, Ramirez et al. observed that 14-3-3ơ, a cell cycle checkpoint gene, is methylated in
one third of NSCLC patients and that methylation is related to better median OS for these
patients (46). It is possible that METTL6 might influence DNA methylation, and further
influence response to platinum-based therapy.
In summary, we have identified several genetic variants that are potentially associated with
the efficacy of platinum-based chemotherapy in patients with lung cancer by using genome-wide
interrogation of LCLs together with genotyping studies using DNA samples from lung cancer
patients, followed by functional validation in lung cancer cells. Our results provide important
insight into novel genes and mechanisms that may contribute to variation in response to platinum
drug therapy. Most of our patients with advanced NSCLC were treated with cisplatin
combination therapy, often with the addition of other chemotherapeutics, radiation or surgery.
Even though the SNPs that we tested were selected based on their association with cisplatin
cytotoxicity in LCLs, we can not exclude the possibility of potential interaction of genetic
variation with other treatments and their effect on cisplatin treatment outcome in patients with
lung cancer. Therefore, it is particularly important that our functional studies confirmed the
functional effects of these genes in cisplatin response in vitro. Obviously, additional replication
studies in independent sample sets of patients with lung cancer treated with platinum-based
chemotherapy would be required to further confirm these findings.
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
19
Acknowledgements
We thank Luanne Wussow for her assistance with the preparation of this manuscript. This study
was supported by U.S. National Institutes of Health research grants, R01 CA80127 (to PY), R01
CA84354 (to PY), K22 CA130828 (to LW), R01 CA138461 (to LW), R25T CA92049 (to XT)
and U19 GM61388 (The Pharmacogenomics Research Network), Mayo Foundation funds (to
PY), an ASPET-Astellas Award (to LW) and a PhRMA Foundation “Center of Excellence in
Clinical Pharmacology” Award (to LW).
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
20
References
1. Chemotherapy in non-small cell lung cancer: a meta-analysis using updated data on
individual patients from 52 randomised clinical trials. Non-small Cell Lung Cancer
Collaborative Group. BMJ. 1995;311:899-909.
2. Gurubhagavatula S, Liu G, Park S, Zhou W, Su L, Wain JC, et al. XPD and XRCC1 genetic
polymorphisms are prognostic factors in advanced non-small-cell lung cancer patients treated
with platinum chemotherapy. J Clin Oncol. 2004;22:2594-601.
3. Wu X, Lu C, Ye Y, Chang J, Yang H, Lin J, et al. Germline genetic variations in drug action
pathways predict clinical outcomes in advanced lung cancer treated with platinum-based
chemotherapy. Pharmacogenet Genomics. 2008;18:955-65.
4. Wei SZ, Zhan P, Shi MQ, Shi Y, Qian Q, Yu LK, et al. Predictive value of ERCC1 and XPD
polymorphism in patients with advanced non-small cell lung cancer receiving platinum-based
chemotherapy: a systematic review and meta-analysis. Med Oncol. 2010;28:315-21.
5. Huang RS, Ratain MJ. Pharmacogenetics and pharmacogenomics of anticancer agents. CA
Cancer J Clin. 2009;59:42-55.
6. Marsh S, McLeod H, Dolan E, Shukla SJ, Rabik CA, Gong L, et al. Platinum pathway.
Pharmacogenet Genomics. 2009;19:563-4.
7. Rabik CA, Maryon EB, Kasza K, Shafer JT, Bartnik CM, Dolan ME. Role of copper
transporters in resistance to platinating agents. Cancer Chemother Pharmacol. 2009;64:133-
42.
8. Booton R, Ward T, Heighway J, Ashcroft L, Morris J, Thatcher N. Glutathione-S-transferase
P1 isoenzyme polymorphisms, platinum-based chemotherapy, and non-small cell lung cancer.
J Thorac Oncol. 2006;1:679-83.
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
21
9. Lu C, Spitz MR, Zhao H, Dong Q, Truong M, Chang JY, et al. Association between
glutathione S-transferase pi polymorphisms and survival in patients with advanced nonsmall
cell lung carcinoma. Cancer. 2006;106:441-7.
10. Moyer AM, Sun Z, Batzler AJ, Li L, Schaid DJ, Yang P, et al. Glutathione pathway genetic
polymorphisms and lung cancer survival after platinum-based chemotherapy. Cancer
Epidemiol Biomarkers Prev. 2010;19:811-21.
11. Kim HT, Lee JE, Shin ES, Yoo YK, Cho JH, Yun MH, et al. Effect of BRCA1 haplotype on
survival of non-small-cell lung cancer patients treated with platinum-based chemotherapy. J
Clin Oncol. 2008;26:5972-9.
12. Li F, Sun X, Sun N, Qin S, Cheng H, Feng J, et al. Association between polymorphisms of
ERCC1 and XPD and clinical response to platinum-based chemotherapy in advanced non-
small cell lung cancer. Am J Clin Oncol. 2010;33:489-94.
13. Liu L, Yuan P, Wu C, Zhang X, Wang F, Guo H, et al. Assessment of XPD Lys751Gln and
XRCC1 T-77C polymorphisms in advanced non-small-cell lung cancer patients treated with
platinum-based chemotherapy. Lung Cancer. 2011;73:110-5.
14. Matakidou A, el Galta R, Webb EL, Rudd MF, Bridle H, Eisen T, et al. Genetic variation in
the DNA repair genes is predictive of outcome in lung cancer. Hum Mol Genet.
2007;16:2333-40.
15. Shiraishi K, Kohno T, Tanai C, Goto Y, Kuchiba A, Yamamoto S, et al. Association of DNA
repair gene polymorphisms with response to platinum-based doublet chemotherapy in
patients with non-small-cell lung cancer. J Clin Oncol. 2010;28:4945-52.
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
22
16. Sun X, Li F, Sun N, Shukui Q, Baoan C, Jifeng F, et al. Polymorphisms in XRCC1 and XPG
and response to platinum-based chemotherapy in advanced non-small cell lung cancer
patients. Lung Cancer. 2009;65:230-6.
17. Tibaldi C, Giovannetti E, Vasile E, Mey V, Laan AC, Nannizzi S, et al. Correlation of CDA,
ERCC1, and XPD polymorphisms with response and survival in gemcitabine/cisplatin-
treated advanced non-small cell lung cancer patients. Clin Cancer Res. 2008;14:1797-803.
18. Yang P, Wentzlaff KA, Katzmann JA, Marks RS, Allen MS, Lesnick TG, et al. Alpha1-
antitrypsin deficiency allele carriers among lung cancer patients. Cancer Epidemiol
Biomarkers Prev. 1999;8:461-5.
19. Yang P, Bamlet WR, Sun Z, Ebbert JO, Aubry MC, Krowka MJ, et al. Alpha1-antitrypsin
and neutrophil elastase imbalance and lung cancer risk. Chest. 2005;128:445-52.
20. Yang P, Sun Z, Krowka MJ, Aubry MC, Bamlet WR, Wampfler JA, et al. Alpha1-antitrypsin
deficiency carriers, tobacco smoke, chronic obstructive pulmonary disease, and lung cancer
risk. Arch Intern Med. 2008;168:1097-103.
21. Li L, Fridley B, Kalari K, Jenkins G, Batzler A, Safgren S, et al. Gemcitabine and cytosine
arabinoside cytotoxicity: association with lymphoblastoid cell expression. Cancer Res.
2008;68:7050-8.
22. Li L, Fridley BL, Kalari K, Jenkins G, Batzler A, Weinshilboum RM, et al. Gemcitabine and
arabinosylcytosin pharmacogenomics: genome-wide association and drug response
biomarkers. PLoS One. 2009;4:e7765.
23. Niu N, Qin Y, Fridley BL, Hou J, Kalari KR, Zhu M, et al. Radiation pharmacogenomics: a
genome-wide association approach to identify radiation response biomarkers using human
lymphoblastoid cell lines. Genome Res. 2010;20:1482-92.
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
23
24. Huang RS, Duan S, Shukla SJ, Kistner EO, Clark TA, Chen TX, et al. Identification of
genetic variants contributing to cisplatin-induced cytotoxicity by use of a genomewide
approach. Am J Hum Genet. 2007;81:427-37.
25.Wigginton JE, Cutler DJ, Abecasis GR. A note on exact tests of Hardy-Weinberg equilibrium.
Am J Hum Genet. 2005;76:887-93.
26. Schaid DJ, Batzler AJ, Jenkins GD, Hildebrandt MA. Exact tests of Hardy-Weinberg
equilibrium and homogeneity of disequilibrium across strata. Am J Hum Genet.
2006;79:1071-80.
27. Wellcome Trust Case Control Consortium. Genome-wide association study of 14,000 cases
of seven common diseases and 3,000 shared controls. Nature. 2007;447:661-78.
28. Pongpanich M, Sullivan PF, Tzeng JY. A quality control algorithm for filtering SNPs in
genome-wide association studies. Bioinformatics. 2010;26:1731-7.
29. Price AL, Patterson NJ, Plenge RM, Weinblatt ME, Shadick NA, Reich D. Principal
components analysis corrects for stratification in genome-wide association studies. Nat Genet.
2006;38:904-9.
30. Li Y, Willer CJ, Ding J, Scheet P, Abecasis GR. MaCH: using sequence and genotype data to
estimate haplotypes and unobserved genotypes. Genet Epidemiol. 2010;34:816-34.
31. Durbin RM, Abecasis GR, Altshuler DL, Auton A, Brooks LD, Gibbs RA, et al. A map of
human genome variation from population-scale sequencing. Nature. 2010;467:1061-73.
32. Zhou W, Gurubhagavatula S, Liu G, Park S, Neuberg DS, Wain JC, et al. Excision repair
cross-complementation group 1 polymorphism predicts overall survival in advanced non-
small cell lung cancer patients treated with platinum-based chemotherapy. Clin Cancer Res.
2004;10:4939-43.
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
24
33. Dolan ME, Newbold KG, Nagasubramanian R, Wu X, Ratain MJ, Cook EH, Jr., et al.
Heritability and linkage analysis of sensitivity to cisplatin-induced cytotoxicity. Cancer Res.
2004;64:4353-6.
34. Shukla SJ, Duan S, Badner JA, Wu X, Dolan ME. Susceptibility loci involved in cisplatin-
induced cytotoxicity and apoptosis. Pharmacogenet Genomics. 2008;18:253-62.
35. Huang RS, Duan S, Kistner EO, Hartford CM, Dolan ME. Genetic variants associated with
carboplatin-induced cytotoxicity in cell lines derived from Africans. Mol Cancer Ther.
2008;7:3038-46.
36. Shukla SJ, Duan S, Wu X, Badner JA, Kasza K, Dolan ME. Whole-genome approach
implicates CD44 in cellular resistance to carboplatin. Hum Genomics. 2009;3:128-42.
37. Choy E, Yelensky R, Bonakdar S, Plenge RM, Saxena R, De Jager PL, et al. Genetic analysis
of human traits in vitro: drug response and gene expression in lymphoblastoid cell lines.
PLoS Genet. 2008;4:e1000287.
38. Morley M, Molony CM, Weber TM, Devlin JL, Ewens KG, Spielman RS, et al. Genetic
analysis of genome-wide variation in human gene expression. Nature. 2004;430:743-7.
39. Emilsson V, Thorleifsson G, Zhang B, Leonardson AS, Zink F, Zhu J, et al. Genetics of gene
expression and its effect on disease. Nature. 2008;452:423-8.
40. Kawai T, Matsumoto M, Takeda K, Sanjo H, Akira S. ZIP kinase, a novel serine/threonine
kinase which mediates apoptosis. Mol Cell Biol. 1998;18:1642-51.
41. Shoval Y, Pietrokovski S, Kimchi A. ZIPK: a unique case of murine-specific divergence of a
conserved vertebrate gene. PLoS Genet. 2007;3:1884-93.
42. Raveh T, Kimchi A. DAP kinase-a proapoptotic gene that functions as a tumor suppressor.
Exp Cell Res. 2001;264:185-92.
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
25
43. Wang WJ, Kuo JC, Yao CC, Chen RH. DAP-kinase induces apoptosis by suppressing
integrin activity and disrupting matrix survival signals. J Cell Biol. 2002;159:169-79.
44. Bialik S, Kimchi A. DAP-kinase as a target for drug design in cancer and diseases associated
with accelerated cell death. Semin Cancer Biol. 2004;14:283-94.
45. Taniguchi T, Tischkowitz M, Ameziane N, Hodgson SV, Mathew CG, Joenje H, et al.
Disruption of the Fanconi anemia-BRCA pathway in cisplatin-sensitive ovarian tumors. Nat
Med. 2003;9:568-74.
46. Ramirez JL, Rosell R, Taron M, Sanchez-Ronco M, Alberola V, de Las Penas R, et al. 14-3-
3sigma methylation in pretreatment serum circulating DNA of cisplatin-plus-gemcitabine-
treated advanced non-small-cell lung cancer patients predicts survival: The Spanish Lung
Cancer Group. J Clin Oncol. 2005;23:9105-12.
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
26
Figure Legends
Figure 1. Genome-wide SNP association with cisplatin IC50 values (A) and the strategies
used to select candidate genes for siRNA validation (B). Genome-wide SNP association
analysis was performed with cisplatin IC50 as the drug-response phenotype (n = 283). The y-
axis represents the -log10(P value) for association of SNPs and IC50 values. SNPs are plotted on
the x-axis on the basis of their chromosomal locations. The cut-off P value of 10-5 is highlighted
with a black line. Detailed strategies for the selection of candidate genes have been described in
the text. OS, overall survival; LCLs, lymphoblastoid cell lines.
Figure 2. Functional characterizations of candidate genes with siRNA knockdown of
DAPK3 and METTL6. Knockdown of DAPK3 and METTL6 in human NSCLC cell lines,
H1437 (A), H1299 (B) and the SCLC cell line, H196 (C), showed increased resistance to
cisplatin. A control was also included in which knockdown of RUFY1 did not significant change
the response to cisplatin as determined by MTS assay. Quantitative RT-PCR (D) and western
blot (E) were performed to determine the knockdown efficiency for the three genes. RT-PCR
results are expressed as % of control. Error bars represent standard error of mean (SEM) values
for 3 independent experiments.
Figure 3. Association between the rs1169748 and rs2440915 SNPs and cisplatin IC50 values
and expression of DAPK3 and METTL6 in LCLs, as well as imputation analyses for these
two SNPs. Genotypes for each SNP are plotted against cisplatin IC50 values as well as gene
expression levels of DAPK3 and METTL6, respectively. (A and C) SNP rs1169748 association
with cisplatin IC50 values and DAPK3 expression; (B and D) SNP rs2440915 association with
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
27
cisplatin IC50 values and METTL6 expression; (E and F) imputation analysis for the rs1169748
and rs2440915 SNPs. Black circles indicate SNPs observed by genotyping, while black triangles
indicate imputed SNPs. The y-axis represents –log10(P value) for the association of each SNP
with cisplatin IC50, and the x-axis represents the chromosome location of the SNPs. No stronger
association with cisplatin IC50 were observed for the imputed SNPs, compared to the genotyped
SNPs. AA, African Americans; CA, Caucasian Americans; and HCA, Han-Chinese Americans.
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
-Log
10(p
val
ue)
Chromosome location
1
6
5
4
3
2
1
0
2 3 4 5 6 7 8 9 10 11 13 15
X o
r Y17 19 22
19 SNPs associated with OS in clinical study (P < 0.05)
Gene expression level >50 in LCLs (7 genes)
Gene expression level >50 in LCLs (8 genes)
15 candidate genes selected for functional validation
Genes within 200 kb up or downstream of the 12 SNPs (39 genes)
SNP vs. expression in LCLs with P <10-5
or P <10-4 with ≥ 2 probe sets (56 genes)
12 SNPs concordantly associated with IC50 and OS phenotype
A
B
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
A B C
DAPK352kDaMETTL630kDaRUFY180kDaβ-actin43kDa
Specific siRNA Negative siRNAE
H1437H1299
H196H1437
H1299H196
-0.5 0.0 0.5 1.0 1.5 2.00.00.20.40.60.81.01.2
DAPK3 siRNAIC50(95%CI): 16.0(15.3-16.6)
Negative siRNAIC50(95%CI): 12.3(11.0-13.8)
Cisplatin concentration, Log (μmol/L)
Cell s
urviv
al
-0.5 0.0 0.5 1.0 1.5 2.00.00.20.40.60.81.01.2
METTL6 siRNAIC50 (95%CI): 14.9(13.5-16.5)
Cisplatin concentration, Log (μmol/L)
Cell s
urviv
al
-0.5 0.0 0.5 1.0 1.5 2.0-0.10.10.30.50.70.91.1
DAPK3 siRNAIC50(95%CI): 26.7(23.8-29.8)
Negative siRNAIC50(95%CI): 15.9(13.6-18.7)
Cisplatin concentration, Log (μmol/L)
Cell s
urviv
al
-0.5 0.0 0.5 1.0 1.5 2.0-0.10.10.30.50.70.91.1
METTL6 siRNAIC50(95%CI): 33.9(26.3-43.7)
Cisplatin concentration, Log (μmol/L)
Cell s
urviv
al
-0.5 0.0 0.5 1.0 1.5 2.0-0.10.10.30.50.70.91.1
RUFY1 siRNAIC50(95%CI): 15.9(13.7-18.5)
Cisplatin concentration, Log (µmol/L)
Cel
l sur
vival
-0.5 0.0 0.5 1.0 1.5 2.00.00.20.40.60.81.01.2
DAPK3 siRNAIC50(95%CI): 8.1(7.4-8.8)
Negative siRNAIC50(95%CI): 6.1(5.6-6.7)
Cisplatin concentration, Log (µmol/L)
Cell s
urviv
al
-0.5 0.0 0.5 1.0 1.5 2.00.00
0.25
0.50
0.75
1.00METTL6 siRNAIC50(95%CI): 8.4(7.9-9.0)
Cisplatin concentration, Log (μmol/L)
Cell s
urviv
al-0.5 0.0 0.5 1.0 1.5 2.0
0.0
0.2
0.4
0.6
0.8
1.0RUFY1 siRNAIC50(95%CI): 6.0(5.6-6.4)
Cisplatin concentration, Log (µmol/L)Ce
ll sur
vival
-0.5 0.0 0.5 1.0 1.5 2.00.0
0.2
0.4
0.6
0.8
1.0
RUFY1 siRNAIC50(95%CI): 11.6(10.9-12.5)
Cisplatin concentration, Log (µmol/L)
Cell s
urviv
al
0.0
0.2
0.4
0.6
0.8
1.0
1.2
NegativesiRNA
DAPK3siRNA
METTL6siRNA
RUFY1siRNA
Rela
tive
mRN
A ex
pres
sion
H1437 H1299 H196
D
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
A B
C D
E F
Adj
uste
d IC
502.0
1.5
1.0
0.5
0.0
-0.5
-1.0
Adj
uste
d IC
50
2.01.51.00.50.0
-0.5-1.0
Adj
uste
d DAPK3
mR
NA
exp
ress
ion
(Log
) 15
10
5
0
Rs1169748 AA AG GG AA AG GG AA AG GG AA AG GGNumber 63 25 3 94 2 0 94 2 0 251 29 3Ethnicity AA CA HCA Total
Adj
uste
d METTL6
mR
NA
exp
ress
ion
(Log
)
121086420
Rs2440915 AA AG GG AA AG GG AA AG GG AA AG GGNumber 63 25 3 94 2 0 96 0 0 253 27 3Ethnicity AA CA HCA Total
Rs1169748 AA AG GG AA AG GG AA AG GG AA AG GGNumber 63 25 3 94 2 0 94 2 0 251 29 3Ethnicity AA CA HCA Total
Imputed SNPGenotyped SNP
-Log
10(P
)
5
4
3
2
1
0
5
4
3
2
1
0
-Log
10(P
)
49700 49800 49900 50000Position (kb)
61200 61300 61400 615500Position (kb)
Imputed SNPGenotyped SNPrs1169748 rs2440915
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
Characteristics of diagnosis and treatment Values and percentages
Mean (SD) 62.3 (10.34)
Median (range) 63.0 (27.0-88.0)
Female 527 (44.5%)
Male 656 (55.5%)
Never 192 (16.2%)
Former smokers 560 (47.3%)
Current smokers 431 (36.5%)
Unknown
SCLC: LIMITED 129 (10.9%)
EXTENSIVE 93 (7.9%)
NSCLC: I 123 (10.4%)
II 82 (6.9%)
III 403 (34.1%)
IV 353 (29.8%)
Adenocarcinoma 550 (46.5%)
Squamous cell carcinoma 194 (16.4%)
Small cell carcinoma 222 (18.8%)
Large cell carcinoma 39 (3.3%)
Mixed and unspecified NSCLC 167 (14.1%)
Carcinoid or salivary gland tumors 11 (0.9%)
Nongradable or unknown 80 (6.8%)
Well differentiated 91 (7.7%)
Histologic cell type
Tumor differentiation grade
Table 1. Description of 1183 patients who were diagnosed with primary lung cancer and
received platinum-based chemotherapy.
Age at diagnosis
Gender, n (%)
Cigarette smoking status
Lung cancer stage a
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
Moderately differentiated 392 (33.1%)
Poor/undifferentiated 620 (52.4)
Only platinum drugs 302 (25.5%)
Surgery and platinum drugs 192 (16.2%)
Radiation and platinum drugs 455 (38.5%)
Surgery, radiation, and platinum drugs 234 (19.8%)a Stage for NSCLC is described on the basis of the tumor-node-metastasis
classification. The small cell lung cancer stage is described as suggested by the
American Cancer Society as either “EXTENSIVE” or “LIMITED.”
Treatment modality
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
MAF R valuea
P value MAF HR (95% CI) b
P value
rs1287276 5 36083458 0.242 -0.247 4.72E-05 0.095 1.36 (1.15-1.61) 0.0004
rs9405302 c
6 8228049 0.394 0.267 1.08E-05 0.211 1.22 (1.08-1.37) 0.001
rs8064630 17 3789240 0.152 0.251 3.61E-05 0.187 0.82 (0.71-0.94) 0.004
rs5970160 c
23 150844654 0.17 0.259 2.25E-05 0.126 1.17 (1.05-1.31) 0.006
rs1946518 11 111540668 0.384 0.249 5.55E-05 0.396 0.87 (0.78-0.96) 0.008
rs2440915 c
10 61343778 0.058 0.249 4.10E-05 0.029 1.41 (1.08-1.83) 0.012
rs5952066 c
23 146692840 0.178 0.242 6.92E-05 0.2 1.14 (1.03-1.26) 0.014
rs901893 c
5 8228049 0.353 0.239 8.53E-05 0.337 1.14 (1.02-1.27) 0.018
rs11169748 c 12 49865438 0.062 0.237 9.60E-05 0.011 1.75 (1.03-2.97) 0.039
rs12669805 c
7 79509055 0.188 0.253 3.32E-05 0.024 3.42 (1.56-7.48) 0.002
rs6092092 c
20 53291685 0.076 0.249 4.28E-05 0.006 7.74 (1.85-32.30) 0.005
rs2833537 21 32135333 0.074 0.254 3.03E-05 0.036 0.40 (0.20-0.79) 0.008
rs7620841 c
3 187425014 0.06 0.242 6.77E-05 0.052 1.80 (1.12-2.89) 0.015
rs1921626 c
2 77025574 0.205 0.238 9.00E-05 0.084 1.57 (1.09-2.26) 0.015
rs17043088 12 76103718 0.104 -0.238 8.88E-05 0.026 2.21 (1.16-4.22) 0.016
rs753921 c
4 35579894 0.231 0.239 8.69E-05 0.274 1.31 (1.05-1.64) 0.018
rs7618373 3 177819713 0.446 -0.255 3.17E-05 0.378 1.27 (1.02-1.57) 0.03
rs5916072 23 5406196 0.343 0.247 5.04E-05 0.442 0.84 (0.70-1.00) 0.046
rs10517364 c
4 35562853 0.246 0.258 2.12E-05 0.31 1.23 (1.00-1.52) 0.05
Table 2. The 19 SNPs with the lowest unadjusted P values that were associated with the overall survival of lung
cancer patients treated with platinum compounds.
SNP Chr. SNP position
LCL GWAS
a R values represent correlation coefficients for the associations between SNPs and cisplatin IC50.
b HR, hazard ratios; if HR > 1, patients carrying the minor allele had worse overall survival.
c SNP showed a concordant association with the IC50 and OS phenotype.
Clinical Study
Among NSCLC patients
Among SCLC patients
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
Gene SNP Distance from the
SNP (< 200 kb)
Expression associated
with the SNP in LCLs
R value P value
DAZAP2 rs11169748 Yes NAa NA
TFCP2 rs11169748 Yes NA NA
LETMD1 rs11169748 Yes NA NA
GALNT6 rs11169748 Yes NA NA
DAPK3 rs11169748 Yes 0.293 1.05E-06
EML3 rs11169748 Yes 0.31 2.22E-07
LRIG1 rs11169748 Yes 0.273 5.66E-06
NENF rs11169748 Yes 0.292 1.15E-06
RUFY1 rs11169748 Yes 0.301 4.92E-07
CCDC6 rs2440915 Yes NA NA
METTL6 rs2440915 Yes 0.267 9.64E-06
FMR1 rs5952066 Yes NA NA
ELOVL5 rs5952066 Yes 0.278 3.75E-06
METTL9 rs5952066 0.250b 3.57E-05
0.261 1.53E-05
ETV5 rs7620841 Yes NA NA
Table 3. Fifteen candidate genes selected for functional validation.
a The gene is located within 200 kb up or downstream of the SNP, and no cis-regulation was identified.
NA, no association between the gene and SNP.b Two probesets were associated with this SNP.
Basis for selection SNP vs. expression in LCLs
Yes
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133
Published OnlineFirst July 20, 2011.Clin Cancer Res Xiang-Lin Tan, Ann M. Moyer, Brooke L. Fridley, et al. platinum-based chemotherapywith overall survival in lung cancer patients receiving Genetic variation predicting cisplatin cytotoxicity associated
Updated version
10.1158/1078-0432.CCR-11-1133doi:
Access the most recent version of this article at:
Material
Supplementary
http://clincancerres.aacrjournals.org/content/suppl/2011/07/20/1078-0432.CCR-11-1133.DC1
Access the most recent supplemental material at:
Manuscript
Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://clincancerres.aacrjournals.org/content/early/2011/07/20/1078-0432.CCR-11-1133To request permission to re-use all or part of this article, use this link
Research. on May 20, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1133