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Molecular Biology ReportsAn International Journal on Molecularand Cellular Biology ISSN 0301-4851 Mol Biol RepDOI 10.1007/s11033-014-3197-3

Thymidylate synthase polymorphismsare associated to therapeutic outcomeof advanced non-small cell lung cancerpatients treated with platinum-basedchemotherapyAurea Lima, Vítor Seabra, SandraMartins, Ana Coelho, António Araújo &Rui Medeiros

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Thymidylate synthase polymorphisms are associated to therapeuticoutcome of advanced non-small cell lung cancer patients treatedwith platinum-based chemotherapy

Aurea Lima • Vıtor Seabra • Sandra Martins •

Ana Coelho • Antonio Araujo • Rui Medeiros

Received: 9 January 2013 / Accepted: 24 January 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Thymidylate synthase (TYMS) has three poly-

morphisms that may modulate thymidylate synthase (TS)

expression levels: (1) 28 base pairs (bp) variable number

tandem repeat (VNTR) (rs34743033); (2) single nucleotide

polymorphism (SNP) C[G at the twelfth nucleotide of the

second repeat of 3R allele (rs2853542); and (3) 6 bp

sequence deletion (1494del6, rs34489327). This study was

conducted to evaluate the influence of TYMS polymor-

phisms on the survival of Portuguese patients with

advanced non-small cell lung cancer (NSCLC) undergoing

platinum-based chemotherapy. Our results showed no sta-

tistically significant differences between VNTR genotypes;

although, considering the SNP C[G, homozygotes 3RG

presented a better prognostic at 36 months (p = 0.004) and

overall survival (p = 0.003) when compared to 2R3RG

patients. Patients with ‘‘median/high expression geno-

types’’ demonstrated a better survival at 12 months

(p = 0.041) when compared to ‘‘low expression

genotypes’’. Furthermore, 6 bp- carriers (p = 0.006)

showed a better survival at 12 months when compared to

6 bp? homozygotes patients. When analyzing TYMS hap-

lotypes, better survival at 12 months was observed for

patients carrying haplotypes with the 6 bp- allele

(2R6 bp-; p = 0.026 and 3RG6 bp-; p = 0.045). This is

the first report that evaluates the three major TYMS poly-

morphisms in the therapeutic outcome of NSCLC in Por-

tugal. According to our results, the TYMS polymorphisms

may be useful tools to predict which advanced NSCLC

patients could benefit more from platinum-based chemo-

therapy regimens.

Keywords NSCLC � Platinum-based chemotherapy �Polymorphisms � Therapeutic outcome � Thymidylate

synthase

A. Lima (&) � V. Seabra

IINFACTS/CESPU, Institute of Research and Advanced

Training in Health Sciences and Technologies, Department of

Pharmaceutical Sciences, Higher Institute of Health Sciences

(ISCS-N), Rua Central de Gandra 1317, 4585-116 Gandra PRD,

Portugal

e-mail: aurea.lima@iscsn.cespu.pt;

aurealima1010@hotmail.com

A. Lima � A. Coelho � A. Araujo � R. Medeiros

Molecular Oncology Group CI, Portuguese Institute of Oncology

of Porto (IPO-Porto), Rua Dr. Antonio Bernardino de Almeida,

4200-072 Porto, Portugal

A. Lima � R. Medeiros

Abel Salazar Institute for the Biomedical Sciences (ICBAS),

University of Porto, Rua de Jorge Viterbo Ferreira 228,

4050-313 Porto, Portugal

S. Martins

Institute of Molecular Pathology and Immunology of the

University of Porto (IPATIMUP), Rua Dr. Roberto Frias,

4200-465 Porto, Portugal

A. Coelho

Faculty of Medicine of University of Porto (FMUP), Al. Prof.

Hernani Monteiro, 4200-319 Porto, Portugal

A. Araujo

Medical Oncology Department, Portuguese Institute of

Oncology of Porto (IPO-Porto), Rua Dr. Antonio Bernardino de

Almeida, 4200-072 Porto, Portugal

R. Medeiros

Research Department, Portuguese League Against Cancer

(LPCC-NRNorte), Estrada Interior da Circunvalacao, 6657,

4200-177 Porto, Portugal

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DOI 10.1007/s11033-014-3197-3

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Introduction

Lung cancer is the most common type of cancer in Europe

[1] and non-small cell lung cancer (NSCLC) accounts for

75–85 % of all histological types. The high mortality rate

(80–85 % within 5 years) results from the lack of effective

screening tools allowing for early-stage diagnosis [2]; the

inability to identify subsets of patients that would benefit

from adjuvant chemotherapy (CT) or adjuvant targeted

therapies; and the slow development of new drug therapies.

More than 70 % of NSCLC patients are diagnosed with

advanced disease and, therefore, are good candidates for

neoadjuvant, adjuvant or palliative systemic treatment with

platinum-based CT [3]. Although the use of cisplatin or

carboplatin has proved to be effective in combination with

non-platinum CT agents, such as paclitaxel or gemcitabine,

considerable variation has been observed in response to

treatment [3]. Pharmacogenetics strives to identify genetic

variations that could be useful in treatment prediction and

has become an important field in cancer treatment.

Recently, genetic polymorphisms have been suggested to

alter drug metabolism and activity leading to differences in

toxicity and/or efficacy on patients’ treatment [4].

Human thymidylate synthase (TS) is a key enzyme in de

novo synthesis of 20-deoxythymidine-50-monophosphate

(dTMP), an essential precursor of deoxyribonucleic acid

(DNA) biosynthesis and important factor to DNA replica-

tion and repair [5]. Inhibition of TS leads to depletion of

dTMP, which contributes for the incorporation of uracil

into DNA leading to chromosome instability [5–7].

Moreover, as TS catalyzes the methylation of 20-deoxyur-

idine-50-monophosphate (dUMP) to dTMP, using 5,10-

methylene-tetrahydrofolate (MTHF), it is important for

several chemotherapeutics [5, 6, 8]. Literature has descri-

bed that three polymorphisms (rs34743033, rs2853542 and

rs34489327) in the untranslated regions (UTRs) of the

thymidylate synthase (TYMS, 18p11.32) seem to influence

TS expression levels: (1) in the TYMS enhancer region

(TSER) there is a variable number tandem repeat (VNTR)

(rs34743033) upstream the ATG initiation codon site [9].

Humans have, most frequently, two and three repeats (2R

and 3R alleles, respectively), with 3R alleles associated to

higher TS expression and translation efficiency when

compared to 2R alleles [7, 10–15]. The 28 base pairs (bp)

repeated element appears to function as a preferential

enhancer linkage site and, therefore, influences TYMS

transcription and TS expression; (2) a functional C[G

single nucleotide polymorphism (SNP) (rs2853542) was

found in the second repeat of 3R alleles (3RC vs. 3RG)

[15]. This SNP changes an upstream stimulatory factor

(USF) binding site, with 3RG alleles allowing its ability to

complex with the USF protein. In vitro, a similar tran-

scriptional activity and translation efficiency has been

found to 3RC and 2R alleles [15–17]; (3) TYMS 1494del6

(rs34489327) is a deletion/insertion polymorphism (DIP)

of 6 bp (CTTTAA) located at nucleotide 1494 in the 30-UTR. Previous in vitro studies suggested that this poly-

morphism is associated with decreased messenger ribonu-

cleic acid (mRNA) stability and lower intratumoral TS

expression [15, 17]. Literature has suggested that TYMS

1494del6 polymorphism is in linkage disequilibrium (LD)

with the TSER polymorphisms (VNTR and SNP) and,

therefore, it has been hypothesized that TYMS polymor-

phisms might have an impact on the efficacy of CT treat-

ment, influencing the overall survival of NSCLC patients

[5, 18, 19]. To evaluate the clinical usefulness of geno-

typing TYMS as a prognostic marker of response to plati-

num-based CT treatment, we analyzed a subset of

advanced NSCLC patients. Moreover, we performed a

haplotype analysis, including all three polymorphisms, in

order to determine if an association that more effectively

predicts clinical outcomes is present, for combined che-

motherapy with platinum plus non-platinum drugs.

Materials and methods

Subjects

In this study, a total of 130 consecutive patients from the

northern region of Portugal with advanced NSCLC (stages

IIIA, IIIB and IV) treated with platinum-based CT were

included without restrictions of age, sex, smoking status or

disease history. All patients included in the study were

Table 1 NSCLC patients variables included in the study

Variable Value

Gender

Male, n (%) 103 (79)

Female, n (%) 27 (21)

Age (years)

Mean ± SD 62.88 ± 9.48

Median 64.00

Smoking status

Current and former, n (%) 100 (77)

Never, n (%) 30 (23)

Tumor stage

III, n (%) 92 (71)

IV, n (%) 38 (29)

Histological type

Squamous cell carcinoma, n (%) 47 (36)

Adenocarcinoma, n (%) 60 (46)

Others, n (%) 23 (18)

NSCLC non-small cell lung cancer

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referred for treatment with platinum derivatives according

to the guidelines of treatments at the date of enrollment.

Clinical stage and pathologic evaluation were classified

according to the International System for Staging Lung

Cancer [20, 21]. Gender, age, smoking status, clinical stage

and histological classification of patients are described in

Table 1.

Prospective clinical follow-up time was available for

patients and defined as the time elapsed between diagnosis

and the last clinical evaluation or patient death. Procedures

were in accordance with the ethical standards of the Hel-

sinki Declaration, the study was approved by the Institu-

tional Ethical Committee and the subjects were included

after providing signed informed consent.

Blood samples were obtained with standard venipunc-

ture technique using ethylenediaminetetraacetic acid

(EDTA) containing tubes and genomic DNA extracted

using a commercial kit (Quiagen� QIAamp� DNA Blood

Mini Kit), according to the manufacturer instructions.

TYMS genotyping

The three polymorphisms (rs34743033, rs2853542 and

rs34489327) in the TYMS untranslated regions were gen-

otyped as following: (1) 28 bp VNTR was amplified by

polymerase chain reaction (PCR) as previously described

by Kawakami and Omura [13]; (2) SNP C[G at the twelfth

nucleotide of the second repeat of 3R allele was genotyped

by PCR-restriction fragment length polymorphism (RFLP)

as previously described by Kawakami and Watanabe [16];

(3) TYMS 1494del6 was genotyped by PCR–RFLP as

previously described by Ulrich and Bigler [17]. For quality

control, 10 % of all TYMS genotypes were confirmed by

automated sequencing in a 3130xl Genetic Analyzer using

the Kit BigDye Terminator v3.1 (Applied� Biosystems)

and results were 100 % concordant.

Statistical analysis

Statistical analyses were performed with the SPSS� soft-

ware (Version 15.0 of 2006, LEAD Technologies�, Inc.;

Chicago). Differences on TYMS genotypes were calculated

by the v2 test with a 5 % statistical significance (p \ 0.05).

To estimates the LD between pairs of alleles at TSER and

TYMS 1494del6 loci, D0 coefficients were calculated in Ar-

lequin 3.11 [22] with 100,000 number of steps in Markov

chain. The measure is interpretable as the proportion of the

maximum possible level of association between two loci,

given the allele frequencies, ranging from 0 (linkage equi-

librium) to 1 (complete LD) [23].

Hazard ratios (HRs) with 95 % confidence intervals

(CIs) were calculated. Genotypes were analyzed as a three-

group categorical variable (codominant model), and they

were also grouped according to the recessive model. TSER

genotypes were classify according to their theoretical TS

functional status as described previously (‘‘low’’: 2R2R,

2R3RC and 3RC3RC, ‘‘median’’: 2R3RG and 3RC3RG

and ‘‘high’’: 3RG3RG expression genotypes) [5, 19, 24].

For the haplotype analysis, allelic phase of genotyped

polymorphisms was inferred by PHASE 2.1 [25].

The influence of the different TYMS genotypes on

patients’ outcome was compared for 12, 36 months and

overall survival (OS) of the patients.

Survival curves were estimated by using the Kaplan–

Meier method. Differences between individual curves were

evaluated by multivariate analyses using Cox proportional

hazards regression models adjusted for disease stage and

NSCLC histological type. For this analysis, survival time

was defined as the time between diagnosis and an event

(either the last clinical evaluation or patient death).

Results

TSER genotypes

Observed genotype frequencies for rs34743033 and

rs2853542 polymorphisms are shown in Table 2. Survival

analysis showed no differences among patients carrying

different VNTR genotypes at 12, 36 months and for OS in

both codominant and recessive models. SNP genotyping of

3R alleles did not provide additional prognostic informa-

tion. Cox regression was not computed for functional 3R at

12 months because all patients in the reference group

(2R3R) were alive at that time. For functional 3R analysis,

3RG homozygotes presented a better prognosis when

compared to 2R3RG patients at both 36 months survival

(HR = 0.03; 95 % CI 0.00–0.34, p = 0.004) and for OS

(HR = 0.03; 95 % CI 0.00–0.31, p = 0.003). Finally,

classification of alleles according to their theoretical TS

functional status has suggested that patients with ‘‘median/

high expression genotypes’’ have a better prognosis than

those with ‘‘low expression genotypes’’, with survival at

12 months (p = 0.041) as shown in Fig. 1.

TYMS 1494del6 genotypes

TYMS 1494del6 observed genotype frequencies are pre-

sented in Table 2. By using a codominant model, a poor

survival rate at 12 months was observed for 6 bp?

homozygotes when compared to 6 bp?6 bp- patients

(p = 0.020) (Fig. 2A). The presence of the 6 bp- allele

was associated with a reduced risk of death. In fact, at

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12 months no deaths were observed in all the 6 bp-

homozygotes. The Cox regression between 6 bp? homo-

zygotes and 6 bp- homozygotes was not computed

because all 6 bp- homozygotes patients were alive at

12 months. In the recessive model, survival curves at

12 months suggested that 6 bp- carriers were associated

with a better prognosis (p = 0.006), as shown in Fig. 2B.

No statistically significant differences were observed at

36 months and for OS in the analyzed models.

Linkage disequilibrium and haplotype analysis

TSER and TYMS 1494del6 polymorphisms are in LD in our

population (p = 0.00058). Alleles 2R and 6 bp?

(D0 = 0.43) as well as 3RG and 6 bp- (D0 = 0.45) are the

most linked ones.

When analyzing TSER-TYMS 1494del6 haplotypes,

different survivals were observed among patients carrying

(1) the 2R6 bp? and 2R6 bp- haplotypes (p = 0.026) and

Table 2 Multivariate analysis of TSER and TYMS 1494del6 polymorphisms in NSCLC patients

Genotype N (%) Survival at 12 months Survival at 36 months Overall survival

HR 95 % CI p* HR 95 % CI p* HR 95 % CI p*

VNTR genotypes (n = 130)

Codominant model

2R2R 26 (20) 1.00 1.00 1.00

2R3R 63 (48) 1.67 (0.45–6.14) 0.445 1.67 (0.66–4.24) 0.275 1.10 (0.50–2.41) 0.812

3R3R 41 (32) 1.05 (0.29–3.76) 0.938 0.98 (0.47–2.34) 0.958 0.78 (0.36–1.67) 0.517

Alleles grouped as a recessive model

2R carriers* 89 (69) 1.00 1.00 1.00

3R3R 41 (31) 0.74 (0.26–2.05) 0.560 0.68 (0.35–1.35) 0.277 0.66 (0.37–1.14) 0.137

TSER genotypes (n = 130)

Functional 2R (n = 81)

2R2R 26 (32) 1.00 1.00 1.00

2R3RC 40 (49) 1.05 (0.29–3.82) 0.942 1.24 (0.48–3.15) 0.657 0.93 (0.41–2.09) 0.856

3RC3RC 15 (19) 0.79 (0.17–3.61) 0.763 0.86 (0.30–2.51) 0.786 0.91 (0.34–2.41) 0.847

Funcional 3R (n = 49)

2R3RG 23 (47) 1.00a 1.00 1.00

3RC3RG 21 (43) 0.50 (0.08–3.15) 0.463 0.33 (0.10–1.09) 0.069

3RG3RG 5 (10) 0.03 (0.00–0.34) 0.004 0.03 (0.00–0.31) 0.003

Alleles grouped according to the functional status** (n = 130)

Low expression 81 (62) 1.00 1.00 1.00

Median/High expression 49 (38) 0.32 (0.09–1.15) 0.041 0.59 (0.28–1.24) 0.067 0.86 (0.49–1.51) 0.605

TYMS 1494del6 genotypes (n = 130)

Codominant model

6 bp?6 bp? 60 (46) 1.00 1.00 1.00

6 bp?6 bp- 54 (42) 0.17 (0.04–0.75) 0.020 0.61 (0.30–1.24) 0.170 0.71 (0.39–1.28) 0.259

6 bp-6 bp- 16 (12) b 0.49 (0.14–1.65) 0.251 0.78 (0.32–1.91) 0.583

Recessive model

6 bp?6 bp? 60 (46) 1.00 1.00 1.00

6 bp- carriers*** 70 (54) 0.12 (0.03–0.55) 0.006 0.59 (0.30–1.15) 0.121 0.74 (0.43–1.28) 0.278

CI confidence interval, HR hazard ratio, NSCLC non-small cell lung cancer, SNP single nucleotide polymorphism, TSER thymidylate synthase

enhancer region, TYMS thymidylate synthase (gene), VNTR variable number tandem repeat

* 2R carriers include homozygotes 2R and heterozygotes 2R3R. ** Low expression genotypes are 2R2R, 2R3RC and 3RC3RC. Median

expression genotypes are 2R3RG and 3RC3RG. High expression genotype is 3RG3RG. *** 6 bp- carriers include homozygotes 6 bp- and

heterozygous 6 bp?6 bp-

p* values correspond to multivariate Cox models adjusted for disease stage and to histological type of NSCLCa Cox regression was not computed because all patients in the reference group (2R3RG) were alive at 12 monthsb Cox regression was not computed because all 6 bp-6 bp- patients were alive at 12 months

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(2) the 2R6 bp? and 3RG6 bp- haplotypes (p = 0.045)

(Table 3; Fig. 3). In addition, a better prognosis was

observed at 12 months for patients carrying haplotypes

with the 6 bp- allele.

Discussion

TS is an essential enzyme for DNA biosynthesis, replica-

tion and repair, hence, it is an important target for CT drugs

and its over-expression has been described as associated

with CT resistance in cancer [6–8]. Currently, despite the

large number of pharmacogenomics studies, there is no

available protocol for selecting cancer patients at risk for

drug resistance prior to CT.

Three polymorphisms (rs34743033, rs2853542 and

rs34489327) on TYMS have been shown to influence TS

expression levels and CT response [26, 27] although results

have not been consistent [28]. Considering the influence of

Fig. 1 Kaplan–Meier plots according to the TS functional status at

12 months. p values correspond to multivariate Cox models adjusted

for disease stage and to histological type of NSCLC

Fig. 2 Kaplan–Meier plots for

NSCLC patients according to

their TYMS 1494del6 genotypes

with survival at 12 months, as a

codominant (A) and recessive

model (B). p values correspond

to multivariate Cox models

adjusted for disease stage and to

histological type of NSCLC

Table 3 Multivariate analysis for TSER-TYMS 1494del6 haplotypes of NSCLC patients

Frequency Survival at 12 months Survival at 36 months Overall survival

N (%) HR 95 % CI p* HR 95 % CI p* HR 95 % CI p*

TSER-TYMS 1494del6 haplotype (n = 130)

2R6 bp? 37 (28) 1.00 1.00 1.00

3RC6 bp? 32 (25) 0.82 (0.37–1.83) 0.629 0.87 (0.47–1.62) 0.668 0.92 (0.55–1.56) 0.769

3RG6 bp? 19 (15) 0.31 (0.65–1.45) 0.135 0.56 (0.20–1.59) 0.278 0.73 (0.32–1.68) 0.462

2R6 bp- 22 (17) 0.23 (0.06–0.84) 0.026 0.55 (0.25–1.20) 0.133 0.55 (0.28–1.08) 0.082

3RC6 bp- 12 (9) 0.24 (0.03–1.85) 0.171 0.64 (0.25–1.62) 0.343 0.79 (0.39–1.61) 0.519

3RG6 bp- 8 (6) 0.12 (0.02–0.95) 0.045 0.54 (0.23–1.25) 0.151 0.69 (0.36–1.34) 0.276

bp base pairs, CI confidence interval, HR hazard ratio, NSCLC non-small cell lung cancer, TSER thymidylate synthase enhancer region, TYMS

thymidylate synthase (gene)

* p values correspond to multivariate Cox models adjusted for disease stage and to histological type of NSCLC

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these polymorphisms on TS levels, it is extremely impor-

tant to study their role in NSCLC, since the degree and

duration of TS inhibition with CT drugs may depend on its

expression levels and, therefore, may influence patients’

response.

TSER genotypes

TSER polymorphisms have been shown to influence the total

amount of active protein, although significant individual

variability is observed [29]. Preliminary data suggested

that 3R homozygotes patients have higher TS mRNA

expression than those homozygotes for 2R allele [12, 30].

Moreover, a poor response to CT is observed in patients with

3R allele [26, 27]. Although published results have not been

consistent, studies have shown that patients with low TS

levels seem to have a worse outcome when treated with

adjuvant CT, probably due to the high incidence of adverse

drug reactions associated with 2R allele, that, consequently,

can lead to poor survival [28, 31]. These inconsistent results

might be explained by the variation of TYMS gene copy

number due to loss of heterozygosis or gene amplification as

showed by others [32–36]. In our study, results showed no

significant differences among patients carrying different

VNTR genotypes at 12, 36 months survival and OS, in both

codominant and recessive models, similarly to the results

shown by Lecomte and Ferraz [18].

To the best of our knowledge, the putative relationship

between the VNTR and the survival time needs further

clarification. Some authors suggested that this could be

improved with the study of the SNP C[G at the second

repeat of the 3R allele, since it is reported that the presence

of 3RG allele is associated with higher transcriptional

activity and translation efficiency due to an increased

ability of this allele to complex with the USF protein [15,

16]. In our study, 3RG homozygotes showed better sur-

vival, corroborating the results of Edler et al. [28]. These

results underpin the importance of 3RG allele SNP’s on TS

functional role. On the other hand, since there is no dif-

ference on survival analysis between patients with 2R3RG

and 3RC3RG genotypes, these results are in accordance

with in vitro studies were a similar transcriptional activity

and translation efficiency was found for those 2R and 3RC

alleles [16]. Moreover, and considering the theoretical

functional status of TS, accordingly with the possible

genotypes for the 50-UTR, our results indicated that

patients with ‘‘median/high expression genotypes’’ have a

better prognosis that those with ‘‘low expression geno-

types’’ (Table 2; Fig. 1). In agreement with others [29, 37]

these results may be explained by the influence of 3RG

allele, since homozygotes 3RG have two 3RG alleles in

comparison to 2R3RG and 3RC3RG patients, both with

only one 3RG allele. However, different results regarding

NSCLC patients treated with pemetrexed were reported,

showing longer progression free survival times for patients

with ‘‘low expression genotypes’’ [38, 39]. According with

these results, it is important to elucidate de influence of

TSER genotypes on the TS target CT outcome.

TYMS 1494del6 genotypes

Another polymorphism described on TYMS consists in a

6 bp DIP in the 30-UTR [17]. Although the function of this

polymorphism is not entirely known, there are evidences

suggesting that the deletion is associated with a decrease in

TS mRNA stability and expression [17, 40, 41]. Similarly

to previous studies, our results suggested that the 6 bp-

allele is associated with a better prognosis [19]. At

12 months, for both models, a better survival time was

observed for 6 bp?6 bp- patients when compared to

6 bp? homozygotes; no deaths were observed in the all

6 bp- homozygotes at that time; and survival curves at

12 months suggested that 6 bp- allele was associated with

a better prognosis. Therefore, it seems that 6 bp- allele is

important to predict the survival time of NSCLC patients.

Fig. 3 Kaplan–Meier plots to

TSER-TYMS 1494del6

haplotypes at 12 months.

A 2R6 bp? and 2R6 bp-

haplotypes. B 2R6 bp? and

3RG6 bp- haplotypes. p values

correspond to multivariate Cox

models adjusted for disease

stage and to histological type of

NSCLC

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Moreover, recent studies reported similar results in NSCLC

patients treated with pemetrexed [39].

Linkage disequilibrium and haplotype analysis

Our study has confirmed that TYMS polymorphisms were in

LD as suggested by others [5, 18, 19, 40, 42, 43]. The associ-

ation was higher with haplotypes harboring the 6 bp- allele,

suggesting a prominent role of the 30-UTR polymorphism in

predicting the prognosis of advanced NSCLC patients. At

12 months, our results suggested that the haplotypes with the

6 bp- allele were associated with a better response to CT,

better prognostic and, consequently, reduced risk of death, as

previously reported in other populations [19, 44, 45].

From the haplotype analysis we can infer that 2R6 bp-

and 3RC6 bp- haplotypes were different when survival at

12 months was considered (Table 3; Fig. 3). This can be

explained by the presence of the 6 bp- allele which can

interact differently with 2R and 3RC alleles, in agreement

with Lurje and Zhang [24]. Thus, our results demonstrated

the importance of locus 6 bp- in a better prognosis and

suggested that TYMS haplotypes analysis needs to be

considered in the evaluation of TYMS polymorphisms on

NSCLC therapeutic response.

Final conclusions

In this study we have addressed the possible role of three

TYMS polymorphisms in the prognosis of Portuguese patients

with advanced NSCLC undergoing platinum-based CT regi-

mens. As a result, we have attempted to establish if TYMS

polymorphisms, using genotype and haplotype-based

approaches, lead to differences in clinical outcome of patients.

This is the first report that evaluates the three major TYMS

polymorphisms related with TS expression in therapeutic

outcome of a NSCLC Portuguese population. According to

our results, the genotyping of TYMS polymorphisms may be a

useful tool to predict which advanced NSCLC patients could

benefit more from platinum-based CT regimens and also

emphasize the importance of analyzing patients’ TYMS

haplotypes. Nevertheless, the clinical utility of analysing

3RG6 bp- and 2R6 bp- haplotypes deserves further eval-

uation, as more work is necessary before coming to a positive

conclusion. Although, this is a matter of controversy and some

caution is required when translating this approach into the

clinic, in order to optimise and individualize therapeutic

options as an approach to predict prognosis and therapy

outcomes.

Acknowledgments The authors wish to acknowledge the Ministry

of Health of Portugal (CFICS-Project 31/2007) and Astrazeneca

Foundation for the financial support; Liga Portuguesa Contra o

Cancro—Centro Regional do Norte (Portuguese League Against

Cancer) for the support to the lab; and to Fundacao para a Ciencia e

Tecnologia (FCT) for the Doctoral Grant (SFRH/BD/64441/2009) for

Aurea Lima. Authors would also like to acknowledge Hugo Sousa

(Ph.D.) for his critics in the final version of the manuscript.

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