Thymidylate synthase polymorphisms are associated

Thymidylate synthase polymorphisms are associated to therapeutic outcome of advanced non-small cell lung cancer patients treated with platinum-based chemotherapy Aurea Lima, Vítor Seabra, Sandra Martins, Ana Coelho, António Araújo & Rui Medeiros Molecular Biology Reports An International Journal on Molecular and Cellular Biology ISSN 0301-4851 Mol Biol Rep DOI 10.1007/s11033-014-3197-3

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Author's personal copy Mol Biol Rep DOI 10.1007/s11033-014-3197-3

Thymidylate synthase polymorphisms are associated to therapeutic outcome of advanced non-small cell lung cancer patients treated with platinum-based chemotherapy Aurea Lima • Vı´tor Seabra • Sandra Martins • Ana Coelho • Antońio Arau´jo • Rui Medeiros

Received: 9 January 2013 / Accepted: 24 January 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Thymidylate synthase (TYMS) has three polymorphisms 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 polymorphisms on the survival of Portuguese patients with advanced non-small cell lung cancer (NSCLC) undergoing platinum-based chemotherapy. Our results showed no statistically 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 genotypes’’ 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 haplotypes, better survival at 12 months was observed for patients carrying haplotypes with the 6 bpallele (2R6 bp-; p = 0.026 and 3RG6 bp-; p = 0.045). This is the first report that evaluates the three major TYMS polymorphisms in the therapeutic outcome of NSCLC in Portugal. According to our results, the TYMS polymorphisms may be useful tools to predict which advanced NSCLC patients could benefit more from platinum-based chemotherapy 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: [email protected]; [email protected]

S. Martins Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Rua Dr. Roberto Frias, 4200-465 Porto, Portugal

A. Lima A. Coelho A. Arau´jo R. Medeiros Molecular Oncology Group CI, Portuguese Institute of Oncology of Porto (IPO-Porto), Rua Dr. Antońio Bernardino de Almeida, 4200-072 Porto, Portugal

A. Arau´jo Medical Oncology Department, Portuguese Institute of Oncology of Porto (IPO-Porto), Rua Dr. Antońio 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

R. Medeiros Research Department, Portuguese League Against Cancer (LPCC-NRNorte), Estrada Interior da Circunvalaçaõ, 6657, 4200-177 Porto, Portugal

A. Coelho Faculty of Medicine of University of Porto (FMUP), Al. Prof. Hernaˆni Monteiro, 4200-319 Porto, Portugal

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Author's personal copy Mol Biol Rep

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 replication 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 -deoxyuridine-50 -monophosphate (dUMP) to dTMP, using 5,10methylene-tetrahydrofolate (MTHF), it is important for several chemotherapeutics [5, 6, 8]. Literature has described 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 transcriptional activity and translation efficiency has been

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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 (%) IV, n (%)

92 (71) 38 (29)

Histological type Squamous cell carcinoma, n (%)

47 (36)

Adenocarcinoma, n (%)

60 (46)

Others, n (%)

23 (18)

NSCLC non-small cell lung cancer

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 polymorphism is associated with decreased messenger ribonucleic 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 polymorphisms might have an impact on the efficacy of CT treatment, influencing the overall survival of NSCLC patients [5, 18, 19]. To evaluate the clinical usefulness of genotyping TYMS as a prognostic marker of response to platinum-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 chemotherapy 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


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 Helsinki Declaration, the study was approved by the Institutional Ethical Committee and the subjects were included after providing signed informed consent. Blood samples were obtained with standard venipuncture technique using ethylenediaminetetraacetic acid (EDTA) containing tubes and genomic DNA extracted using a commercial kit (QuiagenÒ QIAampÒ DNA Blood Mini Kit), according to the manufacturer instructions. TYMS genotyping The three polymorphisms (rs34743033, rs2853542 and rs34489327) in the TYMS untranslated regions were genotyped 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 (AppliedÒ Biosystems) and results were 100 % concordant. Statistical analysis Statistical analyses were performed with the SPSSÒ software (Version 15.0 of 2006, LEAD TechnologiesÒ, 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 Arlequin 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 equilibrium) 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 information. 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 presented 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 bpallele was associated with a reduced risk of death. In fact, at

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Author's personal copy Mol Biol Rep Table 2 Multivariate analysis of TSER and TYMS 1494del6 polymorphisms in NSCLC patients Genotype

N (%)

Survival at 12 months HR

95 % CI

Survival at 36 months p*

HR

95 % CI

Overall survival 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

89 (69) 41 (31)

1.00 0.74

(0.26–2.05)

0.560

1.00 0.68

(0.35–1.35)

0.277

1.00 0.66

(0.37–1.14)

0.137

2R2R

26 (32)

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

2R3RG

23 (47)

1.00a

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

(0.49–1.51)

0.605

Alleles grouped as a recessive model 2R carriers* 3R3R TSER genotypes (n = 130) Functional 2R (n = 81) 1.00

1.00

Funcional 3R (n = 49) 1.00

1.00

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

81 (62)

1.00

Median/High expression

49 (38)

0.32

1.00 (0.09–1.15)

0.041

(0.04–0.75)

0.020

0.59

1.00 (0.28–1.24)

0.067

0.86

0.61

(0.30–1.24)

0.170

0.71

(0.39–1.28)

0.259

0.49

(0.14–1.65)

0.251

0.78

(0.32–1.91)

0.583

(0.30–1.15)

0.121

(0.43–1.28)

0.278

TYMS 1494del6 genotypes (n = 130) Codominant model 6 bp?6 bp?

60 (46)

1.00

54 (42)

0.17

16 (12)

b

6 bp?6 bp?

60 (46)

1.00

6 bp- carriers***

70 (54)

0.12

6 bp?6 bp6 bp-6 bp-

1.00

1.00

Recessive model 1.00 (0.03–0.55)

0.006

0.59

1.00 0.74

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

Cox regression was not computed because all patients in the reference group (2R3RG) were alive at 12 months

b

Cox regression was not computed because all 6 bp-6 bp- patients were alive at 12 months

12 months no deaths were observed in all the 6 bphomozygotes. The Cox regression between 6 bp? homozygotes and 6 bphomozygotes was not computed because all 6 bphomozygotes 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.

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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 bphaplotypes (p = 0.026) and


(2) the 2R6 bp? and 3RG6 bphaplotypes (p = 0.045) (Table 3; Fig. 3). In addition, a better prognosis was observed at 12 months for patients carrying haplotypes with the 6 bpallele.

Discussion

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

TS is an essential enzyme for DNA biosynthesis, replication 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. 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

N (%)

HR

95 % CI

Survival at 36 months p*

HR

Overall survival

95 % CI

p*

HR

95 % CI

p*

TSER-TYMS 1494del6 haplotype (n = 130) 2R6 bp?

37 (28)

1.00

3RC6 bp?

32 (25)

0.82

(0.37–1.83)

0.629

1.00 0.87

(0.47–1.62)

0.668

1.00 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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Author's personal copy Mol Biol Rep Fig. 3 Kaplan–Meier plots to TSER-TYMS 1494del6 haplotypes at 12 months. A 2R6 bp? and 2R6 bphaplotypes. B 2R6 bp? and 3RG6 bphaplotypes. p values correspond to multivariate Cox models adjusted for disease stage and to histological type of NSCLC

these polymorphisms on TS levels, it is extremely important 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 survival, corroborating the results of Edler et al. [28]. These results underpin the importance of 3RG allele SNP’s on TS

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functional role. On the other hand, since there is no difference 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 genotypes’’ (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 bpallele 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 bphomozygotes at that time; and survival curves at 12 months suggested that 6 bpallele was associated with a better prognosis. Therefore, it seems that 6 bpallele is important to predict the survival time of NSCLC patients.


Moreover, recent studies reported similar results in NSCLC patients treated with pemetrexed [39].

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.

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 association was higher with haplotypes harboring the 6 bpallele, 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 bpallele 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 bpand 3RC6 bphaplotypes were different when survival at 12 months was considered (Table 3; Fig. 3). This can be explained by the presence of the 6 bpallele 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 regimens. 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 bphaplotypes deserves further evaluation, 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 Fundaçaõ para a Cieˆncia e

References 1. Alberg AJ, Brock MV, Samet JM (2005) Epidemiology of lung cancer: looking to the future. J Clin Oncol 23(14):3175–3185. doi:10.1200/JCO.2005.10.462 2. Beane J, Spira A, Lenburg ME (2009) Clinical impact of highthroughput gene expression studies in lung cancer. J Thorac Oncol 4(1):109–118. doi:10.1097/JTO.0b013e31819151f8 3. National Comprehensive Cancer Network (2012) NCCN clinical practice guidelines in oncology (NCCN guidelines)—non-small cell lung cancer. Version 2. http://www.tri-kobe.org/nccn/guide line/lung/english/small.pdf. Accessed 29 April 2013 4. Watters JW, McLeod HL (2003) Cancer pharmacogenomics: current and future applications. Biochim Biophys Acta 1603(2):99–111. doi:10.1016/S0304-419X(03)00003-9 5. Lima A, Azevedo R, Sousa H, Seabra V, Medeiros R (2013) Current approaches for TYMS polymorphisms and their importance in molecular epidemiology and pharmacogenetics. Pharmacogenomics 14(11):1337–1351. doi:10.2217/pgs.13.118 6. Carreras CW, Santi DV (1995) The catalytic mechanism and structure of thymidylate synthase. Annu Rev Biochem 64:721–762. doi:10.1146/annurev.bi.64.070195.003445 7. Horie N, Aiba H, Oguro K, Hojo H, Takeishi K (1995) Functional analysis and DNA polymorphism of the tandemly repeated sequences in the 50 -terminal regulatory region of the human gene for thymidylate synthase. Cell Struct Funct 20(3):191–197 8. Danenberg PV (1977) Thymidylate synthetase—a target enzyme in cancer chemotherapy. Biochim Biophys Acta 473(2):73–92. doi:10.1016/0304-419X(77)90001-4 9. Marsh S, McKay JA, Cassidy J, McLeod HL (2001) Polymorphism in the thymidylate synthase promoter enhancer region in colorectal cancer. Int J Oncol 19(2):383–386 10. Kaneda S, Takeishi K, Ayusawa D, Shimizu K, Seno T, Altman S (1987) Role in translation of a triple tandemly repeated sequence in the 50 -untranslated region of human thymidylate synthase mRNA. Nucleic Acids Res 15(3):1259–1270. doi:10.1093/nar/15.3.1259 11. Kawakami K, Salonga D, Park JM, Danenberg KD, Uetake H, Brabender J, Omura K, Watanabe G, Danenberg PV (2001) Different lengths of a polymorphic repeat sequence in the thymidylate synthase gene affect translational efficiency but not its gene expression. Clin Cancer Res 7(12):4096–4101 12. Pullarkat ST, Stoehlmacher J, Ghaderi V, Xiong YP, Ingles SA, Sherrod A, Warren R, Tsao-Wei D, Groshen S, Lenz HJ (2001) Thymidylate synthase gene polymorphism determines response and toxicity of 5-FU chemotherapy. Pharmacogenomics J 1(1):65–70 13. Kawakami K, Omura K, Kanehira E, Watanabe Y (1999) Polymorphic tandem repeats in the thymidylate synthase gene is associated with its protein expression in human gastrointestinal cancers. Anticancer Res 19(4B):3249–3252 14. Kaneda S, Nalbantoglu J, Takeishi K, Shimizu K, Gotoh O, Seno T, Ayusawa D (1990) Structural and functional analysis of the human thymidylate synthase gene. J Biol Chem 265(33):20277–20284 15. Mandola MV, Stoehlmacher J, Muller-Weeks S, Cesarone G, Yu MC, Lenz HJ, Ladner RD (2003) A novel single nucleotide polymorphism within the 50 tandem repeat polymorphism of the thymidylate synthase gene abolishes USF-1 binding and alters transcriptional activity. Cancer Res 63(11):2898–2904 16. Kawakami K, Watanabe G (2003) Identification and functional analysis of single nucleotide polymorphism in the tandem repeat

123


17.

18.

19.

20.

21. 22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

sequence of thymidylate synthase gene. Cancer Res 63(18): 6004–6007 Ulrich CM, Bigler J, Velicer CM, Greene EA, Farin FM, Potter JD (2000) Searching expressed sequence tag databases: discovery and confirmation of a common polymorphism in the thymidylate synthase gene. Cancer Epidemiol Biomark Prev 9(12):1381–1385 Lecomte T, Ferraz JM, Zinzindohoue F, Loriot MA, Tregouet DA, Landi B, Berger A, Cugnenc PH, Jian R, Beaune P, LaurentPuig P (2004) Thymidylate synthase gene polymorphism predicts toxicity in colorectal cancer patients receiving 5-fluorouracilbased chemotherapy. Clin Cancer Res 10(17):5880–5888. doi:10. 1158/1078-0432 Dotor E, Cuatrecases M, Martinez-Iniesta M, Navarro M, Vilardell F, Guino E, Pareja L, Figueras A, Mollevi DG, Serrano T, de Oca J, Peinado MA, Moreno V, Germa JR, Capella G, Villanueva A (2006) Tumor thymidylate synthase 1494del6 genotype as a prognostic factor in colorectal cancer patients receiving fluorouracil-based adjuvant treatment. J Clin Oncol 24(10):1603–1611. doi:10.1200/JCO.2005.03.5253 Mountain CF (1986) A new international staging system for lung cancer. Chest 89(4 Suppl):225S–233S. doi:10.1378/chest.89.4_ Supplement.225S Mountain CF (1997) Revisions in the international system for staging lung cancer. Chest 111(6):1710–1717 Excoffier L, Laval G, Schneider S (2005) Arlequin (version 3.0): an integrated software package for population genetics data analysis. Evol Bioinform Online 1:47–50. doi:10.4137/EBO.S0 Schaid DJ, Rowland CM, Tines DE, Jacobson RM, Poland GA (2002) Score tests for association between traits and haplotypes when linkage phase is ambiguous. Am J Hum Genet 70(2): 425–434. doi:10.1086/338688 Lurje G, Zhang W, Yang D, Groshen S, Hendifar AE, Husain H, Nagashima F, Chang HM, Fazzone W, Ladner RD, Pohl A, Ning Y, Iqbal S, El-Khoueiry A, Lenz HJ (2008) Thymidylate synthase haplotype is associated with tumor recurrence in stage II and stage III colon cancer. Pharmacogenet Genomics 18(2):161–168. doi:10.1097/FPC.0b013e3282f4aea6 Stephens M, Donnelly P (2003) A comparison of bayesian methods for haplotype reconstruction from population genotype data. Am J Hum Genet 73(5):1162–1169. doi:10.1086/379378 Johnston PG, Lenz HJ, Leichman CG, Danenberg KD, Allegra CJ, Danenberg PV, Leichman L (1995) Thymidylate synthase gene and protein expression correlate and are associated with response to 5-fluorouracil in human colorectal and gastric tumors. Cancer Res 55(7):1407–1412 Lenz HJ, Hayashi K, Salonga D, Danenberg KD, Danenberg PV, Metzger R, Banerjee D, Bertino JR, Groshen S, Leichman LP, Leichman CG (1998) p53 Point mutations and thymidylate synthase messenger RNA levels in disseminated colorectal cancer: an analysis of response and survival. Clin Cancer Res 4(5): 1243–1250 Edler D, Glimelius B, Hallstrom M, Jakobsen A, Johnston PG, Magnusson I, Ragnhammar P, Blomgren H (2002) Thymidylate synthase expression in colorectal cancer: a prognostic and predictive marker of benefit from adjuvant fluorouracil-based chemotherapy. J Clin Oncol 20(7):1721–1728 Kawakami K, Ishida Y, Danenberg KD, Omura K, Watanabe G, Danenberg PV (2002) Functional polymorphism of the thymidylate synthase gene in colorectal cancer accompanied by frequent loss of heterozygosity. Jpn J Cancer Res 93(11):1221–1229 DiPaolo A, Chu E (2004) The role of thymidylate synthase as a molecular biomarker. Clin Cancer Res 10(2):411–412. doi:10. 1158/1078-0432 Kristensen MH, Pedersen PL, Melsen GV, Ellehauge J, Mejer J (2010) Variants in the dihydropyrimidine dehydrogenase, methylenetetrahydrofolate reductase and thymidylate synthase

123

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

genes predict early toxicity of 5-fluorouracil in colorectal cancer patients. J Int Med Res 38(3):870–883. doi:10.1177/1473230010 03800313 Brody JR, Hucl T, Gallmeier E, Winter JM, Kern SE, Murphy KM (2006) Genomic copy number changes affecting the thymidylate synthase (TYMS) gene in cancer: a model for patient classification to aid fluoropyrimidine therapy. Cancer Res 66(19):9369–9373. doi:10.1158/0008-5472.CAN-06-2165 Etienne MC, Chazal M, Laurent-Puig P, Magne N, Rosty C, Formento JL, Francoual M, Formento P, Renee N, Chamorey E, Bourgeon A, Seitz JF, Delpero JR, Letoublon C, Pezet D, Milano G (2002) Prognostic value of tumoral thymidylate synthase and p53 in metastatic colorectal cancer patients receiving fluorouracil-based chemotherapy: phenotypic and genotypic analyses. J Clin Oncol 20(12):2832–2843 Uchida K, Hayashi K, Kawakami K, Schneider S, Yochim JM, Kuramochi H, Takasaki K, Danenberg KD, Danenberg PV (2004) Loss of heterozygosity at the thymidylate synthase (TS) locus on chromosome 18 affects tumor response and survival in individuals heterozygous for a 28-bp polymorphism in the TS gene. Clin Cancer Res 10(2):433–439. doi:10.1158/1078-0432.CCR-0200-03 Wang TL, Diaz LA Jr, Romans K, Bardelli A, Saha S, Galizia G, Choti M, Donehower R, Parmigiani G, Shih Ie M, IacobuzioDonahue C, Kinzler KW, Vogelstein B, Lengauer C, Velculescu VE (2004) Digital karyotyping identifies thymidylate synthase amplification as a mechanism of resistance to 5-fluorouracil in metastatic colorectal cancer patients. Proc Natl Acad Sci USA 101(9):3089–3094. doi:10.1073/pnas.0308716101 Ooyama A, Okayama Y, Takechi T, Sugimoto Y, Oka T, Fukushima M (2007) Genome-wide screening of loci associated with drug resistance to 5-fluorouracil-based drugs. Cancer Sci 98(4):577–583. doi:10.1111/j.1349-7006.2007.00424.x Jakobsen A, Nielsen JN, Gyldenkerne N, Lindeberg J (2005) Thymidylate synthase and methylenetetrahydrofolate reductase gene polymorphism in normal tissue as predictors of fluorouracil sensitivity. J Clin Oncol 23(7):1365–1369. doi:10.1200/JCO. 2005.06.219 Li WJ, Jiang H, Fang XJ, Ye HL, Liu MH, Liu YW, Chen Q, Zhang L, Zhang JY, Yuan CL, Zhang QY (2013) Polymorphisms in thymidylate synthase and reduced folate carrier (SLC19A1) genes predict survival outcome in advanced non-small cell lung cancer patients treated with pemetrexed-based chemotherapy. Oncol Lett 5(4):1165–1170. doi:10.3892/ol.2013.1175ol-05-04-1165 Wang X, Wang Y, Cheng J, Ha M (2013) Association of thymidylate synthase gene 30 -untranslated region polymorphism with sensitivity of non-small cell lung cancer to pemetrexed treatment: TS gene polymorphism and pemetrexed sensitivity in NSCLC. J Biomed Sci 20:5. doi:10.1186/1423-0127-20-514230127-20-5 Mandola MV, Stoehlmacher J, Zhang W, Groshen S, Yu MC, Iqbal S, Lenz HJ, Ladner RD (2004) A 6 bp polymorphism in the thymidylate synthase gene causes message instability and is associated with decreased intratumoral TS mRNA levels. Pharmacogenetics 14(5):319–327 Merkelbach-Bruse S, Hans V, Mathiak M, Sanguedolce R, Alessandro R, Ruschoff J, Buttner R, Houshdaran F, Gullotti L (2004) Associations between polymorphisms in the thymidylate synthase gene, the expression of thymidylate synthase mRNA and the microsatellite instability phenotype of colorectal cancer. Oncol Rep 11(4):839–843 Ulrich CM, Bigler J, Bostick R, Fosdick L, Potter JD (2002) Thymidylate synthase promoter polymorphism, interaction with folate intake, and risk of colorectal adenomas. Cancer Res 62(12):3361–3364 Graziano F, Kawakami K, Watanabe G, Ruzzo A, Humar B, Santini D, Catalano V, Ficarelli R, Merriman T, Panunzi S, Testa

Author's personal copy Mol Biol Rep E, Cascinu S, Bearzi I, Tonini G, Magnani M (2004) Association of thymidylate synthase polymorphisms with gastric cancer susceptibility. Int J Cancer 112(6):1010–1014. doi:10.1002/ijc. 20489 44. Kawakami K, Graziano F, Watanabe G, Ruzzo A, Santini D, Catalano V, Bisonni R, Arduini F, Bearzi I, Cascinu S, Muretto P, Perrone G, Rabitti C, Giustini L, Tonini G, Pizzagalli F, Magnani M (2005) Prognostic role of thymidylate synthase polymorphisms in gastric cancer patients treated with surgery and adjuvant

chemotherapy. Clin Cancer Res 11(10):3778–3783. doi:10.1158/ 1078-0432.CCR-04-2428 45. Fernandez-Contreras ME, Sanchez-Hernandez JJ, Gonzalez E, Herraez B, Dominguez I, Lozano M, Garcia De Paredes ML, Munoz A, Gamallo C (2009) Combination of polymorphisms within 50 and 30 untranslated regions of thymidylate synthase gene modulates survival in 5 fluorouracil-treated colorectal cancer patients. Int J Oncol 34(1):219–229. doi:10.3892/ijo_ 00000144

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