G polymorphism: A biomarker for the metastasis of nasopharyngeal carcinoma in a Chinese population

Clinica Chimica Acta 411 (2010) 179–183

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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m

FAS-670A/G polymorphism: A biomarker for the metastasis of nasopharyngeal carcinoma in a Chinese population Qingyao Zhu a, Tao Wang a,⁎, Jinghua Ren a, Kai Hu b, Wei Liu c, Gang Wu a a b c

Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China Department of Radiation Oncology, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Province, China Department of Oncology, Anhui Provincial Hospital, Hefei 230038, Anhui Province, China

a r t i c l e

i n f o

Article history: Received 29 August 2009 Received in revised form 25 October 2009 Accepted 29 October 2009 Available online 4 November 2009 Keywords: Nasopharyngeal carcinoma FAS Polymorphism Apoptosis Metastasis

a b s t r a c t Background: FAS-670 A/G single nucleotide polymorphism has been demonstrated to affect the expression of FAS gene by altering the transcriptional activity of FAS gene promoter. Downregulation of FAS with resultant resistance to death signals has been found in many cancers. We carried out a case-control study to investigate the biological significance of this polymorphism in nasopharyngeal carcinoma (NPC). Methods: FAS-670 A/G polymorphism was examined in a Chinese population of 237 patients with NPC and 264 control subjects using the polymerase chain reaction–restriction fragment length polymorphism (PCRRFLP) technique. Results: There were no significant differences in the genotype or allele distribution of FAS-670 A/G polymorphism between cases and controls. FAS-670 (AG + GG) genotype and G allele showed significant associations with an increasing risk of lymph node metastasis (OR = 2.08, P = 0.01; OR = 1.67, P = 0.011, respectively) and distant metastasis (OR = 3.87, P = 0.015; OR = 1.81, P = 0.03, respectively) of NPC patients. In addition, FAS-670 (AG + GG) genotype showed an increasing incidence of advanced clinical stage, but this finding was not statistically significant (OR = 1.79, P = 0.066). Conclusion: The FAS-670 G allele could be used as a genetic risk marker for the metastasis of NPC patients. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Nasopharyngeal carcinoma (NPC) is a malignant tumor showing a distinctly geographically and ethnically unbalanced distribution. While the incidence of NPC is very high in Southern China, this disease is extremely rare in the Caucasians in Western Europe and North America [1]. Evidence from epidemiology suggests that the occurrence of NPC is associated with many factors, such as infection of Epstein-Barr virus, heredity, diet, smoking, environmental pollution and so on, among which Epstein-Barr virus infection and heredity are the two most important factors [2–4]. Apoptosis is not only closely related to the genesis and development of the embryo, the formation of the body and the homeostasis of cells, but also plays an important role in autoimmune disorders, virus diseases and tumorigenesis [5–7]. FAS (CD95/APO-1) which is known as a member of tumor necrosis factor receptors superfamily is a transmembrane receptor involved in apoptotic signal transmission in many types of cells. The

Abbreviations: NPC, nasopharyngeal carcinoma; SNP, single nucleotide polymorphism; GAS, gamma interferon activation signal; STAT-1, signal transducer and activator of transcription-1. ⁎ Corresponding author. Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China. Tel.: +86 27 65650733. E-mail address: [email protected] (T. Wang). 0009-8981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2009.10.024

death signal cascade is initiated upon cross-linking of FAS with its natural ligand FASL [8]. Decreased expression or mutation of FAS gene has been found in many malignant tumors, which impair the sensitivity of tumor cells to apoptotic signal and cause tumor cells to evade from or weaken the immune elimination of the body through FAS-FASL pathway [9–13]. Single nucleotide polymorphism (SNP) at −670 of FAS gene promoter (A/G) (rs1800682) has been found with potentially different transcriptional efficiency [14], which implies that this SNP may take certain effects in the occurrence and progression of apoptosis related disorders. In fact, many studies have demonstrated that FAS-670 A/G polymorphism was closely associated with autoimmune diseases [14–16], hematological malignancies [17] and solid tumors such as cervical cancer [18], esophageal cancer [19] and squamous cell carcinoma of the head and neck [20], etc. Given the role of the FAS and FAS-670 A/G polymorphism in carcinogenesis, we investigated, in a case-control study of 237 patients with NPC and 264 cancer-free control subjects, the potential association of this SNP with the risk and progression of NPC in a Chinese population. 2. Materials and methods 2.1. Study population The case-control population was composed of 501 adult genetically unrelated ethnic Han Chinese who were selected from the same

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population living in Hubei Province and its surrounding regions in China. A total of 237 incident NPC cases were recruited from Union Hospital and Tongji Hospital of Huazhong University of Science and Technology between June 2008 and April 2009. There was no restriction on gender and age. The diagnosis of NPC was determined by histopathology analysis. The histological type was confirmed by two independent pathologists according to WHO classification (2002). TNM stage designation was referred to the definitions of the sixth edition (2002) of the International Union Against Cancer (UICC) clinical staging criteria. The control group comprised 264 unrelated healthy blood donors who visited the General Health Check-up Division at Union Hospital. Selection criteria for controls were no evidence of any personal or family history of cancer or other serious illness. The control group was comparable to the NPC cases with respect to age, gender and ethnic background. General information about the healthy controls was extracted from a standard questionnaire. Written informed consent was obtained from all subjects.

2.3. Statistical analysis Hardy–Weinberg equilibrium was verified by calculation of expected frequencies and numbers, and significance testing was based on the 1df χ 2. Differences in the distributions of demographic characteristics and selected variables between NPC patients and healthy control subjects were evaluated using the χ 2test and Student's t-test. The association between FAS-670 A/G polymorphism and the risk and clinical characteristics of NPC were estimated by computing the odds ratio (OR) and their 95% confidence interval (CI) from both univariate and multivariate logistic regression analysis. On all statistical tests, a Pb 0.05 was considered significant. All the statistical analyses were performed with Statistical Analysis System software (SPSS 15.0 Inc, Chicago, IL). 3. Results 3.1. Population characteristics

2.2. FAS-670 A/G genotype analysis From each subject, 5 ml peripheral blood was collected in tubes containing EDTA. Genomic DNA was then extracted using a commercially available kit according to the manufacturer's instructions (Blood genomic DNA miniprep kit, Axygen Biosciences, Union City, CA) and preserved at −20 °C. The FAS-670 A/G polymorphism was detected by using a polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method. The PCR primers were designed based on the GenBank reference sequence and primer 5.0. PCR primers for amplification of the FAS promoter region containing the FAS-670 A/G polymorphism were 5′GCTGGGGCTATGCGATTT-3′ (forward) and 5′-GGTACAGGAGCCTTGGCTAATT-3′ (reverse). The PCR reactions were performed in a total volume of 35 μl reaction mixture containing approximately 90 ng genomic DNA samples, 12.5 pmol of each primer, 0.1 mM each dNTP, 1× PCR buffer (50 mmol/l KCl, 10 mmol/l Tris–HCl, and 0.08% Nonidet P40), 1.0 mmol/ l MgCl2, and 1.5 unit of Taq DNA polymerase (Fermentas, Vilnius, Lithuania). Reaction conditions used with the thermal cycler (Biometra, Gϋttingem, Germany) were as follows: an initial incubation at 95 °C for 5 min; 35 cycles of 95 °C for 15 s, 55 °C for 30 s, 72 °C for 30 s, and a final elongation at 72 °C for 5 min. Reaction products were digested overnight at 37 °C with 10U of MvaI (Fermentas, Vilnius, Lithuania) which cut allele G. After an overnight digestion, the products were resolved and separated on a 2% agarose gel stained with ethidium bromide for visualization under UV light. After electrophoresis, homozygous G-alleles were represented by DNA bands with sizes at 133 bp and 110 bp, an uncut fragment of 243 bp indicated the homozygous A-alleles, while heterozygous genotype displayed a combination of 243, 133, and 110 bp (Fig. 1). The fragments of 110 and 133 bp were too close to be identified by naked eye. In order to validate the genotyping assay, more than 10% PCR products were randomly selected and confirmed by direct sequencing using an automated sequencer (ABI model 377 genetic analysis; Perkin-Elmer Applied Biosystems, Foster City, CA). We found no differences between the results from 2 methods.

Fig. 1. PCR-RFLP assay for analyzing the − 670 A/G polymorphism of FAS. PCR product was digested by MvaI and separated on 2% agarose gel electrophoresis. A 243 bp DNA band indicated the homozygous A-alleles. A combination of 133 and 110 bp DNA bands indicated the homozygous G-alleles. A combination of 243, 133 and 110 bp DNA bands indicated the heterozygous genotypes. The fragments of 110 bp and 133 bp were too close to be identified by naked eye.

The general characteristics of 237 NPC cases and 264 controls are summarized in Table 1. The differences of gender and age distribution between NPC patients and controls were not statistically significant. Out of the 237 patients, 95.8% were classified as non-keratinizing carcinoma, and only 4.2% belonged to other types. 23.2% of patients were at clinical stages I–II, the others (76.8%) at clinical stages III–IV. Distant metastasis was found in 31 patients, occupying 13.1% of the total number. As for lymph node metastasis, 35.4% of patients had N0– N1 disease, and 64.6% of patients had N2–N3 disease. 3.2. FAS-670 A/G polymorphism and the risk of NPC The observed genotype frequencies of both the NPC patients and the controls were in Hardy–Weinberg equilibrium (χ2 = 1.735 P = 0.188; χ2 = 0.503 P = 0.478, respectively). Table 2 exhibits the genotype distributions and allele frequencies for FAS-670 in NPC

Table 1 General characteristics of cases and controls. Characteristics

Patients

Controls

P

Gender Male Female

164 (69.2%) 73 (30.8%)

166 (62.9%) 98 (37.1%)

NSa

Age at diagnosis Mean ± SD

46.43 ± 11.81

45.36 ± 9.80

NS b

Tumor size T1–T2 T3–T4

119 (50.2%) 118 (49.8%)

– –

– –

Lymph node metastasis N0–N1 N2–N3

84 (35.4%) 153 (64.6%)

– –

– –

Distant metastasis M0 M1

206 (86.9%) 31 (13.1%)

– –

– –

Clinical stage I–II III–IV

55 (23.2%) 182 (76.8%)

– –

– –

Histological type NKC KSCC NACC Adenocarcinoma

227 (95.8%) 6 (2.5%) 2 (0.8%) 2 (0.8%)

– – – –

– – – –

NKC: non-keratinizing carcinoma; KSCC: keratinizing squamous cell carcinoma; NACC: nasopharyngeal adenoid cystic carcinoma. a P value was calculated using the χ2 square test. b P value was calculated using the Student t-test.

Q. Zhu et al. / Clinica Chimica Acta 411 (2010) 179–183 Table 2 Genotype and allelic frequencies of FAS-670A/G among the cases and controls and their associations with the risk of NPC polymorphism. Genotype

AA AG GG AG + GG A-alleles G-alleles

Patients

Control

Crude (Unadjusted)

N

f(%)

N

f(%)

OR

79 124 34 158 282 192

33.3 52.3 14.4 66.7 59.5 40.5

93 132 39 171 318 210

35.2 50.0 14.8 64.8 60.2 39.8

1 0.90 0.97 0.92 1 0.97

95%CI

P

0.61–1.33 0.56–1.69 0.64–1.33

NS NS NS

0.75–1.25

NS

Table 4 Stratification analyses of FAS-670A/G polymorphism with clinical characteristics in NPC patients.

Adjusted for age and sex OR 1 0.93 0.98 0.94 1 0.98

95%CI

P

0.63–1.37 0.57–1.70 0.65–1.36

NS NS NS

0.76–1.26

NS

181

Logistic regression analysis was employed to assess the associations between genotype or allele distributions and the risk of NPC. OR: odds ratio; CI: confidence interval; f: frequencies.

patients and in the control group. By using the AA genotype or A-allele as references, no significant association was found between any genotype or allele and the risk of developing NPC before and after adjustment of gender and age. Neither was FAS-670 A/G polymorphism found to increase the risk of NPC when the 2 gender groups were analyzed respectively (Table 3).

Characteristics

Patients

Gender AA AG GG AG + GG A-allele G-allele

Male 52 88 24 112 192 136

Female 27 36 10 46 90 56

OR 1 0.79 0.80 0.79 1 0.88

Clinical stage AA AG GG AG + GG A-allele G-allele

I–II 24 25 6 31 73 37

III–IV 55 99 28 127 209 155

1 1.73 2.04 1.79 1 1.46

Tumor size AA AG GG AG + GG A-allele G-allele

T1–T2 44 59 16 75 147 91

T3–T4 35 65 18 83 135 101

1 1.39 1.41 1.39 1 1.21

Lymph node metastasis AA AG GG AG + GG A-allele G-allele

N0–N1 37 39 8 47 113 55

N2–N3 42 85 26 111 169 137

1 1.92 2.86 2.08 1 1.67

Distant metastasis AA AG GG AG + GG A-allele G-allele

M0 75 103 28 131 253 159

M1 4 21 6 27 29 33

1 3.82 4.02 3.87 1 1.81

Negative 70 102 32 134 242 166

Positive 9 22 2 24 40 26

1 1.68 0.49 1.39 1 0.95

95%CI

P

0.43–1.44 0.34–1.92 0.44–1.41

NS NS NS

0.59–1.31

NS

0.90–3.31 0.75–5.56 0.96–3.32

NS NS 0.066

0.94–2.29

NS

0.79–2.44 0.63–3.17 0.81–2.39

NS NS NS

0.84–1.75

NS

1.07–3.44 1.16–7.09 1.19–3.64

0.028 0.023 0.010

1.12–2.47

0.011

1.26–11.60 1.06–15.31 1.30–11.47

0.018 0.042 0.015

1.06–3.10

0.030

0.73–3.86 0.10–2.38 0.61–3.16

NS NS NS

0.56–1.61

NS

3.3. FAS-670 A/G polymorphism and clinical characteristics of NPC We also analyzed the relationship between the FAS-670 A/G polymorphism and the clinical characteristics of NPC patients, including gender, clinical stage, tumor size, lymph node metastasis, distant metastasis and family history of malignant tumor. As shown in the details in Table 4, compared with FAS-670 AA genotype, AG genotype and GG genotype were associated with an increasing risk of lymph node metastasis (OR=1.92, P=0.028, OR=2.86, P=0.023, respectively) and distant metastasis (OR=3.82, P=0.018, OR=4.02, P=0.042, respectively) of NPC patients. The G allele was found to be more frequent among NPC patients at stages N2–N3 (71.35% vs. 59.93%, OR=1.67, P=0.011) or with distant metastasis (17.19% vs. 10.28%, OR=1.81, P=0.03) than allele A. Out of the patients at clinical stages III–IV, the percentage of G allele carriers (AG genotype + GG genotype) increased slightly in comparison with AA genotype carriers (80.38% vs. 69.62%, OR=1.79), but this finding was not statistically significant (P=0.066). No significant association was observed between the genotype or allele distributions and gender, tumor size or family history of malignant tumor.

Family history AA AG GG AG + GG A-allele G-allele

4. Discussion

a

Logistic regression analysis was employed to assess the associations between genotype or allele distributions and the clinical characteristics of NPC patients. P values b 0.05 are shown in bold. OR: odds ratio; CI: confidence interval. a Family history of malignant tumors.

The expression of FAS could be regulated by various cytokines such as interferon-γ derived from many inflammatory cells [21]. FAS-670 A/G polymorphism situates in the nuclear transcription element GAS (γ interferon activation signal) of FAS gene promoter region and is responsible for the signal transmission through interferon-γ-STAT-1 (signal transducer and activator of transcription-1) pathway [22,23]. In normal circumstances, a combination of interferon-γ and its

receptor activates tyrosine kinase 1 and 2, leading to STAT1 tyrosine phosphorylation and then tyrosine-phosphorylated STAT1s form homodimer which moves into the nucleus to integrate with GAS and induce the transcription of genes containing GAS [23,24]. The

Table 3 Associations between FAS-670A/G polymorphism and the risk of NPC polymorphism by gender. Genotye

AA AG GG AG + GG A-alleles G-alleles

Male

Female

Patients

Controls

OR

52 88 24 112 192 136

55 86 25 111 196 136

1 0.92 0.99 0.94 1 0.98

95%CI

P

0.57–1.50 0.50–1.94 0.59–1.49

NS NS NS

0.72–1.34

NS

Patients

Controls

OR

27 36 10 46 90 56

38 46 14 60 122 74

1 0.91 1.00 0.93 1 0.98

Logistic regression analysis was employed to assess the associations between genotype or allele distributions and the risk of NPC. OR: odds ratio; CI: confidence interval.

95%CI

P

0.47–1.75 0.39–2.57 0.50–1.73

NS NS NS

0.63–1.52

NS

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substitution (A to G) in FAS-670 position destructs the binding sequence of STAT-1, weakens the transcriptional activity of FAS gene promoter, and decreases the transcription of FAS gene induced by interferon-γ [14]. Recently, Park JY et al. investigated the expression of FAS in early stage non-small cell lung cancer tissues with different FAS-670 genotypes by immunohistochemistry, and found that strongly positive FAS immunostaining was significantly more frequent in patients with AA genotype than those with (AG + GG) genotype (10.8% vs. 4.5%; P = 0.04) [25]. Moreover, Bel Hadj Jrad B et al. revealed that the FAS-670 GG genotype showed a significantly higher frequency in NPC patients with producing anti-nuclear autoantibodies than in those without [26]. This might be one of the reasons why this genotype could disrupt the immune regulatory system of patients and stimulate the development and progression of tumor. In our study, we analyzed the relationship between FAS-670 A/G polymorphism and NPC by a case-control study, and found that FAS670 A/G single nucleotide polymorphism was significantly associated with the risk of the lymph node metastasis and distant metastasis of NPC patients instead of the susceptibility of NPC. And patients carrying FAS-670 AG genotype or GG genotype were at a higher risk for the lymph node metastasis and distant metastasis than those carrying AA genotype. These findings suggested that FAS-670 G allele was likely to affect the progression of NPC. Similar conclusion was also reached in other malignant tumor. Kang et al. found that FAS-670 polymorphism showed no association with the risk of cervical cancer, but AG genotype or GG genotype could increase the risk of lymph node metastasis of patients in Koreans [27]. The clinical stage, lymph node metastasis and distant metastasis are closely associated with the prognosis of malignant tumor patients [28], which suggests that FAS-670 G allele might become a negative indicator of prognosis for NPC patients. In fact, some findings about the relationship between FAS-670 A/G polymorphism and the survival of patients with malignant tumors have been reported. Hofmann G et al. found that carriers of the homozygous FAS-670 GG genotype had a significantly lower survival rate compared with (AA + AG) genotype carriers in colorectal cancer (OR = 1.76, 95%CI = 1.08–2.87, P = 0.023) [29]. Park et al. drew a similar conclusion in early stage non-small cell lung cancer that the overall survival time of patients with FAS-670 GG genotype or (AG + GG) genotype was apparently lower than that of patients with FAS-670 AA genotype (OR = 1.71, P = 0.03; OR = 1.48, P = 0.047, respectively) [25]. However, the relationship between FAS-670 A/G polymorphism and malignant tumors seems to be controversial. Up to now, several studies addressed that FAS-670 G allele showed a significant association with the risk or progression of malignant tumors, such as cervical cancer [18], esophageal squamous cell carcinoma [19], squamous cell carcinoma of the head and neck [20]. While conflicting results were also reported in many other tumors, even in the same cancer. Farre et al. revealed that FAS-670 AA genotype had a significant association with the susceptibility, aggressiveness and survival of adult T cell leukemia [17]. In agreement with this finding, studies in melanoma indicated FAS-670 AA genotype were related not only with the risk of melanoma [30] but also with the Breslow thickness and Clark level of this disease [31]. Another results from Lai et al. indicated that the frequency of A-allele and AA genotype increased in accordance with the multi-step carcinogenesis from low grade SIL (squamous intraepithelial lesions), high grade SIL to invasive squamous cell in cervical cancer. They stated that A allele and AA genotype conferred immune cells an intact GAS element and more efficient FAS expression which induced these cells death and protected tumor cells from immune elimination [32]. Moreover, several studies have found no significant association between the FAS - 670 A/G polymorphism and the risk or progression of several types of human cancers [33,34]. As for NPC, findings by Bel Hadj Jrad et al. showed that FAS-670 AG genotype and GG genotype were closely associated with an increasing risk of NPC in Tunisians [26] which also did not coincide with our

results. Taking into account the fact that allele and genotype frequencies of FAS-670 A/G polymorphism vary greatly with ethnicity [19,35], we hypothesize that these discrepancies may be mainly due to the different ethnicities of the study populations, which could influence the genetic effect of the FAS polymorphisms on human cancer. In addition, these differences may also be related to different molecular mechanisms underlying the development of different cancers. In conclusion, we did not find a significant association between FAS-670 A/G polymorphism and susceptibility to NPC in a Chinese population, but our results suggested that the G allele of this position was closely associated with increasing risk of lymph node metastasis and distant metastasis of NPC patients. Further studies are necessary to evaluate whether FAS-670 A/G polymorphism has an impact on the survival time of NPC patients. Acknowledgments We are grateful to the staff of the Cancer Center of Union Hospital and Tongji Hospital for providing samples and clinical information. This work was supported by the Natural Science Foundation of Hubei Province (Grant No. 2008CBB399). References [1] McDerrmott AL, Dutt SN, Watkinson JC. The aetiology of nasopharyngeal carcinoma. Clin Otolaryngol Allied Sci 2001;26:82–92. [2] Chen DL, Huang TB. A case-control study of risk factors of nasopharyngeal carcinoma. Cancer Lett 1997;117:17–22. [3] Yu MC, Yuan JM. Epidemiology of nasopharyngeal carcinoma. Semin Cancer Biol 2002;12:421–9. [4] Zheng X, Yan L, Nilsson B, Eklund G, Drettner B. Epstein-Barr virus infection, salted fish and nasopharyngeal carcinoma. A case-control study in southern China. Acta Oncol 1994;33:867–72. [5] Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995;267:1456–62. [6] Lorenz HM, Herrmann M, Winkler T, Gaipl U, Kalden JR. Role of apoptosis in autoimmunity. Apoptosis 2000;5:443–9. [7] Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer. Nature 2001;411:342–8. [8] Nagata S, Golstein P. The Fas death factor. Science 1995;267:1449–56. [9] Peters AM, Kohfink B, Martin H, et al. Defective apoptosis due to a point mutation in the death domain of CD95 associated with autoimmune lymphoproliferative syndrome, T-cell lymphoma, and Hodgkin's disease. Exp Hematol 1999;27: 868–74. [10] Boldrini L, Faviana P, Gisfredi S, et al. Identification of Fas (APO-1/CD95) and p53 gene mutations in non-small cell lung cancer. Int J Oncol 2002;20:155–9. [11] Viard-Leveugle I, Veyrenc S, French LE, Brambilla C, Brambilla E. Frequent loss of Fas expression and function in human lung tumours with overexpression of FasL in small cell lung carcinoma. J Pathol 2003;201:268–77. [12] Keane MM, Ettenberg SA, Lowrey GA, Ruussell EK, Lipkowitz S. Fas expression and function in normal and malignant breast cell lines. Cancer Res 1996;56: 4791–8. [13] Müschen M, Warskulat U, Beckmann MW. Defining CD95 as a tumor suppressor gene. J Mol Med 2000;78:312–25. [14] Kanemitsu S, Ihara K, Saifddin A, et al. A functional polymorphism in Fas (CD95/ Apo-1) gene promoter associated with systemic lupus erythematosus. J Rheumatol 2002;29:1183–8. [15] Liakouli V, Manetti M, Pacini A, et al. The − 670G N A polymorphism in the FAS gene promoter region influences the susceptibility to systemic sclerosis. Ann Rheum Dis 2009;68:584–90. [16] Huang QR, Danis V, Lassere M, Edmonds J, Manolios N. Evaluation of a new Apo-1/ Fas promoter polymorphism in rheumatoid arthritis and systemic lupus erythematosus patients. Rheumatology (Oxford) 1999;38:645–51. [17] Farre L, Bittencourt AL, Silva-Santos G, et al. Fas 670 promoter polymorphism is associated to susceptibility, clinical presentation, and survival in adult T cell leukemia. J Leukoc Biol 2008;83:220–2. [18] Ueda M, Hung YC, Terai Y, et al. Fas gene promoter − 670 polymorphism(A/G) is associated with cervical carcinogenesis. Gynecol Oncol 2005;98:129–33. [19] Sun T, Miao X, Zhang X, Tan W, Xiong P, Lin D. Polymorphisms of death pathway genes FAS and FASL in esophageal squamous-cell carcinoma. J Natl Cancer Inst 2004;96:1030–6. [20] Zhang Z, Wang LE, Sturgis EM, et al. Polymorphisms of FAS and FAS ligand genes involved in the death pathway and risk and progression of squamous cell carcinoma of the head and neck. Clin Cancer Res 2006;12:5596–602. [21] Sayama K, Yonehara S, Watanabe Y, Miki Y. Expression of Fas antigen on keratinocytes in vivo and induction of apoptosis in cultured keratinocytes. J Invest Dermatol 1994;103:330–4.

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