CASP8 polymorphisms contribute to cancer ... - Semantic Scholar

Carcinogenesis vol.31 no.5 pp.850–857, 2010 doi:10.1093/carcin/bgq047 Advance Access publication February 22, 2010

CASP8 polymorphisms contribute to cancer susceptibility: evidence from a meta-analysis of 23 publications with 55 individual studies Ming Yin, Jingrong Yan1, Sheng Wei and Qingyi Wei Department of Epidemiology, The University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA and 1 Department of Pediatrics, Baylor College of Medicine, and Texas Children’s Cancer Center, One Baylor Plaza, Houston, TX 77030, USA To whom correspondence should be addressed. Tel: þ1 713 745 2481; Fax: þ1 713 563 0999; Email: [email protected]

Several potentially functional polymorphisms of CASP8 encoding an apoptotic enzyme, caspase 8, have been implicated in cancer risk, but individually published studies showed inconclusive results. We performed a meta-analysis of 23 publications with a total of 55 174 cancer cases and 59 336 controls from 55 individual studies. We summarized the data on the associations between three studied CASP8 polymorphisms (G>C D302H, –652 6N del and Ex14-271A>T) and cancer risk and performed subgroup analysis by ethnicity, cancer type, study design and etiology. We found that D302H CC and CG variant genotypes were associated with significantly reduced overall risk of cancers using conservative random genetic models [homozygote comparison: odds ratios (OR) 5 0.79; 95% confidence interval (CI): 0.69–0.92; dominant comparison: OR 5 0.93, 95% CI: 0.89–0.98; recessive comparison: OR 5 0.81, 95% CI: 0.71–0.93). In further stratified analyses, the reduced cancer risk remained for subgroups of Caucasians, breast or estrogen-related cancers, and hospital- or population-based studies, except for an elevated risk for brain tumors. Similarly, the –652 6N del polymorphism was also associated with significantly reduced overall risk of cancers (homozygote comparison: OR 5 0.84, 95% CI: 0.75–0.94; dominant comparison: OR 5 0.88, 95% CI: 0.81–0.96; recessive comparison: OR 5 0.90, 95% CI: 0.82–0.99) and all subgroups analyzed. However, the Ex14-271A>T polymorphism did not appear to have an effect on cancer risk. These results suggest that CASP8 D302H and –652 6N del polymorphisms are potential biomarkers for cancer risk.

Introduction Apoptosis, also called programmed cell death, is important to maintain internal homeostasis by removing irreparable damaged cells. The caspase 8 protein regulates apoptosis, and it stimulates cell proliferation, malignant transformation and tumor progression as a result of its dysfunction or reduced activity (1–3). The human CASP8 gene contains at least 11 exons spanning 30 kb on human chromosome 2q33–34 (4), which is highly polymorphic. According to the dbSNP database (http://www.ncbi.nlm.nih.gov /SNP), CASP8 has at least 474 variants. Previous studies have mainly focused on three common CASP8 variants, including D302H (aspartate to histidine, G.C; rs1045485) of exon 10, a promoter six-nucleotide deletion/insertion (–652 6N del; rs3834129) and a nucleotide substitution in the 3#-untranslated region (Ex14-271A.T; rs13113), because they are probably to be functional. These variants may influence the interaction of the caspase 8 protein with other apoptosis-regulating molecules and consequently alter its expression or activation. For example, the aspartate to histidine change at residue 302 on the surface of caspase 8 is hypothesized to influence its autoprocessing or interactions with antiapoptotic molecules, such as the fas-associated Abbreviations: CI, confidence interval; HWE, Hardy–Weinberg equilibrium; OR, odds ratio.

protein with death domain-like apoptosis regulator (CFLAR) (5). The –652 6N del allele has been found to cause destruction of a binding site for the transcriptional activator Sp1, resulting in a reduced expression of the caspase 8 protein (6). The Ex14-271A.T polymorphism leads to an amino acid change within the 3#-untranslated region, which contains regulatory sequences and binding sites that could alter the stability of messenger RNA transcripts (7). Therefore, it is probably that these variants may contribute to the inter-individual difference in apoptotic activation induced by caspase 8 and modulate individual susceptibility to malignancies because of the varied ability to eliminate DNA-damaged cells. Since reports of the association of CASP8 variants with cancer susceptibility from individual studies are not consistent (6,8,9), and some recent meta-analysis analyzed such an association only for breast cancer (10), with some sorts of simple combined analyses buried in several published papers (11–13), we performed a comprehensive meta-analysis by including the most recent and relevant articles to identify statistical evidence of the association between CASP8 variants (i.e. D302H, –652 6N del and Ex14-271A.T) and risk of all cancers that have been investigated. Materials and methods Identification and eligibility of relevant studies We searched for relevant papers published before 24 November 2009 in literature in English by using the electronic MEDLINE database with the following terms ‘CASP8’ or ‘caspase 8’, ‘cancer’, ‘carcinoma’, ‘tumor’ or ‘tumour’, ‘neoplasm’ and ‘polymorphism’ or ‘variant’. References of the retrieved articles were also screened for original studies. We included all the case–control studies and cohort studies that investigated the association between CASP8 polymorphisms and cancer risk with genotyping data for at least one of three polymorphisms, CASP8 D302H, CASP8 652 6N del and CASP8 Ex14-271A.T. Abstracts, unpublished reports and articles written in non-English language were not considered. Investigations in subjects with family cancer risks or cancer-prone disposition were also excluded. Additionally, when the case–control study was included by more than one article using the same case series, we selected the study that included the largest number of individuals. Data extraction We extracted the following information from each manuscript: author, year of publication, country of origin, cancer type, demographics, ethnicity, genotyping information and source of control groups (population-based, hospital-based or mixed controls). For studies including subjects of different ethnicities, data were extracted separately and categorized as Asians, Caucasians and others. Statistical analysis We performed a meta-analysis to estimate the risk [odds ratio (OR)] of cancer associated with respective CASP8 polymorphisms. In addition to the comparison among all subjects, we also performed stratification analyses by cancer type (if one cancer type contained less than three individual studies, it was combined into the ‘other cancers’ group), ethnicity and source of controls. Lung, bladder, esophageal, head and neck, and pancreatic cancers were defined as smoking-related cancers because tobacco smoking is an established risk factor for these cancers (14–17). In addition, given the roles of estrogens in the etiology of breast, cervical and ovarian cancers, these cancers were defined as estrogen related (18,19). We examined whether the CASP8 polymorphisms were associated with the risk of these cancers as a group as well. We investigated the between-study heterogeneity by using the Cochran’s Q-test, and the heterogeneity was considered significant, if P , 0.05 (20). Values from single studies were combined using models of both random effects (DerSimonian Laird) and fixed effects (Mantel–Haenszel). Inverted funnel plots and the Egger’s test were used to examine the influence of publication bias (linear regression analysis) (21). We checked deviation from the Hardy– Weinberg equilibrium (HWE) among controls for each of CASP8 polymorphisms by a v2-test, with one degree of freedom. All the P values were two sided, and all analyses were done in Statistical Analysis System software (v.9.0; SAS Institute, Cary, NC) and Review Manager (v4.2; Oxford, UK).

Ó The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

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CASP8 polymorphisms contribute to cancer susceptibility

D302H polymorphism, 11 articles (including 16 case–control studies) for the CASP8 652 6N del and 4 articles (including four case–control studies) for the CASP8 Ex14-271A.T. Of these, there were 34 population-based studies, 17 hospital-based studies, 3 studies with mixed controls and one case–control study nested in a cohort study (30). There were 21 breast cancer studies, 11 ovary cancer studies, 11 brain tumor studies, 3 colorectal cancer studies, 2 studies with multiple cancer types and the others, which were categorized into the ‘other cancers’ group. Four of 55 case–control studies were conducted in Asians, one was conducted in Indians and the remaining 50 studies were conducted in Caucasians, of which the study by Haiman et al. (9) included multiple ethnicities. Cancers were confirmed histologically or pathologically in all the studies, except for the study by Cybulski, et al. (32), which did not provide this information.

Results Meta-analyses database We identified a total of 47 relevant publications after initial screening (as of 24 November 2009). Among these, 26 publications appeared to have met the inclusion criteria and were subjected to further examination. We excluded four publications because they did not provide detailed genotype data (22), used subjects with family cancer history or cancer-prone disposition (23–25) or included overlapped data with other studies (25). We also included the data from our study in head and neck cancer (26). Therefore, our final data pooling consisted of 23 publications (Figure 1 and Table I), including 55 case–control studies because eight publications provided more than one individual study. Overall, there were 13 articles (including 40 case–control studies) for the CASP8

Fig. 1. Study flow chart for the process of selecting the final 23 publications.

Table I. Characteristics of populations and cancer types of the studies included in the meta-analysis Author

Country

Ethnicity

Cancer type

Case–control

Source of control

Genotype method

Polymorphisms

MacPherson (5) Frank (27) Son (28) Sun (6) Lan (29) Cox (12) Rajaraman (8)

UK Germany Korea China USA Mixed USA

Caucasian Caucasian Asian Asian Caucasian Caucasian Caucasian

Breast cancer Breast cancer Lung cancer Multiple cancers Lymphoma Breast cancer Brain tumor

2802–3046 355–1098 432–432 4938–4919 451–526 13 005–12 792 623–553

Population Population Hospital Population Population Mixed Hospital

Not defined Not defined PCR–RFLP PCR–RFLP PCR sequencing TaqMan TaqMan

Sigurdson (30)

USA

Caucasian

Breast cancer

Nested in cohort

TaqMan

Li (31)

USA

Caucasian

Melanoma

Hospital

PCR–RFLP

Bethke (13) Cybulski (32) Enjuanes (33) Frank (34) Pittman (35)

UK Poland Spain Germany UK

Caucasian Caucasian Caucasian Caucasian Caucasian

Glioma Multiple cancers Leukemia Breast cancer Colorectal cancer

1005–1013 618–965 692–738 7753–7921 4016–3749

Population Population Mixed Mixed Mixed

Illumina PCR–RFLP Illumina Fluorescent analysis PCR

Hosgood (36) Yang (37) Haiman (9) Ramus (38) Wang (39) Bethke (11) Gangwar (40) Lan (41) Li (26)

USA China USA Mixed China UK India USA USA

Caucasian Asian Mixed Caucasian Chinese Caucasian Indian Caucasian Caucasian

Multiple myeloma Pancreatic cancer Multiple cancers Ovarian cancer Bladder cancer Meningioma Bladder cancer Lymphoma Head and neck cancer

120–507 397–907 6801–5044 5317–9092 365–368 639–638 212–250 1946–1808 1023–1052

Population Population Population Population Hospital Population Hospital Population Hospital

Real-time PCR PCR–RFLP TaqMan TaqMan PCR–RFLP Illumina PCR–RFLP Not defined PCR–RFLP

G.C D302H G.C D302H –652 6N del –652 6N del Ex14-271A.T G.C D302H G.C D302H, Ex14-271A.T G.C D302H, Ex14-271A.T G.C D302H, –652 6N del G.C D302H –652 6N del G.C D302H –652 6N del D302H-G.C, –652 6N del Ex14-271A.T –652 6N del –652 6N del G.C D302H –652 6N del G.C D302H –652 6N del G.C D302H G.C D302H, –652 6N del

859–1083 805–835

PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism.

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Different genotyping methods were used in these studies, including the classical polymerase chain reaction–restriction fragment length polymorphism assay or direct polymerase chain reaction sequencing in 11 of 55 studies, the TaqMan assay in 28 studies, the Illumina or fluorescent fragment analysis in the remaining studies, but three publications did not provide information about genotyping methods (Table I).

Overall, the distribution of genotypes in the controls was consistent with HWE, except for the D302H polymorphism in three studies [Kuopio and Singapore and Swedish Breast Cancer Study contained in the study by Cox et al. (12) and New England based case-control study contained in the study by Ramus et al. (38)] and the –652 6N del polymorphism in two studies [multiethnic cohort study and Los Angeles-based case-control studies contained in the study by Haiman

Fig. 2. ORs of overall cancer risks associated with the three most-studied CASP8 polymorphisms under the homozygote model by random effects for each of the published studies. Comparison of the minor allele homozygotes versus the common allele homozygotes. (A) G.C D302H, (B) –652 6N del and (C) Ex14271A.T. For each study, the estimates of OR and its 95% CI were plotted with a box and a horizontal line. The symbol filled diamond indicates pooled OR and its 95% CI.

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et al. (9)]. As some studies used the same control group (32) or selected study subjects from the same population (11,13), they were grouped as one in the meta-analyses of all subjects with exception of those stratified by tumor site. Quantitative synthesis CASP8 G.C D302H The eligible studies included 33 064 cases and 37 127 control subjects, all of whom were Caucasians. The C or H allele frequency in Caucasians was 12.5% with 95% confidence interval (CI) between 11.8 and 13.2%. Overall, there was statistical evidence of an association between the reduced cancer risk and the minor variant C allele, when all eligible studies were pooled into the meta-analysis (homozygote comparison, C/C versus G/G: OR 5 0.78; 95% CI: 0.69–0.88; dominant comparison, C/CþC/G versus G/G: OR 5 0.93; 95% CI: 0.89–0.98; recessive comparison, C/C versus C/GþG/G: OR 5 0.80; 95% CI: 0.71–0.90) (Figure 2 and Table II).

In the stratified analysis by cancer type, as shown in Table II, we found that individuals with the minor variant genotypes had a significantly lower risk of breast cancer but a higher risk of brain tumors. Significantly, reduced risk was also found to be associated with the minor variant genotypes in different genetic models among the estrogen-related cancers (Table II). For the studies with population-based controls, the C allele was associated with a significantly decreased cancer risk in all genetic models (homozygote comparison: OR 5 0.74; 95% CI: 0.64–0.86; P 5 0.326 for heterogeneity, I2 5 9%; dominant model: OR 5 0.95; 95% CI: 0.90–1.00; P 5 0.255 for heterogeneity, I2 5 14%; recessive model: OR 5 0.76; 95% CI: 0.64–0.87; P 5 0.387 for heterogeneity, I2 5 5%; Table II). However, among studies with hospital-based controls, the association was only observed in the dominant model (OR 5 0.95; 95% CI: 0.90–1.00; P 5 0.032 for heterogeneity, I2 5 51%). In terms of subgroup analysis by ethnicity, the result for Caucasian populations was

Fig. 2. Continued

853

854

0.106 0.387 0.103 0.89 (0.69–1.16) 0.76 (0.64–0.87) 0.74 (0.64–0.85)

the same as that of the overall population because these studies were all conducted in Caucasians. There was no substantial between-study heterogeneity among the 40 studies of the CASP8 D302H polymorphism, except for the dominant model (v2 5 65.6, df 5 39, P 5 0.005). The leave-one-out sensitivity procedure indicated that the study by Enjuanes et al. (33) was the main source of heterogeneity, exclusion of which effectively abrogated the heterogeneity (C/CþC/G versus G/G: P 5 0.080 for heterogeneity, I2 5 25%) without significantly influencing the estimates of overall cancer risks. Although the genotype distribution in the studies of Kuopio and Singapore and Swedish Breast Cancer Study [contained in the study by Cox et al. (12)] and New England based case-control study [contained in the study by Ramus et al. (38)] did not follow HWE, the corresponding pooled ORs were not substantially altered with or without including these studies (data not shown). Finally, no publication bias was detected by either the funnel plot (Figure 3) or the Egger’s test (t 5 1.53, P 5 0.134).

Random effects model. Fixed effects model. c Phet: P value of the Q-test for heterogeneity test. b

a

0.91 (0.63–1.30) 0.75 (0.63–0.89) 0.74 (0.62–0.89) 7267–7490 19 829–24 707 22 592–26 955 None

0.87 (0.67–1.13) 0.74 (0.64–0.86) 0.73 (0.63–0.84)

0.107 0.326 0.119

0.95 (0.90–1.00) 0.96 (0.91–1.03) 0.90 (0.86–0.94)

0.94 (0.90–0.98) 0.95 (0.90–1.00) 0.90 (0.86–0.94)

0.032 0.255 0.845

0.93 (0.65–1.33) 0.76 (0.64–0.89) 0.76 (0.63–0.91)

0.242 0.80 (0.71–0.90) 0.79 (0.69–0.92) 33 064–37 127 None

0.78 (0.69–0.88)

0.200

0.93 (0.89–0.98)

0.92 (0.89–0.95)

0.005

0.81 (0.70–0.93)

0.956 0.226 0.099 0.796 1.36 (0.82–2.26) 0.73 (0.62–0.85) 0.80 (0.59–1.10) 0.87 (0.68–1.12) 1.42 (0.85–2.37) 0.70 (0.58–0.85) 0.87 (0.56–1.35) 0.86 (0.66–1.11) 2179–2133 18 794–20 318 3798–6637 8293–8039

1.42 (0.85–2.37) 0.71 (0.601–0.83) 0.81 (0.59–1.10) 0.86 (0.66–1.10)

0.952 0.264 0.106 0.675

1.27 (1.10–1.47) 0.87 (0.83–0.92) 0.99 (0.90–1.09) 0.89 (0.77–1.04)

1.27 (1.10–1.47) 0.87 (0.83–0.92) 0.99 (0.90–1.09) 0.92 (0.86–0.99)

0.460 0.938 0.857 0.001

1.35 (0.81–2.26) 0.72 (0.60–0.87) 0.87 (0.56–1.35) 0.87 (0.68–1.13)

0.242 0.80 (0.71–0.90) 0.81 (0.70–0.93) 0.92 (0.89–0.95) 0.93 (0.89–0.98) 0.200 0.79 (0.69–0.92)

All Tumor site Brain Breast Ovary Other Ethnicity Caucasian Asian Source of control Hospital Population Estrogen-related Smoking-related

33 064–37 127

0.78 (0.69–0.88)

Phet

OR (95% CI) Case–control

OR (95% CI)

0.005

OR (95% CI)b OR (95% CI)a OR (95% CI) OR (95% CI)

a

Dominant (C/CþC/G versus G/G)

c b a

Homozygote (C/C versus G/G) Sample size Variables

Table II. Associations between the CASP8 G.C D302H polymorphism and cancer risk stratified by cancer site

b

Phet

c

Recessive (C/C versus C/GþG/G)

Phetc

M.Yin et al.

CASP8 652 6N del The eligible studies included 27 459 cases and 31 614 control subjects. The minor allele frequency was significantly different between Asian and Caucasian populations (22.4%; 95% CI: 18.4–26.4 and 49.1%; 95% CI: 47.7–50.6, respectively; P , 0.001). Overall, individuals carrying the CASP8 652 del/del genotype had reduced cancer risk than those with CASP8 652 ins/ins genotype in homozygote, dominant and recessive models (del/del versus ins/ins: OR 5 0.84, 95% CI: 0.75–0.94; del/delþdel/ins versus ins/ins: OR 5 0.88, 95% CI: 0.81–0.96 and del/del versus del/insþins/ins: OR 5 0.90, 95% CI: 0.82–0.99, respectively; Table III). In the stratified analysis by tumor site, significantly reduced risks were found for colorectal cancer only in the dominant model (OR 5 0.89, 95% CI: 0.83–0.96, P 5 0.300 for heterogeneity, I2 5 19%), in addition to breast and other cancer types in homozygote model (breast cancer: OR 5 0.87, 95% CI: 0.76–0.98, P 5 0.025 for heterogeneity, I 2 5 56%; other cancers: OR 5 0.76; 95% CI: 0.61–0.95, P , 0.001 for heterogeneity, I2 5 73%). Further analyses demonstrated that the CASP8 652 del allele was associated with significantly reduced cancer risks in nearly all subgroups, including ethnicities (Asians and Caucasians), studies with different sources of controls (hospital based or population based) and estrogen- or smoking-related cancers (Table III). Our meta-analysis indicated substantial heterogeneity among the overall 16 studies of the CASP8 652 6N del polymorphism (del/del versus ins/ins: v2 5 52.79, df 5 15; P , 0.001). The source of heterogeneity mainly came from sample sizes (P , 0.001), ethnicities (P , 0.001), sources of controls (P , 0.001) and tumor types (P , 0.001). The between-study heterogeneity for the CASP8 652 6N del polymorphism in Asians mainly resulted from three independent studies by Sun et al., Yang et al. and Wang et al. (6,37,39), exclusion of which elevated the OR substantially (del/delþdel/ins versus ins/ins: OR 5 1.06, 95% CI: 0.95–1.18, by fixed effects; P 5 0.677 for heterogeneity, I 2 5 0%). Exclusion of the two studies [multiethnic cohort study and Los Angeles-based case-control studies contained in the study by Haiman et al. (9)], whose genotype distribution deviated from HWE, did not significantly change the corresponding pooled ORs. No single study influenced the pooled ORs qualitatively. No publication bias was detected by either the funnel plot (Figure 3) or the Egger’s test (t 5 1.85, P 5 0.086). CASP8 Ex14-271A.T The eligible studies included 1973 cases and 2587 control subjects, all of whom were Caucasians. Our analysis did not provide any statistical evidence of an association between CASP8 Ex14-271A.T and overall cancer risks (Figure 2 and other data not shown). Further stratified analyses were not performed because of limited data for this polymorphism. There was no substantial heterogeneity among the four studies of the CASP8 Ex14-271A.T polymorphism (v2 5 0.83, df 5 3, P 5 0.842). No publication bias was detected by either the funnel plot (Figure 3) or the Egger’s test (t 5 2.51, P 5 0.129).

Fig. 3. Funnel plot analysis to detect publication bias for each of the CASP8 polymorphisms. (A) G.C D302H, (B) –652 6N del and (C) Ex14-271A.T. Each point represents an individual study for the indicated association.

All above-mentioned analyses used crude ORs for comparisons because not all studies either provided information about the adjustment for other confounders or used the same set of confounders. Therefore, in two supplementary tables (supplementary Tables SI and SII are available at Carcinogenesis Online), we also provided the comparisons of the pooling analyses of crude and adjusted ORs obtained from each study, using the homozygous genetic models. Overall, these results did not change the above-mentioned data substantially and did not change the conclusions.

Table III. Associations between the CASP8 652 6N del polymorphism and cancer risk stratified by cancer site Variables

a

Homozygote (del/del versus ins/ins) a

Dominant (del/delþdel/ins versus ins/ins) c

a

b

Recessive (del/del versus del/insþins/ins)

Case–control

OR (95% CI)

OR (95% CI)

Phet

OR (95% CI)

OR (95% CI)

Phet

OR (95% CI)a

OR (95% CI)b

Phetc

27 459–31 614

0.84 (0.75–0.94)

0.89 (0.85–0.94)

,0.001

0.88 (0.81–0.96)

0.89 (0.86–0.92)

,0.001

0.90 (0.82–0.99)

0.95 (0.91–0.99)

,0.001

11 859–12 847 6251–9433 15 144–18 913

0.87 (0.76–0.98) 0.86 (0.70–1.06) 0.76 (0.61–0.95)

0.91 (0.84–0.98) 0.93 (0.84–1.03) 0.84 (0.79–0.91)

0.025 0.036 ,0.001

0.91 (0.82–1.02) 0.88 (0.81–0.96) 0.81 (0.71–0.93)

0.92 (0.87–0.97) 0.89 (0.83–0.96) 0.85 (0.81–0.89)

,0.001 0.300 ,0.001

0.90 (0.81–1.01) 0.92 (0.76–1.12) 0.86 (0.71–1.03)

0.95 (0.89–1.01) 1.00 (0.92–1.09) 0.91 (0.852–0.97)

0.037 0.031 ,0.001

14 701–15 463 8466–9984

0.93 (0.87–1.00) 0.69 (0.51–0.91)

0.94 (0.88–1.00) 0.62 (0.54–0.71)

0.323 0.025

0.93 (0.88–0.98) 0.85 (0.70–1.04)

0.93 (0.88–0.98) 0.81 (0.76–0.86)

0.600 ,0.001

0.97 (0.91–1.04) 0.71 (0.57–0.89)

0.99 (0.94–1.04) 0.66 (0.58–0.77)

0.201 0.135

4129–4681 18 216–21 970 12 173–13 414 3361–3755

0.82 (0.65–1.02) 0.84 (0.70–1.01) 0.85 (0.74–0.98) 0.63 (0.44–0.89)

0.85 (0.74–0.97) 0.89 (0.84–0.95) 0.90 (0.83–0.97) 0.61 (0.49–0.76)

0.061 ,0.001 0.007 0.132

0.82 (0.71–0.96) 0.93 (0.82–1.06) 0.92 (0.82–1.02) 0.75 (0.64–0.89)

0.84 (0.76–0.92) 0.90 (0.86–0.94) 0.91 (0.86–0.96) 0.75 (0.68–0.82)

0.036 ,0.001 ,0.001 0.077

0.91 (0.76–1.08) 0.86 (0.74–1.00) 0.89 (0.79–1.00) 0.68 (0.51–0.92)

0.93 (0.83–1.04) 0.92 (0.87–0.98) 0.94 (0.88–1.00) 0.67 (0.54–0.84)

0.104 ,0.001 0.012 0.228

Random effects model. Fixed effects model. c Phet: P value of the Q-test for heterogeneity test. b

b

c

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All Tumor site Breast Colorectum Other Ethnicity Caucasian Asian Source of control Hospital Population Estrogen related Smoking related

Sample size

M.Yin et al.

Discussion In this meta-analysis, including a total of 55 174 cancer cases and 59 336 controls from 23 independent publications, we examined the association of three well-characterized polymorphisms (D302H, –652 6N del and Ex14-271A.T) of the CASP8 gene with cancer risk. We demonstrated that the minor alleles of the CASP8 D302H and 652 6N del polymorphisms were associated with a significant decrease in overall cancer risks, whereas the CASP8 Ex14-271A.T polymorphism did not appear to have an influence on cancer susceptibility. From stratification analyses, we found an effect modification of cancer risks by tumor site and source of study controls for CASP8 D302H, whereas the 652 6N del polymorphism showed a protective effect of cancer susceptibility in all the subgroups analyzed. As an apical caspase, caspase 8 is central in initiating a caspase cascade upon receipt of apoptosis signaling from a death receptor– ligand interaction (42). The present meta-analysis supports a significant impact of CASP8 polymorphisms on overall cancer risks, particularly showing a protective effect of D302H and 652 6N del polymorphisms against cancer development. Although the 652 6N del variant was associated with a decrease in caspase 8 expression, which might favor an increased cancer susceptibility, one study has shown that T-lymphocytes carrying the 652 6N del variant have significant lower caspase 8 activity and activationinduced cell death upon stimulation with cancer cell antigens (6). Therefore, individuals with the deletion variant may be less susceptible to cancer development, probably because of the powerful and effective immune surveillance of malignant cells by T-lymphocytes. As for the CASP8 D302H polymorphism, its biological functions have not yet been clarified but presumed to affect interaction of caspase 8 with its regulating proteins (5). Tumor origins could have different susceptibility conferred by CASP8 polymorphisms, as demonstrated by the association of the 302H minor allele with an elevated risk of developing brain tumors but lower risk of breast cancer. Recently, inactivation of the caspase 8 expression by hypermethylation of regulatory sequences of the CASP8 gene was found in relapsed glioblastoma multiforme (43), whereas restoration of caspase 8 expression sensitized resistant tumor cells to death receptor or drug-induced apoptosis (44). These findings supported a protective role of the caspase 8 protein in brain tumor development. Since biological function of the CASP8 D302H polymorphism has not yet been clarified, it remains unclear how the D302H polymorphism may produce such distinct opposite effects in different types of tumors. It may involve the mechanisms by which caspase 8 regulates apoptosis sensitivity of different cancer cells. However, it is probably that the opposite finding in brain tumors may be by chance because most of the data of brain tumor came from the same study population of Northern Europe (11,13). Both tobacco and estrogen may cause genetic alterations and promote cell proliferation. Tobacco smoke is a well-characterized risk factor for cancer of different organs (45), whereas estrogen stimulates cell mitotic activity and carcinogenesis, especially for the breasts and ovaries (19,46). In the subgroup analysis, the modification of tumor susceptibility by CASP8 D302H and 652 6N del polymorphisms was more pronounced among smoking- or estrogen-related cancers, compared with other cancer types. This difference could be due to limited statistical power as a result of small sample size of other cancer types or different carcinogenic mechanisms underlying the etiology of different cancer types. It may also suggest that cells with a high apoptotic rate are more probably to be influenced by CASP8 polymorphisms, since both tobacco and estrogen have been found to induce caspase activation and apoptosis through the nuclear factorkappaB pathway (47,48). Further investigations are required to validate the results of the present meta-analysis. Ethnicity may influence tumor susceptibility by different genetic backgrounds and environmental exposures through gene–gene and gene–environmental interactions (49). Studies whose controls were selected from hospitals tended to have underestimated cancer risks because of inherent selection bias due to the fact that such controls

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may not be the representative of the general population. In the present meta-analysis, the association between the CASP8 D302H polymorphism and cancer risk was stronger among population-based studies than that among hospital-based ones. In addition, the protective effect of the CASP 652 6N del polymorphism was more pronounced in Asians than in Caucasians, despite a higher frequency of the del allele in the latter. These results suggest that the characteristics of study subjects, such as ethnicity and source of control subjects, need to be carefully documented and considered when examining the role of genetic polymorphisms in cancer risk. In interpreting our results of the current meta-analysis, some limitations need to be addressed. Firstly, although the funnel plot and Egger’s test did not show any publication bias, selection bias could have occurred because only studies published in English were included. For example, for the CASP8 D302H polymorphism, no published data were available for Asian populations in English literature, probably because this variant is rare in Asian populations (6). Secondly, there was significant between-study heterogeneity from studies of the 652 6N del polymorphism, and the genotype distribution also showed deviation from HWE in some studies. In addition, the numbers of published studies were still not sufficiently large for the analysis of some particular cancer sites. Thirdly, our meta-analysis was based on unadjusted OR estimates because not all published studies presented adjusted ORs or when they did, the ORs were not adjusted by the same potential confounders, such as age, sex, ethnicity and exposures. Without the original data from each published study, we did try to calculate the summary ORs using adjusted ORs if provided in the original papers. There were no substantial changes in the pooled ORs with or without adjustment, except for the stratified analysis of breast and estrogen-related cancers in the 652 6N del polymorphism in the random effect model (homozygote comparison: OR 5 0.87, 95% CI: 0.72–1.05; OR 5 0.85, 95% CI: 0.68–1.05, respectively), which showed slightly wider 95% CIs, possibly owing to the reduced sample size used in the pooled adjusted analysis. Given these results, our conclusions should be interpreted cautiously. In summary, our meta-analysis supports an association between minor variants of the CASP8 D302H and 652 6N del polymorphisms and reduced cancer risks. The influence of the D302H polymorphism on tumor susceptibility may also be tumor site dependent. Future studies with large sample sizes and tissue-specific biological characterization are required to confirm current findings. Supplementary material Supplementary Tables I and II can be found at http://carcin .oxfordjournals.org/ Funding National Institutes of Health (R01 CA131274, R01 ES011740) to Q.W. Acknowledgements Conflict of Interest Statement: None declared.

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