Chronic mastitis is a prolonged inflammatory breast disease, and little is known about its etiology. We identified 85 cases and 112 controls from 5 hospitals in Morocco and Egypt. Cases were women with chronic mastitis . Controls had benign breast disease, including fibroadenoma, benign phyllodes, and adenosis. Both groups were identified from histopathologically diagnosed patients from 2008 to 2011, frequency-matched on age. Patient interviews elicited demographic, reproductive, breastfeeding, and clinical histories. Cases had higher parity than controls  and more reported history of contraception use . Cases were less likely to report wearing a bra  and less often used both breasts for breastfeeding . Chronic mastitis cases were significantly less likely to be employed outside home  and more likely to report mice in their households . This is the largest case-control study reported to date on risk factors for chronic mastitis. Our study highlights distinct reproductive risk factors for the disease. Future studies should further explore these factors and the possible immunological and susceptibility predisposing conditions.  Chronic mastitis (CM) is a group of diseases characterized by chronic inflammation of the breast, affecting mainly women of reproductive age in their fourth decade –3. CM isMycobacterium tuberculosis, Staphylococcus aureus, and Candida albicans, as well as several species of Corynebacterium and other rare syndromes and infections [Known etiologies of CM are diverse and include diabetes, lupus erythematosus, fections –5. Severfections –10. HoweNearly all published case series on CM find that the average age of women at presentation is in their fourth decade –4, 11–14The prevalence of CM is generally reported at less than 1% worldwide among women presenting with breast problems in studied hospitals; however, reports vary greatly by country and ethnic group. Reports from Pakistan show prevalence at less than 1% among women undergoing biopsy for breast diseases , and a sGiven the rarity of CM worldwide, little is known about etiology, risk factors, and treatment. A thorough literature review of PubMed, Scopus, and ISI Web of Knowledge using the search term “breast disease OR mastitis OR chronic mastitis OR granulomatous mastitis” and covering 1960 through 2011 revealed that almost all published studies are hospital-based case-series reports. The largest series we identified involved 54 patients with CM . TherefoThe current state of knowledge on CM is inadequate to inform treatment protocols, prevention efforts, or patient education. No risk factors are statistically shown to be associated with CM, severely limiting the potential prevention efforts. The lack of information, combined with the relatively high estimated prevalence in North Africa, created an optimal setting for the study of a neglected rare disease. The aim of this study was to identify potential risk factors for CM to inform future clinical research and possible prevention interventions. Based on a previous case-series work, we hypothesized that breastfeeding and reproductive factors would have important associations with diagnosis of CM.We preformed a retrospective hospital-based case-control study and identified 85 cases of CM and 112 controls from 5 hospitals in Morocco and Egypt. The hospitals in Morocco included L'Hopital Ibn Rochd, Hassan II University, in Casablanca, and L'Hopital Ibn Tofail in Marrakech. The hospitals in Egypt were Cairo University Medical School in Cairo, the Mansoura University Oncology Center in Mansoura city (in the East Nile Delta Region), and the Tanta Cancer Center in Tanta, in the center of the Nile Delta Region. These hospitals were chosen because of their relatively high reported number of CM cases. Cases were defined as any female patient with histopathological diagnosis of chronic mastitis  seen at the study hospitals between 2008 and 2011. The only exclusion criterion was previous diagnosis of malignancy. Controls were women with histopathologic diagnoses of other forms of benign breast diseases, excluding mastitis and malignancy, and including fibroadenoma, benign phyllodes, and adenosis, diagnosed at the same hospitals during the same period. The control conditions were chosen based on no suspected association with breast cancer. Cases and controls identified from pathology records of each collaborating hospital were frequency age-matched within ±5 years. Pathological records of the study subjects were linked to their medical records, and both were abstracted for relevant information. Any contact information available from medical records was used to contact and obtain consent from patients, as well as to conduct a follow-up interview. The consent process and interview were generally conducted in tandem by an Arabic-speaking interviewer over the telephone. At the Mansoura study site in Egypt, interviews were conducted in a face-to-face format at the hospital. No incentive was provided to interview, and the overall response rate among those contacted was 89%.The 2008–2011 databases of the pathology departments of the participating hospitals included 204 cases and 419 controls meeting the study criteria . Of thesInterviewer-administered questionnaire included questions on demographics, reproductive history, breastfeeding history, hormone use, menstrual and menopause history, and occupational history, as well as a section on description of symptoms and treatment for their breast diseases. This questionnaire was a shortened version of a risk-factor assessment that was pilot-tested for reliability and validity in Morocco and Egypt  and is ct-tests for continuous variables, with a two-sided significance level of α = 0.05. Bivariate analysis was performed using logistic regression, adjusted for age and hospital site in the model to control matching factors, to assess significance of variables as risk factors for case status. Due to the small sample size and the large number of strata created in the presence of two matching factors, conditional logistic regression was not used. Data were compiled and imported into SAS, version 9.1, statistical software  for statistical analysis. Crude associations were assessed by frequency tables and descriptive statistics, and differences between cases and controls were tested by Chi-square tests or Fisher's exact tests for categorical variables and by Student's The dataset was then imputed twenty times to allow for complete-case analysis by logistic regression. Imputation was carried out using IVEware, Imputation and Variance Estimation Version 0.2, Survey Methodology Program . Following imputation, the complete set of twenty datasets was used to conduct bivariate analyses and multivariate analyses, adjusted for age and study site, to model predictors of the dichotomous outcome cases status and create an integrated predictive model. In the crude modeling of variables, 10 factors were shown to be significant at alpha ≤ 0.05. These variables were then included in an adjusted model, and all but 1 variable remained significant. This variable was “breastfed at least one child,” as its significance was accounted for in the variables “breastfed all children” and “did not breastfeed.” Additionally, 3 interaction terms were shown to significantly predict case status in the fully adjusted model, and these were included in the final analysis.P = 0.005). P < 0.0001). Additionally, cases had a higher average parity of 3.46 births than controls, 2.30 births (P = 0.0002). Median ages at first birth (22 years) and last birth (29 years) were the same for cases and controls. Similar frequencies of miscarriage (12%), irregular menstruation (18%), and infertility (13%) were seen among cases and controls. A significantly higher proportion of cases reported past use of contraceptive methods (P = 0.005), but when broken down by type of contraception used, no difference was seen. For those for whom information was available, 76% of cases were pre-menopausal; the distribution between menopausal statuses did not differ significantly for cases and controls. However, average age at menopause was 1.25 years lower for cases than for controls.P = 0.0019. Cases were less likely to wear a bra (78%) than controls (90%); P = 0.071.N = 37, 43.5%), one of the types of chronic mastitis included. In terms of temporal proximity to lactation (defined as problem within 6 months of lactation), no significant differences were seen between cases and controls; however, information was missing for the majority of both cases and controls. The main symptom of the breast problem was most likely to be swelling for both cases (40.0%) and controls (74.6%), but cases were more likely to report abscess, secretions, and redness. Treatment administered to cases was most likely to be antibiotics or radiation; controls reported other treatments or radiation as most common. Too little clinical information was reported for these differences to be significant.Results from the adjusted bivariate analysis and fully adjusted logistic regression model, completing postimputation, are shown in A case of CM was 3.76 times more likely to have breastfed all of her children than a control, and cases who breastfed were 4.40 times more likely to have not alternated breasts when breastfeeding. Cases wore a bra less often than controls . Temporal proximity of problem to pregnancy and lactation was also found to be significant in the final model, with cases being 67% more likely than controls to have experienced their breast problem during pregnancy, lactation, or within 6 months after lactation. A woman with CM was found to have 0.71 times lower odds of maintaining employment outside home than a control subject. Additionally, variables associated with reporting mice in the study subject's neighborhood or household were found to be risk factors; cases had a 47% increase in odds of reporting a campaign to control mice in their neighborhoods and a 63% increase in odds of reporting attempts to control mice in their households.Interactions between parity and contraception were found to be significant; likewise, the effect of whether or not a woman breastfed all of her children varied across levels of parity. Both effects were less than additive , with the effect of breastfeeding all children decreasing with each additional birth. Use of only one breast as a risk factor appeared to have a significant interaction with whether or not the breast problem was associated with lactation . Previous studies have suggested risk factors for CM involving parity, breastfeeding habits, contraception use, socioeconomic status, and treatment patterns; however, ours is the first study to present statistically significant findings , 17, 22.History of contraception has been suggested in previous studies as a potential risk factor , 11, 22.Suspected associations with pregnancy have been reported previously in case-series , 14, 17.Breastfeeding has previously been correlated with CM in several case-series , 17, 22;One of the main expected risk factors, as communicated by clinicians and reported in the literature, is the failure to alternate between breasts when breastfeeding . This prNearly all published reports –4, 11–14We included the condition of having mice in one's household on the basis of a hypothesis relating mouse mammary tumor virus (MMTV) to breast cancer . The resWhile presentation and treatment were not included in the final predictive models, due to temporal occurrence after presentation with disease, differences in presentation and treatment between cases and controls were seen. The main symptom of the breast problem was most likely to be swelling for both cases and controls, but cases were more likely to report abscess, secretions, and redness. This fits within descriptions of CM in the literature. Treatment administered to cases was most likely to be antibiotics or radiation; controls reported other treatments or radiation as most common. From the published literature, the optimal treatment of CM is still unclear , 25. RepData and prior studies on CM are extremely limited; however, when compared with the previously published associations, our results reveal similar findings. The majority of published papers and case reports have come from the developing countries, which has been hypothesized to be due to lower prevalence of CM in the developed countries, overdiagnosis in the developing countries, or underdiagnosis of tuberculosis mastitis in the developing countries , 7. AddiThe study has a limitation. Among those for whom medical records were available, many did not have working contact information. However, response rates were high among those we were able to contact. Conclusions drawn from this study therefore require an assumption of nondifferential missingness patterns between cases and controls, along with an assumption that those who could be contacted were a representative sample of all cases and controls. The case definition used in this study was wider than that commonly used to study CM, as the vast majority of the published literature studies specific types of CM. Due to the rarity of the disease and the commonalities in presentation and treatment, as well as a general lack of knowledge, we felt that it was appropriate to study these conditions together. While recall bias is often a concern in case-control studies, the fact that both our case and control populations experienced past breast disease potentially mitigates this concern. There is still some temporal ambiguity; however, many of the potential risk factors surround pregnancy and lactation, and dates of pregnancy were known, eliminating some ambiguity. Despite these limitations, this study is a landmark picture of the epidemiology of CM. Clinical observations long reported in the literature but never studied broadly now have statistical significance, opening the way for future studies. Chronic mastitis is a rare inflammatory disease of the breast. Little is known about the etiology or risk factors for the disease. Because studies on risks, etiologies, and treatments are lacking, women experiencing CM are often incorrectly treated. Despite its relatively severe presentation and lack of standard treatment protocol, we are unaware of any controlled analytic studies modeling risk factors for CM to date. Therefore, we believe that we have conducted the largest case-control study of CM in the literature, investigating the epidemiology and potential risk factors for the disease. Our findings indicate that the disease typically occurs in the fourth decade of life and has a statistically significant association with pregnancy/parity, lactation, breastfeeding patterns, menopausal status, employment status, history of use of contraception, having mice in one's household, and not wearing a bra. Particularly strong associations were found between CM and breastfeeding patterns and CM and parity. These data provide the first comprehensive statistical analysis of potential risk factors for CM. Further studies are required to parse out understandings of mechanisms, establish temporality, and further understand risks and etiologies of chronic mastitis.


A common single nucleotide polymorphism (SNP), rs10795668, located at 10p14, was first identified to be significantly associated with risk of colorectal cancer (CRC) by a genome-wide association study (GWAS) in 2008; however, another GWAS and following replication studies yielded conflicting results.2χ-based Q statistic test and Egger's test, respectively.We conducted a case-control study of 470 cases and 475 controls in a Chinese population and then performed a meta-analysis, integrating the current study and 9 publications to evaluate the association between rs10795668 and CRC risk. Heterogeneity among studies and publication bias were assessed by the P = 0.017). The following meta-analysis further confirmed the significant association, with per-A-allele OR of 0.91  in European population and 0.86  in Asian population. Besides, sensitivity analyses and publication bias assessment indicated the robust stability and reliability of the results.In the case-control study, significant association between the SNP and CRC risk was observed, with per-A-allele OR of 0.71 (95%CI: 0.54–0.94, Results from our case-control study and the followed meta-analysis confirmed the significant association of rs10795668 with CRC risk. Colorectal cancer(CRC) is the third most commonly cancer in males and the second in females, with over 1.2 million new cases and 608,700 deaths in 2008 worldwideAPC, SMAD4 and MMR genes, are responsible for less than 5% of cases in the pathogenesis of CRCCRC is provoked by interactions between genetic and environmental factors. Among all causes for CRC, inherited genetic factors account for approximately 35% of the disease etiology Genome-wide association studies (GWAS), which made possible the genotyping of hundreds of thousands of single nucleotide polymorphisms (SNPs) that tag linkage disequilibrium (LD) blocks in the genome, have successfully identified novel susceptibility loci for CRCThe study was approved by the institutional review board of Tongji Medical College of Huazhong University of Science and Technology. And all participants provided written informed consent to participate in this study.The study population consisted of 470 CRC cases with newly diagnosed CRC and 475 cancer-free controls. Patients were consecutively recruited between January 1, 2007 and November 31, 2009 at the Eighth Hospital of Wuhan, central China, which is a hospital specializing in colorectal diseases and absorbing majority of colorectal patients in Wuhan and nearby regions. Controls were cancer-free individuals living in Wuhan city and surrounding regions, who were selected from a pool of healthy volunteers visiting the health check-up center at the same hospital during the same period of the recruitment of CRC patients. The inclusion criteria for patients included histopathologically confirmed CRC, without previous chemotherapy or radiotherapy and no restriction in regards to sex, age or disease stage. Cases with pathology report designated familial adenomatous polyposis, inflammatory bowel disease, colorectal adenoma, Lynch syndrome, intestinal tuberculosis and schistosomiasis were not eligible. The selection criteria for controls included cancer-free individuals and frequency matched to cases by sex and age (±5 years). All subjects were unrelated ethnic Han Chinese. Demographic and lifestyle information, and medical data were collected by trained interviewers via direct interview using standardized questionnaires. Clinical data were abstracted from hospital medical records. 5-ml peripheral venous blood was drawn from each participant.Genomic DNA was extracted from peripheral blood sample using the RelaxGene Blood System DP319-02  according to the manufacturer's instructions. The rs10795668 SNP was genotyped by using the Sequenom MassARRAY platform . Genotypes were called using MassARRAY Typer 4.0 software 2χ test to compare the observed genotype frequencies to the expected genotype frequencies in controls. Pearson's 2χ test was used to compare the differences in distribution of categorical variables  and Student's t-test was used for continuous variables [age and body weight index (BMI)]. For the SNP with minor allele frequency (MAF) of 0.35, the power of our sample size was calculated to be 0.72 to detect an OR of 0.7 in case-control study.Hardy–Weinberg equilibrium (HWE) was tested by a goodness-of-fit 2P values were two-tailed tested with a significant level at 0.05.For the main effect of rs10795668, unconditional Logistical Regression was conducted to calculate odds ratios (ORs) and their corresponding 95% confidence intervals (CIs), adjusted for potential confounders . ORs and 95% CIs as the metrics of effect size were calculated for genotypes AG versus GG and AA versus GG. In order to avoid the assumption of genetic models, dominant, recessive and additive models were also analyzed. Two-factor gene-environment interaction analyses were evaluated by using likelihood ratio tests comparing nested models with only main effects and models with main effects plus the relevant interaction term. There environmental factors, such as smoking status, alcohol use and BMI, were included in the interaction models. According to the WHO Expert Consultation's conclusion, the BMI cut-off point for overweight for Chinese was set to be 24 kg/mhttp://www.prisma-statement.org) (Checklist S1).To further investigate the association between rs10795668 and CRC risk, a meta-analysis was conducted in accordance with the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement  based on the same strategyStudies were included if they met the all of the following criteria: (1) original study; (2) assessment of the association between rs10795668 and CRC risk; (3) case-control or cohort study design; (4) providing odds ratio (OR) with corresponding confidence 95% interval (95%CI) or sufficient data to calculate them; (5) studies of humans; (6) the genotypes of rs10795668 in controls are in HWE. We left out investigations in subjects with Lynch syndrome. Case reports, comments, reviews and editorials were also excluded. If the studies had overlapping subjects, only the study with the largest population was finally included. If more than one geographic or ethnic population were included in one report, each population was considered separately. All data were extracted independently by two reviewers (Qin Qin and Li Liu) and any disagreement was adjudicated by a third author (Rong Zhong).2χ test (P<0.01)Q-statistic and considered significant at P<0.052I statistic was then utilized to estimate heterogeneity quantitatively P values were two-tailed tested with a significant level at 0.05.The following information was extracted from each study: first author's name, publication year, study country, ethnicity of study population, study design, study type, genotyping method, numbers of cases and controls, control source, MAF. HWE in controls was estimated again in the meta-analysis by the goodness-of-fit P = 0.652). More smokers were observed in cases than in controls (P<0.001). In addition, BMI in cases was significantly higher than in controls (P = 0.023).The characteristics of the study subjects were detailed in P = 0.433). Significant difference in genotype distribution was observed between cases and controls . In the unconditional Logistical Regression model, individuals with the AA genotype showed a significant decreased CRC risk compared with those carrying the GG genotype . In the allelic model, A allele carriers also showed significantly decreased risk compared to those with the G allele carriers . Additionally, significantly decreased risk of CRC was also found in additive model, with per-A-allele OR of 0.71 (95%CI: 0.54–0.94). Likewise, significant association between this polymorphism and decreased CRC risk was found in dominant model . While no significant associations were found for AG versus GG and recessive model.The genotype data of rs10795668 for cases and controls were shown in P value of the interaction term (P≥0.017). Among individuals with BMI≥24 kg/m2, carries with AA and AG genotypes showed significantly decreased risk of CRC compared with those with GG genotype, with an OR of 0.45 (95%CI: 0.22–0.91). However, the association was not found among individuals with BMI<24 kg/m2.To explore the potential interactions between rs10795668 and smoking status, alcohol use and BMI, we performed two-factor gene-environment interaction analyses by unconditional Logistic Regression. The results were displayed in P>0.01).As shown in heterogeneityP = 0.236 and 0.462, respectively). The pooled frequency using the fixed random-effects model were 0.327 (95%CI: 0.322–0.333) in European controls and 0.063 (95%CI: 0.051–0.075) in African American controls. There was significant heterogeneity among the Asian studies (heterogeneityP<0.001), and the pooled frequency was 0.385 (95%CI: 0.352–0.419) under random-effects model. These pooled frequencies were similar to those reported in dbSNP database of 0.353, 0.390 and 0.065 for Europeans, Asians and African Americans, respectively. Because of the different frequency distribution of the SNP rs10795668 across different populations, the meta-analysis was started in each ethnic group separately.No significant heterogeneity were observed in European and African American groups , therefore ORs in Asian group were pooled under random-effects model, and ORs in European and African American group were both pooled under fixed-effects model. Significant association between the variant and CRC risk was found both in European and Asian groups, with ORs of 0.91 (95%CI: 0.89–0.93) (As shown in heterogeneityP = 0.242) .For the European data sets, additional analysis without the initial study Figure S2, the pooled ORs were similar before and after deletion of each study, indicating the robust stability of the current results.To assess the influence of each individual study on the pooled additive OR, sensitivity analyses was performed by removing the individual study sequentially. As shown in Figure S3, the shape of the funnel plot seemed symmetrical. And the Egger's test did not detect any publication bias  for rs10795668 at 10p14 and increased expression of ATP5C1ATP5C1 encodes the gamma subunit of the catalytic core (F1) of the mitochondrial ATP synthase, the enzyme complex responsible for ATP synthesis, known to play a central role in cellular respiration. A common event in tumor cells is the metabolic switch from respiration (in the mitochondria) to glycolysis (in the cytosol), often referred as “the Warburg effect” ATP5B), leading to the disruption of the catalytic function of the ATP synthase complex, an event that has been previously observed in multiple cancer types ATP5C1 associated with the A allele at rs10795668 would be consistent with maintaining the activities of ATP synthase and cellular respiration and potentially inhibiting tumor progression for colorectal cancer. However, ATP5C1, located at10p15.1, is not the closest neighboring gene. Some studies The SNP rs10795668 maps to an 82-kb LD block (8.73–8.81 Mb) within 10p14 The Meta-analysis results showed significant association between the variant rs10795668 and CRC risk both in European and Asian population, but in African Americans the association was borderline significant and in the opposite direction. The pooled A allele frequency across different ethnic population varied. Besides, it was noted that Kupfer SS et al. also found that there was genetic heterogeneity in CRC associations in Americans of African versus European descentSome limitations in the case-control study and the meta-analysis need to be addressed. First, the sample size of our case-control study was relatively small, resulting in the relatively inadequate statistical power. Second, in the meta-analysis, the number of published studies was still insufficient for the analyses in Asian and African American group, which might mask or exaggerate the possible true association. Third, although significant association between rs10795668 and CRC has been confirmed in this study, the functional experiments have not been done, so whether this variant is protecting remained uncertain.In conclusion, our case–control study in a Chinese population and the meta-analysis verified that the variant rs10795668 was significantly associated with CRC risk. Further functional verification should be conducted to confirm the findings, which may help unravel the underlying mechanisms of this variant in CRC development and progression.Figure S1The forest plot of association of rs10795668 with colorectal cancer for European group without the original study. (TIF)(TIF)Click here for additional data file.Figure S2Sensitivity analyses for European group. (TIF)(TIF)Click here for additional data file.Figure S3Funnel plot of publication bias for European group. (TIF)(TIF)Click here for additional data file.Checklist S1The PRISMA 2009 checklist.(DOC)Click here for additional data file.


Evidence indicates that CYP1A1 MspI polymorphism might be a possible risk factor for several malignancies. A growing body of literature has been devoted to the association of CYP1A1 MspI polymorphism with acute myeloid leukemia (AML). However, the results remain conflicting. The aim of the present study was to derive a more precise estimation of the relationship.Meta-analyses assessing the association of CYP1A1 MspI variation with AML were conducted and subgroup analyses on ethnicity and age groups were further performed. Eligible studies were identified for the period up to May 2012.A total of ten case–control studies including 1330 cases and 3688 controls were selected for analysis. The overall data failed to indicate a significant association of CYP1A1 MspI polymorphism with AML risk . In subgroup analysis stratified by ethnicity, significant AML risk was shown among Asians  but not Caucasians or mixed races. In subgroup analysis regarding age groups, no associations were observed in either the childhood AML or the adult AML subgroups.The results of the present study suggested that CYP1A1 MspI polymorphism might be a risk factor for AML among Asians. Further investigations are needed to confirm the conclusions. Acute myeloid leukemia (AML), also known as acute nonlymphocytic leukemia (ANLL), is the most common acute leukemia mostly affecting adults, characterized by the rapid growth of abnormal white blood cells in the bone marrow and impaired production of normal blood cells. The mechanisms for AML genesis are still rarely understood. Evidence suggests that radiation, smoking, obesity and exposure to chemical carcinogens are considered as its possible risk factors . NeverthSeveral genetic polymorphisms have been determined as possible risk factors for leukemia by meta-analyses. Variations of GSTM1, GSTT1, MTHFR C677T and XRCC1 Arg399Gln have been indicated to raise leukemia susceptibility -4. NeverRecent evidence indicates that carcinogen-metabolizing genes might play critical roles in determining individual susceptibility to cancers . SusceptPublished studies devoted to the relationship between CYP1A1 MspI polymorphism and AML risk have generated controversial results. The issue of whether CYP1A1 MspI polymorphism is a risk factor for AML remains uncertain. Therefore, in this study, we aimed to perform a quantitative meta-analysis that increased statistical power to generate more confidential results.Cytochrome P450 1A1, CYP1A1, T3801C, MspI, acute myeloid leukemia, acute nonlymphocytic leukemia, hematology,malignancy, neoplasm, cancer, variation and polymorphism. All searched studies were retrieved and the bibliographies were checked for other relevant publications. Review articles and bibliographies of other relevant studies identified were hand searched to find additional eligible studies.We carried out a search in the Medline, EMBASE, OVID, Sciencedirect, and Chinese National Knowledge Infrastructure (CNKI) without a language limitation, covering all publications published up to May 2012, with a combination of the following keywords: The following criteria were used for the literature selection: first, studies should concern the association of CYP1A1 MspI polymorphism with AML risk; second, studies must be observational studies (Case—control or cohort); third, papers must offer the size of the sample, odds ratios (ORs) and their 95% confidence intervals (CIs), the genetic distribution or the information that can help infer the results. Accordingly, the following criteria for exclusion were also utilized: first, the design and the definition of the experiments were obviously different from those of the selected articles; second, the source of cases and controls and other essential information were not offered; third, reviews and duplicated publications. After deliberate searching, we reviewed all papers in accordance with the criteria defined above for further analysis.Data were carefully extracted from all eligible publications independently by two of the authors according to the inclusion criteria mentioned above. For conflicting evaluations, an agreement was reached following a discussion. If a consensus could not be reached, another author was consulted to resolve the dispute and then a final decision was made by the majority of the votes. The extracted information was entered into a database.P >0.1, ORs were pooled according to the fixed-effect model (Mantel-Haenszel); otherwise, the random-effect model (DerSimonian and laird) was used. The significance of the pooled ORs was determined by Z-test. The Hardy-Weinberg equilibrium (HWE) was assessed by Fisher’s exact test. Publication bias was assessed by visual inspection of funnel plots [The odds ratio (OR) of CYP1A1 MspI polymorphisms and AML risk was estimated for each study. The pooled ORs were performed for an allelic contrast , a homozygote comparison (CC versus TT) and a dominant model (CC + TC versus TT). For detection of any possible sample size biases, the OR and its 95% confidence interval (CI) to each study was plotted against the number of participants respectively. A Chi-square based Q statistic test was performed to assess heterogeneity. If the result of the Q-test was el plots . StatistRelevant publications were retrieved and screened originally. A total of seventy-eight publications were identified, of which sixty irrelevant papers were excluded. As shown in FigureOf the selected publications, one was written in Chinese  while thThere were four groups of Caucasians ,21,25,26The distributions of CYP1A1 MspI genotype as well as the genotyping methods of the included studies are presented in TableI-square value is another index for the heterogeneity test [I-square values were 71.7%, 55.9% and 65.5 for the overall data of the allelic contrast, homozygote comparison and dominant model, respectively, indicating marked heterogeneities between the studies. Hence, the random-effect models were utilized. However, when subgroup analyses regarding ethnicity and age groups were further conducted, we found loss of heterogeneities in the subgroups regarding Caucasians and childhood AML, respectively. As shown in Tableity test , with vaThe main results of the meta-analysis were listed in TableHowever, in subgroup analysis according to ethnicity, increased risk was shown among Asians  under the dominant model, but not the allele contrast or homozygote comparison models. No increased risk could be observed among Caucasians or mixed races under the three genetic models. The data indicated that Asians who carry variant C allele might have increased AML risk relative to those who harbor wild type TT alleles. . Then, one-way sensitivity analysis  was carrFunnel plots were created to detect possible publication bias. Then, Egger’s linear regression tests were used to assess the symmetries of the plots. The funnel plots appeared to be symmetrical for the overall data Figurea. MoreovFor the overall data, the results showed that CYP1A1 MspI polymorphism might not have a significant correlation with AML risk. Moreover, in subgroup analyses stratified by ethnicity, the data suggested an excess AML risk among Asians but not Caucasians or mixed races.Previously, several meta-analyses have been devoted to the association of CYP1A1 MspI polymorphism with other cancer risk. Nevertheless, the results were conflicting. CYP1A1 MspI genetic variations have been indicated to raise risk for lung cancer, cervical cancer, prostate cancer and laryngeal cancer -34. HoweAs for leukemia, a recent meta-analysis by Zhang et al…  regardinIn subgroup analysis according to ethnicity, significant increased risk was found among Asians, but not Caucasians and mixed races. Notably, this association could be only observed in the dominant model but not the allele contrast and homozygote comparison models, indicating that Asians who bear variant C allele of CYP1A1 MspI polymorphism might have an excess AML risk compared with those who carry wild type TT alleles. Possible racial differences in presentation, treatment patterns and survival with respect to AML might exist . The difIn the subgroup analysis stratified by age groups, no increased risk was shown among either the childhood AML or the adult AML subgroups. Evidence indicates that the etiologies of childhood AML and adult AML might be different . MoreoveThe overall data were not stratified by source of controls because all studies concerned the population-based controls, except for one study with limited sample sizes . HospitaIn the present meta-analysis, evident between-study heterogeneities for the overall data were observed for the three comparisons, respectively, and thus, the random-effect models were utilized. In the subgroup analyses, loss of heterogeneities was also found in the subgroups regarding Caucasian and childhood AML, respectively. Though we tried to minimize the possibility of encountering heterogeneity problems by conducting a careful search of the literature and using rigorous criteria for data pooling, evident heterogeneities still existed in some of the comparisons. Therefore, heterogeneities might be multifactoral. In addition to ethnicity and age groups, other factors such as gender, source of controls, histological types and prevalence of lifestyle factors might also yield the heterogeneities.Several limitations should be concerned in the present meta-analysis. First, the primary articles only provided data about Caucasians, Asians and mixed races. Detailed information regarding other ethnicities such as African should be concerned. Second, subgroup analyses regarding gender and other factors such as smoking, drinking and radiation exposure have not been conducted in the present study because relevant information was insufficient in the primary articles. Third, only studies written in English and Chinese were included in this meta-analysis. Any selection bias should be noted. Furthermore, although the meta-analysis in this study is suggestive, high heterogeneity and lack of significant association in any genetic model among Caucasian and Mixed subgroups or age subgroups observed in this study could also originate from the nature of AML as a genetically heterogeneous disease and further assessment on the relationship between CYP1A1 MspI polymorphism and risk of AML subtypes might provide more instructive information. Additionally, gene-gene and gene-environment interactions should also be considered in the further investigations.In summary, the results of the present meta-analysis suggest that variant C allele of CYP1A1 MspI polymorphism might have an association with increased AML risk among Asians. Further investigations are needed to confirm the conclusions.The authors declare that they have no competing interests.WZand ZC conceived of the study, and carried out the analysis of the literatures and drafted the manuscript. LZ and YW carried out the collection of the literatures. BZ helped with the statistical analysis and manuscript drafting. ZC and WZ conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


PIK3CA genotype as well as a previously reported PI3K/mTOR-pathway gene signature (PIK3CA-GS) and their ability to estimate the level of PI3K pathway activation in two clinical trials of newly diagnosed ER-positive breast cancer patients- a total of 81 patients- one of which was randomized between letrozole and placebo vs letrozole and everolimus. The main objectives were to correlate the baseline PIK3CA genotype and GS with the relative change from baseline to day 15 in Ki67 (which has been shown to be prognostic in breast cancer) and phosphorylated S6 (S240) immunohistochemistry (a substrate of mTOR). In the randomized dataset, the PIK3CA-GS could identify those patients with the largest relative decreases in Ki67 to letrozole/everolimus  compared with letrozole/placebo . In a second dataset of pre-surgical everolimus alone, the PIK3CA-GS was not significantly correlated with relative change in Ki67  but with relative change in phosphorlyated S6 (S240) . PIK3CA genotype was not significantly associated with any endpoint in either datasets. Our results suggest that the PIK3CA-GS has potential to identify those ER-positive BCs who may benefit from the addition of everolimus to letrozole. Further evaluation of the PIK3CA-GS as a predictive biomarker is warranted as it may facilitate better selection of responsive patient populations for mTOR inhibition in combination with letrozole.The phosphatidylinositol 3′ kinase (PI3K) pathway is commonly activated in breast cancer and aberrations such as PI3K mutations are common. Recent exciting clinical trial results in advanced estrogen receptor-positive (ER) breast cancer support mTOR activation is a major means of estrogen-independent tumor growth. Hence the means to identify a responsive breast cancer population that would most benefit from these compounds in the adjuvant or earlier stage setting is of high interest. Here we study  PIK3CA mutations, PTEN loss and HER2 amplification are frequent in breast cancer and whilst data suggest they could predict for greater sensitivity to PI3K pathway inhibition, not all sensitivity to PI3K inhibitors can be explained by these markers The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian Target Of Rapamycin (mTOR) pathway is commonly deregulated in breast cancer and consequently, there is intense interest in the clinical development of agents that target this pathway PIK3CA-GS) derived from patients with estrogen receptor (ER)-positive breast cancer with PIK3CA kinase domain mutations which was able to predict PIK3CA mutation status (genotype) in independent datasets and was also associated with better outcomes in tamoxifen-treated ER-positive breast cancers PIK3CA mutated compared with wild-type genotype is associated with a better prognosis, though a robust prognostic study is yet to be performed in large clinical breast cancer cohorts PIK3CA mutant breast cancers. Hence, high levels of the PIK3CA-GS in estrogen receptor (ER)-positive breast cancer were associated with PIK3CA mutant (genotyped) breast cancers but paradoxically low mTORC1 pathway output. This led us to ask the question if the signature could provide an indication of pathway activation in ER-positive breast cancer patients, how this would compare to mutation status per se and importantly, if it could identify patients who may benefit from addition of a PI3K/mTOR pathway inhibitor as well as their hormonal therapy.We have previously reported a gene signature  after two weeks of therapy. Both the change in percentage positively staining Ki67 measured from baseline to day 15 whilst on therapy, hereby referred to as “relative %Ki67 decrease” and the number of patients achieving a “complete cell cycle response”  were evaluated. Both of these endpoints have been shown to be correlated with long term outcomes in ER-positive breast cancer- ie have potential as early biomarkers of efficacy prediction of new drugs and have been used in clinical trials in this disease for this purpose In this study we evaluated the ability of both PIK3CA-GS and the letrozole/everolimus combination compared with the letrozole alone. A biomarker that can identify individuals with a high chance of benefit from a drug may ultimately have more utility in the clinic.Importantly, one of the data sets examined in this study contained samples from a randomized clinical trial. This allowed us the opportunity to determine whether a predictive association existed between the Two independent datasets (A and B) were used for these analyses and the patient populations have been previously described PIK3CA-mutant associated gene signature (PIK3CA-GS) to predict benefit from the addition of everolimus to endocrine therapy (letrozole).Dataset A was a subset of patients from a randomized, double-blind, placebo-controlled, multi-centre, phase II neoadjuvant trial conducted in operable, previously untreated ER-postiive breast cancer greater than 2 cm at diagnosis in post-menopausal women PIK3CA genotype and the PIK3CA-GS with changes in phosphorylated S6, a target of everolimus, as well as changes in Ki67, though it is unknown how changes in Ki67 induced by a mTOR inhibitor relate to prognosis in ER-positive BC.Dataset B was comprised of 23 post-menopausal patients also diagnosed with operable, previously untreated, ER-positive BC. These patients were enrolled in a single arm, single institution pre-surgical study to receive 5 mg of everolimus daily without therapeutic intent for 14 days prior to definitive surgery. All 23 patients had pre-treatment (baseline) tumor samples profiled using Illumina HumanRef-8 v2 Expression Bead-Chip . This dataset was used to examine the association between The main endpoints for this biomarker study were based on centrally reviewed proliferative measurements of Ki67 expression by immunohistochemistry (IHC) log transformed as previously described by Dowsett and colleagues PIK3CA genotype or GS with objective tumor responses were performed.In the original trial cohort, whilst changes in tumor size  assessed using clinical palpation were significantly different between the letrozole/everolimus vs letrozole/placebo arms  Phosphorylated levels of S6 (S240) were also measured using IHC for dataset A and B. The methodology has been previously described PIK3CA-GS was calculated exactly as previously published and is essentially a weighted average of 278 probe sets using weights of +1 or −1, predefined by the association between the gene and the PIK3CA mutation status in the original training series Gene expression data were normalized and log-transformed prior to analysis. The PIK3CA mutation hot spot sequencing for dataset A and B has been previously described PIK3CA-GS was developed by distinguishing PIK3CA genotyped (hot spot) mutant and WT tumors, we assessed the association between the PIK3CA-GS and PIK3CA genotype in these two datasets. The PIK3CA-GS score was used as a continuous variable. Receiver Operating Characteristic (ROC) curves were constructed and predictive ability was assessed by calculating the area under the curve (AUC) together with 95% confidence intervals (CI) using the trapezoidal rule.Several different analyses were planned for this biomarker study. First, because the PIK3CA gene status on its own, (b) the PIK3CA-GS alone and (c) a combination of the two variables. A linear regression approach was used for this purpose, with log transformed Ki67 values as dependent variable, and treatment arm, PIK3CA mutation status, the continuous PIK3CA gene signature as independent variables. When possible in the dataset A, responders were also defined by “absolute D15 Ki67” values as described above. A test for interaction was used to verify whether the slopes of the regression lines between PIK3CA-GS (or the coefficients of the PIK3CA mutation status) and relative change in Ki67 (log transformed) differed according to treatment arms. For visualization purposes, patients were ranked according to their PIK3CA-GS score and three groups of increasing values were obtained using the tertiles.Secondly, we investigated the association between relative %Ki67 change at day 15 from baseline and (a) PIK3CA genotype and PIK3CA-GS. For the regression line, log transformed values of pS6 were used.As an exploratory analysis, changes in percentage staining of phosphorylated S6, one of the substrates of mTOR (pS6 S240), as measured by IHC Histo-score between baseline and day 15 were correlated with Analyses were performed using SPSS version 20.0 . All p-values were two-sided and values ≤0.05 were considered significant. No correction for multiple hypotheses testing was taken as the results were considered hypothesis generating. The Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) criteria were followed in this study PIK3CA-GS with mutation status and anti-proliferative response to letrozole with or without the mTOR inhibitor everolimus or everolimus alone. Baseline characteristics of these data sets are summarized in Eighty-one patients were represented in the two clinical datasets A and B used to assess the association of Concerning this study’s primary biological endpoint, relative %Ki67 decrease, dataset A showed 85.6±4.1% in the letrozole/everolimus compared with 60.8±8.55% in the letrozole/placebo arm  in the letrozole/everolimus arm compared with 6/27 (22%) in the letrozole/placebo arm, again comparable to the global trial cohort. There were only three absolute D15 Ki67 responders in dataset B.PIK3CA mutations was 38.5% (22/58) in dataset A and 26% (6/23) in dataset B. The PIK3CA-GS was associated with PIK3CA mutation status in both datasets with ROC curves evaluating the PIK3CA-GS as a continuous variable  compared with dataset B where the mean Ki67 was 14.08±3.14% and 21.68±3.08% (p = 0.45) in mutant and WT tumors respectively.Baseline Ki67 expression was not statistically different between PIK3CA genotype and changes in %Ki67 decrease was investigated. In dataset A, no significant differences were observed in the letrozole alone arm between patients with PIK3CA mutant and WT BCs  as dependent variable, with treatment arm and the continuous PIK3CA GS as independent variables (PIK3CA-GS (as a continuous variable) and relative %Ki67 decreases were associated with a significant negative trend for the letrozole/everolimus arm , but not the letrozole/placebo arm  with a significant test for interaction between the arms (p = 0.02). In other words, lower PIK3CA-GS scores had the greatest reductions in %Ki67 in letrozole/everolimus treated patients, consistent with the concept that lower scores identified tumors with higher pathway activation.We investigated the correlations between relative %Ki67 changes and the ariables . A test PIK3CA-GS in dataset B where single agent everolimus (5 mg/daily) was given PIK3CA-GS and %Ki67 decreases in this dataset  were associated with larger reductions in Ki67 with letrozole/everolimus compared with letrozole alone . Again, for improved visualization, tertiles of the PIK3CA-GS signature score were used for both mutant and WT population  detected using IHC in the pre-surgical study cohort of dataset B . The mean change in percentage positive cells staining for pS6 from baseline to day 15 for all patients was 79.9±6.24%. As seen, there was no significant difference seen for percentage change in pS6 according to genotype  PIK3CA mutations have been associated with a relatively better prognosis compared with PIK3CA wild-type BC patients PIK3CAPIK3CA mutant breast cell lines have been associated with sensitivity to tamoxifen PIK3CA mutational activation on signaling and clinical relevance in ER-positive BC is unclear.The efforts invested into developing therapeutics targeted against the PI3K signaling pathway have been immense due to the knowledge that deregulation of this pathway contributes to increased tumor growth and proliferation PIK3CA-GS is associated with the PIK3CA mutant genotype. However, here we provide further data to support that higher values of the PIK3CA-GS (i.e.: the mutant-like phenotype) are associated with low levels of pathway activation PIK3CA wild-type is unknown- in other words, there are probably multiple other means of PI3K pathway activation that exist in this group– or that low as opposed to high levels of pathway activation could be enough in the luminal breast cancer subtype for reasons yet to be fully understood- ie for example, too high levels may result in senescence. Furthermore, in this manuscript we report that those ER-positive BCs with lower PIK3CA-GS scores and therefore higher levels pathway activation had greater %Ki67 reductions with the combination letrozole/everolimus treatment compared with letrozole alone–in other words, a significant interaction is seen between treatment arms. Importantly in dataset A the early Ki67 changes were reported to correspond with the better objective responses observed with the combination arm letrozole/everolimus arm compared with letrozole alone, supporting its use as a potential prognostic marker and surrogate endpoint for neoadjuvant trials in endocrine therapy and highlighting the potential significance of our findings Our results, whilst derived from two small studies have important conclusions. These data support our original observations the high levels of the PIK3CA-GS (but not genotype) was correlated with these changes. In contrast, the PIK3CA-GS (and not genotype) was associated with Ki67 responses but not changes in pS6 in dataset A. Dataset A involved a randomization between endocrine therapy with and without everolimus. In dataset A, changes in Ki67 reflected the primary endpoint results (objective decrease in tumor size), whereas changes in phosphorylated S6 were not correlated with clinical outcomes and seemed to indicate a purely pharmacodynamic effect PIK3CA mutant BC samples– many ER-related genes are present in the gene signature and this could explain why the signature could be conceivably also affected by endocrine therapy and have correlations with anti-proliferative Ki67 reductions in this context (ii) the small sample size in dataset B limited our power to see subtle Ki67 effects with the single agent everolimus, (iii) the combination of letrozole/everolimus altered the effects on pS6 reductions compared with everolimus alone, (iv) different patient populations between datasets (v) the lower dose of everolimus and finally (vi) technical issues could all be contributing factors to these observations Admittedly, the results from the second dataset of single agent everolimus alone are hard to interpret given that prognostic value of early Ki67 changes have only been shown for endocrine therapy and no clinical outcome data is currently available (ie no tumor shrinkage or relapse information). As dataset B was designed to be a pre-surgical study, it can only be concluded that everolimus was indeed “hitting its target” in terms of S6 reductions and the PIK3CA mutation status alone could yield the same prognostic or predictive information as the PIK3CA-GS in larger clinical trial data sets that would have more statistical power, notably there was no interaction seen previously in the whole trial cohort of dataset A involving nearly 300 women PIK3CA mutation status and PIK3CA-GS on more robust clinical endpoints such as survival other than Ki67 changes. It is also possible that PIK3CA genotype may be more predictive of sensitivity to specific PI3K or AKT inhibitors rather than mTOR. Regardless, future phase II/III trials should consider upfront stratification by PIK3CA genotype in order to accurately determine efficacy in this population, especially given its high frequency. Perhaps the combination of mutation status (genotype) and PIK3CA-GS (phenotype) could allow optimal therapy individualization for this particular aberration. Moreover, PIK3CA genotype may have different implications in early vs. metastatic settings,Importantly, whilst we cannot exclude the possibility that HER2 negative vs. overpressing breast cancer subtypes – the latter could imply further dependency on p110α or higher “oncogene addiction” to PI3K signaling.PIK3CA-GS in two small cohorts, as correlative tumor collection is not standard in many clinical studies. Yet, this is the first time a PI3K pathway-related microarray-derived gene signature has been validated in an independent randomized, prospective clinical trial dataset for its potential as a predictive biomarker to PI3K-pathway targeted agents.We acknowledge that none of these analyses were prospectively pre-planned in the study protocols and the cohort sizes were small so the conclusions are hypothesis generating. Nevertheless, the strengths of our analysis derive from the fact that the validation was performed using data from prospectively designed clinical trials - one of which was randomized. Importantly, both trials used the same centrally reviewed Ki67 endpoint. Notably, these results highlight the importance of prospective tumor banking, the use of standard endpoints in clinical trials and collaboration between academia and pharma in order to facilitate biomarker validation. Without concerted efforts in this regard, the hope for individualized cancer treatment will remain elusive. As seen, we could only evaluate the PIK3CA-GS in appropriate randomized datasets as a biomarker that could identify women who may derive benefit from the addition of everolimus to letrozole in ER-positive disease.In conclusion, whilst it is clear that many ER-positive BC patients do well with endocrine therapy alone David.chen@novartis.com. Gene expression data for dataset B is available at https://catissuesuite.ecmc.ed.ac.uk/caArray. We acknowledge the Edinburgh Cancer Medicine Centre and Cancer Research UK for tissue banking support for dataset B.De-identified gene expression data for dataset A is available on request to David Chen, Novartis Table S1Table gives PIK3CA-GS scores and clinical information for both datasets.(XLS)Click here for additional data file.


Genome-wide association studies and subsequent replication studies have shown that single nucleotide polymorphisms (SNPs) in the chromosomal region 8q24 are associated with colorectal cancer susceptibility.We examined 11 SNP markers in the 8q24 region between 128.47 and 128.54 Mb, using a total of 1,987 colon cases and 2,339 controls who self-reported as white from two independent, well-characterized study populations. Analysis was performed separately within each study, and combined using random effects meta-analysis. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs) and to test for effect modification by known colon cancer risk factors. We also performed a meta-analysis combining our results with previous studies.2 ranging from 0.18 to 0.93) localized in a 16.2 kb region defined by rs10505477 and rs1056368. The combined results for our two studies of colon cancer showed an OR of 1.10 , and a meta-analysis of our results with previously reported studies of colon and colorectal cancer strongly support the association for this SNP . We did not observe any notable evidence of effect modification by known colon cancer risk factors, and risk did not differ significantly by tumor site or stage.We observed evidence of association for four SNPs in low to high linkage disequilibrium (rOur study confirms the association between polymorphisms on chromosome 8q24 and colon cancer risk and suggests that the susceptibility locus in region 8q24 is not strongly modified by various lifestyle, environmental, and demographic risk factors for colon cancer. APC) and DNA mismatch-repair genes, account for < 5% of the cases and it is expected that a large proportion of the inherited susceptibility is likely to be explained by numerous low-penetrance variants in combination with susceptibility to diet, lifestyle, and environmental exposures. Numerous candidate gene studies have been performed for colorectal cancer, and a large number have detected associations. However, candidate gene studies have not been consistently replicated [Colorectal cancer is the third leading cause of cancer and cancer death for both men and women in the United States . At leasplicated  and estaplicated .The ability to conduct genome-wide association studies that include hundreds of thousands of single nucleotide polymorphisms (SNPs) has considerably improved this situation. Genome-wide scans and subsequent replication in other observational studies have shown that SNPs in the chromosomal region 8q24 are associated with prostate -14, breaIn this paper, we followed up these intriguing findings by examining 11 SNP markers in the 8q24 region between positions 128.47 and 128.54 Mb in two independent, well-characterized study populations. In addition, we performed a meta-analysis of the rs6983267 variant in relation to risk of colon and colorectal cancer. Further, we characterized the epidemiologic architecture of this susceptibility locus by testing for interactions with known environmental risk factors for colorectal cancer, and by examining associations by tumor stage and site.The study uses colon cancer cases and controls from two previously described study populations: a multi-center population-based case-control study focusing on Diet, Activity and Lifestyle (DALS) and colon cancer  and a neCases and controls in the multi-center DALS were recruited from three sites: the Kaiser Permanente Medical Care Program (KPMCP) of Northern California; an eight-county area in Utah; and the metropolitan Twin Cities area of Minnesota . Cases wControls in DALS were frequency matched to cases by 5-year age groups and sex. Controls from KPMCP were randomly selected from membership lists. In Utah, controls who were under 65 years of age were randomly selected from lists generated using random-digit dialing and driver license lists, and those 65 years of age and older were randomly selected from Health Care Financing Administration lists. In Minnesota, control participants were identified from drivers license or state identification lists. Of the controls asked to participate, 64%  cooperated, of whom 85%  provided a blood sample for DNA extraction. Our primary analysis is restricted to 1,461 cases and 1,813 controls who self-reported as white.The WHI Observational Study (OS), a prospective cohort study conducted in conjunction with the WHI randomized Clinical Trials (CT), recruited 93,676 postmenopausal women living in proximity to 40 clinical centers located across the USA -38. BetwAll participants included in this study provided written informed consent, and the study was approved by the human subjects committee at each participating institution.For the DALS study, in-person interviews were used to collect demographic, dietary, and lifestyle data ,40. StudFor WHI, information on demographics, smoking and alcohol use, medical history, lifestyle/behavioral factors, family history of cancer, reproductive history, and details of physical activity, among other risk factors, were obtained by standardized self-administered questionnaires at baseline. Measurements for weight, height, waist, and hip were ascertained during a baseline physical examination. Details on medication usage, including non-steroidal anti-inflammatory drugs (NSAIDs), postmenopausal hormone (PMH), and vitamin and mineral supplements were collected from an interview-administered questionnaire during the baseline clinical visit. Dietary intake was assessed using a self-administered, 122-item Food Frequency Questionnaire at baseline.st degree relative  with colorectal cancer versus no 1st degree relatives with colorectal cancer. Non-smokers were defined as individuals smoking less than 100 cigarettes in their lifetime, and smokers were classified as either current or former smokers. Body mass index  was calculated based on baseline measurements of height and weight for the WHI study, and on self-report of height and weight on a reference date (~2 years before diagnosis for cases and interview for controls) in DALS. Our examination of effect modification by BMI was restricted to men and estrogen-positive women . For physical activity, subjects were divided into those with one or more hour of vigorous/strenuous leisure physical activity per week, and those without at least one hour of vigorous/strenuous leisure physical activity per week. For NSAID use, we divided subjects into current NSAID users versus not-current NSAID users. Effect modification by PMH was examined only in post-menopausal women and we compared ever versus never use of PMH. Current alcohol consumption was dichotomized to less than one drink per week versus one or more drinks per week.Clinical and epidemiologic data was harmonized across the two studies through a systematic evaluation of individual study questionnaires, protocols, and data dictionaries. The reference date for DALS is two years prior to the date of diagnosis. The reference date for WHI is the baseline questionnaire date. We selected seven risk factors that are consistently shown to be associated with colon cancer risk. For family history, subjects were classified as having at least one 1-4). In all three cases, one of the homozygous genotype calls was missing. The call rate was > 95% for 10 out of the 11 SNPs, and the 11th SNP (rs6983267) had a call rate of 94.8%. Blinded duplicates displayed > 99% concordance for all SNPs. Using an exact test, the allele frequencies among controls for self-reported whites did not deviate from HWE for any SNP at p = 0.01. HWE for all SNPs for both the self-reported whites and the full population are presented in Additional file Genotyping was performed using genomic DNA extracted from peripheral blood lymphocytes or from immortalized cell lines. The genotyping lab was blinded to case/control status and duplicate quality-control samples were interspersed among plates (341 duplicates from 226 different subjects). All genotyping was performed by MALDI-TOF mass spectrometry on the Sequenom MassARRAY Compact platform using the iPLEX Gold (low-plex) reaction. In total, we received clear genotyping results for 11 out of 14 SNPs. The remaining three SNPs  were excluded for having discordances with the HapMap positive-control samples and/or severe departures from HWE  and 95% confidence intervals (95% CIs) using a two-stage pooled analysis, which allow us to combine results for the two studies, even though they have different designs ,43. StagFor SNPs that showed associations at alpha level 0.05 and no evidence of between-study heterogeneity, we explored interactions between genetic variations and known risk factors for colon cancer. Specifically, we conducted stratified analyses and tests for multiplicative interaction with inclusion of cross-product terms in the regression models. The statistical significance of interaction terms was assessed using a likelihood-ratio test comparing the full model including the interaction term to the nested model that included only the main effects and adjustment variables. We also did a combined case-only and case-control analysis to examine evidence for gene-environment interactions .Because risk factors for colon cancer can differ by anatomic site, we used polytomous regression analysis to examine the association between variants and cancer subtypes defined by site  and by SEER staging categories . In addition to polytomous regression, we also used the generalized odds ratio to examine stage, ranking localized, regional and distant disease in that order .2 = 0.93 in the HapMap CEU population), and because some studies have used them as tags for one another [2 metric. I2 takes values between 0 percent and 100 percent, with higher values indicating higher levels of heterogeneity [ad hoc analysis including the study that used a sib-pair analysis and did not meet our initial inclusion criteria [We conducted a meta-analysis including all published studies of the 8q24 variant rs6983267 and colorectal cancer. Because rs6983267 and rs10505477 are in high linkage disequilibrium  was associated with an increased risk in DALS  but showed a trend toward decreased risk in the WHI . This SNP showed high between-study heterogeneity . We did not observe any additional significant SNPs when we considered dominant or recessive models of inheritance. Further, the haplotype frequencies did not show statistically significant differences between cases and controls . Four SNPs  showed an increased risk of colon cancer at the alpha = 0.05 level in the combined analysis of individuals who self-report as white, with ORs ranging from 1.1 to 1.3 (Table 2 range: 0.18-0.93). LD was strongest between rs6983267 and rs10505477 (r2 = 0.93)  and show a range from low to high linkage disequilibrium  than in males and estrogen-positive women with a low BMI ; however, we did not observe any evidence for statistically significant gene-environment interactions ; with no evidence of between-study heterogeneity . There was also no evidence for heterogeneity between racial/ethnic groups (p = 0.76), although the number and size of studies was relatively small for non-European/European American populations. A similar association was found when we also included studies that examined the rs10505477 variant [-53; see Additional file 2 = 0.0%; overall heterogeneity p = 0.540). Our results are also consistent with those reported for the rs7014346 SNP in this region  [post hoc analysis, we also included the study by Poynter and colleagues [-23), but did increase overall heterogeneity  . There w . There -44). We examined 10 additional SNPs in the region defined by 128.47 and 128.54 Mb and found the associated interval at alpha = 0.05 to be localized in a 16.2 kb region defined by rs10505477 and rs1056368. We did not observe any statistically significant interactions between the 8q24 genotypes and known colon and colorectal cancer risk factors, and risk did not differ by tumor site or tumor stage.This study confirms the previously reported association between the 8q24 SNP rs6983267 and risk of colon cancer. The strength of association observed in our combined examination of WHI and DALS is relatively modest ; however it is consistent with other studies as shown in our meta-analysis for rs6983267 . In addition, a study of 438 cases from Utah, 405 from Sheffield and 249 from Leeds found notable differences in the strength of association between the US site and the two UK sites, suggesting potential gene-environment interactions; however, this observation was not explained by differences in family history or early onset of disease. Given the potential for differences between males and females, it is important to note that the study did not observe effect modification by sex [We identified no statistically significant interactions between genetic variants in 8q24 and various known risk factors for colon cancer. Although some trends were observed, we acknowledge that we have examined a large number of factors, and our results should be interpreted in the context of the large number of tests performed. This finding of no significant interactions is consistent with our previous study of 2,587 colorectal adenoma and 547 colorectal cancer cases in European American populations, which also showed no statistically significant interactions between rs6983267 and smoking, family history, age, or sex , as welln by sex . These iPOU5F1, which encodes the transcription factor OCT4 [MYC), located about 350 kb telomeric of rs10505477 and rs6983267 [MYC is an important protooncogene, over-expressed in numerous tumors, including colorectal tumors. Two recent studies have provided several important insights into the functional role of the rs6983267 variant [MYC promoter region. Together, these results point to an impact on Wnt signaling and MYC expression as a strong candidate for the biological mechanisms connecting the rs6983267 variant to risk of colorectal cancer [MYC expression in colon tumors based on 8q24 genotypes. However, the studies many not be sensitive to subtle effects that could be relevant for disease risk. Further, the studies do not capture gene expression throughout cancer development [The 8q24 region is typically described as a gene desert . The 8q2tor OCT4 . Howevers6983267 . MYC is  variant ,60. Firsl cancer ,59,60. Ielopment .A major strength of this analysis was that we examined the genetic variation in the chromosome 8q24 region between 128.47 and 128.54 Mb in two well-characterized studies with rich environmental data. This allowed us to thoroughly investigate potential gene-environment interactions and to examine the association by tumor site and location. The two studies differ on several key features. Most notably, the WHI is a matched nested case-control study of older women, whereas DALS is a population-based case-control study of both women and men. However, with the exception of rs9297756, the results were very consistent across the two studies. Further, the rs9297756 finding does not appear to be replicated in other studies, indicating that it is likely to be a spurious finding in DALS rather than a true heterogeneous finding across the two populations. The studies also differed on how they assessed information on environmental risk factors. Notably, for WHI we used prospective ascertainment of baseline characteristics, where as the DALS study had retrospective ascertainment for a period two years before the diagnosis (cases) or selection (controls) date. There were also some differences in how variables were ascertained. For example, for NSAID use, DALS had detailed information on past use and duration from questionnaire data on all participants, while the WHI had information only for current NSAID users. We were able to compare data across studies by restricting our analysis to a comparison of current users at baseline or reference date and current non-users at baseline or reference date. It is possible that misclassification or lack of comparability is responsible for our null findings for gene-environment interactions. Future studies should also investigate potential gene-environment interactions in other well-characterized population-based studies.Our results from a multi-center case-control study and the WHI cohort replicate previous findings of an association between polymorphisms in a 16.2 kb region located between 128.4766 and 128.4928 Mb on chromosome 8q24 and colon cancer risk. Taken as a whole, the evidence for this association is consistent across studies and populations. Most studies performed to date have focused on European and European American populations. Additional replication in other ethnic and racial groups is merited; because of differences in LD patterns across populations, such studies should initially examine variation across the entire 8q24 region. Our results do not suggest evidence for gene-environment interactions underlying this association.The authors declare that they have no competing interests.CMH carried out the statistical analyses, coordinated the study and drafted the manuscript. MLS was a lead in the conception, design and coordination of the DALS study and provided substantive input into the analysis and manuscript writing. DJD oversaw the SNP selection and genotyping. JM performed the genotyping. KC coordinated the data management and delivery of the DALS data and assisted with manuscript preparation. LH participated in the design and analysis of the statistical methods. SAAB, AR, GES, JRM and NH participated in the design and coordination of the WHI study, and provided feedback on the design for this analysis and manuscript. RW participated in the design and coordination of the WHI study, and provided substantive input into the manuscript writing. KWM performed the DNA extraction, oversaw sample handing and management and assisted with SNP selection. RLP leads the WHI clinical coordinating center, and provided substantive input into the statistical methods and manuscript writing. BJC was a lead in the conception, design and coordination of the DALS study, participated in the design and coordination of the WHI study, and provided feedback on the design for this analysis and manuscript. JDP was a lead in the conception, design and coordination of the DALS study, helped conceive of the study and design for this analysis and manuscript, and also helped to draft the manuscript. UP conceived of the study and participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final version of this manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/670/prepubSupplemental table S1: Exact Hardy-Weinberg p-value in controls for self-reported whites and full population for each study. Presents the HWE p-values for controls in WHI and DALS separately, showing both the full population  and subjects who self report as white.Click here for fileSupplemental table S2: Haplotype frequencies for cases and controls. Presents the haplotype frequencies for cases, controls and full population for both DALS and WHI.Click here for fileSupplemental table S3a: Associations between rs10956368 and colorectal cancer stratified by CRC risk factors, by study population. Supplemental table S3b: Associations between rs6983267 and colorectal cancer stratified by CRC risk factors, by study population. Supplemental table S3c: Associations between rs10956368 and colorectal cancer stratified by CRC risk factors, by study population. Each worksheet gives information on our investigation of gene-environment interactions using stratified analysis for a different SNP . Results are presented for WHI, DALS and the combined study.Click here for fileSupplemental table S4: Associations between 8q24 SNPs and colon cancer/colorectal cancer risk stratified by tumor stage. Presents results by tumor stage . Results are presented for WHI, DALS and the combined study.Click here for fileSupplemental table S5: Associations between 8q24 SNPs and colorectal cancer risk stratified by tumor site. Presents results by tumor site . Results are presented for WHI, DALS and the combined study.Click here for fileSupplemental figure S1: Meta-analysis of the association between colorectal cancer and the 8q24 SNPs rs6983267 and rs10505477 including sib-pair analysis. Forest plot of effect sizes and inverse-variance pooled estimates of the association between colorectal cancer risk and 8q24 SNPs for a) both rs6983267 and rs10505477; and b) including the study by Poynter et al which used sib-pair analysis.Click here for fileSupplemental figure S2. Funnel plot and cumulative meta-analysis plots for meta-analysis. Funnel plot of odds ratio (OR) vs. standard error of OR for studies included in the meta-analysis and cumulative meta-analysis plots for rs6983267 in European/European American populations. These diagnostic plots can be used to assess biases.Click here for file


A consensus has not been reached regarding the association of several different gene polymorphisms and susceptibility to obstructive sleep apnea syndrome (OSAS). We performed a meta-analysis to better evaluate the associations between 5-HT2A, 5-HTT, and LEPR polymorphisms, and OSAS.5-HT2A, 5-HTT, and LEPR polymorphisms and OSAS were identified in PubMed and EMBASE. The pooled odd rates (ORs) with 95%CIs were estimated using a fixed-effect or random-effect models. The associations between these polymorphisms and OSAS risk were assessed using dominant, recessive and additive models.Twelve publications were included in this study. The -1438 “A” allele of 5-HT2A was identified as a candidate genetic risk factor for OSAS . Individuals carrying the -1438 “G” allele had a nearly 70% reduced risk of OSAS when compared with AA homozygotes . There was no significant association between 5-HT2A 102C/T and OSAS risk, using any model. The “S” allele of 5-HTTLPR conferred protection against OSAS , while the “10” allele of 5-HTTVNTR contributed to the risk of OSAS . The “GG” genotype of LEPR was associated with a reduced risk of OSAS .The meta-analysis demonstrated that 5-HTR-1438 “A” and 5-HTTVNTR “10” alleles were significantly associated with OSAS. The “S” allele of 5-HTTLPR and the “GG” genotype of LEPR conferred protection against OSAS. Further studies, such as Genome-Wide Association study (GWAS), should be conducted in a large cohort of OSAS patients to confirm our findings. Obstructive sleep apnea syndrome (OSAS) is a respiratory disorder characterized by upper airway obstruction during sleep, breathing pauses with oxygen desaturation, and arousal from sleep. OSAS is the third most prevalent respiratory disease after asthma and chronic obstructive pulmonary disease Although the pathogenetic mechanism of OSAS remains unclear, many factors have been incriminated. A combination of genetic makeup and environmental factors could contribute to the development of OSAS. The high concordance of first-degree relatives with OSAS, family clustering, and ethnic differences show that genetic factors may play an important role in the pathogenesis of OSAS Evaluation of the genetic contribution to the occurrence of OSAS is of interest We searched PubMed and EMBASE in all languages for relevant studies. The final date for inclusion was March 10, 2013. Comprehensive search themes included Medical Subject Headings (MeSH) terms and keywords: “sleep apnea”; “obstructive sleep apnea Syndrome”; “sleep apnea/hypopnea syndrome” and “serotonin 2A receptor(5-HT2A)”; “serotonin transporter protein(5-HTT)”; “leptin receptor (LEPR)”; “polymorphism”; ”variant”; and “genotype”.Inclusion criteria for this study were: (1) OSAS patients must be clearly diagnosed; (2) the study must be a case-control study; (3) the study should report enough genotyping information to extract and estimate odds ratios (ORs) with a 95% confidence interval (CI). Studies were excluded if: (1) the diagnosis was unclear; (2) the study did not contain sufficient data for extraction; (3) the study design was based on case, family or sibling pairs; or (4) no control group was included in the study. When overlapping reports occurred, the study with largest number of patients was selected.All included studies were retrieved and the required information was extracted separately, in duplicate, by two authors (BDQ and SZ). Any discrepancies were resolved by discussion and agreement. The characteristics collected from each study were: author, year of publication, country, age, AHI, sample size, diagnosis criteria, genotype, allele frequency, and specific technique of analysis. If the allele frequency was not given, it was calculated from the corresponding genotype distributions. The OR with its 95%CI was extracted or calculated for each study.There is no standard quality criteria for single nucleotide polymorphism (SNP) studies, so we employed a modified Newcastle-Ottawa scale (NOS) score system to assess the quality of these non-randomized studies 2-based Q test and I2 test. P<0.10 was considered to be representative of significant heterogeneity with the Q test. The I2 statistic represented quantification of heterogeneity ranging from 0% to 100%. Hundred percent represented a high degree of heterogeneity and 50%-<100% represented substantial heterogeneity 2>50% for I2 test), a random-effects model was used to pool the data. Otherwise, a fixed-effects model was used. A funnel plot was employed to visually assess potential publication bias in meta-analyses including more than five studies. The Egger's regression asymmetry test and Begg's rank correlation test were used to statistically examine publication bias. A sensitivity analysis was performed to assess the impact of each individual study upon the overall ORs, using the one-study removal approach We performed a systematic review and meta-analysis following a predetermined protocol in accordance with the Meta-analysis of Observational Studies in Epidemiology (MOOSE) reporting guidelines Two hundred fifty-six non-overlapping articles were identified in the initial published work search. Two hundred eighteen articles were excluded based on screening of abstracts or titles. Thirty-eight full-text articles were retrieved and assessed for eligibility. Twenty-six of these articles were excluded Figure 1Table S1).Nine of the twelve studies included were in English and three in were Chinese. One thousand twenty OSAS patients and one thousand eighty-three control subjects were reported Six studies evaluated the association between 5-HT2A 102C/T and OSAS. The pooled data revealed no significant association between 5-HT2A 102C/T polymorphism and OSAS risk, using any model Table 3.vs. AG; OR: 4.22, 95%CI 2.38–7.50 for AA vs. GG). AA carriers had an increased risk of OSAS compared with individuals having an AG or GG genotype. Significant heterogeneity was observed in the recessive and additive models (2 = 82.60%)  had a nearly 70% reduced risk for the development of OSAS, when compared with AA homozygotes . Individuals with the “A” allele were more susceptible to OSAS than GG homozygotes . Using the additive model, a significant association was found  Figure 2Three studies evaluated the relationship between LEPR- Gln223Arg and OSAS. The “GG” genotype” was associated with a low risk for OSAS, when compared with the “AA” homozygote  Figure 2Sensitivity analyses were performed when more than 3 studies were involved. The stability and reliability of pooled ORs were examined using the leave-one-out method. This method repeated the meta-analysis after sequential exclusion of each study. The estimated ORs were not significantly influenced by any individual study.Data not shown). Both Begg's test and Egger's tests demonstrated no publication bias .The NOS scoring system was used to evaluate study quality. Five studies scored 8 stars, 5 studies scored 7 stars, 1 study scored 6 stars, and 1 study scored 5 stars . Bayazit et al. did not identify this positive association between “AA” genotype and OSAS risk. Yin found that the “AA' genotype was over-expressed in patients with LSaOThe LPR VNTR polymorphism in the 5-HTT gene correlated with the occurrence of OSAS in some, but not all, studies. A meta-analysis was performed to better evaluate the association between LPR VNTR polymorphisms and OSAS. The “S” allele of 5-HTT-LPR and “SS” genotype was associated with a lower frequency of OSAS. The frequency of the “10” allele in the 5-HTT-VNTR was significantly higher in OSAS patients, suggesting that it was a risk factor for OSAS. These findings were consistent with those of Chen et al. and Yue et al., but not Yilmaz et al. Another observation made here was that the frequency of the “GG” genotype in exon 6 of the LEPR gene was significantly lower in OSAS patients than controls, suggesting this genotype was low risk factor.In vitro studies have shown that polymorphisms of the 5-HTR 2A/2C genes influence receptor expression Polymorphisms related to the development of OSAS mainly lie in the serotoninergic system, an important component of sleep and airway function during sleep. The GWAS has given better insight into the genetic nature of human disease, and identified several disease specific genes. The small sample size of previous studies and of this meta-analysis show the need for GWASs of large numbers of OSAS patients in order to better detect genetic associations. The GWAS approach could detect new disease genes, give clues to the pathogenesis of OSAS, and provide indicators for diagnosis, treatment, and prevention of the disease.There were several limitations to this study. The number of included studies was relatively small, restricting the power of the study. Previous studies have shown associations between gender, SaO2, severity of disease and gene polymorphisms. The small number of patients identified in our literature review did not allow subgroup meta-analysis. The publications included in this study designed to evaluate the roles of different gene polymorphisms in OSAS. These different study designs could be a potential source of bias. Ongoing, unpublished and missing studies from the literature review could have contributed to publication bias, although the funnel plots, Egger' test and Begg's test did not identify any bias. Finally, a combination of susceptibility genes and environmental factors could contribute to the development of OSAS. Our study could not evaluate gene-gene and gene–environment interactions due to the small number of patients identified. These findings need to be studied in a large cohort of OSAS patients in the future.In conclusion, our study demonstrated that 5-HT2R, 5-HTT, and LEPR polymorphisms were significantly associated with the development of OSAS. 5-HT2A-1438 “A” and 5-HTT-VNTR “10” were independent risk factors for OSAS. The “S” allele of 5-HTT-LPR and the “GG” genotype of Gln223Arg in the LEPR gene conferred protection against OSAS. The small sample size may have affected the accuracy of our findings. GWAS is needed to better detect candidate disease genes and to expand our understanding of the genetic background of OSAS. Future research should also focus on the clinical relevance of these findings.Table S1The HWE test for ACE(I/D), TNF-α-308 A/G, 5-HT2A-102C/T, 5-HT2A-1438G/A, 5-HTT LPR, 5-HTT VNTR, LEPR genotype distribution in included studies.(DOC)Click here for additional data file.Table S2Methodological quality of included studies according to the NEWCASTLE-OTTAWA Quality Assessment Scale.(DOCX)Click here for additional data file.Checklist S1PRISMA Checklist.(DOC)Click here for additional data file.