Novel polymorphisms in caspase-8 are associated with breast cancer risk in the California Teachers Study

Ethics statement

This study was carried out in compliance with the Helsinki Declaration and approved
by the Institutional Review Boards at each study center, namely, the City of Hope
(COH), the University of Southern California (USC), the Cancer Prevention Institute
of California (CPIC), the University of California at Irvine (UCI), and by the California
State Committee for the Protection of Human Subjects, in accordance with assurances
filed with and approved by the US Department of Health and Human Services. All study
participants provided written informed consent to participate in the study.

Participants

The CTS is a well-established prospective cohort study of 133,479 female California
public school teachers and administrators who were enrolled in the California State
Teachers Retirement System. A detailed account of the methods employed by the CTS
has been published previously 30]. Briefly, participants completed a baseline questionnaire and returned it by mail
in 1995–1996. The baseline survey, which collected information on demographics, personal
and family cancer history, height, weight, history of hormone use, and behavioral
factors including physical activity and alcohol consumption, is available on the CTS
website (www.calteachersstudy.org). New diagnoses of first primary invasive breast cancer among cohort members were
identified through annual linkages with California Cancer Registry (CCR), a legally
mandated statewide population-based cancer reporting system in which cancer data are
obtained from cancer patients’ pathology reports at the hospital in which the patient
was initially diagnosed. CCR ascertainment of newly diagnosed cancers is estimated
to be 99 % complete 31].

For this nested, breast cancer case control study, biospecimens were collected between
2005-2009 from breast cancer cases diagnosed under age 80 years and unaffected controls
in the cohort, all of whom had continued residence in California during the study
period (1995 to time of blood draw). Cases were women who had a histologically confirmed
invasive first primary carcinoma of the breast (International Classification of Disease
for Oncology code C50 restricted to morphology codes under 8590) after 1998. Unaffected
control participants were selected from the cohort and frequency matched to the cases
based on age at baseline (within 5-year age groups), self-reported race/ethnicity
(white, African American, Latina, Asian, and other), and three broad geographic regions
in California (surrounding the three CTS specimen collection centers: CPIC, USC/COH,
and UCI).

Collection of biological specimens and DNA extraction

The collection of specimens has been described previously 32]. Briefly, cases and controls provided a blood sample and completed a brief questionnaire
at the time of blood draw, which updated breast and reproductive and gynecologic history
and several lifestyle factors. Women who declined providing blood provided saliva
in Oragene DNA self-collection kits (DNA Genotek, Kanata, ON, Canada). All biological
specimens were sent overnight to the UCI laboratory. DNA was extracted from blood
clots using Qiagen Clotspin Baskets and DNA QIAmp DNA Blood Maxi Kits (Qiagen, Inc.,
Valencia, CA, USA) in accordance with Qiagen protocols. DNA was extracted from saliva
samples using the Oragene protocol (DNA Genotek).

Genotyping

The 12 tagging SNPs included in this analysis were selected to capture all common
linkage disequilibrium tagging SNPs [minor allele frequency (MAF) of at least 5 %],
20 kb upstream of the 5? untranslated region (UTR) and 10 kb downstream of the 3?
UTR, in individuals of European ancestry with minimum pairwise r ²
of at least 0.80, using data from the International HapMap Project for the white CEPH
(Utah residents with ancestry from northern and western Europe) population [HapMap
release 21, July 2006, genotype build 36 (http://hapmap.ncbi.nlm.nih.gov)] 32].

DNA samples from 1,751 cases and 1,697 controls were plated for genotyping. A random
sample of 193 duplicates (105 cases and 88 controls) was included for quality control.
The samples were genotyped using the Illumina Golden Gate Assay (Illumina, Inc., San
Diego, CA USA) at the University of Southern California Core Facility. Twelve haplotype-tagging
SNPs in CASP8 were included and genotyped. Samples with genotype call rates 90 % were excluded.
Among the remaining samples, 160 randomly selected duplicates exhibited a genotype
concordance rate of 99.9 %. Additional details were described previously 32]. Because the majority of participants were non-Hispanic whites, we restricted analyses
to 2,737 non-Hispanic white women (1,353 cases and 1,384 controls).

Statistical analyses

All statistical tests were two-sided. We used unconditional logistic regression models
to estimate the odds ratios (ORs), 95 % confidence intervals (CIs), and p-values for the association of invasive breast cancer and each SNP, using log-additive
models. Allele frequencies are shown in Additional file 1: Table S1. We adjusted for potential confounding by study center and other known
risk factors, namely, age at baseline, family history (having a first-degree relative
with history of breast cancer), body mass index (25, 25.0-29.9, ?30 kg/m
²
), alcohol consumption in the past year (none, 20 g/day, ?20 g/day), physical activity
in the past 3 years (0-0.5 hrs/wk/yr, 0.51–4.0 hr/wk/yr, 4.0 hr/wk/yr), and menopausal
and hormone therapy (HT) status (premenopausal, postmenopausal and never used HT,
postmenopausal and used HT in the past, postmenopausal and using estrogen only at
baseline, postmenopausal and using estrogen and progesterone at baseline, and unknown)
at baseline. To potentially improve power by increasing subgroup homogeneity, we stratified
our analysis by estrogen receptor (ER) and human epidermal receptor (HER2) status
of breast cancer. We evaluated the association for ER-positive (n?=?1,046), ER-negative (n?=?155), HER2-positive (n?=?159), and HER2-negative (n?=?662) subtype. Some breast cancers were not included in any specific receptor (ER
or HER2) subtype analysis because they were missing either ER or HER2 status. PR status
was not included since PR expression usually follows ER expression 33] and the clinical rationale to determine associations with PR-specific breast cancer
risk was lacking since no chemotherapies or preventive therapies are being studied
for PR status-specific subtypes. While therapies targeting triple-negative breast
cancer are being considered, the number of triple-negative cancers in our subset of
cases and controls was too small for analysis (n?=?60). We used the conservative Bonferroni correction to correct for multiple testing
(n?=?60, 12 SNPs x 5 outcomes). Statistical significance was set to p??0.0008. All analyses were done using SAS software version 9.2.

Recombination rates and linkage disequilibrium across the CASP8 gene was evaluated using the HapMap database (http://hapmap.ncbi.nlm.nih.gov) and r
²
values were computed from the pairwise SNP genotype counts of the generated genotype
data.