This study was approved by the Institutional Review Boards of the University of California, Berkeley and all collaborating institutions, and a written informed consent was obtained from the parents or guardians of all participating subjects.
Detailed recruitment process has been described previously (5
). Briefly, subjects of this analysis were recruited by the Northern California Childhood Leukemia Study (NCCLS). The NCCLS is an ongoing case-control study that started in 1995 and recruits subjects from 35 counties in Northern and Central California. Case subjects newly diagnosed with leukemia are recruited from nine hospitals usually within 72 hours of diagnosis. Birth certificate information obtained from California Office of Vital Records is used to select one or two controls for each case, matching on age, sex, Hispanic ethnicity, and maternal race. The eligibility criteria for all subjects are: 1) being a resident of the study area; 2) being younger than 15 years old at case diagnosis (reference date for the matched controls); 3) having at least one English- or Spanish-speaking parent or guardian; and 4) having not been previously diagnosed with cancer. We published previously on the association between maternal IgE and childhood leukemia using 139 ALL cases and 193 controls recruited by the NCCLS between August 2000 and December 2007 (5
). We selected the study subjects from these subjects in order to investigate the correlation between neonatal cytokine and maternal IgE in addition to evaluating the association between neonatal cytokine profiles and childhood ALL. 116 of the 139 ALL cases had an archived neonatal blood spot available to be included in the current study. 116 of the 193 controls were selected to match the 116 ALL cases on age (birthdate), sex, and race/ethnicity.
For each subject, 1/8 of an archived newborn blood spot was excised, placed in an Eppendorf tube with 160 uL of extraction buffer (phosphate buffered saline, pH 7.4, 0.5% Tween-20, 1% BSA, and 1X complete protease inhibitor cocktail [Roche]), shaken at 600rpm under room temperature for 1-1.5 hour, then incubated at 4°C overnight. Extracts were assayed in duplicate and block randomized on 96-well plates, with each plate containing equal number of cases and controls and the same proportion of racial/ethnic groups along with a seven point standard curve per plate. Eleven cytokines (IL2, IL4, IL5, IL6, IL10, IL12, IL13, IL17, GM-CSF, IFN-γ, TNF-α) were measured using a Luminex bead-based assay (Bio-Rad). Protein content of each extract was determined with the Bradford Assay (Bio-Rad). Cytokine measurements for each duplicate were averaged and normalized to protein concentrations.
Demographic characteristics were compared between cases and controls using chi-squared tests or t-tests. Univariate analyses were first performed to compare the neonatal cytokine levels between cases and controls using Wilcoxon rank-sum tests and t-tests. In addition, cytokine levels were divided into tertiles based on levels among the controls, and the proportions of cases and controls in different tertiles were compared using chi-squared tests. Multivariable analyses were performed using unconditional logistic regression with case/control status as outcome and each cytokine as the independent variable adjusted for child's age, sex, race/ethnicity, and annual household income. Annual household income, a proxy measure of socioeconomic status, was included as a covariate because its distribution was different between cases and controls. Finally, all cytokines were included in the same statistical model to assess for the independent effect of each cytokine on the risk of childhood ALL.