BMC Cancer (2001) 1:8 http://www.biomedcentral.com/1471-2407/1/8 id-phase extraction and clean-up followed by dual-col- umn gas-chromatographic separation and electron capture detection [26]. To test for quality, bovine serum was used to which three concentrations of 9 commercial analytes were added. With every batch of 10 samples one quality control and one reagent-blank sample was ana- lyzed. All quality control results were compared with lim- its established in the laboratory. If a quality sample was not within control limits the samples in this batch were discarded. Only completely resolved chromatographic peaks area ratios were compared with a linear calibration curve generated by analysis of standards of 6 different concentrations in triplet. Recoveries were calculated by subtracting the background level in the blank serum from the level in the spiked serum and dividing by the original spike concentration. The detection limit for all analytes was based on two SD of the result from the low- est control samples over the course of the analysis. The detection limits on the evaluated compounds are: HCB 0.05 ng/mL, dieldrin 0.15 ng/mL, p,p'-DDE 0.41 ng/mL, and varied for the PCB congeners from 0.13 ng/mL to 0.30ng/mL. For lipid adjustment the total serum lipid concentration (mg/dl) was calculated as: 2.27 × cholesterol (mg/dl) + triglycerides (mg/dl) + 0.623 [27]. A total of 161 cases (67.1%) and their matched controls (318 breast cancer free women) were eligible for analysis of breast cancer risk according to ER. The case group (161) served as a cohort in the survival analysis. Organochlorine concentrations were categorized in four levels of exposure using quartiles as cut-points. Associa- tions between organochlorine exposure and breast can- cer risk were examined by conditional logistic regression. ERP and ERN tumors were analyzed sepa- rately. The Cox proportional hazard method was used to investigate tumor characteristics, and the influence of organochlorine exposure on over all breast cancer sur- vival [28]. The proportional hazard assumption was checked by log (-log) plots from stratified analyses. All variables included in the analyses complied with the as- sumption. Odds ratios (OR), relative risks (RR), and 95% confidence intervals (CI) were computed using SAS sta- tistical software [29]. Body weight (kg, in quartiles), par- ity (0, 1, ≥ 2 children), menopausal status (pre- and postmenopausal), hormone replacement therapy (never, ever), smoking (never, ever), alcohol consumption (nev- er or hardly ever, sometimes each month, sometimes each week, every day), household income (<4000DKK, 4000-10000DKK, >10000DKK per month before tax), and school education (<7, 7-10, >10 years) were included in the risk analysis. Tumor size (actual size in mm, or <50 mm, > 50 mm) degree of spread (axillary lymph nodes with metastatic involvement: 0, > 1 nodes), and stage of disease (early stage: tumor size <50 mm and de- gree of spread 0 nodes; advanced stage: all other cases) were included in the survival analyses as potential con- founders. Backward step-wise procedures were used to evaluate the modifying effect of the potential confound- ers and other organochlorines. Only covariates that reached statistical significance, were allowed to remain in the model. Due to the observations with non-detecta- ble organochlorine concentrations, a test for trend in risk and prognosis was done using quartiles of organochlo- rine concentrations. All performed tests of statistical sig- nificance were two sided with a level of significance on 5%. The study has been performed in accordance with the Helsinki Declaration and approved by the Scientific Eth- ical Committee serving Copenhagen and Frederiksberg Municipality (KF) 01-157/94. Computerized linkage be- tween Danish registries was performed according to Danish legislation. Results The immunohistochemical estrogen receptor analysis showed that 72.0% (116 cases) had developed an ERP breast cancer and 28.0% (45 cases) an ERN cancers. Ta- ble 1 shows the measured lipid adjusted serum organo- chlorine concentration among these breast cancer cases. Table 1: Lipid adjusted serum concentrations of organochlorines among breast cancer cases with available estrogen receptor status. Organochlorine Min – Max concentration Median concentration (ppb)a (ppb) HCB Dieldrin ΣPCB p,p'-DDE LODb – 1,901.50 LOD – 345.69 156.84 – 2,621.21 162.01 – 5,072.56 269.75 28.30 1,076.04 1,129.75 a Parts per billion; b Detection limit High body weight, nulliparity, and use of hormone re- placement therapy were identified as overall breast can- cer risk factors (data not shown) and were therefore included as covariates in the analyses of organochlorine compounds. The excess risk of breast cancer associated with high body weight and hormone replacement thera- py after menopause derives from women who developed and ERN breast cancer, but was not statistical significant (table 2).