Deutscher et al. BMC Cancer 2010, 10:519 http://www.biomedcentral.com/1471-2407/10/519 405 nm. A standard curve was performed on each plate for either TF or Tn. A range of known concentrations of TF or Tn displaying proteins, asialofetuin (TF) or ovine salivary mucin (Tn), were assayed. This allowed us to compare results from plate to plate and account for varia- tion between plates. The data were reported as absorbance (Abs) at 405 nm per microliter of ND. The overall average between batch % coefficients of variation (%CVs) for TF and Tn were 5.5 and 8.9, respectively. The within batch average %CVs were 4.3 and 5.9, respectively. The intraclass correlation coefficient (ICC) for TF is .73 (95% CI .63 - .80) and for Tn it is .68 (95% CI .58 - .77). Statistical analysis Biomarker levels for TF and Tn in the ND samples were heavily skewed and not normally distributed. We there- fore described and analyzed TF and Tn biomarker levels using medians and log-transformed (Log10) means to achieve normal distributions. Univariate analyses using the Mann-Whitney-Wilcoxon signed ranks test were performed to determine median biomarker levels while t-tests were calculated to determine log10 means of the biomarkers. Logistic regression analyses are based on log10 transformed data. Three logistic regression models were used to examine the effects of TF, Tn, and meno- pausal status (pre-vs. post-menopausal) in predicting (a) cancer vs. no cancer diagnosis; (b) cancer vs. benign diagnosis; and (c) abnormal vs. benign diagnosis. Because menopausal status was a near significant pre- dictor in all three overall logistic models, follow-up logistic models were conducted separately for premeno- pausal women and for postmenopausal women, control- ling for age. ROC curves were calculated based on the logistic regression results for the postmenopausal women’s data, and AUC values are presented. Results Recruitment Of 137 women, we successfully collected NAF in 111 of 124 (90%) and PND in 13/13 (100%). Among the 124 women evaluated, 72 (58%) were post- menopausal. Ages ranged from 19-86 years, with a med- ian age in pre- and postmenopausal women of 45 and 61, respectively. Thirty-seven postmenopausal women took hormone replacement therapy (HRT) at one point in their lives, with nine taking HRT at the time of NAF collection. HRT use did not significantly influence the ability of TF or Tn to predict either breast abnormal pathology or cancer. Eighty-three (67%) of the partici- pants were found to have breast cancer (11 DCIS and 72 invasive), and 41 (33%) benign disease (7 AH, 28 H, and 6 normal pathology). Page 3 of 7 Fluid Volume and Biomarker expression not different among women with vs. those without PND ND volume ranged from 0.5-546 microliters, with a mean of 53 and median of 25 microliters. ND volume did not vary by pathologic diagnosis nor by fluid type (NAF vs. PND). We first determined if TF and Tn expression in women requiring diagnostic biopsy due to PND differed from expression in women requiring biopsy who did not have PND. Results of logistic regres- sion analyses conducted with women with PND (n = 13) and NAF (n = 111) were equivalent to results obtained from analyses conducted with only women providing NAF. Therefore, all reported analyses include both PND and NAF samples (N = 124). Biomarker expression increases with the development of precancerous changes in the breast Median concentrations of TF and Tn were 7 and 3 fold higher, respectively (Figure 1), in women with atypical compared to those with usual hyperplasia (H). The dif- ference in TF expression was significant (p = .043), despite limited sample size in the atypical hyperplasia group. The highest median concentration of TF and Tn were in women with ductal carcinoma in situ (DCIS). TF expression trended higher in women with DCIS compared to those with usual hyperplasia (p = .09) and normals (p = .07). TF Concentration is associated with breast atypia and cancer Univariate analysis indicated that TF concentration was higher in women with abnormal (atypia or cancer) than benign pathology (p = .016, Table 1). TF was also higher in women with cancer (DCIS or invasive) than in women with 1) no cancer: atypia or benign pathology (p = .048), and 2) benign pathology (p = .018). Tn concen- tration was not significantly associated with either abnormal pathology or cancer. TF and Tn expression based on menopausal status We analyzed samples based on menopausal status to determine if these markers had greater predictive ability in one or the other population of women. Univariate analyses indicated that TF was a greater predictor of breast atypia and cancer among post- than among pre- menopausal women. TF concentration was higher in postmenopausal women with abnormal than benign pathology (Table 1, p = .007), and in cancer (DCIS or invasive) than in women with 1) no cancer (p = .025), and 2) benign pathology (p = .007). Tn concentration was not significantly associated with abnormal pathology or cancer in either pre- or postmenopausal women.