Additional Supporting Information may be found in the online version of this article.

IJC_24292_sm_SuppFig1.tif831KSupporting Information Figure 1. Preliminary data on ALCAM in breast cancer. ALCAM was initially identified in the condition media of cancer cell lines, BT474 and MDA-MB-468. Analyzing some pooled serum samples by an ALCAM-specific immunoassay (a) showed elevated levels of ALCAM in breast cancer and prostate cancer compared with their control groups of normal female and male, respectively. In the second phase of the screening process, the pools were broken down into individual serum samples (b) and ALCAM levels continued to be increased in breast and prostate cancers compared to their controls. When comparing the values between normal women (n = 9) and patients with breast cancer (n = 35) by the nonparametric Mann Whitney test (two-tailed), the medians were significantly different (median normals = 56 μg/L; median cancer = 84 μg/L; p < 0.0002). For ALCAM, at 90% specificity (cutoff point of 62 μg/L) the sensitivity for breast cancer diagnosis was 91%. CA 15-3 levels were measured in the serum of breast cancer patients and the Spearman correlation coefficient between ALCAM and CA 15-3 was 0.63 for 35 samples with a p-value of <0.0001 (data not shown). Finally, the sensitivity of the test for breast cancer diagnosis in patients where CA 15-3 was normal (<30 U/mL) was 78% (data not shown). This promising preliminary data prompted a more rigorous validation study.
IJC_24292_sm_SuppFig2.tif1433KSupporting Information Figure 2. The assay trueness was verified by the dilution and recovery tests. (a) Dilution test consisted of 4 serum samples that were serially diluted and analyzed for ALCAM. The results were analyzed as a regression of the Expected vs. Observed values. (b) Recovery test spiking in different concentrations of recombinant ALCAM into serum samples. The observed values were then compared to the expected values to determine the % recovery.
IJC_24292_sm_SuppFig3.tif1035KSupporting Information Figure 3. Scatter plot of individual markers for cases and female controls versus age. Solid lines and dashed lines are loess fit for cancer and normal patients, respectively. Slopes are based on the fit of linear regression models of log(marker) with age as the predictor.
IJC_24292_sm_SuppFig4.tif1406KSupporting Information Figure 4. Scatter plot of ALCAM (y-axis) distribution by tumor grade (x-axis) of the primary breast carcinoma cases examined. The solid horizontal line indicates the median value for each of the groups.
IJC_24292_sm_SuppTable1.xls34KSupporting Information Table 1. Clinical information on 150 breast cancer specimens used in this study.

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