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The location of DIG incorporated into PCR amplicons was detected by an immunoperoxidase assay using anti-DIG antibodies (1:100 dilution) reacted for 1 hour at room temperature. The chromogen was diaminobenzidine, and no counterstain was used initially so that visualization of the signal would not be obscured. A semiquantitative judgment of color density of the cellular reactions was the outcome measurement using a scale from 1 to 4+, with 1+ indicating light tan, 2+ indicating medium tan, 3+ indicating dark tan, and 4+ indicating almost black. Only ratings ��2+ were counted as positive in this study. Controls, #links# which were run simultaneously and under identical conditions as the experimental samples, included 1) a smear of the CaSki cell line reacted with complete PCR mix (positive control), and 2) a smear of CaSki cells and an adjacent serial section of each tissue reacted with the PCR mix minus the primers (negative controls). All specimens were evaluated by our team pathologist, S. Krishnamurthy, as part of the clinical diagnosis of the patients. The tumors were graded by using the combined Nottingham histologic grading system. All HPV-positive specimens were reviewed by Dr. Krishnamurthy to evaluate which cell types (normal, premalignant, and/or malignant) were positive for HPV.21 Cells were classified as premalignant according to the consensus guidelines of the Cancer Committee #links# of the College of American Pathologists based on the relative risk of developing breast cancer for women diagnosed with particular nonmalignant breast changes.22 All statistical analyses were performed in SAS version 9.2 (SAS Institute Inc., Cary NC). We used the following procedure (PROC) terms for all analyses: 1) PROC MEANS to calculate the mean, median, standard error, and standard deviation for all continuous variables (age, estrogen receptor status, progesterone receptor status, #links# and patient tumor size); 2) PROC FREQ (frequency) with option CHISQ to perform chi-square tests to test whether women who were positive for ��any�� virus were more likely to have a specific tumor grade after categorizing the tumors as NEWGRADE = 0 (low, low/intermediate), NEWGRADE = 1 (intermediate), or NEWGRADE = 2 (high, intermediate/high); 3) PROC NPAR1WAY (Wilcoxon option) to compare the means of estrogen receptor and progesterone receptor values between women who were positive for ��any�� virus and women without virus (this test was stratified further by race to check whether racial/ethnic differences may factor into the comparison); and 4) PROC TTEST also was used to compare the means of tumor sizes between women who were positive for ��any�� virus and women without virus. All comparisons tested the null hypotheses that the populations were the same using a 2-tailed P value < .05 as the level of significance. The characteristics of the study population and their malignant breast tumors are summarized in Table 1.