Introduction 1 2 3 4 5 6 7 8 10 11 15 16 Methods Study population 17 18 19 Diagnosis of BBD In the NBSS, women who had clinical or radiologic evidence of breast lesion underwent either a needle aspiration or a biopsy. Diagnosis of BBD was performed by a reference pathologist. Our study was restricted to women who had no evidence of either in situ or invasive breast cancer on their initial surgical biopsy. Women with a history of BBD were not excluded from the analyses. During the follow-up period, we identified 4,888 women with a histopathologic diagnosis of BBD, who were followed up for the subsequent development of breast cancer. Selection of cases and controls 18 19 Questionnaire Upon enrollment in the NBSS, all participants completed a questionnaire that sought information on demographic characteristics and risk factors for breast cancer, including menstrual and reproductive histories and family history of breast cancer. Morphometry 20 Statistical analysis t P Results 1 Table 1. Distribution of selected characteristics among breast cancer cases and non-cases   N P Cases Controls Age at menarche <13 30 (49) 26 (37) 0.29 13 13 (21) 22 (31)  14+ 18 (30) 23 (32)  Age at first live birth Nulliparous 11 (18) 9 (13) 0.84 <23 22 (36) 29 (41)  23–26 19 (31) 23 (32)  27+ 9 (15) 10 (14)  Menopausal status Pre- 30 (49) 31 (44) 0.71 Peri- 9 (15) 14 (20)  Post- 22 (36) 26 (36)  Ever used oral contraceptives Yes 35 (57) 42 (60) 0.76 No 26 (43) 28 (40)  Missing 0 1  Ever used postmenopausal estrogens Yes 15 (25) 15 (22) 0.70 No 46 (75) 54 (78)  Missing 0 2  2 <25 32 (53) 41 (58) 0.42 25– < 30 27 (44) 25 (35)  30+ 2 (3) 5 (7)  Family history of breast cancer Yes 23 (38) 28 (39) 0.84 No 38 (62) 43 (61)  Hyperplasia in benign tissue Absent 34 (59) 47 (68) 0.27 Present 24 (41) 22 (32)  Missing 3 2  2 Table 2. Comparison of nuclear morphometric features in benign breast tissue between breast cancer cases and non-cases Morphometric measurements Mean (standard deviation) P n n 2 26.8 (7.5) 25.3 (7.2) 0.25 2 5.2 (1.8) 5.0 (1.6) 0.43 Nuclear perimeter (μm) 19.7 (2.7) 19.3 (2.7) 0.37 Nuclear diameter (μm) 5.8 (0.8) 5.6 (0.8) 0.23 Shortest nuclear axis (μm) 4.8 (0.7) 4.6 (0.7) 0.16 Longest nuclear axis (μm) 7.1 (1.0) 7.0 (1.0) 0.53 Axis ratio 1.5 (0.1) 1.6 (0.2) 0.15 Form_AR 0.984 (0.005) 0.981 (0.007) 0.0089 Form_PE 0.844 (0.037) 0.831 (0.045) 0.083 Form_NCI 3.874 (0.095) 3.909 (0.122) 0.071 Contour 1.198 (0.061) 1.221 (0.080) 0.068 Roundness 1.093 (0.027) 1.103 (0.034) 0.071 3 Table 3. a Morphometric measurements Cut-off value OR (95% CL) b c 2 31.2 1.28 (0.73, 2.25) 0.94 (0.50, 1.78) 2 6.1 1.33 (0.76, 2.31) 1.11 (0.59, 2.07) Nuclear perimeter (μm) 21.4 1.14 (0.70, 1.93) 0.85 (0.47, 1.55) Nuclear diameter (μm) 6.3 1.29 (0.73, 2.27) 0.95 (0.50, 1.79) Shortest nuclear axis (μm) 5.2 1.62 (0.92, 2.86) 1.18 (0.62, 2.26) Longest nuclear axis (μm) 8.0 1.34 (0.75, 2.39) 0.95 (0.50, 1.81) Axis ratio 1.6 0.59 (0.30, 1.17) 0.71 (0.33, 1.54) Form_AR 0.986 2.45 (1.42, 4.22) 3.07 (1.61, 5.84) Form_PE 0.867 1.22 (0.71, 2.08) 1.57 (0.83, 2.97) Form_NCI 3.935 1.07 (0.58, 1.98) 1.18 (0.61, 2.27) Contour 1.236 1.13 (0.61, 2.10) 1.22 (0.63, 2.35) Roundness 1.110 1.07 (0.58, 1.98) 1.18 (0.61, 2.27) a b c Discussion We found that breast cancer risk in women with BBD was positively associated with the shape factor Form_AR, a measurement that takes the shortest nuclear axis and the longest nuclear axis into consideration simultaneously. In contrast, there was no alteration in risk in association with nuclear area, SD of nuclear area, nuclear perimeter, nuclear diameter, shortest nuclear axis, longest nuclear axis, and other shape factors. Although subjects with hyperplasia had greater measures of Form_AR than did subjects without hyperplasia, we adjusted for hyperplasia, suggesting that the association with Form_AR is independent of that due to the presence of hyperplasia. 21 23 24 25 26 27 28 29 16 Our case-control study was nested in a cohort of patients with histopathologically confirmed BBD and our findings are likely to be internally valid. Biased measurement of the study exposures was not likely a source of error, given that the morphometric features were assessed without knowledge of the patient outcome status. Our study power, however, was limited by the relatively small sample size, due to which we were not able to evaluate modifying effects by well-documented risk factors of breast cancer. Moreover, residual confounding might still exist, although to minimize confounding we controlled for menstrual and reproductive history, exogenous estrogen use, body mass index, and family history of breast cancer in multivariate analyses. In conclusion, our study results suggest that the shape factor that takes both shortest nuclear axis and longest nuclear axis into consideration might be of value to predict subsequent development of breast cancer among patients with BBD. Given the limitations of our study, larger studies are warranted to confirm our study results.