Abstract P3-03-27: Assessment of the proliferative activity of breast tissue after the administration of combined oral hormonal contraceptive, associated or not with estriol
Authors: Pellegrini, Jr. Osmar , Nazário Afonso , Waitzberg Angela Flavia L. 
Published: March 2023

Abstract
The molecular mechanisms related to the breast cancer development are poorly understood, particularly those associated with estrogen. Evidence is mounting that developmental differences may influence the risk of cancer. However, understanding breast development, morphology and the biochemical factors that influence them is pertinent to study pre-malignant and malignant conditions that affect the breasts.
Objective: To evaluate the proliferative activity of normal breast epithelium, through the c-myc expression, after the administration of combined oral hormonal contraceptive, associated or not with estriol. Retrospective double-blind study, in which the effect of combined oral contraceptives on the normal breast epithelium in women with fibroadenoma was evaluated. We analyzed 33 women In a cohort of 70 selected (37 excluded). The control group (1) received a compound containing levonorgestrel 0.15mg (LNG) and ethinylestradiol 0.03mg (EE), associated with 2mg of placebo (PLC) manufactured in the same capsule ingested daily for 21 days with an interval of 7 days, during four cycles. The study group (2) received a compound containing levonorgestrel 0.15 mg (LNG) and ethinylestradiol 0.03 mg (EE), associated with estriol 2 mg (E3) manufactured in the same capsule ingested daily for 21 days with an interval of 7 days, up to four cycles, when was proceeded the lumpectomy plus normal breast tissue sample. The UNIFESP-EPM Department of Pathology received the samples collected and fixed in 10% buffered formalin. We used an automation device to perform the Immunohistochemistry reactions, using c-myc antibodies, added DAB developer (diaminobenzidine) that provides a bright brown color that provides good contrast. We used conventional optical microscopy to read TMA slides. We considered positive the nuclei stained with dark brown in contrast to the blue negative to the c-myc reactions. We evaluated the epithelial areas at least five acinar units were present in the sample at 40X magnification. In the statistical analysis, the initial stages of consolidation as well as the analysis were performed in Software R (2020) and Rstudio (version 4.1), using the packages Tidyverse (2016), Psyco, SummaryTools, Janitor and DataExplorer. After verifying the assumptions of normality, independence and linearity, Student’s T test was performed for independent samples. An alpha of 0.05 was chosen for rejection of the null hypothesis, that is, a confidence interval of the test results of 95%. An effect size measure of the difference between the groups was also performed, called Cohen’s D; it ranges from -1 to 1, and the closer to 0, the smaller the effect size of the difference between the groups. Regarding the control group (30.30%) and the case group (69.70%). The table 2 presents the results for group 1 (COC + Placebo), while table 3 presents the results for group 2 (COC + Estriol). The T test was performed to verify a possible significant difference in the levels of cell proliferation in patients with or without Estriol. The normality was verified by the Shapiro-Wilk test, with data following a normal distribution (W = 0.901 and 0.96, p value &gt; 0.3). The equitable variance was verified by the Levine test, which verified the equitable variance in both groups (F (1) = 0.24, p value = 0.62). A mean positive nucleus of 14 (SD = 10.65) was observed in the group of patients with contraceptive plus placebo and a mean of positive nuclei of 20.81 (SD = 9.88) in the group of patients with contraceptive plus Estriol. The ratio of positive nuclei to total was higher in the estriol group than in the control group. However, it was not possible to verify a significant difference in the two groups (T (14.57) = 1.3, p value = 0.215, d Cohen = 0.53). Cohen’s d pointed to a moderate difference effect (table 4).
Table 2. Number of positive, negative and total normal epithelial cell nuclei in immunohistochemical investigation of c-myc antigens in Group 1 (COC + Placebo) (N = 10)
Table 3. Number of positive, negative and total normal epithelial cell nuclei in immunohistochemical investigation of c-myc antigens in Group 2 (COC + Estriol) (N = 23)
Table 4. Inferential results of the ratio of positive normal epithelial cell nuclei as a function of the control and case groups
Citation Format: Osmar Pellegrini, Jr., Afonso Nazário, Angela Flavia L. Waitzberg. Assessment of the proliferative activity of breast tissue after the administration of combined oral hormonal contraceptive, associated or not with estriol [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-03-27.