Ac Nazario

Assessment of the proliferative, apoptotic and cellular renovation indices of the human mammary epithelium during the follicular and luteal phases of the menstrual cycle

IntroductionDuring the menstrual cycle, the mammary gland goes through sequential waves of proliferation and apoptosis. In mammary epithelial cells, hormonal and non-hormonal factors regulate apoptosis. To determine the cyclical effects of gonadal steroids on breast homeostasis, we evaluated the apoptotic index (AI) determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining in human mammary epithelial cells during the spontaneous menstrual cycle and correlated it with cellular proliferation as determined by the expression of Ki-67 during the same period.MethodsNormal breast tissue samples were obtained from 42 randomly selected patients in the proliferative (n = 21) and luteal (n = 21) phases. Menstrual cycle phase characterization was based on the date of the last and subsequent menses, and on progesterone serum levels obtained at the time of biopsy.ResultsThe proliferation index (PI), defined as the number of Ki-67-positive nuclei per 1,000 epithelial cells, was significantly larger in the luteal phase (30.46) than in the follicular phase (13.45; P = 0.0033). The AI was defined as the number of TUNEL-positive cells per 1,000 epithelial cells. The average AI values in both phases of the menstrual cycle were not statistically significant (P = 0.21). However, the cell renewal index (CRI = PI/AI) was significantly higher in the luteal phase (P = 0.033). A significant cyclical variation of PI, AI and CRI was observed. PI and AI peaks occurred on about the 24th day of the menstrual cycle, whereas the CRI reached higher values on the 28th day.ConclusionsWe conclude that proliferative activity is dependent mainly on hormonal fluctuations, whereas apoptotic activity is probably regulated by hormonal and non-hormonal factors.

Effects of tamoxifen on the breast in the luteal phase of the menstrual cycle

Objectives: The effect of tamoxifen on cyclic mastalgia and on chemoprophylaxis against breast cancer is little known, mainly due to the difficulties in studying the normal human gland. We proposed to evaluate the mitotic index and the nuclear volume of the lobule of women medicated with tamoxifen only during the luteal phase of the menstrual cycle in order to observe the effect of tamoxifen on the normal human mammary gland. Methods: Twenty‐four premenopausal women with fibroadenoma diagnosed via biopsy were studied. The phase of the cycle was determined by the date of menstruation and serum progesterone level in the luteal phase (≥3 ng/ml). The patients admitted to the study and were given written informed consent to participate in the investigation, which was previously approved of by the hospital Ethics Committee. Patients were divided at random into two groups: Group I consisted of 12 untreated women (control) and Group II consisted of 12 patients treated with 20 mg/day tamoxifen for 10 consecutive days beginning on the 13th day of the menstrual cycle. In both groups, the patients were submitted to biopsies of the nodule and of a 1‐cm3 fragment of adjacent mammary parenchyma between the 23rd and 26th day of the cycle. The mitotic index (number of mitoses/1000 nuclei counted) and mean nuclear volume (mean of 10 nuclear volumes for each case) were measured. Results: No mitosis was observed in Group II. There was a reduction in the mean nuclear volume in Group II (Mann–Whitney test). Conclusions: Tamoxifen, when administered only during the luteal phase of the menstrual cycle, significantly reduces the nuclear volume and mitotic activity of the epithelium. This data demonstrates an antagonistic action of tamoxifen on estrogen even when administered for short periods of time.

Hysteroscopic evaluation of the endometrium of post-menopausal patients with breast cancer before and after tamoxifen use

Objective: To evaluate by hysteroscopy and histopathology the influence of tamoxifen in the endometrium of post‐menopausal women with previous breast cancer. Method: Out of 46 patients studied, 20 of them had been using tamoxifen for an average length of 12 months, and are still being followed‐up. Hysteroscopy with endometrial biopsy was performed before and after the use of the drug. Results: The prevalence of endometrial activity before and after this hormoniotherapy was the same, i.e. 10.0%, showing a non‐significant variation. Conclusion: The hormoniotherapy with tamoxifen has not increased the endometrial proliferactive activity of postmenopausal patients with breast cancer. The most common hysteroscopical finding was numerous vesicles disseminated throughout the uterine cavity probably due to atrophy of the endometrium.

Analysis of human mammary fibroadenoma by Ki‐67 index in the follicular and luteal phases of menstrual cycle

Objectives:  Fibroadenoma is the most common benign mammary condition among women aged 35 or younger. Expression of Ki‐67 antigen has been used to compare proliferative activity of mammary fibroadenoma epithelium in the follicular and luteal phases of the menstrual cycle.

The relationship between estrogen receptor gene polymorphism and mammographic density in postmenopausal women

ABSTRACT Objective To assess the relationship between the presence of PVUII and XBAI polymorphisms in the estrogen receptor α gene and mammographic density in postmenopausal women. Methods For the present analysis, 189 postmenopausal women who had never used hormonal therapy and who did not have clinical or mammographic features were selected. Based on the ACR-BIRADS® 2003 classification, the mammographic density was determined by three independent readers (two subjective ratings and one computerized). Blood samples were available to extract DNA according to KIT GFX® protocol. PCR-RFLP was then used to identify the polymorphisms. Results There was a high degree of agreement among the three readers to determine the mammographic density (κ > 0.75). Sixty women (32%) had dense breasts and 129 (68%) had non-dense breasts. The PVUII polymorphism was found in 132 (69.8%) of 189 women, while the XBAI polymorphism was found in 135 (71.4%) women. Parity (p = 0.02) and body mass index (p < 0.0001) were associated with mammographic density. It was observed that, for the XBAI polymorphism, women with two mutated alleles were approximately 2.5 times more likely to be classified in the dense breasts group (p = 0.003) and the presence of both wild alleles was associated with fibroglandular tissue replacement by fat (p = 0.02). Conclusions There was no significant association of the PVUII polymorphism in the estrogen receptor α gene with mammographic density (p = 0.34). However, the XBAI polymorphism was observed at a higher mutated homozygous frequency in women with dense breasts and there was an increased frequency of wild-type homozygous and heterozygous women with fat-replaced breasts (p = 0.01).

Abstract P3-09-13: Assessment of muscle strenght and range of motion of shoulder women undergoing surgery for breast cancer

Background: Surgery for breast cancer can cause various deficiencies in upper limb and the surgery type can influence, normally the radical surgeries are more harmful. The presence of pain, reduced range of motion and decrease of muscle strength of the upper limb in the early postoperative period are some of the major deficiencies of these patients. The aim of this study was to evaluate muscle strength and range of motion of the shoulder in patients undergoing partial or total mastectomy to treat breast cancer preoperatively, 7 and 30 days after surgery. Methods: We included patients with breast cancer and surgical indication in a prospective study in the Gynecological Department of Sao Paulo Federal University, Brazil. Exclusion criteria were previous breast surgeries, immediate reconstruction, bilateral surgery, orthopedic diseases in the shoulder and neurological diseases. A total of 114 women were evaluated preoperatively (Pre), seven (PO7) and thirty days (PO30) after surgery, being 70 partial mastectomies (P) and 44 total mastectomies (T). For the assessment of muscle strength, it was used a Hand Held Dynamometer, model 01163 from Lafayette Instrument Company®, using the peak force in kilogram. It was calibrated to isometric contraction for five seconds. Each movement was measured twice and the average was used for the final rating. To measure the range of motion in degrees was used a goniometer. For both assessments cited the movements were: flexion, extension, adduction, abduction, internal and external rotation of the shoulder. Results: We used the test with two-factor ANOVA and multiple comparisons with the Tukeys test to analyze the results. In the assessment of muscle strength, every movement had a statistically significant difference between the periods Pre, PO7 and PO30 (p < 0.001). Thereby, a reduction of strength in PO7 improvement in PO30, but did not reach the initial period of Pre. The internal rotation, beyond the difference between the periods, showed a difference in muscle strength between the types of surgery (p = 0.005) being greater reduction in group T, on PO7 and PO30. In evaluating range of motion of shoulder, extension and internal rotation showed no statistical differences for periods or for the surgical groups. The flexion, abduction and adduction had statistical differences between the three periods (p < 0.001) and also between surgical types, always with smaller values of range of movements in group T compared to P (flexion p = 0.013; abduction p < 0.001 and adduction p < 0.001). However, the adduction in partial mastectomies had returned its values to the Pre period on PO30 (p < 0.001). And finally the external rotation showed difference only between the periods pre and post-surgery (p < 0.001), but not between PO7 and PO30. Conclusion: Surgery for breast cancer causes reduction in muscle strength of the shoulder that does not return after 30 days of surgery in both surgical types (P and T). It also reduces the range of motion of this joint in flexion, abduction, adduction and external rotation, with deficits of partial always lower than the total mastectomy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-09-13.

Abstract 3031: Angiotensin 1-7 up regulates apoptosis related genes and it preferentially targets differentiated epithelial breast cells

Introduction: Renin Angiotensin System (RAS) components are not only related to breast cancer but also to other cancer types. Angiotensin-(1-7) [Ang-(1-7)] is an endogenous 7-amino acid peptide hormone of the renin-angiotensin system that has antiproliferative properties. The aim of this work is to analyze the action of Ang-(1-7) treatment in MCF-10F (normal) and in SKBR3 (tumoral) epithelial breast cells in their capacity to be droven to apoptosis, and also to assess the expression of the membrane receptors CD24 and CD44 in these cells. Methods: We used the SA Biosciences Human Apoptosis RT *2 Profiler PCR Array profiles to analyze the expression of 84 key genes involved in programmed cell death. Furthermore a flow cytometer (GUAVA) was used for quantification of the membrane proteins CD24 and CD44 in the MCF10F and in the SKBR3 breast cells after 24 hours of Angiotensin 1-7 stimulation. Results: Flow cytometric analysis using MCF10F cells showed that a 24h Ang1-7 stimulation period down regulates CD24 gene expression (about 43%) and it up regulates CD44 gene expression (about 16%). On the other hand there was no difference in the expression of both CD24 and CD44 proteins after Ang1-7 stimulation in SKBR3 tumoral cells. Furthermore, after the gene expression analysis by PCR Array, we found differential expression in MCF10F cells after Ang 1-7 treatment and we observed that the genes had their expression changed about 30% (2.1 to 514 fold increases) and 11% (2.6 to 3.8 fold decreases), respectively on a panel of 84 genes related to apoptosis. For SKBR3 cells, we found that the genes were altered in about 5.9% (2.6 to 5.4 fold increases) and 12% (2.1 to 3.3 fold decreases), respectively. Conclusion: Ang 1-7 appears to modulate the expression of membrane proteins CD24 and CD44 in MCF10F cells, also this hormone seems to induce apoptosis by inducing the expression of apoptosis related genes, such as, Abl1, Akt, Bax, Bcl2 (fold change, 3, 12, 6, and 6, respectively). Another important gene is the Hrk (harakiri, BCL2 interacting protein), that regulates apoptosis through interaction with death-repressor proteins Bcl-2 and Bcl-X(L), it displayed a fold change of 514. On the other hand in SKBR3 cells, comprised almost entirely by mammary stem cells, we found no regulation of CD24 and CD44 proteins by Ang1-7. Moreover, it was observed a very low differential expression in the genes related to the apoptosis pathway in these cells. However, the peptide Ang 1-7 seems to act preferentially in differentiated cells of the normal breast epithelium. Financial Support: FAPESP. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3031. doi:1538-7445.AM2012-3031

P4-02-10: The Relationship between Estrogen Receptor Gene Polymorphism and Mammographic Density in Postmenopausal Women.

Background Assess the relation between the presence of PVUII and XBAI polymorphisms in the estrogen receptor alpha gene and mammographic density in postmenopausal women. Methods For the present analysis, 189 postmenopausal women who had never used hormonal therapy and who did not have clinical or mammographic features were selected. Based on the ACR-BIRADSâ 2003 classification, the mammographic density was determined by three independent readers (two subjective ratings and one computerized - Adobe Photoshop â 7.0 software). Blood samples were available to extract DNA according to KIT GFX â protocol. PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism) was then used to identify the polymorphisms. Results There was a high degree of agreement among the three readers to determine the mammographic density (Kappa>0.75). Sixty women (32%) had dense breasts and 129 (68%) had non-dense breasts. The PVUII polymorphism was found in 132 (69.8%) of 189 women, while the XBAI was found in 135 (71.4%) of women. Parity (p=0.02) and body mass index (p Conclusions There was no significant association of the PVUII polymorphism in the estrogen receptor alpha gene with mammographic density (p=0.34). However, the XBAI polymorphism was observed at a higher mutated homozygous frequency in women with dense breasts and there was an increased frequency of wild-type homozygous and heterozygous women with fat-replaced breasts (p=0.01). Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-02-10.

Prevalence of fibroadenomas in kidney-transplanted patients using cyclosporine A.

Abstract #6078 Background: Fibroadenoma is the commonest benign tumor of female breast, classified as fibroepithelial neoplasia. It is frequent during reproductive years mainly in the 2nd and 3rd decades of life, coinciding with follicular maturation and steroidogenesis. With the beginning of the era of transplants the use of cyclosporine A was accepted by the medical community as potential lymphocyte T suppressant. In 1980 the first report on multiple breast fibroadenomas in renal transplant patients taking cyclosporine A was published. The imaging standard of fibroadenomas also seems to differ from usually found in asymptomatic patients: diameters are notably larger, lower longitudinal / anteroposterior diameter ratio and are more hyperechogenic.
 Objetive: To define the prevalence of breast fibroadenomas in kidney-transplanted patients taking cyclosporine A.
 Materials and Methods: Prevalence of breast fibroadenomas was determined in three groups of patients: 50 women with renal transplant taking cyclosporine A (Group 1); 51 women with renal transplant not taking cyclosporine A (Group 2) and 181 women without renal transplant (Control Group). Evaluation was performed by anamnesis, physical examination and breast ultrasonography. In the case of clinical or imaging suspicion of fibroadenoma, the diagnosis was confirmed by core needle biopsy or lumpectomy.
 Results: The groups were homogeneous regarding age, menstrual status and former breast cancer family history. Fourteen percent fibroadenomas were found in the renal transplant patient group taking cyclosporine A versus 2.0% in the renal transplant patient group not taking cyclosporine A, and 2.8% in the control group. Prevalence of breast fibroadenomas in renal transplant patients taking cyclosporine A was significantly higher than in those not taking the drug and than in the control group (p=0.001). Frequency of fibroadenomas of the control group and renal transplant patients not taking cyclosporine was similar (p=0.073). Multiplicity, bilaterality and greater nodule dimensions were observed more in renal transplant patients taking cyclosporine A, but there was no statistical significance among the groups.
 Conclusion: Breast fibroadenomas are more prevalent in the renal transplant patients taking cyclosporine A. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6078.

Association of estrogen receptor gene alpha polymorphisms with postmenopausal mammary density.

Abstract #5003 Background: With the human genome studies, knowledge about polymorphisms started raising interest in a variety of fields and, in medicine, the evidence of direct action of polymorphisms on the arising and progression of diseases, disclosing the possibility of using them as disease predisposition markers. Substitutions, insertions or deletions which are transmitted through generations and reach frequencies equal or superior to 1% in the population are named polymorphisms. Knowing that the mammographic pattern is a multifactorial character, the objectives of this study were to evaluate a possible association of clinical characteristics and polymorphisms HaeIII, MspI and XbaI of the estrogen receptor gene alpha with postmenopausal mammary density. Materials and Methods: A prospective evaluation was made of 120 women who were not hormone therapy users and had no clinically or mammographically identified breast lesions. All of them underwent bilateral mammography, and the radiological density was determined by three independent observers, with two subjective evaluations based on the ACR-BIRADS ® classification of mammographic patterns, 2003, and one computerized evaluation – the grey-scale histogram tool of the Adobe Photoshop ® 7.0 software. Peripheral blood samples were obtained for DNA extraction, performed according to the GFX ® Kit protocol from Amersham-Pharmacia. After DNA extraction, PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism) was carried out for an analysis of the polymorphisms present in intron 1 (HaeIII and XbaI) and in exon 1 (MspI) of the estrogen receptor gene. Results: There was a high degree of concordance among the observers in the determination of mammary density (Kappa, Pearson and Spearman - p Conclusion: Polymorphism XbaI and the clinical factors age, menarche and body mass index showed to be associated with postmenopausal mammary density. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5003.