Transformation Potential of E2 Exposed Breast Cancer Susceptibility Gene Mutation Heterozygous Epithelial Breast Cells

NCT ID: NCT01907438

Last Updated: 2013-07-25

Basic Information

• Recruitment Status: UNKNOWN
• Total Enrollment: 30 participants
• Study Classification: OBSERVATIONAL
• Study Start Date: 2013-09-30
• Study Completion Date: 2015-09-30

Brief Summary

Susceptibility to breast cancer is related to the combination of genetic, hormonal and multiple other environmental risk factors, such as mutations in the BRCA gene and excess exposure to exogenous estrogen, respectively. BRCA is a nuclear protein that maintains genome stability, by acting as a key player in the DNA repair complex. Recently, evidence has emerged that BRCA mutation heterozygosis itself enhances aborted DNA repair and can contribute to breast cancer initiation after exposure to irradiation. In our preliminary results on short-term lymphocyte cultures, we found additional evidence that healthy heterozygous BRCA1 and BRCA2 mutation carriers have a different response to DNA damage than do non-carriers.

The main aim of our ongoing project is to identify the transcriptional modulation and transformation potential of normal BRCA1 and BRCA2 mutation heterozygous epithelial breast cells following irradiation and to examine how it is affected by exposure to estrogen. Our hypotheses will be investigated by RNA-seq and microRNA-seq in order to identify a unique molecular expression profile of the estrogen exposed cells following ionizing irradiation.

Understanding the role of BRCA heterozygosity in cell response to exposure to estrogen and to irradiation may facilitate the development of more appropriate diagnostic and therapeutic strategies for these individuals.

Detailed Description

To study the different cell lineages, we plan to isolate and propagate luminal progenitors from a normal human breast tissue. To this end, 10 human breast tissue samples will be obtained from risk-reducing mastectomy in healthy premenopausal women with BRCA1 mutations. Tissues from 10 premenopausal women undergoing esthetic breast surgery with no family history of breast or ovarian cancers will serve as a control group. In order to isolate primary epithelial cells, human mammary tissue will be minced and enzymatically digested overnight in collagenase and hyaluronidase to yield suspension of epithelial organoids. These organoids will be collected and further digested with trypsin, dispase and deoxyribonuclease 1 (DNAse), will be filtered to generate a single cell suspension, resuspended in Hank's + 2% fetal bovine serum (FBS) and 0.1 mg/mL DNAse, and also incubated with a blocking antibody for 15 minutes on ice.

Cells will be treated with estrogen (E2) (10 nM) for 48 h and then will be irradiated for inducing double-strand break. Cells will be irradiated with 8 Gray (Gy) using a Co60 source.

To accomplish our study, estrogen exposed and unexposed BRCA mutation heterozygous epithelial breast cells will be irradiated and 1 h later RNA will be extracted from the cells using Tri-reagent (Sigma). The RNA will be converted to a library of cDNA (complementary DNA) fragments and will be sent for deep sequencing in the illumina Hi-Seq platform.

Conditions

BRCA1 Gene Mutation; BRCA2 Gene Mutation

Study Design

• Observational Model Type: CASE_CONTROL
• Study Time Perspective: CROSS_SECTIONAL

Study Groups

non-carriers

Tissues from premenopausal women undergoing esthetic breast surgery with no family history of breast or ovarian cancers will be obtained.epithelial breast cells will be isolated, treated with estrogen for 48 h and then will be irradiated.

No interventions assigned to this group

BRCA1 mutation carriers

Tissues from risk-reducing mastectomy in healthy premenopausal women with BRCA1 mutations undergoing.epithelial breast cells will be isolated, treated with estrogen for 48 h and then will be irradiated.

No interventions assigned to this group

BRCA2 mutation carriers

Tissues from risk-reducing mastectomy in healthy premenopausal women with BRCA2 mutations undergoing.epithelial breast cells will be isolated, treated with estrogen for 48 h and then will be irradiated.

No interventions assigned to this group

Eligibility Criteria

Inclusion Criteria

• healthy
• premenopausal
• women
• BRCA1 mutations
• BRCA2 mutations

Exclusion Criteria

• women with breast or ovarian cancer

• Minimum Eligible Age: 25 Years
• Maximum Eligible Age: 50 Years
• Eligible Sex: FEMALE
• Accepts Healthy Volunteers: Yes

Sponsors

Hadassah Medical Organization

OTHER

Sponsor Role: lead

Responsible Party

Asher Salmon

Asher Salmon

Responsibility Role: PRINCIPAL_INVESTIGATOR

Principal Investigators

Asher Y Salmon, MD, PhD

Role: PRINCIPAL_INVESTIGATOR

Hadassah Medical Organization

Locations

Hadassah medical center

Jerusalem, , Israel

Countries

Israel

Central Contacts

Asher Y Salmon, MD, PhD

Role: CONTACT

asalmon@hadassah.org.il

Facility Contacts

Tamar Zahavi, PhD

Role: primary

tamar.harel1@mail.huji.ac.il

Other Identifiers

ISF-1702/12

Identifier Type: -

Identifier Source: org_study_id