The Effect of the Menstrual Cycle on the Normal Human Breast

The Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center [www.komentissuebank.iu.edu] (KTB) was established expressly for the prospective collection of normal, healthy breast tissue from volunteer donors. Blood is also obtained from donors at the time of donation and is processed for serum, plasma and peripheral blood leukocyte DNA. Specimens are annotated and data includes age, race, ethnicity, personal health history, family cancer history, medication usage at the time of donation, and breast cancer risk factors. Premenopausal donors to the KTB were identified by a query of the Bank's database. Hematoxylin and eosin stained sections of the formalin-fixed paraffin-embedded tissue of the identified donors were reviewed and tissue was graded on the basis of the abundance of epithelium within the section. Only tissue containing abundant epithelium was considered for this study. Based on dates, the specimens of nine women in the follicular phase of the menstrual cycle and five in the luteal phase were chosen. Six donors using hormonal contraception at the time of donation were also included. Whole blood obtained from 19 of the 20 donors at the time of tissue donation was available and it was processed for serum. Estradiol, estriol, luteinizing hormone and progesterone concentrations were determined by the Indiana University Health Pathology Laboratory using a Beckman Unicel DxI 800 Immunoassay System. The phase of the menstrual cycle was verified by serum progesterone concentration. The epithelium of these 20 specimens was microdissected from multiple 8 micron frozen tissue sections. Total RNA extracted from the tissue was subsequently depleted of rRNA via locked nucleic acid probes. This enabled profiling of both poly-A and non-poly-A RNA species. Barcoded cDNA libraries from the 20 normal breast epithelia were prepared and sequenced on an Applied Biosystems (AB) SOLiD3 or SOLiD 4 platform. RNA-seq reads for each sample were then mapped to the human genome (hg19) using the LifeScope software version 2.5.1 (Life Technologies, Foster City, CA) and BAM (Binary Alignment/Map) files generated. Read counts for each gene were derived from the output BAM files using the RefSeq database (UCSC Genome Brower) as the gene model.]]>
For donation to the KTB: Inclusion Criteria: Women ages ≥ 18 years without known active breast cancer. Women ages ≥ 18 years with Stage 0-IV breast cancer. In these women, tissue collection will be limited to blood, salvia, etc. The cancer patient's malignant tissue (breast or metastatic site) will not be included in this bank. Ability to understand and the willingness to sign an informed consent document. Exclusion Criteria: Dementia, altered mental status, or any psychiatric condition that would prohibit the understanding or rendering of informed consent. For biopsy of breast tissue only: History of serious or life-threatening allergic reaction to local anesthetics (i.e. lidocaine, xylocaine). Anticoagulation: For the purpose of invasive breast biopsies, women may not be receiving therapeutic anticoagulants. For blood draws, anticoagulants will be permitted. Any other condition, which in the opinion of the physician performing the biopsy procedure would make participation in this protocol unreasonably hazardous for the subject. For the purpose of invasive breast biopsies, women may not have had breast implants or breast reduction surgery. For inclusion in this study: The hematoxylin and eosin section had to demonstrate sufficient epithelium so that it would be reasonable to predict that a sufficient amount of RNA would be isolated for sequencing.]]>
April 2007 - The Susan G. Komen for the Cure Tissue Bank at the IU Simon Cancer Center founded January - June 2009 Collection of the tissue samples 1-10 May 2009 - January 2010 Collection of tissue samples 11-20 March - September 2009 Epithelium of specimens 1-10 microdissected using Acturus and RNA isolated March 2010 - March 2011 Epithelium of specimens 11-20 microdissected using Leica and RNA isolated April and September 2009 - RNA of specimens 1-10 sequenced by Cofactor Genomics using Life Technologies SOLiD 3 sequencing platform, 50 bp read length December 2010 - RNA of specimens 11-18 and 20 sequenced by Cofactor Genomics using Life Technologies SOLiD 4 sequencing platform, 50 x 35 bp Paried-End October 2011 - RNA of specimen 19 sequenced by The Indiana University Center for Medical Genomics using Life Technologies SOLiD 4 sequencing platform, 50 bp read length ]]>