Next-Generation Transcriptome Sequencing of the Normal Breast

from poster presented at SABCS 2010
Background: Our efforts to prevent and treat breast cancer are significantly impeded by a lack of knowledge of the biology and developmental genetics of the normal mammary gland. The Susan G. Komen for the Cure Tissue Bank at the IU Simon Cancer Center (KTB) was established expressly to address and remedy this deficiency. The KTB acquires and banks normal breast tissue, that is, breast tissue from volunteer donors with no clinical evidence of breast malignancy. This tissue is NOT from reduction mammoplasties or from histologically normal tissue adjacent to a malignancy.
The breast is one of the most complex genetic organs within the body. This is because the expression of its genes is under the control and influence of the hormonal milieu present in the circulating plasma, which changes as a function of age; for premenopausal women as a function of the menstrual cycle; and as a consequence of pregnancy. Therefore, there is unlikely to be a singular “normal” breast. We propose to produce a molecular encyclopedia of the normal breast which covers the entire spectrum of normal: puberty to menopause, low risk to high risk, nulliparous and parous.
Materials and Methods: The epithelial compartment of fresh frozen tissue from 10 premenopausal donors to the KTB, 5 women who were in the follicular phase of the menstrual cycle and 5 who were in the luteal, was isolated using laser capture microdissection. Total RNA extracted from the cells was subsequently depleted for ribosomal RNA. RNA was sequenced on an Applied Biosystems SOLiD3 sequencer using 50bp runs. Reads were mapped to the human genome. Whole blood was collected at the time of tissue donation and uniformly processed into serum.

Results: RNA sequencing of the 10 samples produced 596 million reads of which 386 million (62%) mapped to the human genome. Setting the p-value at <0.05 for the comparison of follicular versus luteal, there were 3395 differentially expressed RefSeq genes, 35 differentially expressed pre-miRNAs, 297 differentially expressed lincRNA exons and 40 differentially expressed UCRs (Ultra Conserved Regions). There were 1394 novel transcribed regions which were significantly differentially expressed. The serum estradiol at the time of donation was determined for 9 of the 10 donors. The gene expression of 901 genes was strongly correlated with serum estradiol concentration.
Proliferating Cell Nuclear Antigen (PCNA), nucleosome assembly genes and genes involved with mitosis have greater expression during the luteal phase of the menstrual cycle. Genes associated with development, e.g., NOTCH2, PAX3, DKK3 and TWIST1, are more abundantly expressed during the follicular phase. Many of the differentially expressed genes have been implicated in breast oncogenesis.

Conclusions: The Komen Tissue Bank has completed the first ever next-generation transcriptome sequencing of epithelial compartment of ten normal human breast specimens. This work has produced the most comprehensive catalog to date of the differences in the expression of protein encoding genes, pre-miRNAs, lincRNA exons, UCRs and novel transcribed regions as a function of the phase of the menstrual cycle. Additionally, this effort has identified a relatively significant number of genes whose expression is very likely under the control of estrogen.

Clare SE, Pardo I, Mathieson T, Lillemoe HA, Goulet, Jr. RJ, Henry JE, Sun J, Mitchum P, Parsons E, Jackson VP, Rager EL, Kennedy PR, Willimas-Bowling M, Savader B, Westphal SM, Pennington RE, Walker KH, Ritter HE, Berg, Jr RC, Bangs R, Badve S, Liu Y, Radovich M, Rufenbarger CA, Storniolo AMV.

Indiana Univeristy School of Medicine, Indianapolis; Susan G. Komen for the Cure Tissue Bank at the IU Simon Cancer Center, Indianapolis, IN; Clarian Arnett Cancer Center, Lafayette, IN; Indiana University School of Medicine, Indianapolis [poster]