Abstract 2296: Sequencing of breast cancer stem cells and corresponding primary bulk tumor reveals dominantly overlapping mutational spectra.
Authors: Rosin Gustaf , Klevebring Daniel , Ma Ran , Lindberg Johan , Viberg Lisa , Kere Juha , Czene Kamila , Lindh Claes , Fredriksson Irma , Bergh Jonas , Hartman Johan 
Published: August 2014

Breast cancer cells with phenotypical similarities to adult mammary stem cells have been shown to initiate and maintain tumor growth and therefore named cancer stem cells (CSC). Phenotypic markers, such as CD44+/CD24-, ALDH1 High and PKH26High are all characterizing breast cancer cells with stem-like traits and ability to initiate tumor growth. There is substantial overlap of these markers with the biological capacity to form non-adherent cell spheres in vitro, named mammospheres. Based on the CSC concept, two alternative and opposing explanations have been proposed to describe the tumorigenic process governed by CSC; (1) the CSCs are an early-tumor initiating clone with capacity of asymmetric cell-divisions and subsequent differentiation to yield intratumoral heterogeneity; (2) the CSC phenotype is dynamic and reversible and can be adopted by any cancer epithelial cell through epigenetic de-differentiation.

To understand which of these models is the predominant, governing the development of breast cancer, we collected paired bulk tumor samples, in vitro purified mammospheres and normal leukocyte DNA from ten patients. DNA from mammospheres were amplified to increase the input DNA amount and in duplicates to control for the introduction of novel mutations. The sequencing was run with Illumina Hiseq 2000 using an exome capture kit. The sequence raw data was aligned to the human genome (hg19). Average target coverage was 53x. Mutations found in only one of the sequenced mammosphere amplicons and neither in the bulk tumor were considered artifacts by the DNA amplification and excluded from further analysis.

Across all patients, 80% of the mutations are on average shared between the tumor and CSC-fraction (range 45%-91%). We hypothesize that if the two cell populations, differentiated cells and CSC, were strictly separated, the number of mutations in the CSC would be very low due to low replication rate of that cell type compared to the continuously proliferating epithelial cells. In such a scenario, very few mutations would also be shared between CSC and bulk tumor cells. In contrast, we see a large degree of shared mutations. A cell-state model where inter-conversion between cell types occurs, would explain the large degree of shared mutations. As mutations occur in the rapidly dividing epithelial cells, they are propagated to daughter cells that are occasionally converted into CSCs. We also investigated the allele frequencies of the mutations discovered. There is no trend to larger frequencies in bulk tumor or in CSC, instead the frequencies are mostly equal in the two cellular components, pointing towards an inter-conversion of bulk tumor cells and CSC. Our data points towards the support of a model where the epithelial cells of the bulk tumor in vivo are able to phenotypically revert into CSC with stem cell properties and vise versa (model 2).

Citation Format: Gustaf Rosin, Daniel Klevebring, Ran Ma, Johan Lindberg, Lisa Viberg, Juha Kere, Kamila Czene, Claes Lindh, Irma Fredriksson, Jonas Bergh, Johan Hartman. Sequencing of breast cancer stem cells and corresponding primary bulk tumor reveals dominantly overlapping mutational spectra. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2296. doi:10.1158/1538-7445.AM2013-2296