Abstract 3082: Isolation and identification of disseminated tumor cells from bone marrow
Authors: Zhu Peixuan , Adams Daniel , Aft Rebecca L. , Pillai Sreeraj G. , Watson Mark A. , Li Shuhong , Makarova Olga V. , Amstutz Platte T. , Tang Cha-Mei 
Published: May 2015

Abstract
Background: Disseminated tumor cells (DTCs), detected by immunohistological staining for cytokeratins, found in the bone marrow (BM) of breast cancer patients portend a poor prognosis. Study of these cells has been hindered by their rarity in BM specimens. We have tested a microfiltration technique for the enrichment of DTCs.
Methods: BM aspirates from healthy volunteers were collected and mixed with defined numbers SKBR3 breast cancer cells at a concentration of 1,000 to 10,000 tumor cells per 1x10^6 nucleated BM cells., BM was then diluted 1:10 with PBS and 7 ml was placed in CellSaveTM tubes to simulate a patient BM collection. Each specimen was processed within 24 hours of collection. Specimens were pre-filtered using a 70 µm mesh sieve (BD cat#352350) to remove large particles. The effluent was filtered through CellSieveTM microfilters, which have 7 µm diameter pores in a uniform array, of 160,000 pores in a 9 mm diameter area. Filters containing cells were post-fixed, cells permeabilized, and stained with DAPI and fluorescent antibodies specific to epithelial cytokeratins 8, 18, and 19 (FITC), EpCAM (PE), and CD45, a negative control (Cy5). Five representative 1% areas of each filter were imaged and used to estimate total cell and tumor cell counts, based on fluorescent DAPI and cytokeratin antibody staining.
Results: From each BM specimen (7 ml) containing a total of 14x10^6 nucleated BM cells, post filtration samples contained only 17-21x10^3 residual, nucleated BM cells. For specimens spiked at 10 tumor cells per 1,000 BM cells, approximately 30x10^3 positively staining tumor cells were recovered (20% tumor cell recovery with approximately 170 fold enrichment). For specimens spiked at 1 tumor cell per 1,000 BM cells, approximately 7x10^3 total tumor cells were recovered (50% tumor cell recovery with approximately 400-fold enrichment). Captured cells co-stained with cytokeratins and EpCAM, but not CD45. Control BM specimens with no added tumor cells demonstrated only rare, staining cells (4 per sample), likely due to background or epithelial cell contamination.
Conclusions: We have tested a microfiltration technique for enrichment of BM DTCs. Microfiltration of BM is rapid without filter clogging. Recovered DTCs can be immuno-stained, and are easily identified and distinguished from BM cells. The DTC capture efficiency varied with cancer cell concentration and ranged from 20-50% with up to 400 fold enrichment when samples contained lower numbers of tumor cells. The microfiltration enrichment assay continues to be optimized for isolation and molecular characterization of DTCs from patient BM samples, where the concentration of DTCs seldom exceeds 10 cells per 1x10^6 BM cells.
Citation Format: Peixuan Zhu, Daniel Adams, Rebecca L. Aft, Sreeraj G. Pillai, Mark A. Watson, Shuhong Li, Olga V. Makarova, Platte T. Amstutz, Cha-Mei Tang. Isolation and identification of disseminated tumor cells from bone marrow. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3082. doi:10.1158/1538-7445.AM2014-3082