Abstract P6-05-11: DEPArray™ enables recovery of pure tumor cells from heterogeneous fine needle aspirates for routine downstream NGS analysis
Authors: Sero V , Forcato C , Bolognesi C , Buson G , Medoro G , Yazdani M , Blevins A , Manaresi N , Bischoff FZ 
Published: May 2016

Abstract
Introduction: We have previously shown reliability in isolating pure populations of cells from complex tissues using the DEPArray™. Fine Needle Aspiration (FNA) is a quick and simple procedure often performed to make a diagnosis or rule out conditions such as cancer. Although FNA is also used to assess response to treatment, the procedure is often deemed insufficient in yield and purity of tumor cells. Here we provide preliminary results showing 100% efficiency in recovering pure tumor cell populations from FNA samples of patients affected by Metastatic Breast Cancer and known to have low tumor burden (&lt;20%) prior to using the DEPArray™ platform.
Method: FNA paraffin embedded sections (50 microns thickness) from metastases originating from breast (n=3) primary tumors were evaluated. Each FFPE curl was processed to yield single cells followed by DEPArray™ sorting based on cytokeratin (Ker), vimentin (Vim) and nuclear staining. The recovered cell populations were directly lysed in the collection tube prior to PCR-based target enrichment for next generation sequencing using Ion AmpliSeq™ CHPv2.
Results: DEPArray™ analysis allowed identification of 3 well separated cell populations, including tumor (Ker+/Vim-), stromal (Vim+/Ker) and putative EMT (Ker+/Vim+) cells. Overall, only 21% (4.3% to 42.7% range) of the total (mean of 6335) cells analyzed were of tumor (KER+/Vim-) origin. Groups of pure cells (mean 105 cells, range 15-200) for each population were recovered for sequence analysis. In one breast cancer FNA sample, we observed TP53 LoH but only in the recovered tumor (KER+) cells and not in the unsorted, stromal (VIM+), or EMT (KER+/VIM+) populations. In addition, a PIK3CA missense somatic heterozygous variant was identified in both the tumor and putative EMT populations but not in stromal cells, confirming this as a somatic mutation.
Conclusion: DEPArray™ allows resolution of two main limitations associated with FNA samples obtained for genomic analysis: too few target cells and unwanted admixture of normal cells. DEPArray™ allows for phenotypic distinction between the sorted cells prior to recovery; thus, enabling sequence analysis that is suitable for detecting genomic aberrations such as CNVs and LoH, which cannot be evaluated as precisely in an unsorted sample. Clearly, the DEPArray™ platform brings precision to detection, quantification and recovery of pure target cells that are suitable for subsequent downstream molecular analysis that can improve cancer diagnosis and personalized treatment strategies for breast cancer patients.
Citation Format: Sero V, Forcato C, Bolognesi C, Buson G, Medoro G, Yazdani M, Blevins A, Manaresi N, Bischoff FZ. DEPArray™ enables recovery of pure tumor cells from heterogeneous fine needle aspirates for routine downstream NGS analysis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-05-11.