ABSTRACT
OBJECTIVE: The purpose of this study were to develop a methodology of isolating fetal cells from maternal blood and use deep sequence demonstrating the promise for complete and accurate genetic screening compared to other non-invasive prenatal testing. METHODS: Here in this study, we developed a double negative selection (DNS) procedure to unbiasedly enrich fetal cells. After validated by short tandem repeat (STR), the isolated circulating fetal cells (CFCs) were subjected to deep whole genome sequencing analysis. RESULTS: Our DNS protocol significantly increasing the purity of the mimic fetal cells from 1 in 1 million nucleated cells in whole blood to 1:8 to 1:30 (12.5%-3.33%) after 2 steps of enrichment. Isolated single fetal cell obtained a coverage rate (86.8%) and allelic dropout rate (24.90%) comparative to the reported results of human cell line. Several disease-associated variants were identified in the whole genome sequencing data of isolated CFCs and further confirmed in the sequencing data of unamplified gDNA. CONCLUSION: In conclusion, the robustness of DNS and STR to collect CFCs from peripheral maternal blood for the first time coupled with deep sequencing technique demonstrates the possibility of comprehensive non-invasive prenatal testing of genetic disorders using isolated CFCs.
Subject(s)
Cell Separation/methods , Maternal Serum Screening Tests/methods , Whole Genome Sequencing , Feasibility Studies , Female , Humans , Microsatellite Repeats , Paternity , PregnancyABSTRACT
PURPOSE: We generated a humanized antibody, HuLuc63, which specifically targets CS1 (CCND3 subset 1, CRACC, and SLAMF7), a cell surface glycoprotein not previously associated with multiple myeloma. To explore the therapeutic potential of HuLuc63 in multiple myeloma, we examined in detail the expression profile of CS1, the binding properties of HuLuc63 to normal and malignant cells, and the antimyeloma activity of HuLuc63 in preclinical models. EXPERIMENTAL DESIGN: CS1 was analyzed by gene expression profiling and immunohistochemistry of multiple myeloma samples and numerous normal tissues. HuLuc63-mediated antimyeloma activity was tested in vitro in antibody-dependent cellular cytotoxicity (ADCC) assays and in vivo using the human OPM2 xenograft model in mice. RESULTS: CS1 mRNA was expressed in >90% of 532 multiple myeloma cases, regardless of cytogenetic abnormalities. Anti-CS1 antibody staining of tissues showed strong staining of myeloma cells in all plasmacytomas and bone marrow biopsies. Flow cytometric analysis of patient samples using HuLuc63 showed specific staining of CD138+ myeloma cells, natural killer (NK), NK-like T cells, and CD8+ T cells, with no binding detected on hematopoietic CD34+ stem cells. HuLuc63 exhibited significant in vitro ADCC using primary myeloma cells as targets and both allogeneic and autologous NK cells as effectors. HuLuc63 exerted significant in vivo antitumor activity, which depended on efficient Fc-CD16 interaction as well as the presence of NK cells in the mice. CONCLUSIONS: These results suggest that HuLuc63 eliminates myeloma cells, at least in part, via NK-mediated ADCC and shows the therapeutic potential of targeting CS1 with HuLuc63 for the treatment of multiple myeloma.