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Objective The goal was to develop methods for detection of chromosomal and subchromosomal abnormalities in fetal cells in the mother's circulation at 10–16 weeks' gestation using analysis by array comparative genomic hybridization (CGH) and/or next‐generation sequencing (NGS). Method Nucleated cells from 30 mL of blood collected at 10–16 weeks' gestation were separated from red cells by density fractionation and then immunostained to identify cytokeratin positive and CD45 negative trophoblasts. Individual cells were picked and subjected to whole genome amplification, genotyping, and analysis by array CGH and NGS. Results Fetal cells were recovered from most samples as documented by Y chromosome PCR, short tandem repeat analysis, array CGH, and NGS including over 30 normal male cells, one 47,XXY cell from an affected fetus, one trisomy 18 cell from an affected fetus, nine cells from a trisomy 21 case, three normal cells and one trisomy 13 cell from a case with confined placental mosaicism, and two chromosome 15 deletion cells from a case known by CVS to have a 2.7 Mb de novo deletion. Conclusion We believe that this is the first report of using array CGH and NGS whole genome sequencing to detect chromosomal abnormalities in fetal trophoblastic cells from maternal blood. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd., What's already known about this topic? Analysis of cell‐free DNA for noninvasive prenatal testing (NIPT) is widely practiced, and the frequency of amniocentesis and CVS has decreased.However, cell‐free NIPT is not adequate for detecting smaller deletions and duplications with high specificity, sensitivity, and positive predictive value.Although fetal nucleated red blood cells and trophoblastic cells are known to be present in the maternal circulation, it has not been possible to develop a reliable cytogenetic cell‐based form of NIPT. What does this study add? Fetal cytotrophoblasts were successfully recovered from maternal blood.Although a clinical test has not been validated, for the first time, the feasibility of using array comparative genomic hybridization and next generation sequencing to detect chromosomal and subchromosomal abnormalities is demonstrated.The results suggest the possibility of developing a cell‐based form of NIPT with ability to detect abnormalities with a similar accuracy as can currently be obtained with amniocentesis and CVS.

Objective The goal was to develop methods for detection of chromosomal and subchromosomal abnormalities in fetal cells in the mother's circulation at 10–16 weeks' gestation using analysis by array comparative genomic hybridization (CGH) and/or next-generation sequencing (NGS). Method Nucleated cells from 30 mL of blood collected at 10–16 weeks' gestation were separated from red cells by density fractionation and then immunostained to identify cytokeratin positive and CD45 negative trophoblasts. Individual cells were picked and subjected to whole genome amplification, genotyping, and analysis by array CGH and NGS. Results Fetal cells were recovered from most samples as documented by Y chromosome PCR, short tandem repeat analysis, array CGH, and NGS including over 30 normal male cells, one 47,XXY cell from an affected fetus, one trisomy 18 cell from an affected fetus, nine cells from a trisomy 21 case, three normal cells and one trisomy 13 cell from a case with confined placental mosaicism, and two chromosome 15 deletion cells from a case known by CVS to have a 2.7 Mb de novo deletion. Conclusion We believe that this is the first report of using array CGH and NGS whole genome sequencing to detect chromosomal abnormalities in fetal trophoblastic cells from maternal blood. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Background. Analysis of circulating tumor cells (CTC) allows non-invasive investigation of prostate cancer biology and response to treatment. The primary level of analysis is the CTC count, which has been demonstrated to be prognostic of outcome. Deeper characterization of CTC phenotype and pertinent biomarkers can confirm cancer lineage and identify drug targets or drug-resistance markers. Single cell analysis of individual CTCs can provide genomic insight into cancer heterogeneity. RareCyte has developed AccuCyte® - CyteFinder® (AC-CF), an integrated technology platform for highly sensitive visual identification and retrieval of rare cells in blood by both immunohistochemistry (IHC) and immunofluorescence (IF) staining. Recently we have developed technology allowing 6-marker assays for broader phenotypic analysis. Methods. Normal human whole blood samples were spiked with prostate cancer lines as model CTCs (mCTCs). Blood samples from University of Washington patients with advanced prostate cancer were collected under an IRB-approved protocol. Blood was processed using AccuCyte and the nucleated cell fraction was collected and spread onto microscope slides. Slides were stained on an automated stainer using (1) an IHC assay for cytokeratin, (2) a standard 4-wavelength IF assay (DAPI, CD45, cytokeratin and EpCAM) or (3) a novel 6-parameter IF assay using SYTOX-Orange (nuclear stain), cytokeratin, EpCAM, androgen receptor (AR), prostate-specific membrane antigen (PSMA) and CD45. An assay for AR variant 7 (ARv7) was applied to samples with mCTCs with the ARv7 splice variant. Percent recovery of IHC-stained slides (by blinded pathologist review) was compared to IF-stained slides (by CyteFinder image analysis). Individual IHC-stained CTCs were retrieved after on-slide visual identification and re-visualized after dispensing for confirmation. Whole genome amplification (WGA) of retrieved cells was performed, followed by X- and Y-chromosome gene-specific PCR. Results. There was strong linear correlation between IF and IHC counts of mCTCs over a range of ∼25 - 100 cells/mL (R2 = 0.99). The 6-parameter IF assay was successfully applied to mCTC and clinical samples. AR and PSMA were co-expressed in the majority of epithelial-marker positive clinical CTCs. The ARv7 assay identified mCTCs that express the splice variant. Individual IHC-stained mCTCs spiked into female donor blood were demonstrated to be male after WGA and PCR. Conclusions. Light microscope identification of IHC cytokeratin-positive mCTCs approximated IF identification. 6-parameter phenotyping of prostate cancer CTCs is feasible and allows identification of lineage-specific markers. IHC-stained cells can be individually retrieved from slides for genome amplification and molecular analysis. Citation Format: Daniel Campton, Rachel Needham, Josh Nordberg, Arturo Ramirez, Nick Drovetto, Alisa Clein, Daniel E. Sabath, Jackie Stilwell, Eric Kaldjian. Multi-level analysis of prostate cancer circulating tumor cells allowing IHC-based identification, 6-parameter fluorescence phenotyping, and individual cell molecular analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4959.