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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.

Gene silencing by RNA interference has become a powerful tool to help identify genes that regulate biological processes. However, the complexity of the biology probed and the incomplete validation of the reagents used make it difficult to interpret the results of genome-wide siRNA screens. To address this challenge and maximize the return on the efforts required for validating genomic screen hits, the screening strategy must be designed to increase the robustness of the primary screening hits and include assays that inform on the mechanism of action of the knocked-down transcripts. Here, we describe the implementation of a small interfering RNA (siRNA) screen to identify genes that sensitize the effect of poly-(ADP ribose)-polymerase (PARP) inhibitor on cell survival. In the strategy we designed for the primary screen, two biological activities, apoptosis and cell viability, were measured simultaneously at different time points in the presence and absence of a PARP inhibitor (PARPi). The multiplexed assay allowed us to identify PARPi sensitizers induced by both caspase-dependent and independent mechanisms. The multiplexed screening strategy yielded robust primary hits with significant enrichment for DNA repair genes, which were further validated using relevant high-content imaging assays and confirmation of transcript knockdown by real-time PCR (rtPCR).

Group B streptococcus (GBS) remains a major cause of morbidity and mortality among newborn children. The bacterium is a commensal organism colonizing the rectum and the gastrointestinal and urogenital tracts of adults, but it can be transmitted to neonates by an ascending infection of the maternal genital tract or during parturition. We previously reported that a transposon insertion disrupting rpoE resulted in the decreased survival of the mutant in the neonatal rat sepsis model of GBS infection. rpoE encodes the δ protein, a subunit of RNA polymerase (RNAP) that has been characterized in Bacillus species. In this study, we confirm the association of the δ protein with purified GBS RNAP and show that it is expressed in strains representing all nine serotypes. Flow cytometric analysis of a reporter strain containing a transcriptional fusion of the rpoE promoter to gfp revealed that, in vitro, this gene is continuously expressed. Analysis of δ expression in the transposon mutant by quantitative Western blotting revealed a 10-fold reduction in relative abundance (which was linked to the attenuation in virulence that was observed for this mutant) compared to that for the wild-type strain. These data suggest that a minimum intracellular concentration of δ is necessary for this organism to cause disease.

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.

siRNAs mediate sequence-specific gene silencing in cultured mammalian cells but also silence unintended transcripts. Many siRNA off-target transcripts match the guide-strand “seed region,” similar to the way microRNAs match their target sites. The extent to which this seed-matched, microRNA-like, off-target silencing affects the specificity of therapeutic siRNAs in vivo is currently unknown. Here, we compare microRNA-like off-target regulations in mouse liver in vivo with those seen in cell culture for a series of therapeutic candidate siRNAs targeting Apolipoprotein B (APOB). Each siRNA triggered regulation of consistent microRNA-like off-target transcripts in mouse livers and in cultured mouse liver tumor cells. In contrast, there was only random overlap between microRNA-like off-target transcripts from cultured human and mouse liver tumor cells. Therefore, siRNA therapeutics may trigger microRNA-like silencing of many unintended targets in vivo, and the potential toxicities caused by these off-target gene regulations cannot be accurately assessed in rodent models.

Gene silencing by RNA interference is a powerful genetic tool to identify genes involved in specific biological processes in model organisms and human cells (3, 14, 18, 38, 42, 56). It is now possible to perform unbiased genetic screens using cultured human cells. Recently, small-scale siRNA (small interfering RNA) and shRNA (small hairpin RNA) screens in human cells have successfully identified genes that modulate cell growth, apoptosis, chemoresistance, and chemosensitivity (4, 44, 52). While establishing proof-of-concept, most of these early screens tested gene sets too limited in size to provide a comprehensive view of the genes involved in the cellular phenotypes being tested. Several groups have employed nearly genome-wide screens of shRNAs expressed from vectors to identify genes that modify certain phenotypes (6, 40, 49, 59). To date, these shRNA screens have been limited to phenotypes amenable to positive selection, but other approaches like bar code screening have shown promise (6). Systematic well-by-well testing of genomic-scale siRNA or shRNA libraries for modulation of nonselectable phenotypes in human cells has not been widely attempted. Cancer cells undergo numerous genetic changes that drive cellular transformation from normal cell progenitors. Anticancer drug target discovery is now frequently directed toward understanding and exploiting genetic alterations that exist in tumor cells. Knowledge of genetic alterations in tumors may lead to better use of conventional therapeutics or development of new therapeutics that offer better therapeutic windows. Loss-of-function genetic screens can identify genes whose loss of function enhances cytotoxicity of chemotherapeutics in cells with defined genetic lesions. The TP53 transcription factor is a central mediator of the cellular response to DNA damage and a variety of other stresses and is one of the most frequently mutated genes in human cancers (19, 57). TP53 status can also effect the chemosensitivity of tumor cells (15). The identification of genes that, when silenced, selectively enhance the chemosensitivity of TP53 mutant cancer cells but not TP53 wild-type cells would make attractive drug targets. Drugs developed to these genes have the potential to selectively increase the toxicity of the chemotherapeutic in the cancer cell. Genetic screens in model organisms have led to identification of drug enhancers whose loss increases sensitivity to the drug (43, 54, 56, 61). We hypothesized that identification of enhancers for commonly used cancer therapeutics would lead to new strategies for therapeutic application of these drugs and/or identification of new targets for the development of combination chemotherapies. Here we used small- and genome-scale siRNA screens to identify genes that enhance the sensitivity of human tumor cells to the cancer chemotherapeutic cisplatin. The screen hits included known genes that function in DNA repair, cell cycle checkpoints, and survival signaling, as well as genes with no annotated functions. Selected hits from these screens were validated and then tested on matched TP53-positive and TP53-deficient cells to identify enhancers selective for tumor cells that have lost TP53 function. We show that silencing of BRCA1, BRCA2, and certain genes whose products interact with BRCA1/2, selectively enhances cisplatin cytotoxicity in TP53-deficient cells.

Group B streptococci (GBS) remain the most significant bacterial pathogen causing neonatal sepsis, pneumonia, and meningitis in the United States despite the chemoprophylaxis strategies for preventing infection recommended by the Centers for Disease Control and Prevention. Using signature-tagged transposon mutagenesis to screen for novel virulence factors, we identified the rpoE gene as essential for development of sepsis in a neonatal rat model of GBS infection. An rpoE allelic replacement mutant displayed attenuated virulence in the sepsis model of infection identical to that of the transposon mutant, confirming linkage of the phenotype to the mutation in rpoE . The rpoE mutants also displayed increased sensitivity to killing in whole-blood bactericidal assays, which may explain the attenuated virulence. The mutants were otherwise phenotypically identical to the wild-type strain, including growth rate in plasma, indicating that a growth defect is not responsible for the attenuated virulence. rpoE is found only in gram-positive bacterial species and encodes the delta peptide, a subunit of RNA polymerase. Previous in vitro studies in other bacteria suggest that the delta peptide plays a role in maintaining transcriptional fidelity by blocking RNA polymerase binding at all but the strongest promoters, thereby inhibiting initiation of transcription. Despite the availability of rpoE mutants for several gram-positive bacterial species, a role for the peptide in vivo has not been defined, though it has been postulated that the delta peptide may be important for long-term survival in vitro or during growth phase transitions. Our data represent the first report of a phenotype relevant to virulence for rpoE mutants.