Single HER2-positive tumor cells are detected in initially HER2-negative breast carcinomas using the DEPArray™-HER2-FISH workflow.

BACKGROUND: In breast cancer (BC), overexpression of HER2 on the primary tumor (PT) is determined by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) to stratify samples as negative, equivocal and positive to identify patients (pts) for anti-HER2 therapy. CAP/ASCO guidelines recommend FISH for analyzing HER2/neu (ERBB2) gene amplification and for resolving equivocal HER2 IHC results. However, pre-analytical and analytical aspects are often confounded by sample related limitations and tumor heterogeneity and HER2 expression may differ between the PT and circulating tumor cells (CTCs), the precursors of metastasis. We used a validation cohort of BC patients to establish a new DEPArray™-PT-HER2-FISH workflow for further application in a development cohort, characterized as PT-HER2-negative but CTC-HER2/neu-positive, to identify patients with PT-HER2 amplified cells not detected by routine pathology. METHODS: 50 µm FFPE tumor curls from the validation cohort (n = 49) and the development cohort (n = 25) underwent cutting, deparaffinization and antigen retrieval followed by dissociation into a single-cell suspension. After staining for cytokeratin, vimentin, DAPI and separation via DEPArray™, single cells were processed for HER2-FISH analysis to assess the number of chromosome 17 and HER2 loci signals for comparison, either with available IHC or conventional tissue section FISH. CTC-HER2/neu status was determined using the AdnaTest BreastCancer (QIAGEN, Hilden, Germany). RESULTS: Applying CAP/ASCO guidelines for HER2 evaluation of single PT cells, the comparison of routine pathology and DEPArray™-HER2-FISH analysis resulted in a concordance rate of 81.6% (40/49 pts) in the validation cohort and 84% (21/25 pts) in the development cohort, respectively. In the latter one, 4/25 patients had single HER2-positive tumor cells with 2/25 BC patients proven to be HER2-positive, despite being HER2-negative in routine pathology. The two other patients showed an equivocal HER2 status in the DEPArray™-HER2-FISH workflow but a negative result in routine pathology. Whereas all four patients with discordant HER2 results had already died, 17/21 patients with concordant HER2 results are still alive. CONCLUSIONS: The DEPArray™ system allows pure tumor cell recovery for subsequent HER2/neu FISH analysis and is highly concordant with conventional pathology. For PT-HER2-negative patients, harboring HER2/neu-positive CTCs, this approach might allow caregivers to more effectively offer anti-HER2 treatment.

Dasatinib, paclitaxel, and carboplatin in women with advanced-stage or recurrent endometrial cancer: A pilot clinical and translational study.

PURPOSE: To evaluate the effect of dasatinib therapy on EphA2 signaling in cancers of women with measurable (biopsy amenable) advanced-stage, chemo-naïve primary or recurrent endometrial cancer. Preliminary efficacy was also assessed. PATIENTS AND METHODS: We performed a pilot study of single-agent dasatinib lead-in, followed by triplet dasatinib, paclitaxel, and carboplatin. We measured the downstream effectors of EphA2 signaling in pre- and post-dasatinib treatment biopsy tissue samples; we also determined the severity of adverse events and patients' progression-free survival and overall survival durations. RESULTS: Eighteen patients were recruited and given dasatinib (150 mg orally daily for 14 days), followed by paclitaxel, carboplatin and dasatinib (daily) for six cycles (21-day cycles). Seventeen patients were evaluable for toxicity and 11 patients for response. A reverse phase protein array and proximity ligation assay revealed that CRAF/BRAF dimerization, caveolin-1 level, and Notch pathway signaling were predictive of response and resistance to dasatinib. Overall, the objective response rate was 45% (95% CI: 17%-77%), with median progression-free survival duration of 10.5 months and median overall survival duration of 30.4 months. The most common grade 3 or 4 adverse events were neutropenia (76%), thrombocytopenia (53%), anemia (53%), and fatigue (12%). CONCLUSIONS: Caveolin-1 expression, in combination with CRAF/BRAF heterodimerization, is associated with resistance to EphA2 targeting by dasatinib. The triplet combination showed interesting clinical activity in endometrial cancer with acceptable toxicity. Pretreatment with dasatinib may accentuate combination therapy toxicity.

Multiparametric liquid biopsy analysis in metastatic prostate cancer.

Molecular profiling of prostate cancer with liquid biopsies, such as circulating tumor cells (CTCs) and cell-free nucleic acid analysis, yields informative yet distinct data sets. Additional insights may be gained by simultaneously interrogating multiple liquid biopsy components to construct a more comprehensive molecular disease profile. We conducted an initial proof-of-principle study aimed at piloting this multiparametric approach. Peripheral blood samples from men with metastatic castrate-resistant prostate cancer were analyzed simultaneously for CTC enumeration, single-cell copy number variations, CTC DNA and matched cell-free DNA mutations, and plasma cell-free RNA levels of androgen receptor (AR) and AR splice variant (ARV7). In addition, liquid biopsies were compared with matched tumor profiles when available, and a second liquid biopsy was drawn and analyzed at disease progression in a subset of patients. In this manner, multiparametric liquid biopsy profiles were successfully generated for each patient and time point, demonstrating the feasibility of this approach and highlighting shared as well as unique cancer-relevant alterations. With further refinement and validation in large cohorts, multiparametric liquid biopsies can optimally integrate disparate but clinically informative data sets and maximize their utility for molecularly directed, real-time patient management.

Comprehensive Mutation and Copy Number Profiling in Archived Circulating Breast Cancer Tumor Cells Documents Heterogeneous Resistance Mechanisms.

Addressing drug resistance is a core challenge in cancer research, but the degree of heterogeneity in resistance mechanisms in cancer is unclear. In this study, we conducted next-generation sequencing (NGS) of circulating tumor cells (CTC) from patients with advanced cancer to assess mechanisms of resistance to targeted therapy and reveal opportunities for precision medicine. Comparison of the genomic landscapes of CTCs and tissue metastases is complicated by challenges in comprehensive CTC genomic profiling and paired tissue acquisition, particularly in patients who progress after targeted therapy. Thus, we assessed by NGS somatic mutations and copy number alterations (CNA) in archived CTCs isolated from patients with metastatic breast cancer who were enrolled in concurrent clinical trials that collected and analyzed CTCs and metastatic tissues. In 76 individual and pooled informative CTCs from 12 patients, we observed 85% concordance in at least one or more prioritized somatic mutations and CNA between paired CTCs and tissue metastases. Potentially actionable genomic alterations were identified in tissue but not CTCs, and vice versa. CTC profiling identified diverse intra- and interpatient molecular mechanisms of endocrine therapy resistance, including loss of heterozygosity in individual CTCs. For example, in one patient, we observed CTCs that were either wild type for ESR1 (n = 5/32), harbored the known activating ESR1 p.Y537S mutation (n = 26/32), or harbored a novel ESR1 p.A569S (n = 1/32). ESR1 p.A569S was modestly activating in vitro, consistent with its presence as a minority circulating subclone. Our results demonstrate the feasibility and potential clinical utility of comprehensive profiling of archived fixed CTCs. Tissue and CTC genomic assessment are complementary, and precise combination therapies will likely be required for effective targeting in advanced breast cancer patients.Significance: These findings demonstrate the complementary nature of genomic profiling from paired tissue metastasis and circulating tumor cells from patients with metastatic breast cancer. Cancer Res; 78(4); 1110-22. ©2017 AACR.

Hematogenous metastasis of ovarian cancer: rethinking mode of spread.

Ovarian cancer has a clear predilection for metastasis to the omentum, but the underlying mechanisms involved in ovarian cancer spread are not well understood. Here, we used a parabiosis model that demonstrates preferential hematogenous metastasis of ovarian cancer to the omentum. Our studies revealed that the ErbB3-neuregulin 1 (NRG1) axis is a dominant pathway responsible for hematogenous omental metastasis. Elevated levels of ErbB3 in ovarian cancer cells and NRG1 in the omentum allowed for tumor cell localization and growth in the omentum. Depletion of ErbB3 in ovarian cancer impaired omental metastasis. Our results highlight hematogenous metastasis as an important mode of ovarian cancer metastasis. These findings have implications for designing alternative strategies aimed at preventing and treating ovarian cancer metastasis.

Discordance in HER2 gene amplification in circulating and disseminated tumor cells in patients with operable breast cancer.

Human epidermal growth factor receptor 2 (HER2) gene amplification in circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) might be useful for modifying Herceptin therapy in breast cancer. In the process of investigating the utility of a microfluidic platform for detecting HER2 gene amplification in these cells, we observed novel results on discordance of HER2 status. Peripheral blood (8.5 mL) and bone marrow (BM) (7.5-10 mL) were collected prospectively from patients with clinical stages I-IV breast cancer. Mononuclear cells were recovered, stained with cytokeratin (CK), CD45, and DAPI, and processed through microfluidic channels for fluorescence in situ hybridization (FISH). A ratio of HER2:CEP17 >2 in any CK+/CD45 or CK-/CD45 cell was regarded as positive for HER2 gene amplification. Peripheral blood from 95 patients and BM from 78 patients were studied. We found CK+/CD45-/DAPI+ CTCs in 27.3% of patients. We evaluated HER2 gene amplification by FISH in 88 blood and 78 BM specimens and found HER2+ CTCs in 1 of 9 (11.1%) and HER2+ DTCs (27.2%) in 3 of 11 patients with HER2+ primary tumor. Among patients with a HER2- primary tumor, 5 of 79 had HER2+ CTCs (6.3%) and 14 of 67 had HER2+ DTCs (20.8%). The overall rate of discordance in HER2 status was 15% between primary tumor and CTCs and 28.2% between primary tumor and DTCs. HER2 was amplified in CTCs and DTCs in a portion of both HER2+ and HER2- primary tumors. HER2 discordance was more frequent for DTCs. The clinical implications of evaluating HER2 status in CTCs and DTCs in breast cancer needs to be established in prospective clinical trials. The cell enrichment and extraction microfluidic technology provides a sensitive platform for evaluation of HER2 gene amplification in CTCs and DTCs.

FISH-based determination of HER2 status in circulating tumor cells isolated with the microfluidic CEE™ platform.

Determination of HER2 status in breast cancer patients is considered standard practice for therapy selection. However, tumor biopsy in patients with recurrent and/or metastatic disease is not always feasible. Thus, circulating tumor cells (CTCs) are an alternative source of tumor cells for analysis of HER2. An antibody cocktail for recovery of variable, high- and low-, EpCAM-expressing tumor cells was developed based on FACS evaluation and then verified by CTC enumeration (based on CK and CD45 staining) with comparison to EpCAM-only and with CellSearch® (n=19). HER2 fluorescence in situ hybridization (FISH) on all (CK+ and CK-) captured cells was compared to HER2 status on the primary tumors (n=54) of patients with late stage metastatic/recurrent breast cancer. Capture of low EpCAM-expressing tumor cells increased from 27% to 76% when using the cocktail versus EpCAM alone, respectively. Overall, CTC detection with the OncoCEE™ platform was better compared to CellSearch® (68% vs. 89%, respectively), and a 93% concordance in HER2 status was observed. HER2 FISH analysis of CK+ and CK- CTCs is feasible using the CEE™ platform. Although larger clinical studies are warranted, the results demonstrate adequate sensitivity and specificity as needed for incorporation into laboratory testing.

A novel platform for detection of CK+ and CK- CTCs.

UNLABELLED: Metastasis is a complex, multistep process that begins with the epithelial-mesenchymal transition (EMT). Circulating tumor cells (CTC) are believed to have undergone EMT and thus lack or express low levels of epithelial markers commonly used for enrichment and/or detection of such cells. However, most current CTC detection methods target only EpCAM and/or cytokeratin (CK) to enrich epithelial CTCs, resulting in failure to recognize other, perhaps more important, CTC phenotypes that lack expression of these markers. Here, we describe a population of complex aneuploid CTCs that do not express CK or CD45 antigen in patients with breast, ovarian, or colorectal cancer. These cells were not observed in healthy subjects. We show that the primary epithelial tumors were characterized by similar complex aneuploidy, indicating conversion to an EMT phenotype in the captured cells. Collectively, our study provides a new method for highly efficient capture of previously unrecognized populations of CTCs. SIGNIFICANCE: Current assays for CTC capture likely miss populations of cells that have undergone EMT. Capture and study of CTCs that have undergone EMT would allow a better understanding of the mechanisms driving metastasis.

Detection of EpCAM-Negative and Cytokeratin-Negative Circulating Tumor Cells in Peripheral Blood.

Enrichment of rare circulating tumor cells (CTCs) in blood is typically achieved using antibodies to epithelial cell adhesion molecule (EpCAM), with detection using cytokeratin (CK) antibodies. However, EpCAM and CK are not expressed in some tumors and can be downregulated during epithelial-to-mesenchymal transition. A micro-fluidic system, not limited to EpCAM or CK, was developed to use multiple antibodies for capture followed by detection using CEE-Enhanced (CE), a novel in situ staining method that fluorescently labels the capture antibodies bound to CTCs. Higher recovery of CTCs was demonstrated using antibody mixtures compared to anti-EpCAM. In addition, CK-positive breast cancer cells were found in 15 of 24 samples (63%; range 1-60 CTCs), while all samples contained additional CE-positive cells (range 1-41; median = 11; P = .02). Thus, antibody mixtures against a range of cell surface antigens enables capture of more CTCs than anti-EpCAM alone and CE staining enables the detection of CK-negative CTCs.

Efficient capture of circulating tumor cells with a novel immunocytochemical microfluidic device.

Ability to perform cytogenetic interrogations on circulating tumor cells (CTCs) from the blood of cancer patients is vital for progressing toward targeted, individualized treatments. CTCs are rare compared to normal (bystander) blood cells, found in ratios as low as 1:10(9). The most successful isolation techniques have been immunocytochemical technologies that label CTCs for separation based on unique surface antigens that distinguish them from normal bystander cells. The method discussed here utilizes biotin-tagged antibodies that bind selectively to CTCs. The antibodies are introduced into a suspension of blood cells intending that only CTCs will display surface biotin molecules. Next, the cell suspension is passed through a microfluidic channel that contains about 9000 transverse, streptavidin coated posts. A CTC making contact with a post has the opportunity to engage in a biotin-streptavidin reaction that immobilizes the cell. Bystander blood cells remain in suspension and pass through the channel. The goal of the present study is to establish the technical performance of these channels as a function of antigen density and operating conditions, especially flow rate. At 18 µL/min, over 70% of cells are captured at antigen densities greater than 30 000 sites/cell while 50% of cells are captured at antigen densities greater than 10 000. It is found that lower flow rates lead to decreasing cell capture probabilities, indicating that some streamlines develop which are never close enough to a post to allow cell-post contact. Future modeling and streamline studies using computational fluid dynamics software could aid in optimization of channel performance for capture of rare cells.

Plasma cell-free DNA in ovarian cancer: an independent prognostic biomarker.

BACKGROUND: Cell-free DNA reflects both normal and tumor-derived DNA released into the circulation through cellular necrosis and apoptosis. The authors sought to determine the role of preoperative total plasma cell-free DNA levels in predicting clinical outcome in patients with ovarian cancer. METHODS: After institutional review board consent, DNA was extracted from plasma of 164 women with invasive epithelial ovarian carcinoma (EOC), 49 with benign ovarian neoplasms, and 75 age-matched controls. The samples were randomly divided into training (n = 144) and validation (n = 144) sets. Quantification of cell-free DNA was performed using real-time polymerase chain reaction for beta-globin, and the number of genome equivalents (GE) per milliliter of plasma was determined. Cell-free DNA was correlated with clinicopathologic parameters. RESULTS: The training and validation sets were similar in terms of demographic features. In the training set, EOC patients had a median preoperative cell-free DNA level of 10,113 GE/mL, compared with patients with benign ovarian neoplasms (median, 2365 GE/mL; P < .0001) and controls (median, 1912 GE/mL, P < .0001). Cell-free DNA >22,000 GE/mL was significantly associated with decreased patient survival (P < .001). After adjusting for other clinical variables, preoperative cell-free DNA >22,000 GE/mL was an independent predictor (P = .02) for disease-specific survival. Analysis of the validation set confirmed significantly higher cell-free DNA levels in EOC (median, 13,672 GE/mL) and that cell-free DNA >22,000 GE/mL was associated with a 2.83-fold increased risk of death from disease (P < .001). CONCLUSIONS: Preoperative plasma total cell-free DNA levels are significantly elevated in patients with EOC. Elevated plasma cell-free DNA is an independent predictor for death from disease in ovarian cancer.

Improving enrichment of circulating fetal DNA for genetic testing: size fractionation followed by whole gene amplification.

OBJECTIVE: Among the pitfalls of using cell-free fetal DNA in plasma for prenatal diagnosis is quality of the recovered DNA fragments and concomitant presence of maternal DNA (>95%). Our objective is to provide alternative methods for achieving enrichment and high-quality fetal DNA from plasma. METHODS: Cell-free DNA from 31 pregnant women and 18 controls (10 males and 8 females) were size separated using agarose gel electrophoresis. DNA fragments of 100-300, 500-700 and 1,500-2,000 bp were excised and extracted, followed by whole genome amplification (WGA) of recovered fragments. Levels of beta-globin and DYS1 were measured. RESULTS: Distribution of beta-globin size fragments was similar among pregnant women and controls. Among control male cases, distribution of size fragments was the same for both beta-globin and DYS1. Among maternal cases confirmed to be male, the smallest size fragment (100-300 bp) accounted for nearly 50% (39.76 +/- 17.55%) of the recovered DYS1-DNA (fetal) and only 10% (10.40 +/- 6.49%) of beta-globin (total) DNA. After WGA of plasma fragments from pregnant women, DYS1 sequence amplification was best observed when using the 100-300 bp fragments as template. CONCLUSIONS: Combination of electrophoresis for size separation and WGA led to enriched fetal DNA from plasma. This novel combination of strategies is more likely to permit universal clinical applications of cell-free fetal DNA.

Stability of placental RNA using dried maternal blood spots.

Having demonstrated successful recovery and detection of placental transcripts from dried blood spots (DBS), various preanalytical conditions were examined to determine optimal handling of samples. The role of several factors was explored, including temperature (4 degrees C versus 25 degrees C), processing time (24 h to 8 weeks), and addition of preservatives (RNA later and formalin) that may interfere with stability and detection of placental transcripts in DBS. mRNA transcripts encoding human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; internal control) and beta-human chorionic gonadotrophin (beta HCG; placental) were analysed by real-time-polymerase chain reaction using DBS from 23 pregnant women. GAPDH and beta HCG transcripts were detected in all samples 24 h after collection. Although treatment of blood with RNA later did not affect RNA recovery, formalin treatment negatively affected RNA recovery from DBS. Temperature did not have a significant effect on levels of either transcript. Storage time caused a significant decrease in GAPDH after 4 weeks (P = 0.014) and beta HCG after 1 week (P = 0.007). Decrease of beta HCG levels after 1 week followed by steady detectable levels for up to 4 weeks suggests two populations of circulating placental transcript exist, a population susceptible to degradation in blood versus a more stable form. Therefore, defining proper parameters for collection and storage of DBS further reinforces reliable analysis of target sequences for clinical testing.

Membrane protected apoptotic trophoblast microparticles contain nucleic acids: relevance to preeclampsia.

Microparticles (MPs) that circulate in blood may be a source of DNA for molecular analyses, including prenatal genetic diagnoses. Because MPs are heterogeneous in nature, however, further characterization is important before use in clinical settings. One key question is whether DNA is either bound to aggregates of blood proteins and lipid micelles or intrinsically associated with MPs from dying cells. To test the latter hypothesis, we asked whether MPs derived in vitro from dying cells were similar to those in maternal plasma. JEG-3 cells model extravillous trophoblasts, which predominate during the first trimester of pregnancy when prenatal diagnosis is most relevant. MPs were derived from apoptosis and increased over 48 hours. Compared with necrotic MPs, DNA in apoptotic MPs was more fragmented and resistant to plasma DNases. Membrane-specific dyes indicated that apoptotic MPs had more membranous material, which protects nucleic acids, including RNA. Flow cytometry showed that MPs derived from dying cells displayed light scatter and DNA staining similar to MPs found in maternal plasma. Quantification of maternal MPs using characteristics defined by MPs generated in vitro revealed a significant increase of DNA(+) MPs in the plasma of women with preeclampsia compared with plasma from women with normal pregnancies. Apoptotic MPs are therefore a likely source of stable DNA that could be enriched for both early genetic diagnosis and monitoring of pathological pregnancies.

Elevated levels of total (maternal and fetal) beta-globin DNA in maternal blood from first trimester pregnancies with trisomy 21.

BACKGROUND: Elevated levels of circulating fetal DNA have been observed in maternal plasma when a trisomy 21 fetus is confirmed. However, these studies have been limited to pregnancies carrying a male fetus. We sought to quantify total (fetal and maternal) DNA from dried blood spots (DBS) for use as an additional factor in multi-parameter prenatal screening for aneuploidy. METHODS: Maternal DBS were obtained from the NICHD-sponsored multi-center cohort (BUN) study. Seventeen confirmed trisomy 21 (mean gestational age 12.23 +/- 0.77 weeks) cases were each matched by gestational age to euploid controls (n = 30). DNA was extracted and quantitative PCR was performed to measure four non-chromosome 21 loci, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (12p13), beta-globin (11p15.5), beta-actin (7p15-12) and p53 (17p13.1). RESULTS: Beta-globin DNA levels were significantly elevated (P = 0.003) in 13 of 17 trisomy 21 cases (4.08 +/- 1.78 Geq/ml x 10(5)) compared with matched controls (2.35 +/- 1.84 Geq/ml x 10(5)). Following conversion of beta-globin concentrations into multiples of the median (MoM), MoM for trisomy 21 cases was 2.8 compared with 1.0 in euploid cases. No significant differences in levels of circulating GAPDH, beta-actin and p53 sequences were detected. CONCLUSIONS: This work demonstrates differential levels of circulating beta-globin DNA in maternal blood of euploid and trisomy 21 cases. Sequence-specific quantification could provide an additional measure to improve non-invasive methods of prenatal screening to detect trisomy 21 using dried blood. Beta-globin in particular is an attractive biomarker that could contribute to enhance multiple serum parameter testing in the first trimester.

Metronomic chemotherapy enhances the efficacy of antivascular therapy in ovarian cancer.

Metronomic chemotherapy is the frequent administration of low doses of chemotherapeutic agents targeting tumor-associated endothelial cells. We examined the efficacy of metronomic taxanes alone and in combination with AEE788-a dual epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) inhibitor-in an orthotopic mouse model of ovarian cancer. Growth-modulating effects of metronomic and maximum tolerated dose (MTD) regimens on overall survival were tested in vivo using both chemotherapy-sensitive (HeyA8 and SKOV3ip1) and chemotherapy-resistant (HeyA8-MDR) models. Treated tumors were stained for microvessel density (CD31), proliferation index (proliferating cell nuclear antigen), and apoptosis (terminal deoxyribonucleotide transferase-mediated nick-end labeling). The cytotoxic effects of MTD and metronomic dosing were tested with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Effects of metronomic regimens on circulating endothelial precursors (CEP) and tumor-specific cell-free DNA levels were assessed. In vivo, metronomic docetaxel resulted in significant reduction of tumor growth in the taxane-sensitive cell lines, whereas metronomic docetaxel plus AEE788 had an additive effect resulting in significant prolongation in survival. Combination therapy was effective even in the taxane-resistant model. Metronomic chemotherapy alone and combined with AEE788 resulted in a decrease in the proliferative index and microvessel density of treated tumors, whereas combination therapy increased the apoptotic index (P < 0.001). In vitro, metronomic taxanes caused endothelial cell toxicity at 10- to 100-fold lower concentrations compared with MTD dosing. Metronomic regimens inhibited mobilization of CEPs (P < 0.05) and led to a decrease in cell-free DNA levels (P < 0.05). Our results suggest that metronomic taxane chemotherapy with dual EGFR and VEGFR inhibition is highly efficacious and should be considered for future clinical trials.

Hypoxia-induced membrane-bound apoptotic DNA particles: potential mechanism of fetal DNA in maternal plasma.

Fetal DNA is found in the plasma of pregnant women that appears to be stable for PCR amplification. Although the underlying mechanism giving rise to this DNA in plasma remains unclear, the source of these fragments may be from apoptotic bodies (Apo-Bodies) created from dying cells. Trophoblast apoptosis is essential for normal placental development, given the enormous amount of proliferation, differentiation, and migration during pregnancy. Through flow cytometric analysis coupled with real-time PCR, our lab has shown that aggregates of acridine orange (AO)-stained material (apoptotic particles) are resistant to DNase treatment, disrupted by sodium dodecyl sulfate (SDS), and contain fetal DNA. Because the placenta continuously re-models in an hypoxic environment, our hypothesis is that fetal DNA in maternal plasma comes from hypoxia-induced dying trophoblasts and that this DNA circulates predominantly in the form of Apo-Bodies. We have developed a model culture system for analysis of Apo-Bodies derived from JEG-3 cells, an extravillous trophoblastic cell line, undergoing various methods of cell death: hypoxia-induced, etoposide-induced, and heat stress (necrosis like)-induced cell death. Under conditions of similar propidium iodide (PI) uptake, suggesting comparable levels of death, both hypoxia- and etoposide-induced Apo-Bodies increase in concentration over time, whereas heat-induced levels of particles remain fairly constant, indicating that production of DNA-associated Apo-Bodies is a continuous process. Hypoxia, which is likely to be responsible for trophoblast cell death in vivo, produced membrane-bound Apo-Bodies containing DNA. Our results are consistent with the characteristics of membrane-bound particles containing fetal DNA found in maternal plasma.

Quantification of total plasma cell-free DNA in ovarian cancer using real-time PCR.

Our objective was to compare the levels of total circulating plasma cell-free DNA (CfDNA) using real-time PCR in patients with late-stage ovarian cancer with those in unaffected controls. Following IRB consent, DNA was extracted from archived frozen plasma of 19 patients with primary ovarian carcinoma and 12 age-matched controls using Qiagen DNA Isolation Kits. Quantification of total CfDNA was performed using real-time PCR with the TaqMan Assay for GAPDH, beta-actin and beta-globin and the number of genome equivalents (GE/mL) were determined from a standard curve. CfDNA levels of these loci were compared between the groups with Student's t-test, with P < 0.05 being statistically significant. The mean age of the patients was 61.6 years (+/-9.6) and of the controls was 54 years (+/-12.2). All patients had high-grade, advanced stage (III or IV) serous ovarian carcinomas. Preoperative CA-125 levels ranged from 43 to 15,626 IU/mL (mean 2487.2 +/- 3686 IU/mL). Total CfDNA in ovarian cancer was higher among patients with ovarian cancer as compared to controls at all three loci: GAPDH (P = 0.022), beta-actin (P = 0.025), and beta-globin (P = 0.0089). CfDNA is elevated in advanced stage disease compared to controls. These preliminary results suggest that total CfDNA in the plasma of patients with ovarian cancer may be useful for noninvasive screening and disease surveillance.

Quantity versus quality: optimal methods for cell-free DNA isolation from plasma of pregnant women.

PURPOSE: Methods to isolate cell-free fetal DNA from maternal plasma are critical in developing noninvasive fetal DNA testing strategies. Given that plasma consists of heterogeneous DNA-size fragments in a complex mix of proteins, recovery and analysis of this DNA are understandably inefficient. To facilitate recovery, we performed qualitative and quantitative analysis of DNA isolated from maternal plasma. METHODS: DNA isolated from maternal blood (n = 15) was compared using five different DNA isolation protocols: two conventional, two column-based, and one magnetic-bead based. Purity and concentration of DNA recovered were determined with a NanoDrop spectrophotometer. Real-time polymerase chain reaction quantification of the beta-globin and DYS1 loci was performed to determine total and fetal-specific genome equivalents, respectively. RESULTS: DNA quality and quantity were different among the five methods tested. Although purity and concentration of total DNA were greatest with the conventional boiling-lysis approach, correct detection of a male fetus was achieved in only 62.5% of cases. DNA isolation using the magnetic beads yielded the highest quantity of total DNA (2018.83 +/- 4.09 GEq/mL), with 100% fetal DNA detection. CONCLUSIONS: Optimal plasma DNA recovery protocols must take into account DNA purity and concentration. We confirm that the magnetic-beads method provides a fast, simple, sensitive, and specific approach to purify plasma DNA. The resulting high-quality DNA facilitates efficient examination of fetal DNA sequences.