Circulating Cell-Free DNA Yield and Circulating-Tumor DNA Quantity from Liquid Biopsies of 12 139 Cancer Patients.

BACKGROUND: The amounts of circulating cell-free DNA (cfDNA) and circulating-tumor DNA (ctDNA) present in peripheral blood liquid biopsies can vary due to preanalytic/analytic variables. In this study, we examined the impact of patient age, sex, stage, and tumor type on cfDNA yield, ctDNA fraction, and estimated ctDNA quantity from a large cohort of clinical liquid biopsy samples. METHODS: We performed a retrospective analysis of 12 139 consecutive samples received for liquid biopsy (FoundationOne® Liquid) clinical testing. RESULTS: Significant differences in both cfDNA yield and estimated ctDNA quantity were observed based on the underlying tumor type that initiated the liquid biopsy analysis and the stage of the patient (P < 0.001). In addition, significant differences in ctDNA quantity were present based in both the patient age and sex (P < 0.001). Importantly, we saw a significantly higher success rate of issuing a clinically useful report in patients with higher levels of cfDNA yield and ctDNA quantity (P < 0.001). CONCLUSIONS: In this study, we show that ctDNA quantity varied significantly based on patient age, sex, stage, and tumor type, which could offer an explanation as to why certain liquid biopsy specimens are more likely to fail sequencing or provide clinically meaningful results. In addition, this could affect future clinical decisions on the blood sample volumes required to allow successful liquid biopsy testing.

A homing mechanism for bone marrow-derived progenitor cell recruitment to the neovasculature.

CD34+ bone marrow-derived progenitor cells contribute to tissue repair by differentiating into endothelial cells, vascular smooth muscle cells, hematopoietic cells, and possibly other cell types. However, the mechanisms by which circulating progenitor cells home to remodeling tissues remain unclear. Here we show that integrin alpha4beta1 (VLA-4) promotes the homing of circulating progenitor cells to the alpha4beta1 ligands VCAM and cellular fibronectin, which are expressed on actively remodeling neovasculature. Progenitor cells, which express integrin alpha4beta1, homed to sites of active tumor neovascularization but not to normal nonimmune tissues. Antagonists of integrin alpha4beta1, but not other integrins, blocked the adhesion of these cells to endothelia in vitro and in vivo as well as their homing to neovasculature and outgrowth into differentiated cell types. These studies describe an adhesion event that facilitates the homing of progenitor cells to the neovasculature.

Efficiency of the APTIMA HPV Assay for detection of HPV RNA and DNA targets.

BACKGROUND: The APTIMA HPV Assay (AHPV) is designed to detect HPV E6/E7 mRNA from 14 high-risk types in Cytyc PreservCyt liquid-based cytology specimens. OBJECTIVES: To compare AHPV analytical sensitivity for RNA and DNA; to compare the sensitivity of AHPV and Hybrid Capture 2 (HC2) assays for HPV DNA detection; to compare assay performance with and without sample denaturation; to compare assay results with cytology. STUDY DESIGN: Analytical sensitivity of AHPV for detecting E6/E7 RNA was assessed by spiking samples with various quantities of HPV 16 E6/E7 in vitro RNA transcript or HPV 16-positive SiHa cells. AHPV and HC2 analytical sensitivity for HPV 16 DNA was evaluated by spiking samples with various quantities of a plasmid vector containing cloned HPV 16 DNA, or with purified SiHa cell genomic DNA containing integrated HPV 16 genome. Samples were tested using standard AHPV and HC2 protocols. Endocervical samples from 568 women were collected in Digene Specimen Transport Medium. Non-denatured and denatured samples were tested in AHPV and denatured samples with HC2. Assay results were compared to each other, and to cytology. RESULTS: AHPV had substantially higher (2 4 log(10)) analytical sensitivity for HPV 16 RNA than for HPV 16 DNA. AHPV also had substantially lower (3 log(10)) analytical sensitivity for HPV 16 DNA compared to HC2. The overall agreement between assay results in clinical specimens was 94.2%, but AHPV had fewer positives than HC2 (48.4% positive agreement). In denatured samples, the number of samples testing positive in AHPV increased two-fold, yielding a positive agreement rate of 88.7%. When assay results were compared with cytology, AHPV had fewer positives in samples with normal or ASC-US diagnoses than did HC2. CONCLUSIONS: AHPV is much more efficient at detecting HPV 16 RNA than DNA. Selective capture, amplification, and detection of HPV RNA by AHPV may improve the specificity of molecular HPV testing.

The PIAS-like protein Zimp10 is essential for embryonic viability and proper vascular development.

Members of the PIAS (for protein inhibitor of activated STAT) family play critical roles in modulating the activity of a variety of transcriptional regulators. Zimp10, a novel PIAS-like protein, is a transcriptional coregulator and may be involved in the modification of chromatin through interactions with the SWI/SNF chromatin-remodeling complexes. Here, we investigate the biological role of Zimp10 in zimp10-deficient mice. Homozygosity for the Zimp10-targeted allele resulted in developmental arrest at approximately embryonic day 10.5. Analysis of knockout embryos revealed severe defects in the reorganization of the yolk sac vascular plexus. No significant abnormality in hematopoietic potential was observed in zimp10 null mice. Microarray and quantified reverse transcription-PCR analyses showed that the expression of the Fos family member Fra-1, which is involved in extraembryonic vascular development, was reduced in yolk sac tissues of zimp10 null embryos. Using fra-1 promoter/reporter constructs, we further demonstrate the regulatory role of Zimp10 on the transcription of Fra-1. This study provides evidence to demonstrate a crucial role for Zimp10 in vasculogenesis.

Loss of Apaf-1 leads to partial rescue of the HAND2-null phenotype.

HAND2 is an essential transcription factor for cardiac, pharyngeal arch, and limb development. Apoptosis in the HAND2-null embryo causes hypoplasia of the right ventricle and pharyngeal arches leading to lethality by embryonic day (E)10.0 from heart failure. In order to investigate the role of apoptosis in inducing the HAND2-null phenotype, we generated mouse embryos lacking both HAND2 and Apaf-1, a central downstream mediator of mitochondrial damage-induced apoptosis. In contrast to HAND2-/- embryos, HAND2-/-Apaf-1-/- embryos at E10.5-11.0 had well-developed pharyngeal arches, aortic arch arteries, and no signs of cardiac failure. TUNEL analysis through pharyngeal arches of HAND2-/-Apaf-1-/- embryos revealed decreased apoptosis and the embryos had clearly patent aortic arch arteries. However, ventricular hypoplasia and cell death were unchanged in these animals compared to HAND2-/- embryos, resulting in growth arrest at E11.0. Our study suggests that loss of HAND2 in the pharyngeal arch mesenchyme leads to apoptosis in an Apaf-1-dependent fashion and that, while loss of aortic arch integrity contributes to the early lethality, the ventricular defects are independent of arch development.

Genetic and comparative mapping of genes dysregulated in mouse hearts lacking the Hand2 transcription factor gene.

The helix-loop-helix transcription factor HAND2 plays a vital role in the development of the heart, limb, facies, and other neural crest-derived structures. We used differential display analysis to identify 33 putative HAND2-regulated ESTs that are differentially expressed in Hand2(-/-) vs wild-type mice. We determined the positions on mouse and human genetic maps of 29 of these by using the T31 mouse Radiation Hybrid panel, comparison to human genomic sequence, and comparative mapping. We examined the conserved chromosomal locations for phenotypes that involve development of heart, face, and limb structures that are affected by HAND2. One EST mapped to a region of conserved synteny between mouse chromosome 2 and human chromosome 10p. RACE analysis extended the sequence and identified this cDNA as the mouse ortholog of human nebulette, an actin-binding protein expressed in fetal heart. Nebulette was shown to be deleted in DiGeorge Syndrome 2 patients with the proximal deletion of human 10p13-p14 that is associated with cardiac and craniofacial abnormalities.