Discordant and heterogeneous clinically relevant genomic alterations in circulating tumor cells vs plasma DNA from men with metastatic castration resistant prostate cancer

Circulating tumor cell (CTC) and cell‐free (cf) DNA‐based genomic alterations are increasingly being used for clinical decision‐making in oncology. However, the concordance and discordance between paired CTC and cfDNA genomic profiles remain largely unknown. We performed comparative genomic hybridization (CGH) on CTCs and cfDNA, and low‐pass whole genome sequencing (lpWGS) on cfDNA to characterize genomic alterations (CNA) and tumor content in two independent prospective studies of 93 men with mCRPC treated with enzalutamide/abiraterone, or radium‐223. Comprehensive analysis of 69 patient CTCs and 72 cfDNA samples from 93 men with mCRPC, including 64 paired samples, identified common concordant gains in FOXA1, AR, and MYC, and losses in BRCA1, PTEN, and RB1 between CTCs and cfDNA. Concordant PTEN loss and discordant BRCA2 gain were associated with significantly worse outcomes in Epic AR‐V7 negative men with mCRPC treated with abiraterone/enzalutamide. We identified and externally validated CTC‐specific genomic alternations that were discordant in paired cfDNA, even in samples with high tumor content. These CTC/cfDNA‐discordant regions included key genomic regulators of lineage plasticity, osteomimicry, and cellular differentiation, including MYCN gain in CTCs (31%) that was rarely detected in cfDNA. CTC MYCN gain was associated with poor clinical outcomes in AR‐V7 negative men and small cell transformation. In conclusion, we demonstrated concordance of multiple genomic alterations across CTC and cfDNA platforms; however, some genomic alterations displayed substantial discordance between CTC DNA and cfDNA despite the use of identical copy number analysis methods, suggesting tumor heterogeneity and divergent evolution associated with poor clinical outcomes.     

Feasibility of integrating high-spatial-resolution 3D breath-hold coronary MR angiography with myocardial perfusion and viability examinations

The study was institutional review board approved and Health Insurance Portability and Accountability Act compliant. All subjects provided informed consent. Three-dimensional breath-hold coronary magnetic resonance (MR) angiography with use of steady-state free precession was performed in 12 patients up to 20 minutes after 0.2 mmol gadolinium-based contrast material per kilogram of body weight was administered. Within 24 heartbeats, a spatial resolution of up to 1.0 x 1.2 x 2.0 mm was achieved. Sixty-five (82%) of the 79 visualized coronary artery segments had a grade of 3 or 4 on a four-point scale of depiction in which grade 4 indicated excellent depiction. Twenty-seven percent (n = 21) of the 79 segments were assigned a grade of 4; 56% (n = 44), a grade of 3; 16% (n = 13), a grade of 2; and 1% (n = 1), a grade of 1. Coronary MR angiography performed as part of a first-pass myocardial perfusion and viability assessment MR imaging examination is feasible and does not involve additional imaging time.     

Population sub-structuring among <Emphasis Type="Italic">Trypanosoma evansi</Emphasis> stocks

To investigate the population genetic structure of Trypanosoma evansi from domesticated animals, we have analysed 112 stocks from camels, buffaloes, cattle and horses using the tandemly repeated coding sequence (MORF2) and minisatellite markers 292 and cysteine-rich acidic integral membrane protein (CRAM). We recorded a total of six alleles at the MORF2 locus, seven at 292 and 12 at the CRAM loci. Nei’s genetic distance showed reduced allelic diversity between buffaloes and cattle stocks (1.2) as compared to the diversity between camels and buffaloes (3.75) and camels and cattle stock (1.69). The mean index of association (I _A = 0.92) significantly deviated from zero, and the average number of multilocus genotypes (G/N ratio) was 0.21. Twenty-four multilocus genotypes were defined from the combination of alleles at the three loci. The Kenyan sub-populations showed F _st = 0.28 and analysis of molecular variance showed significant divergence (22.7%) between the Laikipia, Kulal and Galana regions. The regional and host distribution of multi-locus genotypes significant population differentiation and high Nei’s genetic distances suggest existence of genetic sub-structuring within T. evansi stocks while the few multi-locus genotypes and deviation of association index from zero indicate the lack of recombination. In conclusion, this study reveals that some genetic sub-structuring does occur within T. evansi, which has a clonal population structure.     

Identification of human CD4 T-cell epitopes on the VP1 capsid protein of enterovirus 71

The identification of human CD4 T-cell epitopes within a protein vaccine candidate is of great interest,as it provides a better understanding of the mechanisms involved in protective immunity and may therefore help in the design of effective vaccines and diagnostic tools. The entire amino acid sequence of the VP1 capsid protein from enterovirus 71 (EV 71) strain 41 was submitted to analysis by the ProPred algorithm for the identification of potential promiscuous human CD4 T-cellepitopes. Three regions spanning amino acids 66-77, 145-159, and 247-261 of VP1 were predicted to bind more than 25 HLA-DR alleles. The corresponding synthetic peptides (SP1 to SP3) were then tested for their abilities to induce proliferation of CD4 T cells isolated from five human volunteers screened positive for previous EV 71 exposure and one EV 71-negative volunteer. Upon stimulation with either peptide, CD4 T-cell proliferative responses were observed for all EV 71-positive volunteers,indicating the presence of EV 71-specific memory CD4 T cells. The amplitude of the proliferative responses was peptide- and HLA-DR-dependent, and correlated well with the ProPredpredicted binding efficiencies. Moreover, CD4 T cells from EV 71-positive volunteers produced significant levels of IL-2 and IFN- upon stimulation, indicative of a T-cell differentiation into Th-1-type subset. Among the three peptides, SP2 induced the highest proliferative response and cytokine production. Moreover, SP2-induced proliferative response could be inhibited with anti-major histocompatibility complex (MHC) class II antibody, indicating that SP2 represents a MHC class II-restricted CD4 T-cell epitope. This study demonstrates that the ProPred algorithm can accurately predict the presence of human CD4 T-cell epitopes within the VP1 capsid protein of EV 71, and therefore represents a useful tool for the design of subunit vaccines against EV 71.     

Modulation of spontaneous outgrowth of Epstein-Barr virus immortalized B-cell clones by granulocyte-macrophage colony-stimulating factor and interleukin-3

Spontaneous outgrowth of immortalized Epstein-Barr virus (EBV) infected B-cell clones will occur from cultures of peripheral blood mononuclear cells (PBMNCs) of some persons with a history of EBV infection. We determined that outgrowth of such clones may be reproducibly modulated by supplementation of cultures with the hematopoietic growth factors GM- CSF and interleukin-3 (IL-3). Continuous supplementation of cultures with GM-CSF facilitates emergence of immortalized B-cell clones, whereas supplementation with IL-3 completely prevents their emergence. The effect of GM-CSF may be direct, at least in part, as the proliferation of pure clones of EBV-transformed B lymphocytes was augmented in response to GM-CSF. An indirect mechanism appears to be responsible for the inhibition of transformed B-cell outgrowth in response to IL-3, as IL-3 had no inhibitory effect on proliferation of pure transformed B-cell clones and IL-3-mediated inhibition could be eliminated by antibody neutralization of gamma-interferon (gamma-IFN) or tumor necrosis factor-alpha (TNF-alpha) early in culture. The mechanisms of these effects deserve further study and may have clinical relevance to use of hematopoietic growth factors for support of bone marrow (BM) function in immunocompromised patients.     

Expansion in vitro of retrovirally marked totipotent hematopoietic stem cells

A large number of biologic, technological, and clinical studies await the development of procedures that will allow totipotent hematopoietic stem cells to be expanded in vitro. Previous work has suggested that hematopoiesis can be reconstituted using transplants of cells from long- term marrow cultures. We have used retrovirus mediated gene transfer to demonstrate that marked totipotent hematopoietic stem cells are both maintained and can be amplified in such cultures, and then subsequently regenerate and sustain lympho-myeloid hematopoiesis in irradiated recipients. Marrow cells from 5-fluorouracil-treated male mice were infected with a recombinant virus carrying the neomycin resistence gene and seeded onto irradiated adherent layers of pre-established, long- term marrow cultures of female origin. At 4 weeks, cells from individual cultures were transplanted into single or multiple female recipients. Southern blot analysis of hematopoietic tissue 45 days posttransplantation showed retrovirally marked clones common to lymphoid and myeloid tissues in 14 of 23 mice examined. Strikingly, for 3 of 4 long-term cultures, multiple recipients of cells from a single flask showed marrow and thymus repopulation with the same unique retrovirally marked clone. These results establish the feasibility of retroviral-marking techniques to demonstrate the maintenance of totipotent lympho-myeloid stem cells for at least 4 weeks in the long- term marrow culture system and provide the first evidence of their proliferation in vitro. Therefore, such cultures may serve as a starting point for identifying factors that stimulate totipotent hematopoietic stem cell expansion.     

Pre-exposure of murine macrophages to lipopolysaccharide inhibits the induction of nitric oxide synthase and reduces leishmanicidal activity

Murine macrophages produce nitric oxide (NO) from L-arginine on stimulation with lipopolysaccharide (LPS), alone or with interferon-γ (IFN-γ). The effect of incubation of macrophages with low concentrations of LPS on NO synthesis on subsequent stimulation was investigated, using a murine macrophage cell line, J774, and peritoneal macrophages from CBA mice. Cells which had been incubated with LPS produced significantly lower amounts of NO, and expressed lower levels of NO synthase activity, following stimulation with IFN-γ and LPS, or with a high concentration of LPS. This effect was not reversed by tumor necrosis factor-α. The ability of CBA macrophages to kill the intracellular parasite Leishmania major was markedly reduced by pre-incubation with LPS. Reduced NO production by macrophages previously exposed to LPS is a manifestation of endotoxin tolerance, and may represent an important means of regulation of NO synthesis and thus a survival mechanism for intracellular parasites.