A prospective, observational, multicenter cohort study

Background Graft-derived cell-free DNA (GcfDNA), which is released into the blood stream by necrotic and apoptotic cells, is a promising noninvasive organ integrity biomarker. In liver transplantation (LTx), neither conventional liver function tests (LTFs) nor immunosuppressive drug monitoring are very effective for rejection monitoring. We therefore hypothesized that the quantitative measurement of donor-derived cell-free DNA (cfDNA) would have independent value for the assessment of graft integrity, including damage from acute rejection. Methods and findings Traditional LFTs were performed and plasma GcfDNA was monitored in 115 adults post-LTx at three German transplant centers as part of a prospective, observational, multicenter cohort trial. GcfDNA percentage (graft cfDNA/total cfDNA) was measured using droplet digital PCR (ddPCR), based on a limited number of predefined single nucleotide polymorphisms, enabling same-day turn-around. The same method was used to quantify blood microchimerism. GcfDNA was increased >50% on day 1 post-LTx, presumably from ischemia/reperfusion damage, but rapidly declined in patients without graft injury within 7 to 10 d to a median <10%, where it remained for the 1-y observation period. Of 115 patients, 107 provided samples that met preestablished criteria. In 31 samples taken from 17 patients during biopsy- proven acute rejection episodes, the percentage of GcfDNA was elevated substantially (median 29.6%, 95% CI 23.6%–41.0%) compared with that in 282 samples from 88 patients during stable periods (median 3.3%, 95% CI 2.9%–3.7%; p < 0.001). Only slightly higher values (median 5.9%, 95% CI 4.4%–10.3%) were found in 68 samples from 17 hepatitis C virus (HCV)–positive, rejection-free patients. LFTs had low overall correlations (r = 0.28–0.62) with GcfDNA and showed greater overlap between patient subgroups, especially between acute rejection and HCV+ patients. Multivariable logistic regression modeling demonstrated that GcfDNA provided additional LFT-independent information on graft integrity. Diagnostic sensitivity and specificity were 90.3% (95% CI 74.2%–98.0%) and 92.9% (95% CI 89.3%–95.6%), respectively, for GcfDNA at a threshold value of 10%. The area under the receiver operator characteristic curve was higher for GcfDNA (97.1%, 95% CI 93.4%–100%) than for same-day conventional LFTs (AST: 95.7%; ALT: 95.2%; γ-GT: 94.5%; bilirubin: 82.6%). An evaluation of microchimerism revealed that the maximum donor DNA in circulating white blood cells was only 0.068%. GcfDNA percentage can be influenced by major changes in host cfDNA (e.g., due to leukopenia or leukocytosis). One limitation of our study is that exact time-matched GcfDNA and LFT samples were not available for all patient visits. Conclusions In this study, determination of GcfDNA in plasma by ddPCR allowed for earlier and more sensitive discrimination of acute rejection in LTx patients as compared with conventional LFTs. Potential blood microchimerism was quantitatively low and had no significant influence on GcfDNA value. Further research, which should ideally include protocol biopsies, will be needed to establish the practical value of GcfDNA measurements in the management of LTx patients

BTK and PI3K Inhibitors Reveal Synergistic Inhibitory Anti-Tumoral Effects in Canine Diffuse Large B-Cell Lymphoma Cells

Bruton’s tyrosine kinase (BTK) and phosphoinositide 3-kinase (PI3K) in the B-cell receptor (BCR) signaling pathway are considered potential therapeutic targets for the treatment of B-cell lymphomas, among which, diffuse large B-cell lymphoma (DLBCL) is the most common type. Herein, we comparatively evaluated the single and combined application of the BTK inhibitor ibrutinib and the selective PI3Kγ inhibitor AS-605240 in the canine DLBCL cell line CLBL-1. For further comparison, key findings were additionally analyzed in canine B-cell leukemia GL-1 and human DLBCL cell line SU-DHL-4. While ibrutinib alone induced significant anti-proliferative effects on all cell lines in a dose-dependent manner, AS-605240 only induced anti-proliferative effects at high concentrations. Interestingly, ibrutinib and AS-605240 acted synergistically, reducing cell proliferation and increasing apoptosis/necrosis in all cell lines and inducing morphological changes in CLBL-1. Moreover, the combined application of ibrutinib and AS-605240 reduced relative phosphorylation and, in some instances, the levels of the BTK, AKT, GSK3β, and ERK proteins. Comparative variant analysis of RNA-seq data among canine B- and T-lymphoid cell lines and primary B-cell lymphoma samples revealed potentially high-impact somatic variants in the genes that encode PI3K, which may explain why AS-605240 does not singly inhibit the proliferation of cell lines. The combination of ibrutinib and AS-605240 represents a promising approach that warrants further in vivo evaluation in dogs, potentially bearing significant value for the treatment of human DLBCL