Monitoring of Measurable Residual Disease Using Circulating DNA after Allogeneic Hematopoietic Cell Transplantation.

Relapse of the underlying disease is a frequent complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In this study, we describe the clinical utility of measurable residual disease (MRD) and mixed chimerism (MC) assessment in circulating cell-free DNA (cfDNA) analysis to detect earlier relapse in patients with hematological malignancies after allo-HSCT. A total of 326 plasma and peripheral blood mononuclear cell (PBMCs) samples obtained from 62 patients with myeloid malignancies were analyzed by droplet-digital PCR (median follow-up: 827 days). Comparison of MC in patients at relapse and in complete remission identified an optimal discriminating threshold of 18% of recipient-derived cfDNA. After performing a targeted next-generation sequencing (NGS) panel, 136 mutations in 58 patients were detected. In a total of 119 paired samples, the putative mutations were detected in both cfDNA and PBMCs in 73 samples (61.3%). In 45 samples (37.8%) they were detected only in cfDNA, and in only one patient (0.9%) were they detected solely in DNA from PBMCs. Hence, in 6 out of 23 patients (26%) with relapse after allo-HSCT, MRD positivity was detected earlier in cfDNA (mean 397 days) than in DNA derived from PBMCs (mean 451 days). In summary, monitoring of MRD and MC in cfDNA might be useful for earlier relapse detection in patients with myeloid malignancies after allo-HSCT.

Colon and liver tissue damage detection using methylated SESN3 and PTK2B genes in circulating cell-free DNA in patients with acute graft-versus-host disease.

Cell-free DNA (cfDNA) has been investigated in acute graft-versus-host disease (aGvHD) following allogeneic cell transplantation (HSCT). Identifying the tissue of origin of cfDNA in patients with aGvHD is relevant particularly when a biopsy is not feasible. We investigate the cfDNA tissue of origin in patients with aGvHD using methylated gene biomarkers. Patients with liver, colon, or skin aGvHD (n = 28) were analyzed. Liver- and colon-derived cfDNA was measured using a colon- (SESN3) and liver (PTK2B)-specific methylation marker with digital droplet PCR. A statistically significant difference (p < 0.001) in PTK2B and SESN3 concentration was observed between patients with colon or liver GvHD and the control group. For SESN3 and PTK2B the area under the curve in the receiver-operating characteristic (ROC) space was 0.952 (95% CI, 0.888-1 p < 0.001) and 0.971 (95% CI, 0.964-1 p < 0.001), respectively. Thresholds to differentiate aGvHD from non-aGvHD in colon were 0 (sensitivity: 0.905; specificity: 0.989) and liver 1.5 (sensitivity: 0.928; specificity: 0.910). Clinical improvement of liver or colon aGvHD resulted in PTK2B and SESN3 reduced concentration. Whereas, in those patients without improvement the PTK2B and SESN3 level remained stable or increased. The PTK2B liver-specific marker and the SESN3 colon-specific marker and their longitudinal analysis might improve aGvHD detection.

CD44 engagement enhances acute myeloid leukemia cell adhesion to the bone marrow microenvironment by increasing VLA-4 avidity.

Adhesive properties of leukemia cells shape the degree of organ infiltration and the extent of leukocytosis. CD44 and the integrin VLA-4, a CD49d/CD29 heterodimer, are important factors of progenitor cell adhesion in bone marrow (BM). Here, we report their cooperation in acute myeloid leukemia (AML) by a novel non-classical CD44-mediated way of inside-out VLA-4 activation. In primary AML BM samples from patients and the OCI-AML3 cell line, CD44 engagement by hyaluronan induced inside-out activation of VLA-4 resulting in enhanced leukemia cell adhesion on VCAM-1. This was independent from VLA-4 affinity regulation but based on ligand-induced integrin clustering on the cell surface. CD44-induced VLA-4 activation could be inhibited by the Src family kinase inhibitor PP2 and the multikinase inhibitor midostaurin. In further consequence, the increased adhesion on VCAM-1 allowed AML cells to strongly bind stromal cells. Thereby VLA-4/VCAM-1 interaction promoted activation of Akt, MAPK, NF-kB and mTOR signaling and decreased AML cell apoptosis. Collectively, our investigations provide a mechanistic description of an unusual CD44 function in regulating VLA-4 avidity in AML, supporting AML cell retention in the supportive BM microenvironment.

Oncogenic JAK2V617F causes PD-L1 expression, mediating immune escape in myeloproliferative neoplasms.

Recent evidence has revealed that oncogenic mutations may confer immune escape. A better understanding of how an oncogenic mutation affects immunosuppressive programmed death ligand 1 (PD-L1) expression may help in developing new therapeutic strategies. We show that oncogenic JAK2 (Janus kinase 2) activity caused STAT3 (signal transducer and activator of transcription 3) and STAT5 phosphorylation, which enhanced PD-L1 promoter activity and PD-L1 protein expression in JAK2V617F-mutant cells, whereas blockade of JAK2 reduced PD-L1 expression in myeloid JAK2V617F-mutant cells. PD-L1 expression was higher on primary cells isolated from patients with JAK2V617F-myeloproliferative neoplasms (MPNs) compared to healthy individuals and declined upon JAK2 inhibition. JAK2V617F mutational burden, pSTAT3, and PD-L1 expression were highest in primary MPN patient-derived monocytes, megakaryocytes, and platelets. PD-1 (programmed death receptor 1) inhibition prolonged survival in human MPN xenograft and primary murine MPN models. This effect was dependent on T cells. Mechanistically, PD-L1 surface expression in JAK2V617F-mutant cells affected metabolism and cell cycle progression of T cells. In summary, we report that in MPN, constitutive JAK2/STAT3/STAT5 activation, mainly in monocytes, megakaryocytes, and platelets, caused PD-L1-mediated immune escape by reducing T cell activation, metabolic activity, and cell cycle progression. The susceptibility of JAK2V617F-mutant MPN to PD-1 targeting paves the way for immunomodulatory approaches relying on PD-1 inhibition.