Jurgen Langenhorst

Simultaneous quantification of busulfan, clofarabine and F-ARA-A using isotope labelled standards and standard addition in plasma by LC–MS/MS for exposure monitoring in hematopoietic cell transplantation conditioning

In allogeneic hematopoietic cell transplantation (HCT) it has been shown that over- or underexposure to conditioning agents have an impact on patient outcomes. Conditioning regimens combining busulfan (Bu) and fludarabine (Flu) with or without clofarabine (Clo) are gaining interest worldwide in HCT. To evaluate and possibly adjust full conditioning exposure a simultaneous analysis of Bu, F-ARA-A (active metabolite of Flu) and Clo in one analytical run would be of great interest. However, this is a chromatographical challenge due to the large structural differences of Bu compared to F-ARA-A and Clo. Furthermore, for the bioanalysis of drugs it is common to use stable isotope labelled standards (SILS). However, when SILS are unavailable (in case of Clo and F-ARA-A) or very expensive, standard addition may serve as an alternative to correct for recovery and matrix effects. This study describes a fast analytical method for the simultaneous analysing of Bu, Clo and F-ARA-A with liquid chromatography-tandem mass spectrometry (LC-MS/MS) including standard addition methodology using 604 spiked samples. First, the analytical method was validated in accordance with European Medicines Agency guidelines. The lower limits of quantification (LLOQ) were for Bu 10μg/L and for Clo and F-ARA-A 1μg/L, respectively. Variation coefficients of LLOQ were within 20% and for low medium and high controls were all within 15%. Comparison of Bu, Clo and F-ARA-A standard addition results correspond with those obtained with calibration standards in calf serum. In addition for Bu, results obtained by this study were compared with historical data analysed within TDM. In conclusion, an efficient method for the simultaneous quantification of Bu, Clo and F-ARA-A in plasma was developed. In addition, a robust and cost-effective method to correct for matrix interference by standard addition was established.

Filgrastim enhances T-cell clearance by antithymocyte globulin exposure after unrelated cord blood transplantation

Residual antithymocyte globulin (ATG; Thymoglobulin) exposure after allogeneic hematopoietic (stem) cell transplantation (HCT) delays CD4+ T-cell immune reconstitution (CD4+ IR), subsequently increasing morbidity and mortality. This effect seems particularly present after cord blood transplantation (CBT) compared to bone marrow transplantation (BMT). The reason for this is currently unknown. We investigated the effect of active-ATG exposure on CD4+ IR after BMT and CBT in 275 patients (CBT n = 155, BMT n = 120; median age, 7.8 years; range, 0.16-19.2 years) receiving their first allogeneic HCT between January 2008 and September 2016. Multivariate log-rank tests (with correction for covariates) revealed that CD4+ IR was faster after CBT than after BMT with <10 active-ATG × day/mL (P = .018) residual exposure. In contrast, >10 active-ATG × day/mL exposure severely impaired CD4+ IR after CBT (P < .001), but not after BMT (P = .74). To decipher these differences, we performed ATG-binding and ATG-cytotoxicity experiments using cord blood- and bone marrow graft-derived T-cell subsets, B cells, natural killer cells, and monocytes. No differences were observed. Nevertheless, a major covariate in our cohort was Filgrastim treatment (only given after CBT). We found that Filgrastim (granulocyte colony-stimulating factor [G-CSF]) exposure highly increased neutrophil-mediated ATG cytotoxicity (by 40-fold [0.5 vs 20%; P = .002]), which explained the enhanced T-cell clearance after CBT. These findings imply revision of the use (and/or timing) of G-CSF in patients with residual ATG exposure.

Innate immune recovery predicts CD4+ T-cell reconstitution after Hematopoietic Cell Transplantation

Innate immune cells are the first to recover after allogeneic hematopoietic cell transplantation (HCT). Nevertheless, reports of innate immune cell recovery and their relation to adaptive recovery after HCT are largely lacking. Especially predicting CD4+ T cell reconstitution is of clinical interest, because this parameter directly associates with survival chances after HCT. We evaluated whether innate recovery relates to CD4+ T cell reconstitution probability and investigated differences between innate recovery after cord blood transplantation (CBT) and bone marrow transplantation (BMT). We developed a multivariate, combined nonlinear mixed-effects model for monocytes, neutrophils, and natural killer (NK) cell recovery after transplantation. A total of 205 patients undergoing a first HCT (76 BMT, 129 CBT) between 2007 and 2016 were included. The median age was 7.3years (range, .16 to 23). Innate recovery was highly associated with CD4+ T cell reconstitution probability (P < .001) in multivariate analysis correcting for covariates. Monocyte (P < .001), neutrophil (P < .001), and NK cell (P < .001) recovery reached higher levels during the first 200days after CBT compared with BMT. The higher innate recovery after CBT may be explained by increased proliferation capacity (measured by Ki-67 expression) of innate cells in CB grafts compared with BM grafts (P = .041) and of innate cells in vivo after CBT compared with BMT (P = .048). At an individual level, patients with increased innate recovery after either CBT or BMT had received grafts with higher proliferating innate cells (CB; P = .004, BM; P = .01, respectively). Our findings implicate the use of early innate immune monitoring to predict the chance of CD4+ T cell reconstitution after HCT, with respect to higher innate recovery after CBT compared with BMT.