Stephan Mielke

Association of CYP2C8, CYP3A4, CYP3A5, and ABCB1 Polymorphisms with the Pharmacokinetics of Paclitaxel

Purpose: To retrospectively evaluate the effects of six known allelic variants in the CYP2C8, CYP3A4, CYP3A5, and ABCB1 genes on the pharmacokinetics of the anticancer agent paclitaxel (Taxol). Experimental Design: A cohort of 97 Caucasian patients with cancer (median age, 57 years) received paclitaxel as an i.v. infusion (dose range, 80-225 mg/m2). Genomic DNA was analyzed using PCR RFLP or using Pyrosequencing. Pharmacokinetic variables for unbound paclitaxel were estimated using nonlinear mixed effect modeling. The effects of genotypes on typical value of clearance were evaluated with the likelihood ratio test within NONMEM. In addition, relations between genotype and individual pharmacokinetic variable estimates were evaluated with one-way ANOVA. Results: The allele frequencies for the CYP2C8*2, CYP2C8*3, CYP2C8*4, CYP3A4*3, CYP3A5*3C, and ABCB1 3435C>T variants were 0.7%, 9.2%, 2.1%, 0.5%, 93.2%, and 47.1%, respectively, and all were in Hardy-Weinberg equilibrium. The population typical value of clearance of unbound paclitaxel was 301 L/h (individual clearance range, 83.7-1055 L/h). The CYP2C8 or CYP3A4/5 genotypes were not statistically significantly associated with unbound clearance of paclitaxel. Likewise, no statistically significant association was observed between the ABCB1 3435C>T variant and any of the studied pharmacokinetic variables. Conclusions: This study indicates that the presently evaluated variant alleles in the CYP2C8, CYP3A4, CYP3A5, and ABCB1 genes do not explain the substantial interindividual variability in paclitaxel pharmacokinetics.

Ruxolitinib in corticosteroid-refractory graft-versus-host disease after allogeneic stem cell transplantation: A multicenter survey

Despite major improvements in allogeneic hematopoietic cell transplantation over the past decades, corticosteroid-refractory (SR) acute (a) and chronic (c) graft-versus-host disease (GVHD) cause high mortality. Preclinical evidence indicates the potent anti-inflammatory properties of the JAK1/2 inhibitor ruxolitinib. In this retrospective survey, 19 stem cell transplant centers in Europe and the United States reported outcome data from 95 patients who had received ruxolitinib as salvage therapy for SR-GVHD. Patients were classified as having SR-aGVHD (n=54, all grades III or IV) or SR-cGVHD (n=41, all moderate or severe). The median number of previous GVHD-therapies was 3 for both SR-aGVHD (1–7) and SR-cGVHD (1–10). The overall response rate was 81.5% (44/54) in SR-aGVHD including 25 complete responses (46.3%), while for SR-cGVHD the ORR was 85.4% (35/41). Of those patients responding to ruxolitinib, the rate of GVHD-relapse was 6.8% (3/44) and 5.7% (2/35) for SR-aGVHD and SR-cGVHD, respectively. The 6-month-survival was 79% (67.3–90.7%, 95% confidence interval (CI)) and 97.4% (92.3–100%, 95% CI) for SR-aGVHD and SR-cGVHD, respectively. Cytopenia and cytomegalovirus-reactivation were observed during ruxolitinib treatment in both SR-aGVHD (30/54, 55.6% and 18/54, 33.3%) and SR-cGVHD (7/41, 17.1% and 6/41, 14.6%) patients. Ruxolitinib may constitute a promising new treatment option for SR-aGVHD and SR-cGVHD that should be validated in a prospective trial.

Rapid natural killer cell recovery determines outcome after T-cell-depleted HLA-identical stem cell transplantation in patients with myeloid leukemias but not with acute lymphoblastic leukemia

Natural killer (NK) cells are the first lymphocytes to recover after allogeneic stem cell transplantation (SCT) and can exert powerful graft-versus-leukemia (GVL) effects determining transplant outcome. Conditions governing NK cell alloreactivity and the role of NK recovery in sibling SCT are not well defined. NK cells on day 30 post-transplant (NK30) were measured in 54 SCT recipients with leukemia and donor and recipient killer immunoglobulin-like receptor (KIR) genotype determined. In univariate analysis, donor KIR genes 2DL5A, 2DS1, 3DS1 (positive in 46%) and higher numbers of inhibitory donor KIR correlated with higher NK30 counts and were associated with improved transplant outcome. NK30 counts also correlated directly with the transplant CD34 cell dose and inversely with the CD3+ cell dose. In multivariate analysis, the NK30 emerged as the single independent determinant of transplant outcome. Patients with NK30 >150/μl had less relapse (HR 18.3, P=0.039), acute graft-versus-host disease (HR 3.2, P=0.03), non-relapse mortality (HR 10.7, P=0.028) and improved survival (HR 11.4, P=0.03). Results suggest that T cell-depleted SCT might be improved and the GVL effect enhanced by selecting donors with favorable KIR genotype, and by optimizing CD34 and CD3 doses.

Association of Paclitaxel pharmacokinetics with the development of peripheral neuropathy in patients with advanced cancer.

Purpose: The shortening of infusion time from 3 to 1 hour decreases the systemic exposure (area under the curve, AUC) of total and unbound paclitaxel but increases the AUC of its vehicle Cremophor EL, whereas the time above total paclitaxel concentrations of 0.05 μmol/L (T>0.05) remains almost constant. As both Cremophor EL and paclitaxel are neurotoxic, we evaluated their pharmacodynamic effects on the development of peripheral neuropathy as the most important nonhematologic toxicity. Experimental Design: Patients with advanced cancer of different origin were randomized to receive a maximum of 12 weekly-given 1- or 3-hour infusions of 100 mg/m2 paclitaxel (Taxol). Twenty-four patients were assessable for both pharmacokinetics and peripheral neuropathy development evaluated by a clinical scoring system including sensory symptoms, strength, tendon reflexes, and vibratory sense. Results: Patients with peripheral neuropathy development (n = 14) received more weeks of therapy (P = 0.056) and showed significantly higher T>0.05 (P = 0.022) and overall systemic drug exposures (weeks of therapy × AUC) for total paclitaxel (P = 0.002) and unbound paclitaxel (P = 0.003) than those without peripheral neuropathy. In Kaplan-Meier analyses, T>0.05 ≥ 10.6 hours (P = 0.023), AUC of total paclitaxel ≥ 4.7 μg/mL × hour (P = 0.047), and AUC of unbound paclitaxel ≥ 0.375 μg/mL × hour (P = 0.095) were identified as being potential factors for peripheral neuropathy development. In a Cox regression analysis, only T>0.05 ≥ 10.6 hours remained as an independent risk factor (relative risk, 18.43; P = 0.036) after adjusting for prior vincamycin (relative risk, 11.28; P = 0.038). Conclusions: From the results obtained in this study, it is concluded that exposure to paclitaxel but not Cremophor EL is associated with peripheral neuropathy development.

Ex vivo characterization of polyclonal memory CD8 + T-cell responses to PRAME-specific peptides in patients with acute lymphoblastic leukemia and acute and chronic myeloid leukemia

Preferentially expressed antigen of melanoma (PRAME) is aberrantly expressed in hematologic malignancies and may be a useful target for immunotherapy in leukemia. To determine whether PRAME is naturally immunogenic, we studied CD8(+) T-cell responses to 4 HLA-A*0201-restricted PRAME-derived epitopes (PRA100, PRA142, PRA300, PRA425) in HLA-A*0201-positive patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and healthy donors. CD8(+) T cells recognizing PRAME peptides could be detected ex vivo in 4 of 10 ALL, 6 of 10 AML, 3 of 10 CML patients, and 3 of 10 donors by HLA-A2 tetramer analysis and flow cytometry for intracellular interferon-gamma. The frequency of PRAME-specific CD8(+) T cells was greater in patients with AML, CML, and ALL than healthy controls. All peptides were immunogenic in patients, while responses were only detected to PRA300 in donors. High PRAME expression in patient peripheral blood mononuclear cells was associated with responses to greater than or equal to 2 PRAME epitopes compared with low PRAME expression levels (4/7 vs 0/23, P = .001), suggesting a PRAME-driven T-cell response. PRAME-specific T cells were readily expanded in short-term cultures in donors and patients. These results provide evidence for spontaneous T cell reactivity against multiple epitopes of PRAME in ALL, AML, and CML. The potential for developing PRAME as a target for immunotherapy in leukemia deserves further exploration.

Repeated PR1 and WT1 peptide vaccination in Montanide-adjuvant fails to induce sustained high-avidity, epitope-specific CD8+ T cells in myeloid malignancies

Background We previously showed that vaccination with one dose of PR1 and WT1 peptides induces transient anti-leukemia immunity. We hypothesized that maintenance of a sustained anti-leukemia response may require frequent boost injections. Design and Methods Eight patients with myeloid malignancies were enrolled in this phase II study, and 6 completed 6 injections of PR1 and WT1 peptides in Montanide-adjuvant with GM-CSF, every two weeks. Results Both high- and low-avidity PR1 or WT1-specific CD8+ T cells were detected in all evaluable patients after the first vaccine dose. Repeated vaccination led to selective deletion of high avidity PR1- and WT1-specific CD8+ T cells and was not associated with significant reduction in WT1-expression. Additional boosting failed to increase vaccine-induced CD8+ T-cell frequencies further and in all patients the response was lost before the 6th dose. PR1- or WT1-specific CD8+ T cells were not detected in bone marrow samples, excluding their preferential localization to this site. Following a booster injection three months after the 6th vaccine dose, no high-avidity PR1 or WT1-specific CD8+ T cells could be detected, whereas low-avidity T cells were readily expanded. Conclusions These data support the immunogenicity of PR1 and WT1 peptide vaccines. However, repeated delivery of peptides with Montanide-adjuvant and GM-CSF leads to rapid loss of high-avidity peptide-specific CD8+ T cells. These results may offer an explanation for the lack of correlation between immune and clinical responses observed in a number of clinical trials of peptide vaccination. New approaches are needed to induce long-term high-avidity memory responses against leukemia antigens. (ClinicalTrials.gov Identifier: NCT00499772)

Regulatory B cells are enriched within the IgM memory and transitional subsets in healthy donors but are deficient in chronic GVHD

A subset of regulatory B cells (Bregs) in mice negatively regulate T-cell immune responses through the secretion of regulatory cytokines such as IL-10 and direct cell-cell contact and have been linked to experimental models of autoimmunity, inflammation, and cancer. However, the regulatory function of Bregs in human disease is much less clear. Here we demonstrate that B cells with immunoregulatory properties are enriched within both the CD19(+)IgM(+)CD27(+) memory and CD19(+)CD24(hi)CD38(hi) transitional B-cell subsets in healthy human donors. Both subsets suppressed the proliferation and interferon-γ production of CD3/CD28-stimulated autologous CD4(+) T cells in a dose-dependent manner, and both relied on IL-10 secretion as well as cell-cell contact, likely mediated through CD80 and CD86, to support their full suppressive function. Moreover, after allogeneic stem cell transplantation, Bregs from patients with chronic graft-versus-host disease (cGVHD) were less frequent and less likely to produce IL-10 than were Bregs from healthy donors and patients without cGVHD. These findings suggest that Bregs may be involved in the pathogenesis of cGVHD and support future investigation of regulatory B cell-based therapy in the treatment of this disease.

Comparative Pharmacokinetics of Unbound Paclitaxel During 1- and 3-Hour Infusions

PURPOSE The paclitaxel vehicle Cremophor EL (CrEL) profoundly influences the cellular distribution of paclitaxel in human blood in vitro by a concentration-dependent decrease of the unbound drug fraction. Because CrEL clearance increases by extending the infusion duration from 3 to 24 hours, we hypothesized that exposure to unbound paclitaxel might also be schedule-dependent. PATIENTS AND METHODS CrEL and unbound paclitaxel pharmacokinetics were prospectively analyzed in 29 patients with advanced solid tumors treated with paclitaxel 100 mg/m(2) given as a 1-hour (n = 15) or 3-hour (n = 14) intravenous infusion. RESULTS The systemic exposure (area under the curve [AUC]) to CrEL was significantly higher with the 1-hour as compared with the 3-hour schedule (80.2 +/- 24.2 v. 48.5 +/- 24.1 microL x h/mL; P =.002). In contrast, the AUC of unbound paclitaxel was substantially reduced after the 1-hour infusion (0.50 +/- 0.10 v. 0.62 +/- 0.12 micromol/L x h; P =.009). Similarly, clearance and volume of distribution were significantly dependent on infusion duration (P <.005). A trend was observed toward more severe hematologic toxicity with the 3-hour schedule (P =.053), consistent with increased exposure to unbound drug. CONCLUSION Overall, these findings explain, at least in part, previous observations that short-infusion schedules of paclitaxel lack significant myelotoxicity, whereas potentially CrEL-related side effects, including peripheral neuropathy, are augmented.

Leukemia-induced phenotypic and functional defects in natural killer cells predict failure to achieve remission in acute myeloid leukemia.

The majority of patients with acute myeloid leukemia will relapse, and older patients often fail to achieve remission with induction chemotherapy. We explored the possibility that leukemic suppression of innate immunity might contribute to treatment failure. Natural killer cell phenotype and function was measured in 32 consecutive acute myeloid leukemia patients at presentation, including 12 achieving complete remission. Compared to 15 healthy age-matched controls, natural killer cells from acute myeloid leukemia patients were abnormal at presentation, with downregulation of the activating receptor NKp46 (P=0.007) and upregulation of the inhibitory receptor NKG2A (P=0.04). Natural killer cells from acute myeloid leukemia patients had impaired effector function against autologous blasts and K562 targets, with significantly reduced CD107a degranulation, TNF-α and IFN-γ production. Failure to achieve remission was associated with NKG2A overexpression and reduced TNF-α production. These phenotypic and functional abnormalities were partially restored in the 12 patients achieving remission. In vitro co-incubation of acute myeloid leukemia blasts with natural killer cells from healthy donors induced significant impairment in natural killer cell TNF-α and IFN-γ production (P=0.02 and P=0.01, respectively) against K562 targets and a trend to reduced CD107a degranulation (P=0.07). Under transwell conditions, the inhibitory effect of AML blasts on NK cytotoxicity and effector function was still present, and this inhibitory effect was primarily mediated by IL-10. These results suggest that acute myeloid leukemia blasts induce long-lasting changes in natural killer cells, impairing their effector function and reducing the competence of the innate immune system, favoring leukemia survival.

EBV-induced post transplant lymphoproliferative disorders: a persisting challenge in allogeneic hematopoetic SCT

EBV-induced post transplantation lymphoproliferative disorder (EBV-PTLD) is a life-threatening complication after allogeneic hematopoietic cell transplantation. Profound T-cell depletion of the allograft represents a major risk factor for EBV-PTLD. With regard to the increasing use of alternative stem cell sources such as cord blood or purified haploidentical stem cell grafts both associated with impaired immune reconstitution, the frequent occurrence of EBV-PTLD demands particular vigilance on laboratory changes and early symptoms. Here we have summarized today’s knowledge about EBV-PTLD in a comprehensive review explaining the underlying mechanisms of EBV-based transformation, EBV-PTLD development, clinical presentation, incidence, diagnosis, screening, therapy and prognosis. In this context, we emphasize on the necessity of regularly applied screening tools and pre-emptive treatment strategies including anti-CD20 Abs particularly in high-risk patients to avoid disease progression to malignant lymphoma. Although EBV-PTLD has always been associated with a high mortality rate, novel immunotherapeutic approaches such as the transfer of EBV-specific T cells nowadays offer improved chances of disease control even at late stages.