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    • br Materials and methods The study was designed

    2019-05-22


    Materials and methods The study was designed as an open label, single institution phase I study at the National Heart, Lung, Blood Institute, National Institutes of Health and was approved by Institutional Review Board (NCT01629082). All patients signed an informed consent prior to enrollment and the study was conducted in compliance with the Declaration of Helsinki. The eligibility criteria included patients with a diagnosis of high risk MDS, CMML, and AML who were not candidates for standard intensive chemotherapy or allo-SCT and had failed at least one prior therapy. Subjects received a single course of intravenous low-dose clofarabine 5mg/m2/day for five days. At 28 days after induction therapy, oral lenalidomide therapy was initiated with dose escalation from 25mg to 50mg daily for 21 days of 28 days for up to 12 cycles. Response assessment was performed according to International Working Group (IWG) response criteria [6]. Detailed study design was described in Supplementary material. Flow cytometric analysis was performed to characterize T cell and natural killer (NK) cell subsets, with functional markers of T cell exhaustion and activating and inhibitory NK cell receptors. Antibodies used in the panel are listed in Supplementary Table 1. Relative changes in RNA expressions of various Dihydromyricetin related to cancer immunology were evaluated by custom made 384 well PrimePCR™ Assay Panels for Real-Time PCR (Bio-Rad Laboratories, Hercules, CA, USA).
    Results Four subjects with relapsed or refractory high-risk MDS (IPSS risk score>intermediate 2) or AML were enrolled. Clinical and hematological characteristics are summarized in Table 1. Two subjects achieved stable disease with lenalidomide maintenance for 4–6 months, but were taken off study on days 162 and 206 respectively due to progression of disease. One subject had a partial response in the erythroid lineage after clofarabine induction. This subject was taken off study on day 69 due to asymptomatic transient grade 3 liver toxicity during lenalidomide therapy. One subject had disease progression during lenalidomide therapy and was taken off study on day 70. The clinical trial is now closed without dose escalation beyond the first cohort for reasons of poor accrual and lack of durable response. In 3 subjects with partial response (PR) and stable disease (SD), Clofarabine reduced the count of all lineages of lymphocytes, T cells (CD4 and CD8), NK cells, and B cells. However, in the subject with refractory disease, CD4, CD8, and NK cells increased after clofarabine treatment (Fig. 1). T cell subset analysis demonstrated shift in memory subsets after clofarabine. Fractions of central memory (CM) T cells in CD4 cells decreased in responders (0.61 fold change) and increased in partial responder and non-responder (1.22 fold change). Fractions of effector memory (EM) decreased in all but one subject (stable responder). Fractions of naive cells in both CD4 and CD8 T cells increased in all subjects following clofarabine treatment. Before treatment, the exhaustion markers PD1, LAG3, and TIM3 in peripheral blood were moderately increased in both CD4 and CD8 T cells in comparison to healthy volunteers. After clofarabine, absolute numbers of PD1+ CD4 cells decreased in the subjects with stable disease (UPN1 and UPN2) and partial response (UPN3). Absolute numbers of PD1+ CD4 cells were increased in UPN4 with refractory disease. Similar changes were seen in the exhaustion markers LAG3 and TIM3. FoxP3+CD25+ regulatory T (Tregs) cells were largely unchanged but rose in UPN4 (Supplementary Figure 1). Thus the failure of lymphodepletion and rise of exhaustion markers and Tregs following clofarabine correlated with treatment failure. In NK cells, CD57+ and LIR1+ populations fell among the subjects with stable disease and partial response, while NK cells in the subjects with refractory disease increased their CD56dim population expressing CD57 and LIR1 (Fig. 2). These findings indicate restoration of NK cell repertoires after clofarabine and a switch to a less inhibitory NK profile with response or stable disease. The CD4 and NK cell immune profiles in two subjects (UPN1 and UPN2) demonstrated dynamic immune reconstitutions in the subsequent lenalidomide therapy (Fig. 3). Targeted RNA expression profiles in UPN 2 were analyzed and significant up-regulations of genes were observed in IL-15, IFNγ, CSF1, IL7R, and IL-2 after lenalidomide therapy (Supplementary Fig. 2). This finding indicates that lenalidomide induced expressions of genes related to cytotoxic T cell and NK cell activation.