Dynamic changes in the clonal structure of MDS and AML in response to epigenetic therapy.

Traditional response criteria in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are based on bone marrow morphology and may not accurately reflect clonal tumor burden in patients treated with non-cytotoxic chemotherapy. We used next-generation sequencing of serial bone marrow samples to monitor MDS and AML tumor burden during treatment with epigenetic therapy (decitabine and panobinostat). Serial bone marrow samples (and skin as a source of normal DNA) from 25 MDS and AML patients were sequenced (exome or 285 gene panel). We observed that responders, including those in complete remission (CR), can have persistent measurable tumor burden (that is, mutations) for at least 1 year without disease progression. Using an ultrasensitive sequencing approach, we detected extremely rare mutations (equivalent to 1 heterozygous mutant cell in 2000 non-mutant cells) months to years before their expansion at disease relapse. While patients can live with persistent clonal hematopoiesis in a CR or stable disease, ultimately we find evidence that expansion of a rare subclone occurs at relapse or progression. Here we demonstrate that sequencing of serial samples provides an alternative measure of tumor burden in MDS or AML patients and augments traditional response criteria that rely on bone marrow blast percentage.

Contribution of chemotherapy mobilization to disease control in multiple myeloma treated with autologous hematopoietic cell transplantation.

In patients with multiple myeloma (MM) undergoing autologous hematopoietic cell transplantation (auto-HCT), peripheral blood progenitor cells may be collected following mobilization with growth factor alone (GF) or cytotoxic chemotherapy plus GF (CC+GF). It is uncertain whether the method of mobilization affects post-transplant outcomes. We compared these mobilization strategies in a retrospective analysis of 968 patients with MM from the Center for International Blood and Marrow Transplant Research database who received an auto-HCT in the US and Canada between 2007 and 2012. The kinetics of neutrophil engraftment (⩾0.5 × 10(9)/L) was similar between groups (13 vs 13 days, P=0.69) while platelet engraftment (⩾20 × 10(9)/L) was slightly faster with CC+GF (19 vs 18 days, P=0.006). Adjusted 3-year PFS was 43% (95% confidence interval (CI) 38-48) in GF and 40% (95% CI 35-45) in CC+GF, P=0.33. Adjusted 3-year OS was 82% (95% CI 78-86) vs 80% (95% CI 75-84), P=0.43 and adjusted 5-year OS was 62% (95% CI 54-68) vs 60% (95% CI 52-67), P=0.76, for GF and CC+GF, respectively. We conclude that MM patients undergoing auto-HCT have similar outcomes irrespective of the method of mobilization and found no evidence that the addition of chemotherapy to mobilization contributes to disease control.

Allogeneic hematopoietic cell transplant for AML: no impact of pre-transplant extramedullary disease on outcome.

The impact of extramedullary disease (EMD) in AML on the outcomes of allogeneic hematopoietic cell transplantation (alloHCT) is unknown. Using data from the Center for International Blood and Marrow Transplant Research, we compared the outcomes of patients who had EMD of AML at any time before transplant, with a cohort of AML patients without EMD. We reviewed data from 9797 AML patients including 814 with EMD from 310 reporting centers and 44 different countries, who underwent alloHCT between and 1995 and 2010. The primary outcome was overall survival (OS) after alloHCT. Secondary outcomes included leukemia-free survival (LFS), relapse rate and treatment-related mortality (TRM). In a multivariate analysis, the presence of EMD did not affect either OS (hazard ratio 1.00, 95% confidence interval (CI) 0.91-1.09), LFS (0.98, 0.89-1.09), TRM (relative risk 0.92, 95% CI 0.80-1.16, P=0.23) or relapse (relative risk=1.03, 95% CI, 0.92-1.16; P=0.62). Furthermore, the outcome of patients with EMD was not influenced by the location, timing of EMD, or intensity of conditioning regimen. The presence of EMD in AML does not affect transplant outcomes and should not be viewed as an independent adverse prognostic feature.

A phase II study of V-BEAM as conditioning regimen before second auto-SCT for multiple myeloma.

High-dose melphalan has been the standard conditioning regimen for auto-SCT in multiple myeloma (MM) for decades. A more effective conditioning regimen may induce deeper responses and longer remission duration. It is especially needed in the setting of second auto-SCT, which rarely achieves comparable results with the first auto-SCT using the same conditioning regimen. Here we conducted a phase II study to investigate the efficacy and safety of a conditioning regimen V-BEAM (bortezomib-BEAM) before second auto-SCT for multiple myeloma. Ten patients were enrolled from September 2012 to May 2013. The CR rate at day +100 after auto-SCT was 75%; all except for one patient remained in remission after a median follow-up of 6 months. Three patients developed Clostridium difficile infection. Two patients died within the first 30 days of auto-SCT from neutropenic colitis and overwhelming sepsis, respectively. Due to the high rate of morbidity and mortality, the study was terminated after 10 patients. In summary, although the conditioning regimen V-BEAM before second auto-SCT for MM provided promising responses, it was associated with unexpected treatment-related toxicity and should not be investigated further without modifications.

Oral valganciclovir versus ganciclovir as delayed pre-emptive therapy for patients after allogeneic hematopoietic stem cell transplant: a pilot trial (04-0274) and review of the literature.

BACKGROUND: Cytomegalovirus (CMV) infection is an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplant (HSCT). This pilot prospective randomized clinical trial compares valganciclovir (VGV) to ganciclovir (GCV) as pre-emptive therapy for CMV viremia in the post-allogeneic HSCT population. METHODS: Patients undergoing allogeneic HSCT who were at risk for CMV viremia were monitored post HSCT by weekly quantitative whole blood polymerase chain reaction. Pre-emptive therapy was delayed until the viral load (VL) was >10,000 copies/mL once, or >5000 copies/mL twice. Patients were randomized to either GCV 5 mg/kg twice a day (b.i.d.) for 7 days followed by daily GCV 5 mg/kg for up to 21 days, or VGV 900 mg b.i.d. for 7 days followed by 900 mg daily for up to 21 days. The primary endpoint was clearance of viremia (VL <5000 copies/mL) within 28 days of initiation of therapy. RESULT: In total, 37 patients were enrolled; 19 patients received treatment with VGV and 18 patients received treatment with GCV. The VGV was not inferior in efficacy to GCV as pre-emptive therapy, with rates of viral clearance at 28 days of 89.5% and 83%, respectively (P-value for non-inferiority = 0.030). Toxicities were similar between the 2 arms. No patients developed CMV disease. CONCLUSIONS: In this trial, the rates of clearance of viremia appear to be similar with VGV and GCV.

Allo-SCT conditioning for myelodysplastic syndrome and acute myeloid leukemia with clofarabine, cytarabine and ATG.

The application of myeloablative Allo-SCT is limited by its associated morbidity and mortality. Reduced-intensity conditioning regimens attempt to diminish these, but are associated with a higher risk of disease relapse. Given the evidence of activity of clofarabine and cytarabine in myelodysplastic syndrome/acute myeloid leukemia (MDS/AML), we explored a novel reduced-intensity conditioning regimen based on this backbone. Patients received clofarabine 40 mg/m(2) i.v. on days -6 to -2, cytarabine 1 g/m(2) i.v. on days -6 to -2 and anti-thymocyte globulin (ATG) 1 mg/kg on day -4 and 2.5 mg/kg x 2 days on days -3 and -2. Seven patients were enrolled. Their median age was 54 years; three were with MDS and four with AML. The median duration of neutropenia was 14 days and that of thrombocytopenia was 22 days. Toxicities included hand-foot syndrome (57% grade 2), elevated alanine aminotransferase (ALT) (57% grade 3), elevated aspartate aminotransferase (AST) (86% grade 3) and hyperbilirubinemia (29% grade 3-5). No acute GVHD was observed. Enrollment to the trial was halted after three of the first seven patients expired on days +15, +26 and +32. Three of the four surviving patients have relapsed with a median TTP of 152 days. This regimen was not sufficiently immunosuppressive to ensure engraftment, and was associated with substantial morbidity and mortality.

Bortezomib administered pre-auto-SCT and as maintenance therapy post transplant for multiple myeloma: a single institution phase II study.

The appropriate induction therapy before and the role of maintenance therapy after auto-SCT for patients with multiple myeloma remain areas of active investigation. We conducted a study in 40 patients with bortezomib given sequentially pre-auto-SCT and as maintenance therapy post auto-SCT. Pre-transplant bortezomib was administered for two cycles followed by high-dose melphalan 200 mg/m(2) with auto-SCT of G-CSF-mobilized PBMCs. Post transplant bortezomib was administered weekly for 5 out of 6 weeks for six cycles. No adverse effects were observed on stem cell mobilization or engraftment. An overall response rate of 83% with a CR+very good partial remission (VGPR) of 50% was observed with this approach. Three-year Kaplan-Meier estimates of disease-free survival and overall survival (OS) were 38.2 and 63.1%, respectively. Bortezomib reduced CD8(+) cytotoxic T cell and CD56(+) natural killer cell PBL subsets and was clinically associated with high rates of viral reactivation to varicella zoster.

Localization of human Cdc25C is regulated both by nuclear export and 14-3-3 protein binding.

Entry into mitosis requires activation of the Cdc2 protein kinase by the Cdc25C protein phosphatase. The interactions between Cdc2 and Cdc25C are negatively regulated throughout interphase and in response to G2 checkpoint activation. This is accomplished in part by maintaining the Cdc25 phosphatase in a phosphorylated form that binds 14-3-3 proteins. Here we report that 14-3-3 binding regulates the intracellular trafficking of Cdc25C. Although primarily cytoplasmic, Cdc25C accumulated in the nuclei of leptomycin B (LMB)-treated cells, indicating that Cdc25C is actively exported out of the nucleus. A mutant of Cdc25C that is unable to bind 14-3-3 was partially nuclear in the absence of LMB and its nuclear accumulation was greatly enhanced by LMB-treatment. A nuclear export signal (NES) was identified within the amino terminus of Cdc25C. Although mutation of the NES did not effect 14-3-3 binding, it did cause nuclear accumulation of Cdc25C. These results demonstrate that 14-3-3 binding is dispensable for the nuclear export of Cdc25C. However, complete nuclear accumulation of Cdc25C required loss of both NES function and 14-3-3 binding and this was accomplished both pharmacologically and by mutation. These findings suggest that the nuclear export of Cdc25C is mediated by an intrinsic NES and that 14-3-3 binding negatively regulates nuclear import.

The Drosophila grapes gene is related to checkpoint gene chk1/rad27 and is required for late syncytial division fidelity.

BACKGROUND: Cell cycle checkpoints maintain the fidelity of the somatic cell cycle by ensuring that one step in the cell cycle is not initiated until a previous step has been completed. The extent to which cell cycle checkpoints play a role in the initial rapid embryonic divisions of higher eukaryotes is unclear. The initial syncytial divisions of Drosophila embryogenesis provide an excellent opportunity to address this issue as they are amenable to both genetic and cellular analysis. In order to study the relevance of cell cycle checkpoints in early Drosophila embryogenesis, we have characterized the maternal-effect grapes (grp) mutation, which may affect feedback control during early syncytial divisions. RESULTS: The Drosophila grp gene encodes a predicted serine/threonine kinase and has significant homology to chk1/rad27, a gene required for a DNA damage checkpoint in Schizosaccharomyces pombe. Relative to normal embryos, embryos derived from grp-mutant mothers exhibit elevated levels of DNA damage. During nuclear cycles 12 and 13, alignment of the chromosomes on the metaphase plate was disrupted in grp-derived embryos, and the embryos underwent a progression of cytological events that were indistinguishable from those observed in normal syncytial embryos exposed to X-irradiation. The mutant embryos also failed to progress through a regulatory transition in Cdc2 activity that normally occurs during interphase of nuclear cycle 14. CONCLUSION: We propose that the primary defect in grp-derived embryos is a failure to replicate or repair DNA completely before mitotic entry during the late syncytial divisions. This suggests that wild-type grp functions in a developmentally regulated DNA replication/damage checkpoint operating during the late syncytial divisions. These results are discussed with respect to the proposed function of the chk1/rad27 gene.