Mutations of the NPM1 gene (NPM1mut) are among the most common genetic alterations in acute myeloid leukemia and are suitable for minimal residual disease detection. We retrospectively investigated ...the prognostic impact of NPM1mut-based minimal residual disease detection from bone marrow for development of relapse by using a newly developed real-time polymerase chain reaction based on locked nucleic acid–containing primers in 174 patients, 155 of whom were treated within prospective protocols. The prognostic value of 5 cutoff values after completion of treatment or after allogeneic transplantation was studied by using cause-specific hazard models. Subsequent validation using cross-validated partial likelihood analysis revealed that an increase of more than 1% NPM1mut/ABL1 was most prognostic for relapse after chemotherapy, whereas an increase of more than 10% NPM1mut/ABL1 was most prognostic for relapse after allogeneic transplantation. Univariate and multivariate analysis of disease-free survival and overall survival revealed a significantly worse outcome in patients with >1% NPM1mut/ABL1 and >10% NPM1mut/ABL1, respectively, which remained significant after adjustment for FLT3–internal tandem duplication status. Our results in a large data set define and optimize cutoff values for early diagnosis of molecular relapse. These results may be especially important for defining triggers for early therapeutic intervention.
• NPM1 RT-PCR levels >1% are associated with poor overall and disease-free survival in AML patients treated with chemotherapy.• NPM1 MRD levels >10% are associated with poor overall and disease-free survival in AML patients after allogeneic transplantation.
Abstract Background aims Mouse models indicate that adoptive transfer of regulatory T cells (Treg) may suppress graft-versus-host-disease (GvHD) while preserving graft-versus-leukemia reactions. We ...aimed to develop a protocol for the efficient isolation and in vitro expansion of donor-derived Treg and to establish the proof-of-concept for the clinical application of ex vivo –generated Treg preparations in five patients with otherwise treatment-refractory chronic GvHD (cGvHD). Methods Allogeneic Treg were isolated from unstimulated leukapheresis products of the corresponding human leukocyte antigen–matched donors by use of clinical-grade magnetic-activated bead sorting. To increase the amount and purity, Treg were cultivated for 7–12 days and infused after a median time of 35 months after allogeneic hematopoietic cell transplantation. Results Final products contained Treg with a median purity of 84.1% CD4+ CD25high CD127low FOXP3+ of CD45+ cells and a mean quantity of 2.4 × 106 Treg per kg body wt. All isolated cell products showed in vitro suppressive activity. On transfusion, two of five patients showed a clinical response with improvement of cGvHD symptoms. The other three patients showed stable cGvHD symptoms for up to 21 months. In four of five patients, increased counts of Treg were detectable on Treg transfusion, immunosuppressive treatment could be reduced and suppression of CD69 activation marker expression on T-effector cells was observed. However, one patient had development of malignant melanoma and another patient had Bowen skin cancer 4 months and 11 months after Treg transfusion, respectively. Conclusions We demonstrate a feasible and reproducible approach of isolating functional Treg in high quantity and purity for clinical application and show opportunities and risks of adoptive Treg transfer into patients with cGvHD.
Abstract Quantitative real-time PCR (qPCR) has been proposed as a highly sensitive method for monitoring hematopoietic chimerism and may serve as a surrogate marker for the detection of minimal ...residual disease minimal residual disease in myelodysplastic syndrome (MDS), until specific methods of detection become available. Because a systematic comparison of the clinical utility of qPCR with the gold standard short tandem repeat (STR)-PCR has not been reported, we retrospectively measured chimerism by qPCR in 54 children transplanted for MDS in a previous study. Results obtained by STR-PCR in the initial study served as comparison. Because the detection limit of qPCR was sufficiently low to detect an autologous background, we defined the sample as mixed chimera if the proportion of recipient-derived cells exceeded .5%. The true positive rates were 100% versus 80% (qPCR versus STR-PCR, not significant), and mixed chimerism in most cases was detected earlier by qPCR than by STR-PCR (median, 31 days) when chimerism was quantified concurrently in peripheral blood and bone marrow. Both methods revealed a substantial rate of false positives (22.7% versus 13.6%, not significant), indicating the importance of serial testing of chimerism to monitor its progression. Finally, we propose criteria for monitoring chimerism in pediatric MDS with regard to the subtypes, specimens, PCR method, and timing of sampling.
Summary
Both immunosuppressive and cytoreductive effects of γ‐irradiation contribute to engraftment of allogeneic haematopoietic stem and progenitor cells. We hypothesized that a release of host stem ...and progenitor cells from the niche prior to conditioning would permit engraftment after less intensive conditioning. Administration of AMD3100 and SEW2871 on days −4 to −2 followed by irradiation on day −1 in a non‐myeloablative zebrafish transplant model resulted in a reduced radiation minimum dose of 10 Gy from 15 Gy being sufficient for engraftment. Targeting the SDF‐1 (CXCL12)/CXCR4‐ and S1P/S1P1‐axis increased the efficacy of allografting in an experimental transplant model.
Hematopoietic stem and progenitor cells (HSPCs) generate all cell types of the blood and are crucial for homeostasis of all blood lineages in vertebrates. Hematopoietic stem cell transplantation ...(HSCT) is a rapidly evolving technique that offers potential cure for hematologic cancers, such as leukemia or lymphoma. HSCT may be autologous or allogenic. Successful HSCT depends critically on the abundance of engraftment-competent HSPCs, which are currently difficult to obtain in large numbers. Therefore, finding compounds that enhance either the number or the activity of HSPCs could improve prognosis for patients undergoing HSCT and is of great clinical interest. We developed a semiautomated screening method for whole zebrafish larvae using conventional liquid handling equipment and confocal microscopy. Applying this pipeline, we screened 550 compounds in triplicate for proliferation of HSPCs in vivo and identified several modulators of hematopoietic stem cell activity. One identified hit was valproic acid (VPA), which was further validated as a compound that expands and maintains the population of HSPCs isolated from human peripheral blood ex vivo. In summary, our in vivo zebrafish imaging screen identified several potential drug candidates with clinical relevance and could easily be further expanded to screen more compounds.
The identification of small molecules that either increase the number and/or enhance the activity of human hematopoietic stem and progenitor cells (hHSPCs) during ex vivo expansion remains ...challenging. We used an unbiased in vivo chemical screen in a transgenic (c-myb:EGFP) zebrafish embryo model and identified histone deacetylase inhibitors (HDACIs), particularly valproic acid (VPA), as significant enhancers of the number of phenotypic HSPCs, both in vivo and during ex vivo expansion. The long-term functionality of these expanded hHSPCs was verified in a xenotransplantation model with NSG mice. Interestingly, VPA increased CD34
cell adhesion to primary mesenchymal stromal cells and reduced their in vitro chemokine-mediated migration capacity. In line with this, VPA-treated human CD34
cells showed reduced homing and early engraftment in a xenograft transplant model, but retained their long-term engraftment potential in vivo, and maintained their differentiation ability both in vitro and in vivo. In summary, our data demonstrate that certain HDACIs lead to a net expansion of hHSPCs with retained long-term engraftment potential and could be further explored as candidate compounds to amplify ex-vivo engineered peripheral blood stem cells.
Summary
Both immunosuppressive and cytoreductive effects of γ‐irradiation contribute to engraftment of allogeneic haematopoietic stem and progenitor cells. We hypothesized that a release of host stem ...and progenitor cells from the niche prior to conditioning would permit engraftment after less intensive conditioning. Administration of
AMD
3100 and
SEW
2871 on days −4 to −2 followed by irradiation on day −1 in a non‐myeloablative zebrafish transplant model resulted in a reduced radiation minimum dose of 10 Gy from 15 Gy being sufficient for engraftment. Targeting the
SDF
‐1 (
CXCL
12)/
CXCR
4‐ and S1P/S1P
1
‐axis increased the efficacy of allografting in an experimental transplant model.
Scleromyxedema is a rare disorder of connective tissue with unknown etiology. Its manifestation includes a generalized mucin deposition, which is frequently associated with paraproteinemia. The ...course of scleromyxedema is progressive and often lethal. As a result of its poorly understood pathogenesis, there is no causative treatment option. The efficacy of cytoreductive agents and autologous stem cell transplantation has been reported, but so far allografting as a treatment option has not yet been documented. Herein, we report on a patient with severe neurologic involvement and refractory course attaining durable remission after receiving an allogeneic hematopoietic cell transplant from an human leukocyte antigen-matched sibling. This case not only illustrates a potential new treatment option for selected patients, but also provides insights into the pathogenesis of this rare disease.
Abstract 4866
The first pandemic of the 21st century was caused by the novel influenza A (H1N1) virus, now known as pandemic H1N1-09 virus. Its first wave reached Germany in autumn 2009. Though for ...the German public health sector this pandemic was a challenge, a state of national emergency could not be recognized. A popular current believe is that the threat by this pandemic had been overestimated by national and international health care experts. Here, we report our experience with the pandemic H1N1-09 virus in hematologic patients when the first wave hit Germany between December 2009 and February 2010.
Viral diagnostic for all patients was performed in a central virologic laboratory. All patients with at least one sample tested positive by PCR for H1N1-09 between October 2009 and March 2010 were included into the analysis. Samples were obtained by nasal wash, nasopharyngeal swab or broncho-alveolary lavage. The medical charts of all patients with H1N1-09 infection were reviewed systematically.
15 patients with underlying hematologic diseases (10 male and 5 female) with a median age of 52 years (range 18–70 years) were tested positive for H1N1-09 virus by PCR in our department. Notably, in 12 Patients H1N1-09 was nosocomially acquired after a median of 16 days hospitalization (range 6 – 42 days). 13 patients (87%) got infected between December 2 and December 29. At the time of infection all patients were immunocompromised with 11 patients being cytopenic after chemotherapy and four patients after allogeneic hematopoietic cell transplantation (HCT) among the latter two patients were in aplasia after allogeneic HCT (day +5 and day +11 after HCT). CT scan was performed in 8 patients at the time of diagnosis. Seven patients presented with signs of atypical pneumonia on CT scan. Ground glass opacity, consolidation, airway wall thickening, airway dilatation, pleural effusion and lymphadenopathy were common findings.
In 10 patients viral clearance was monitored by RT-PCR. The median duration of viral shedding was 10 days (range 4 – 41 days). Prolonged viral persistence was associated with severe lung injury. All patients received Oseltamivir as first-line therapy, except three patients, who died prior to the confirmation of the diagnosis by RT-PCR. Simultaneously broad spectrum antibiotics and antimycotics were administered.
5 patients (33%) with respiratory failure needed invasive mechanical ventilation (MV) at the time of the H1N1-09 infection. Three out of these patients died.
Within a follow up of 6 months eight patients have died. Six patients (40%) have died from the infection. Among these three patients died from fulminant pulmonary failure whereas three patients died several weeks after H1N1-09 infection from subsequent respiratory or multiorgan failure. The impact on indirect mortality cannot yet be fully assessed, since in some patients the infection caused significant delay of anti-leukemic therapy and acquired comorbidities resulted in dose-reductions of chemotherapy.
In contrast to largely mild infections in the healthy German population pandemic H1N1-09 pneumonia represented a life-threatening infection for hematologic patients associated with a high mortality due to acute respiratory failure, late pulmonary complications and delay of antitumor treatment.
One alarming finding was the frequency of nosocomial infections. This observation points to the possibility of transmission of the virus from patient to patient, visitors to patient or even from medical staff to patient. With this observation in our institution patients admitted to hospital were put under quarantine before they were allowed to be accommodated in a double room in the hematologic unit. Transmission of the virus from asymptomatic staff or visitors to patient is another major concern. The suspected vaccination rate of medical staff in Germany was less than 20%. Especially, when asymptomatic or mild H1N1 infections occur - as it was the case in Germany – the medical staff and visitors could become important vectors of infection. The most effective measure against this threat is active immunization of visitors prior to patient contact and medical staff in hematologic units.
No relevant conflicts of interest to declare.
Infections and acute graft-versus-host disease (GvHD) represent major complications of allogeneic stem-cell transplantation (SCT). Dendritic cells (DCs) display an extraordinary capacity to induce ...innate and adaptive immune responses. Therefore, they play a crucial role in the elimination of pathogens and in the pathogenesis of acute GvHD. 6-Sulfo LacNAc DCs (slanDCs) are a major subpopulation of human blood DCs with a high proinflammatory capacity. We investigated for the first time the reconstitution of slanDCs in the blood of patients after SCT and the modulation of their frequency by bacterial infection, cytomegalovirus (CMV) reactivation, and acute GvHD.
The frequency of slanDCs, CD1c myeloid DCs (mDCs), and plasmacytoid DCs (pDCs) in the peripheral blood was quantified by flow cytometry in 80 patients after SCT. To assess individual DC subsets, we used pregating of the HLADRLin subset and antibodies against slanDCs, blood DC antigen 1 (CD1c mDCs), and blood DC antigen 2 (pDCs).
SlanDCs showed the slowest reconstitution in the first month after SCT compared with CD1c mDCs and pDCs. Interestingly, in the second and third months after SCT, their percentage steadily increased, and slanDCs were the most abundant DC subset. In addition, we observed a markedly reduced frequency of slanDCs in the blood of patients with bacterial infection, CMV reactivation, or severe acute GvHD. Furthermore, slanDCs showed the most prominent reduction after steroid treatment of acute GvHD.
These results indicate that SCT-associated complications such as bacterial infection, CMV reactivation, and acute GvHD can significantly modulate the frequency of slanDCs.
PDF
