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Allogeneic hematopoietic cell transplantation (alloHCT) as a postremission therapy in patients with FLT3-ITD-positive intermediate-risk acute myeloid leukemia (AML) remains controversial. FLT3-ITD mutations are heterogeneous with respect to allelic ratio, location, and length of the insertion, with a high mutant-to-wild-type ratio consistently associated with inferior prognosis. We retrospectively analyzed the role of alloHCT in first remission in relationship to the allelic ratio and presence or absence of nucleophosmin 1 mutations (NPM1) in the Study Alliance Leukemia AML2003 trial. FLT3-ITD mutations were detected in 209 patients and concomitant NPM1 mutations in 148 patients. Applying a predefined cutoff ratio of .8, AML was grouped into high- and low-ratio FLT3-ITD AML (HR(FLT3-ITD) and LR(FLT3-ITD)). Sixty-one patients (29%) were transplanted in first remission. Overall survival (OS) (HR, .3; 95% CI, .16 to .7; P = .004) and event-free survival (EFS) (HR, .4; 95% CI, .16 to .9; P = .02) were significantly increased in patients with HR(FLT3-ITD) AML who received alloHCT as consolidation treatment compared with patients who received consolidation chemotherapy. Patients with LR(FLT3-ITD) AML and wild-type NPM1 who received alloHCT in first remission had increased OS (HR, .3; 95% CI, .1 to .8; P = .02) and EFS (HR, .2; 95% CI, .1 to .8; P = .02), whereas alloHCT in first remission did not have a significant impact on OS and EFS in patients with LR(FLT3-ITD) AML and concomitant NPM1 mutation. In conclusion, our results provide additional evidence that alloHCT in first remission improves EFS and OS in patients with HR(FLT3-ITD) AML and in patients with LR(FLT3-ITD) AML and wild-type NPM1., (Copyright © 2016 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Determination of donor chimerism profiles in blood or bone marrow from patients with allogeneic stem cell transplantation (SCT) is useful for monitoring engraftment or predicting relapse, when specific molecular markers are lacking. CD34+ donor cell chimerism (DCC) analysis in peripheral blood samples from CD34+ acute myeloid leukemia (AML) and myleodysplastic syndrome (MDS) patients proved to be a highly sensitive diagnostic tool that is useful to detect imminent relapse significantly earlier compared to total white blood cell donor cell chimerism monitoring. However, flow-cytometric enrichment of CD34+ cells requires high efforts to human resources and equipment. We present a novel semi-automated CD34+ DCC analysis procedure-employing a magnetic cell-enrichment device, DNA extraction, and short tandem repeat profiling-without the need for flow-cytometric cell sorting. Monitoring 85 patients with AML and MDS over a period of 4 years 24 relapses were detected. Semi-automated peripheral blood CD34+ DCC was diminished below 80 % in all cases of systemic relapse. Significant decrease of the CD34+ DCC value was detected 29-42 days before overt cytological relapse. Our method provides a rapid and sensitive tool for monitoring AML and MDS patients after allogeneic SCT with regard to engraftment and early detection of relapse. Here, we propose a novel semi-automated procedure for CD34+ DCC analysis after allogeneic SCT that is simple, reliable, and therefore applicable in all hematologic laboratories.

Background: 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., Methods: 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)., Results: 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., Conclusions: These results indicate that SCT-associated complications such as bacterial infection, CMV reactivation, and acute GvHD can significantly modulate the frequency of slanDCs.

Allogeneic stem cell transplantation (ASCT) after reduced-intensity conditioning has become a reasonable treatment option for patients with advanced myelofibrosis. The role of characteristic molecular genetic abnormalities, such as JAK2V617F on outcome of ASCT, is not yet elucidated. In 139 of 162 myelofibrosis patients with known JAK2V617F mutation status who received ASCT after reduced-intensity conditioning, the impact of JAK2 genotype, JAK2V617F allele burden, and clearance of mutation after ASCT was evaluated. Overall survival was significantly reduced in multivariate analysis in patients harboring JAK2 wild-type (hazard ratio = 2.14, P = .01) compared with JAK2 mutated patients. No significant influence on outcome was noted for the mutated allele burden analyzed either as continuous variable or after dividing into quartiles. Achievement of JAK2V617F negativity after ASCT was significantly associated with a decreased incidence of relapse (hazard ratio = 0.22, P = .04). In a landmark analysis, patients who cleared JAK2 mutation level in peripheral blood 6 months after ASCT had a significant lower risk of relapse (5% vs 35%, P = .03). We conclude that JAK2V617F-mutated status, but not allele frequency, resulted in an improved survival and rapid clearance after allografting reduces the risk of relapse.

Mesenchymal stem cells (MSC) obtained from human bone marrow have been described as adult stem cells with the ability of extensive self-renewal and clonal expansion, as well as the capacity to differentiate into various tissue types and to modulate the immune system. Some data indicate that leukapheresis products may also contain non-hematopoietic stem cells, as they occur in whole bone marrow transplantation (BMT). However, there is still controversy whether MSC expand in the host after transplantation like blood progenitor cells do. Therefore, we were interested in finding out if graft MSC can be detected in leukapheresis products and in bone marrow after BMT and peripheral blood stem cell transplantation (PBSCT). Every sample from total bone marrow transplants exhibited growth of MSC after in vitro culture, but not one of nine leukapheresis products did. In addition, bone marrow aspirates of 9 patients receiving BMT and of 18 patients after PBSCT were examined for origin of MSC. Almost all MSC samples exhibited a complete host profile, whereas peripheral blood cells were of donor origin. We conclude that even if trace amounts of MSC are co-transplanted during PBSCT or BMT, they do not expand significantly in the host bone marrow.

Analysis of chimerism after allogeneic hematopoietic cell transplantation is important for assessing engraftment and the early detection of graft failure. In addition, the monitoring of minimal residual disease and early detection of imminent relapse has also become an important issue. Novel transplant procedures, for example dose-reduced conditioning protocols, rely on chimerism analysis to guide intervention, i.e. the reduction of immunosuppression or infusion of donor lymphocytes. During the last 30 years, several methods for the analysis of chimerism after hematopoietic cell transplantation have been published. Currently, fluorescent in situ hybridization (XY-FISH) analysis of sex chromosomes after transplantation from a sex-mismatched donor or analysis of polymorphic DNA sequences, i.e. short tandem repeats (STR) or variable number of tandem repeats (VNTR), are the most widely used procedures used in the assessment of chimerism. Two major diagnostic fields can be defined for chimerism analysis: the period of engraftment and the detection of minimal residual disease. Although STR-PCR and FISH analysis are very useful in the diagnosis of engraftment and graft failure, they are only of limited use in the monitoring of minimal residual disease, largely because of its limited level of sensitivity (1-5% for the minor population). Several novel procedures to improve this level of detection have been reported in recent years. One focus has been the use of real-time PCR techniques based on analysis of the Y-chromosome or, more recently, single nucleotide polymorphism (SNPs). These procedures combine quantitative analysis with high sensitivity (10(-4) to 10(-6)), and hold great potential for the future. In addition, the combination of cell sorting based on leukemia-specific immunophenotype and STR-PCR has been successfully used for minimal residual disease detection. First clinical data using these procedures indicate that intervention (e.g. the reduction of immunosuppression or donor lymphocyte infusion) may be effective in the minimal residual disease situation, even in high risk diseases like acute myeloid leukemia and acute lymphoblastic leukemia. The optimal timing of these diagnostic interventions is a critical issue and has to be further optimized. Whether this will ultimately improve the survival of patients with leukemia after transplantation has to be shown in prospective studies.

It is hypothesised that an effective graft-vs.-leukaemia reaction contributes substantially to the therapeutic effect of reduced intensity conditioning stem cell transplantation in chronic myeloid leukaemia. However, kinetic data on eradication of leukaemia cells and stem cell engraftement which could support this assumption are lacking. Thus, we investigated bcr/abl fusion transcripts and haematopoietic chimerism in 14 patients undergoing such a transplantation protocol. Ten of them obtained a complete molecular remission, and two patients achieved haematologic remissions but remained bcr/abl positive. Weekly determinations of bcr/abl transcript numbers by qualitative and quantitative polymerase chain reaction and donor chimerism revealed that 10 responders cleared bcr/abl positive cells from the peripheral blood within a median of 9 wk (range 3-22 wk). The close relation (P = 0.0075) between the first occurrence of graft-vs.-host disease and the complete clearance of bcr/abl positive blood cells argues in favour of an effective graft-vs.-leukaemia reaction.

Summary:A total of 22 patients with acute myeloid leukemia (AML) in first complete remission receiving autologous blood stem cell transplantation (ABSCT) were investigated in order to determine factors affecting outcome. All but two patients had a normal karyotype and received the same high-dose chemotherapy followed by G-CSF-mobilized peripheral blood stem cells after the second (n=5) or third (n=17) course of induction and post-remission chemotherapy, respectively. With a median follow-up of 30 months, the median disease-free survival is 24.1 months. Univariate analysis showed that three chemotherapy cycles before ABSCT were associated with a significant better disease-free survival (P=0.0018) and overall survival (P=0.0033), whereas the presence of an FLT3-mutation (n=6) showed no impact. The number of megakaryocytic progenitors (CFU-MK) infused tended to correlate with primary platelet engraftment (P=0.07) and were predictive for neutrophil (P=0.011) and platelet counts (P=0.009) 180 days after transplantation. Patients receiving a higher amount of CFU-MK had a better event-free survival (P=0.02). Our data suggest that the content of CFU-MK within the graft predicts the quality of hematological recovery and long-term disease control. Additionally, a minimum of three chemotherapy cycles before ABSCT seems to be associated with an improved outcome.Bone Marrow Transplantation (2005) 36, 1083–1088. doi:10.1038/sj.bmt.1705176; published online 10 October 2005 [ABSTRACT FROM AUTHOR]

Summary:It is unknown whether the addition of antithymocyte globulin (ATG) to reduced-intensity conditioning with busulfan (BU) and fludarabine (FLU) is beneficial in HLA-identical sibling transplantation. Therefore, we analyzed retrospectively data on 83 patients, who received peripheral blood stem cells from HLA-identical siblings after conditioning with either 8?mg/kg BU and 150?mg/m2 FLU (n=45) or 8?mg/kg BU, 180?mg/m2 FLU and 40?mg/kg ATG (n=38). Graft-versus-host disease (GVHD) prophylaxis consisted of CSA alone (n=32) or a combination with either MTX or MMF (n=51). The median age was 52 years. Graft failure occurred in two patients after BU/FLU and in three after BU/FLU/ATG (P=0.66). After conditioning with BU/FLU, platelet recovery was significantly faster (P=0.017), and less platelet (P<0.001) and red blood cell (P=0.002) support was needed. Incidences of acute GVHD grades II and IV were 46 and 49%, respectively. Limited chronic GVHD occurred more often after BU/FLU compared to BU/FLU/ATG (54 vs 23%, P=0.02). The overall survival, non-relapse and relapse mortality did not differ significantly. We conclude that in peripheral blood stem cell transplantation from HLA-identical siblings after reduced-intensity conditioning with BU and FLU, ATG has no major impact on the rate of graft rejection and acute GVHD, but it reduces the incidence of limited chronic GVHD.Bone Marrow Transplantation (2004) 33, 483-490. doi:10.1038/sj.bmt.1704384 Published online 29 December 2003 [ABSTRACT FROM AUTHOR]

We report the response to the ABL kinase inhibitor imatinib mesylate (STI571) in a patient with chronic myeloid leukemia (CML) who relapsed twice after dose-reduced allogeneic stem cell transplantation (alloSCT) for B lymphoid blast crisis (BC) and failed to develop an antileukemic response despite grade 3 graft-versus-host disease (GvHD). Complete hematologic, cytogenetic and molecular responses were achieved within 9 weeks of therapy and are maintained after 27 months. Extensive chronic skin GvHD necessitating immunosuppressive therapy developed after 14 months. This case illustrates the ability of imatinib to induce sustained hematologic and molecular remissions in some patients relapsing with advanced stage CML after alloSCT.Bone Marrow Transplantation (2003) 31, 611-614. doi:10.1038/sj.bmt.1703885 [ABSTRACT FROM AUTHOR]

We report the case of a 42-year-old male patient who was diagnosed with a large tumor of the right thoracic aperture 30 months after unrelated hematopoietic stem cell transplantation (HSCT) for accelerated phase of Philadelphia chromosome (Ph)-positive chronic myeloid leukemia (CML). Biopsy revealed an immature lymphoid neoplasia with blastic tumor cell morphology and immunoreactivity for CD34, CD79a, CD43, and CD30 as well as slight positivity for TdT and CD20. Bcr-Abl rearrangement was found in interphase tumor cell nuclei by fluorescence in situ hybridization (FISH). Furthermore, a translocation t(14;18)(q32;q21) was amplified by polymerase chain reaction (PCR). Bone marrow (BM) examination showed regular hematopoiesis including a negative FISH analysis for Bcr-Abl and complete donor chimerism. Nested PCR from peripheral blood (PB), but not conventional PCR, was positive for the b3a2 Bcr-Abl transcript. Neither radiation nor intensive chemotherapy was capable of achieving a tumor remission, and the patient died from progressive disease 6 months later. Postmortem examinations showed a shift of immunophenotype with appearance of myeloperoxidase-positive tumor cells and loss of lymphoid antigens. In addition, there were characteristic cytogenetic findings of multiple Ph chromosomes and a clonal loss of P53 tumor suppressor gene. The latter was already deleted before HSCT. We conclude that lymphoid neoplasia occurring in our patient should be interpreted as an extramedullary, very immature blast crisis of CML expressing lymphoid differentiation markers rather than a true de novo NHL. [ABSTRACT FROM AUTHOR]

Between February 1998 and October 1999, 24 patients with advanced leukemia, lymphoma or solid tumors received G-CSF mobilized peripheral blood stem cells (PBSC) from HLA-matched sibling donors after dose-reduced conditioning therapy. Only patients with reduced performance status or major infectious complications, not eligible for standard transplant procedures, were included. The 5-day conditioning therapy consisted of 3.3 mg/kg intravenous busulphan × 2 days and 30 mg/m2fludarabine × 5 days. GVHD prophylaxis was performed with either CsA alone (n = 5), CsA combined with short course methotrexate (n = 5) or mycophenolate mofetil (n = 14). The day 100 survival was 95.2% for the whole group. All patients engrafted after a median of 15 days (range, 11–19) and 12.5 days (range, 10–19) for neutrophils and platelets, respectively. The median time to a neutrophil count of <0.5 × 109/l was 7 days (range, 2 to 12). acute gvhd >I was observed in six patients, whereas eight patients have signs of chronic GVHD. The prospective 12 month overall survival with a median follow-up of 7 months is 63%. Relapse of disease and toxicity associated with chronic GVHD were the main causes of death. The treatment-related mortality was 12.5%. Dose-reduced conditioning using intravenous busulphan and fludarabine allows stable engraftment without ATG in related transplants and leads to a reduction of transplant-related mortality. Bone Marrow Transplantation (2000) 26, 119–125. [ABSTRACT FROM AUTHOR]

A 17-year-old male with AML FAB M4 relapsed 4 months after myeloablative conditioning and peripheral blood stem cell transplantation (PBSCT) from an HLA-identical unrelated donor. A second PBSC harvest was infused 2 days after completion of cytoreductive therapy with mitoxantrone 7 mg/m2/day i.v. for 3 days (total dose 21 mg/m2), fludarabine 30 mg/m2/day i.v. for 6 days (total dose 180 mg/m2) and Ara-C 125 mg/m2/day i.v. for 5 days (total dose 625 mg/m2). Neutrophil recovery occurred on day +10 and was associated with GVHD grade III of the skin which was treated with cyclosporin A (CsA) and prednisone. Because of fever of unknown origin and progressive fatigue combined with hypotension on day +15 after second PBSCT, echocardiography was performed which revealed a dramatic decrease in systolic function compared to the status pre-transplant. On the same day acute heart failure with consecutive ventricular fibrillation occurred. Although resuscitation was performed immediately the patient died. The autopsy revealed massive infiltration by donor CD8-positive lymphocytes with concomitant extensive damage of the heart tissue. Acute myocarditis of viral origin was excluded by in situ hybridization and nested PCR techniques. In this patient, myocardial involvement by acute GVHD seems to have triggered a fatal arrhythmia and heart failure. Bone Marrow Transplantation (2001) 27, 107–109. [ABSTRACT FROM AUTHOR]

The article presents a case study of a 60-year-old patient with recurrent fever and general debilitation and who had a 16-year history of polycythaemia vera. He was presented with leukocytosis and thrombocytosis, hepatomegaly and generalized lymphadenopathy. He was treated with chemotherapy, cytarabine, and prednisolone, which results to lower serum procalcitonin (PCT) levels. After stem cell transplantation, the patient has recovered with remission of the disease.