Postremission Consolidation by Autologous Hematopoietic Cell Transplantation (HCT) for Acute Myeloid Leukemia in First Complete Remission (CR) and Negative Implications for Subsequent Allogeneic HCT in Second CR: A Study by the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation (EBMT).

After autologous hematopoietic cell transplantation (HCT) in the first complete remission (CR1), patients with acute myeloid leukemia (AML) may relapse and undergo allogeneic HCT in the second complete remission (CR2). The aim of this study was to analyze the outcome of allogeneic HCT performed in CR2 comparing patients with prior consolidation by autologous HCT versus patients with chemotherapy consolidation. Included were 2619 adults with allogeneic HCT in CR2 from 2000 to 2017 with (n = 417) or without (n = 2202) prior autologous HCT. Patient groups were not entirely comparable; patients with prior autologous HCT were younger, had less often a favorable cytogenetic profile, had more commonly donors other than matched siblings, and more often received reduced-intensity conditioning. In multivariate analysis, nonrelapse mortality risks in patients with prior autologous HCT were 1.34 (1.07 to 1.67; P = .01) after adjustment for age, cytogenetic risk, transplant year, donor, conditioning intensity, sex matching, interval diagnosis-relapse, and relapse-allogeneic HCT as compared with chemotherapy consolidation. Similarly, risks of events in leukemia-free survival and graft-versus-host disease, relapse-free survival were higher with prior autologous HCT, 1.17 (1.01 to 1.35), P = .03 and 1.18 (1.03 to 1.35), P = .02, respectively. Risk of death was also higher, 1.13 (0.97 to 1.32), P = .1, but this was not significant. Postremission consolidation with autologous HCT for AML in CR1 increases toxicity of subsequent allogeneic HCT in CR2.

Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), in acute myeloid leukemia.

The transcription factor C/EBPalpha (for CCAAT/enhancer binding protein-alpha; encoded by the gene CEBPA) is crucial for the differentiation of granulocytes. Conditional expression of C/EBPalpha triggers neutrophilic differentiation, and no mature granulocytes are observed in Cebpa-mutant mice. Here we identify heterozygous mutations in CEBPA in ten patients with acute myeloid leukemia (AML). We found that five mutations in the amino terminus truncate the full-length protein, but did not affect a 30-kD protein initiated further downstream. The mutant proteins block wild-type C/EBPalpha DNA binding and transactivation of granulocyte target genes in a dominant-negative manner, and fails to induce granulocytic differentiation. Ours is the first report of CEBPA mutations in human neoplasia, and such mutations are likely to induce the differentiation block found in AML.

Addition of the nuclear export inhibitor selinexor to standard intensive treatment for elderly patients with acute myeloid leukemia and high risk myelodysplastic syndrome.

Treatment results of AML in elderly patients are unsatisfactory. In an open label randomized phase II study, we investigated whether addition of the XPO1 inhibitor selinexor to intensive chemotherapy would improve outcome in this population. 102 AML patients > 65 years of age (median 69 (65-80)) were randomly assigned to standard chemotherapy (3 + 7) with or without oral selinexor 60 mg twice weekly (both arms n = 51), days 1-24. In the second cycle, cytarabine 1000 mg/m2 twice daily, days 1-6 with or without selinexor was given. CR/CRi rates were significantly higher in the control arm than in the investigational arm (80% (95% C.I. 69-91%) vs. 59% (45-72%; p = 0.018), respectively). At 18 months, event-free survival was 45% for the control arm versus 26% for the investigational arm (Cox-p = 0.012) and overall survival 58% vs. 33%, respectively (p = 0.009). AML and infectious complications accounted for an increased death rate in the investigational arm. Irrespective of treatment, MRD status after two cycles appeared to be correlated with survival. We conclude that the addition of selinexor to standard chemotherapy does negatively affect the therapeutic outcome of elderly AML patients. (Netherlands Trial Registry number NL5748 (NTR5902), www.trialregister.nl ).

High levels of circulating CD34+ cells at autologous stem cell collection are associated with favourable prognosis in multiple myeloma.

BACKGROUND: High-dose chemotherapy with autologous stem cell transplantation is a cornerstone in the first-line treatment of multiple myeloma patients. However, only few factors have been identified affecting the outcome in such patients. We hypothesised that varying levels of mobilised CD34+ cells confer prognostic information in myeloma patients undergoing high-dose chemotherapy. METHODS: We determined circulating CD34+ cells at the day of peripheral stem cell collection in 158 consecutive myeloma patients between January 2001 and August 2010. Patients were stratified into two groups (super vs normal mobilisers) with a cutoff of 100,000 peripheral CD34+ cells per ml. RESULTS: We found that patients with more than 100,000 peripheral CD34+ cells per ml had a better overall survival (P=0.005) and a prolonged time to progression (P=0.0398) than patients with CD34+ cell counts below 100,000 CD34+ cells per ml. High levels of CD34+ cells were an independent marker for better overall survival and time to progression in a multivariate analysis that included disease stage, response at transplant, light-chain subtype, age, sex, and height. CONCLUSION: Our results suggest that high levels of mobilised peripheral CD34+ cells are associated with favourable outcome in myeloma patients undergoing autologous transplantation.

AML1-ETO downregulates the granulocytic differentiation factor C/EBPalpha in t(8;21) myeloid leukemia.

The transcription factor CCAAT/enhancer binding protein alpha, or C/EBPalpha, encoded by the CEBPA gene, is crucial for the differentiation of granulocytes. Conditional expression of C/EBPalpha triggers neutrophilic differentiation, and Cebpa knockout mice exhibit an early block in maturation. Dominant-negative mutations of CEBPA have been found in some patients with acute myeloid leukemia (AML), but not in AML with the t(8;21) translocation which gives rise to the fusion gene RUNX1-CBF2T1 (also known as AML1-ETO) encoding the AML1-ETO fusion protein. RUNX1-CBF2T1 positive-AML blasts had eight-fold lower CEBPA RNA levels and undetectable C/EBPalpha protein levels compared with other subgroups of AML patients. Conditional expression of RUNX1-CBF2T1 in U937 cells downregulated CEBPA mRNA, protein and DNA binding activity. AML1-ETO appears to suppress C/EBPalpha expression indirectly by inhibiting positive autoregulation of the CEBPA promoter. Conditional expression of C/EBPalpha in AML1-ETO-positive Kasumi-1 cells results in neutrophilic differentiation. We suggest that restoring C/EBPalpha expression will have therapeutic implications in RUNX1-CBF2T1-positive leukemias.

Prevalence and significance of bacterial contamination of autologous stem cell products.

There is limited and conflicting information on the prevalence of contamination of haematopoietic stem and progenitor cell products (HPCPs), and their optimal management remains unclear. The authors reviewed the microbial surveillance data of HPCPs collected between January 2002 and December 2019 for autologous transplantation at the study institution to determine the prevalence of microbial contamination and the potential infectious complications among recipients. Among 3935 HPCPs, 25 (0.6%) were contaminated. Ultimately, 22 patients received contaminated grafts, with pre-emptive antimicrobial therapy initiated in six of these patients. No patients developed subsequent infectious complications. These data suggest that microbial contamination of autologous HPCPs and associated adverse outcomes are rare.

The tumour-suppressive miR-29a/b1 cluster is regulated by CEBPA and blocked in human AML.

BACKGROUND: CCAAT/enhancer-binding protein-alpha (CEBPA) is crucial for normal granulopoiesis and is frequently disrupted in acute myeloid leukaemia (AML). Increasing evidence suggests that CEBPA exerts its effects, in parts, by regulating specific microRNAs (miRNAs), as previously shown for miR-223. The aim of this study was to investigate the genome-wide pattern of miRNAs regulated by CEBPA in myeloid cells. METHODS: In Kasumi-1 cells, conditionally expressing CEBPA, we assessed the expression of 470 human miRNAs by microarray analysis. We further investigated the microarray results by qRT-PCR, luciferase reporter assays, and chromatin immunoprecipitation assays. RESULTS: In all, 18 miRNAs were more than two-fold suppressed or induced after CEBPA restoration. Among these 18 miRNAs, we focused on CEBPA-mediated regulation of the tumour-suppressive miR-29b. We observed that miR-29b is suppressed in AML patients with impaired CEBPA function or loss of chromosome 7q. We found that CEBPA selectively regulates miR-29b expression on its miR-29a/b1 locus on chromosome 7q32.3, whereas miR-29b2/c on chromosome 1q32.2 is not affected. CONCLUSION: This study reports the activation of the tumour-suppressive miR-29b by the haematopoietic key transcription factor CEBPA. Our data provide a rationale for miR-29b suppression in AML patients with loss of chromosome 7q or CEBPA deficiency.

Correction: Lenalidomide added to standard intensive treatment for older patients with AML and high-risk MDS.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Lenalidomide added to standard intensive treatment for older patients with AML and high-risk MDS.

More effective treatment modalities are urgently needed in patients with acute myeloid leukemia (AML) of older age. We hypothesized that adding lenalidomide to intensive standard chemotherapy might improve their outcome. After establishing a safe lenalidomide, dose elderly patients with AML were randomly assigned in this randomized Phase 2 study (n = 222) to receive standard chemotherapy ("3 + 7") with or without lenalidomide at a dose of 20 mg/day 1-21. In the second cycle, patients received cytarabine 1000 mg/m2 twice daily on days 1-6 with or without lenalidomide (20 mg/day 1-21). The CR/CRi rates in the two arms were not different (69 vs. 66%). Event-free survival (EFS) at 36 months was 19% for the standard arm versus 21% for the lenalidomide arm and overall survival (OS) 35% vs. 30%, respectively. The frequencies and grade of adverse events were not significantly different between the treatment arms. Cardiovascular toxicities were rare and equally distributed between the arms. The results of the present study show that the addition of lenalidomide to standard remission induction chemotherapy does not improve the therapeutic outcome of older AML patients. This trial is registered as number NTR2294 in The NederlandsTrial Register (www.trialregister.nl).

Heterogeneity within AML with CEBPA mutations; only CEBPA double mutations, but not single CEBPA mutations are associated with favourable prognosis.

CCAAT/enhancer binding protein alpha (CEBPA) mutations in AML are associated with favourable prognosis and are divided into N- and C-terminal mutations. The majority of AML patients have both types of mutations. We assessed the prognostic significance of single (n=7) and double (n=12) CEBPA mutations among 224 AML patients. Double CEBPA mutations conferred a decisively favourable overall (P=0.006) and disease-free survival (P=0.013). However, clinical outcome of patients with single CEBPA mutations was not different from CEBPA wild-type patients. In a multivariable analysis, only double -- but not single -- CEBPA mutations were identified as independent prognostic factors. These findings indicate heterogeneity within AML patients with CEBPA mutations.

Nelfinavir and lenalidomide/dexamethasone in patients with lenalidomide-refractory multiple myeloma. A phase I/II Trial (SAKK 39/10).

The antiretroviral agent nelfinavir has antimyeloma activity and can overcome resistance to bortezomib. Our phase I/II trial investigated whether adding nelfinavir to lenalidomide-dexamethasone can overcome lenalidomide resistance in lenalidomide-refractory multiple myeloma (MM). Twenty-nine patients were included (high-risk cytogenetic aberrations 31%; ≥2 prior therapy lines 93%; lenalidomide-bortezomib double-refractory 34%). Twenty-four patients (83%) had prior bortezomib and 10 (34%) were lenalidomide-bortezomib double-refractory. They received four cycles of nelfinavir 2500 mg/day with standard-dose lenalidomide (25 mg days 1-21) and dexamethasone (40/20 mg days 1, 8, 15, 22). Minor response or better was achieved in 16 patients (55%; 95% CI 36-74%), including 40% of those who were lenalidomide-bortezomib double-refractory, and partial response or better in nine patients (31%; 95% CI 15-51%). Median progression-free survival was 3.4 (95% CI 2.0-4.9) months and median overall survival 21.6 (13.0-50.1) months. Lenalidomide-related pneumonitis, pneumonia, and neutropenic fever occurred, but there were no unexpected adverse events. Peripheral blood mononuclear cells showed a 45% (95% CI 40-51%) reduction in total proteasome activity from baseline and significant induction of unfolded protein response and autophagy. Thus, nelfinavir-lenalidomide-dexamethasone is an active oral combination in lenalidomide-refractory MM.

Transcriptional dysregulation during myeloid transformation in AML.

The current paradigm on leukemogenesis indicates that leukemias are propagated by leukemic stem cells. The genomic events and pathways involved in the transformation of hematopoietic precursors into leukemic stem cells are increasingly understood. This concept is based on genomic mutations or functional dysregulation of transcription factors in malignant cells of patients with acute myeloid leukemia (AML). Loss of the CCAAT/enhancer binding protein-alpha (CEBPA) function in myeloid cells in vitro and in vivo leads to a differentiation block, similar to that observed in blasts from AML patients. CEBPA alterations in specific subgroups of AML comprise genomic mutations leading to dominant-negative mutant proteins, transcriptional suppression by leukemic fusion proteins, translational inhibition by activated RNA-binding proteins, and functional inhibition by phosphorylation or increased proteasomal-dependent degradation. The PU.1 gene can be mutated or its expression or function can be blocked by leukemogenic fusion proteins in AML. Point mutations in the RUNX1/AML1 gene are also observed in specific subtypes of AML, in addition to RUNX1 being the most frequent target for chromosomal translocation in AML. These data are persuasive evidence that impaired function of particular transcription factors contributes directly to the development of human AML, and restoring their function represents a promising target for novel therapeutic strategies in AML.

[MRI after renal transplantation with dysfunctional allograft: are there typical diagnoses in the early and late postoperative phases?]. / MRT bei Nierentransplantatdysfunktion: Gibt es typische Diagnosen im frühen und späten postoperativen Verlauf?

PURPOSE: The aim of this study was to evaluate the different MRI diagnoses in the early and late post-operative period after renal transplantation with dysfunctional allograft. MATERIALS AND METHODS: Due to unknown transplant dysfunction, 49 patients (30-male, 19 female) received a total of 74 MRI studies. According to the date of examination all MRI studies were divided in an early (< or = 60 days, ETP) and a late post-transplant time period (> 60 days, LTP). All MRI studies were performed on 1.5 T MRI systems using a standardised imaging protocol consisting of a morphological (pre- and post-contrast enhanced T (1)- and T (2)-weighted TSE sequences), a vascular (contrast-enhanced 3D MRA) and a urographical part (Flash 3D sequences). Frequencies of diagnoses in ETP and LTP, and diagnoses within each transplant time period were analysed. RESULTS: 44/74 MRI studies were performed in ETP, 30/74 in LTP. In total 80 diagnoses were obtained: Renal artery stenosis (ETP, n = 21; LTP, n = 5), renal vein stenosis (ETP, n = 2), renal vein thrombosis (ETP, n = 2), renal perfusion defect (ETP, n = 11), rejection (ETP, n = 1; LTP, n = 2), abscess (ETP, n = 1), urinary outflow obstruction (LTP, n = 4), without MRI pathology (ETP, n = 11; LTP, n = 20). Renal artery stenosis was the most prevalent diagnosis in ETP, and a more frequent finding in ETP compared to LTP (p < 0.05). Renal perfusion defects were more frequent in ETP than in LTP (p < 0.05). In the ETP vascular diseases (34/49 diagnoses) were more frequent (p < 0.05) than uropathological diseases (0/49 diagnoses). CONCLUSIONS: Our results indicate that vascular diseases are a more frequent occurrence in the early post-operative course after renal transplantation than uropathological diseases. However, a transplant follow-up MRI study needs to contain a morphological, vascular and functional imaging part to answer combined clinical questions.

NSAID treatment with meloxicam enhances peripheral stem cell mobilization in myeloma.

Chemotherapy with G-CSF is used to mobilize peripheral stem cells in multiple myeloma (MM) patients, with plerixafor as a rescue strategy for poorly mobilizing patients. Preclinical studies suggested that the nonsteroidal anti-inflammatory drug meloxicam enhances the mobilization of CD34+ cells. In this single-center study, we evaluated whether adding meloxicam to chemotherapy/G-CSF mobilization increases peripheral hematopoietic CD34+ cell levels and reduces the need of using plerixafor. We prospectively compared two consecutive cohorts of MM patients in first remission mobilized with G-CSF and non-myelosuppressive chemotherapy with vinorelbine or gemcitabine. The second cohort additionally received oral meloxicam. The cohorts comprised 84 patients without meloxicam (-M) and 66 patients with meloxicam (+M). Meloxicam was well tolerated and associated with similar hematologic engraftment after transplantation and equal survival rates. However, the meloxicam group had higher CD34+ cell levels on day 8 of the mobilization procedure (53 200 versus 35 600 CD34+ cells/mL; P=0.007), and fewer patients needed >1 collection day (+M: 6 (9%) patients versus -M: 16 (19%) patients; P=0.04). This resulted in reduced plerixafor administrations (+M: 7 (11%) patients versus -M: 18 (21%) patients; P=0.03) and less costs. Our data suggest that meloxicam enhances the mobilization of hematopoietic CD34+ blood cells in MM patients.

Dichotomy of AML1-ETO functions: growth arrest versus block of differentiation.

The fusion gene AML1-ETO is the product of t(8;21)(q22;q22), one of the most common chromosomal translocations associated with acute myeloid leukemia. To investigate the impact of AML1-ETO on hematopoiesis, tetracycline-inducible AML1-ETO-expressing cell lines were generated using myeloid cells. AML1-ETO is tightly and strongly induced upon tetracycline withdrawal. The proliferation of AML1-ETO(+) cells was markedly reduced, and most of the cells eventually underwent apoptosis. RNase protection assays revealed that the amount of Bcl-2 mRNA was decreased after AML1-ETO induction. Enforced expression of Bcl-2 was able to significantly delay, but not completely overcome, AML1-ETO-induced apoptosis. Prior to the onset of apoptosis, we also studied the ability of AML1-ETO to modulate differentiation. AML1-ETO expression altered granulocytic differentiation of U937T-A/E cells. More significantly, this change of differentiation was associated with the down-regulation of CCAAT/enhancer binding protein alpha (C/EBPalpha), a key regulator of granulocytic differentiation. These observations suggest a dichotomy in the functions of AML1-ETO: (i) reduction of granulocytic differentiation correlated with decreased expression of C/EBPalpha and (ii) growth arrest leading to apoptosis with decreased expression of CDK4, c-myc, and Bcl-2. We predict that the preleukemic AML1-ETO(+) cells must overcome AML1-ETO-induced growth arrest and apoptosis prior to fulfilling their leukemogenic potential.

Long-term survival of sorafenib-treated FLT3-ITD-positive acute myeloid leukaemia patients relapsing after allogeneic stem cell transplantation.

BACKGROUND: Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD)-positive acute myeloid leukaemia (AML) relapsing after allogeneic stem cell transplantation (allo-SCT) has a dismal prognosis with limited therapeutic options. FLT3-ITD kinase inhibition is a reasonable but palliative experimental treatment alternative in this situation. Information on long-term outcome is not available. METHODS: We performed a long-term follow-up analysis of a previously reported cohort of 29 FLT3-ITD-positive AML patients, which were treated in relapse after allo-SCT with sorafenib monotherapy. FINDINGS: With a median follow-up of 7.5 years, 6 of 29 patients (21%) are still alive. Excluding one patient who received a second allo-SCT, five patients (17%) achieved sustained complete remissions with sorafenib. Four of these patients are in treatment-free remission for a median of 4.4 years. INTERPRETATION: Sorafenib may enable cure of a proportion of very poor risk FLT3-ITD-positive AML relapsing after allo-SCT.

Comparative value of post-remission treatment in cytogenetically normal AML subclassified by NPM1 and FLT3-ITD allelic ratio.

Post-remission treatment (PRT) in patients with cytogenetically normal (CN) acute myeloid leukemia (AML) in first complete remission (CR1) is debated. We studied 521 patients with CN-AML in CR1, for whom mutational status of NPM1 and FLT3-ITD was available, including the FLT3-ITD allelic ratio. PRT consisted of reduced intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (alloHSCT) (n=68), myeloablative conditioning (MAC) alloHSCT (n=137), autologous hematopoietic stem cell transplantation (autoHSCT) (n=168) or chemotherapy (n=148). Favorable overall survival (OS) was found for patients with mutated NPM1 without FLT3-ITD (71±4%). Outcome in patients with a high FLT3-ITD allelic ratio appeared to be very poor with OS and relapse-free survival (RFS) of 23±8% and 12±6%, respectively. Patients with wild-type NPM1 without FLT3-ITD or with a low allelic burden of FLT3-ITD were considered as intermediate-risk group because of similar OS and RFS at 5 years, in which PRT by RIC alloHSCT resulted in better OS and RFS as compared with chemotherapy (hazard ratio (HR) 0.56, P=0.022 and HR 0.50, P=0.004, respectively) or autoHSCT (HR 0.60, P=0.046 and HR 0.60, P=0.043, respectively). The lowest cumulative incidence of relapse (23±4%) was observed following MAC alloHSCT. These results suggest that alloHSCT may be preferred in patients with molecularly intermediate-risk CN-AML, while the choice of conditioning type may be personalized according to risk for non-relapse mortality.

Expression and regulation of G1 cell-cycle inhibitors (p16INK4A, p15INK4B, p18INK4C, p19INK4D) in human acute myeloid leukemia and normal myeloid cells.

In hematological malignancies, structural alterations of genes for G1-specific cyclin-dependent kinases inhibitors (CKIs) have been extensively investigated. G1-CKIs might play an important role not only as tumor suppressor genes but also in cellular differentiation. We examined constitutive and differentiation-induced expression and regulation of the four members of the G1-CKI family p16INK4A, p15INK4B, p18INK4C and p19INK4D in acute myeloid leukemia as well as their expression in normal granulocytes and monocytes. p18INK4C and p19INK4D mRNA were expressed constitutively at high levels in seven myeloid cell lines and 16 AML patient samples, whereas expression of p15INK4B mRNA was very low and only detectable by nested RT-PCR analysis. During phorbol ester-induced monocytic differentiation of leukemic HL-60 cells expression of particular G1-CKIs was disparately regulated. This process was associated with growth arrest of the majority of the cells (> or = 80%) in G1/G0, and in parallel p15INK4B were upregulated whereas p18INK4C and p19INK4D expression was downregulated. In contrast, granulocytic differentiation induced by DMSO was accompanied by an increase of p18INK4C and p19INK4D expression only. PMA treatment of blast cells from two AML patients confirmed these cell line results. Disparate regulation of p15INK4B and p18INK4C mRNA was dependent on intermediary protein synthesis and occurred at the post-transcriptional level as shown by nuclear run-on analysis and mRNA half-life studies. In normal granulocytes and monocytes low constitutive p15INK4B and p18INK4C mRNA expression was detectable by RT-PCR only, but p19INK4D transcripts were noted by Northern blotting in both cell types. Disparate expression of G1-specific cell cycle inhibitors indicates complex and divergent roles of particular CKIs during normal and leukemic myeloid hematopoiesis.

Varicella zoster virus reactivation after autologous SCT is a frequent event and associated with favorable outcome in myeloma patients.

The occurrence of varicella zoster virus (VZV) reactivation is increased after allogeneic transplantation, whereas limited data are available for herpes zoster (HZ) after autologous SCT (ASCT). We determined the incidence and the prognostic significance of HZ and its correlation with VZV serology in 191 consecutive myeloma patients undergoing high-dose melphalan chemotherapy with ASCT. We found that VZV reactivation occurred in 57 (30%) patients, in 8.5% during induction and in 21.5% after ASCT peaking at 8 months after ASCT. Disease burden due to HZ was assessed as high or rather high in 70% of the patients. By immune fluorescence and Serion Elisa VZV IgG assessment, 90.8% of all patients had specific anti-VZV antibodies at ASCT. Lower specific antibody titers at transplantation were observed in patients with HZ after ASCT than in those without reactivation (P=0.009). Finally, OS was better in myeloma patients with HZ after ASCT compared with patients without HZ (P=0.007). Our data indicate that VZV reactivation after ASCT is a frequent event carrying a significant disease burden and it is associated with improved survival. Low levels of specific VZV antibodies at ASCT suggest increased vulnerability for VZV reactivation.