Measurable residual disease (MRD) status before allogeneic hematopoietic cell transplantation impact on secondary acute myeloid leukemia outcome. A Study from the Acute Leukemia Working Party (ALWP) of the European society for Blood and Marrow Transplantation (EBMT).

Measurable residual disease (MRD) assessment before allogeneic hematopoietic cell transplantation (HCT) may help physicians to identify a subgroup of patients at high risk of relapse for de novo acute myeloid leukemia (AML) but its relevance among patients affected by secondary AML (sAML) is still unknown. We assessed the impact of MRD among 318 adult patients with sAML who received an allogeneic HCT in first complete remission. At the time of HCT, a total of 208 (65%) patients achieved MRD negativity, while 110 (35%) had positive MRD. 2-year overall survival (OS) was 58.8 % (95% CI 52.2-64.9) with leukemia-free survival (LFS) of 50.0 % (95% CI 43.7-56.1), relapse incidence of 34.2% (95% CI 28.4-40.1) and non-relapse mortality (NRM) of 23.3 % (95% CI 19-27.7) for the entire cohort. In multivariate analysis, HCT recipients with KPS ≥ 90 experienced less disease recurrence (HR 0.61, 95% CI 0.4-0.94) with better LFS (HR 0.63, 95% CI 0.44-0.89) and OS (HR 0.58, 95% CI 0.39-0.86). There were no differences in major clinical endpoints between patients with MRD-positive and MRD-negative status at the time of HCT. Pre-transplantation assessment of MRD was not informative on post-HCT outcomes in this retrospective registry-based analysis among patients affected by sAML.

Gilteritinib monotherapy as a transplant bridging option for high risk FLT3-mutated AML with t(6;9)(p23;q34.1);DEK-NUP214 in morphological but not cytogenetic or molecular remission following standard induction chemotherapy.

We report a case of FLT3-mutated AML with t(6;9) in which induction chemotherapy with DA and midostaurin failed to achieve complete cytogenetic or molecular remission. Due to the COVID-19 pandemic and co-existing cellulitis, monotherapy with the selective FLT3-inhibitor gilteritinib was used as an alternative consolidation treatment option rather than further intensive chemotherapy. Gilteritinib was able to achieve complete molecular and cytogenetic remission despite the additional cytogenetic abnormality. This case provides supporting evidence for the use of single agent gilteritinib in high-risk primary refractory FLT3-mutated AML with t(6;9) prior to transplantation.

Two-Stage Priming of Allogeneic Natural Killer Cells for the Treatment of Patients with Acute Myeloid Leukemia: A Phase I Trial.

UNLABELLED: Human Natural Killer (NK) cells require at least two signals to trigger tumor cell lysis. Absence of ligands providing either signal 1 or 2 provides NK resistance. We manufactured a lysate of a tumour cell line which provides signal 1 to resting NK cells without signal 2. The tumor-primed NK cells (TpNK) lyse NK resistant Acute Myeloid Leukemia (AML) blasts expressing signal 2 ligands. We conducted a clinical trial to determine the toxicity of TpNK cell infusions from haploidentical donors. 15 patients with high risk AML were screened, 13 enrolled and 7 patients treated. The remaining 6 either failed to respond to re-induction chemotherapy or the donor refused to undergo peripheral blood apheresis. The conditioning consisted of fludarabine and total body irradiation. This was the first UK trial of a cell therapy regulated as a medicine. The complexity of Good Clinical Practice compliance was underestimated and led to failures requiring retrospective independent data review. The lessons learned are an important aspect of this report. There was no evidence of infusional toxicity. Profound myelosuppression was seen in the majority (median neutrophil recovery day 55). At six months follow-up, three patients treated in Complete Remission (CR) remained in remission, one patient infused in Partial Remission had achieved CR1, two had relapsed and one had died. One year post-treatment one patient remained in CR. Four patients remained in CR after treatment for longer than their most recent previous CR. During the 2 year follow-up six of seven patients died; median overall survival was 400 days post infusion (range 141­910). This is the first clinical trial of an NK therapy in the absence of IL-2 or other cytokine support. The HLA-mismatched NK cells survived and expanded in vivo without on-going host immunosuppression and appeared to exert an anti-leukemia effect in 4/7 patients treated. TRIAL REGISTRATION: ISRCTN trial registry ISRCTN11950134.

Does BCR/ABL1 positive acute myeloid leukaemia exist?

The BCR/ABL1 fusion gene, usually carried by the Philadelphia chromosome (Ph) resulting from t(9;22)(q34;q11) or variants, is pathognomonic for chronic myeloid leukaemia (CML). It is also occasionally found in acute lymphoblastic leukaemia (ALL) mostly in adults and rarely in de novo acute myeloid leukaemia (AML). Array Comparative Genomic Hybridization (aCGH) was used to study six Ph(+)AML, three bi-lineage and four Ph(+)ALL searching for specific genomic profiles. Surprisingly, loss of the IKZF1 and/or CDKN2A genes, the hallmark of Ph(+)ALL, were recurrent findings in Ph(+)AML and accompanied cryptic deletions within the immunoglobulin and T cell receptor genes. The latter two losses have been shown to be part of 'hot spot' genome imbalances associated with BCR/ABL1 positive pre-B lymphoid phenotype in CML and Ph(+)ALL. We applied Significance Analysis of Microarrays (SAM) to data from the 'hot spot' regions to the Ph(+)AML and a further 40 BCR/ABL1(+) samples looking for differentiating features. After exclusion of the most dominant markers, SAM identified aberrations unique to de novo Ph(+)AML that involved relevant genes. While the biological and clinical significance of this specific genome signature remains to be uncovered, the unique loss within the immunoglobulin genes provides a simple test to enable the differentiation of clinically similar de novo Ph(+) AML and myeloid blast crisis of CML.

The hOCT1 SNPs M420del and M408V alter imatinib uptake and M420del modifies clinical outcome in imatinib-treated chronic myeloid leukemia.

Although the prognosis of chronic myeloid leukemia (CML) patients treated with imatinib is good, many fail to develop an optimal response or lose one. This heterogeneity could be attributed to the presence of human organic cation transporter-1 (hOCT1) single nucleotide polymorphisms (SNPs). In the present study, we analyzed the effect of 23 hOCT1 SNPs on imatinib treatment outcome in newly diagnosed CML patients using MassARRAY sequencing and pyrosequencing. The only SNP associated with outcome was M420del (rs35191146), with patients with the M420del demonstrating an increased probability of imatinib treatment failure. In CML cell lines transfected with M420del and/or M408V, M420del significantly decreased imatinib uptake, but this effect was countered if the M408V (rs628031) SNP was also present. A similar effect was seen for the uptake of the hOCT1 substrates TEA(+) and ASP(+). Finally, apparent hOCT1 mRNA levels were studied using both our earlier primers covering the M420del and another set that did not. Different mRNA expression was observed, explaining the disparity in published data on the prognostic importance of hOCT1 mRNA and highlighting the importance of avoiding common SNP sites in primer design. These data demonstrate that the common M420del SNP can modulate the outcome of imatinib treatment.

Incidence and dynamics of Epstein-Barr virus reactivation after alemtuzumab-based conditioning for allogeneic hematopoietic stem-cell transplantation.

BACKGROUND: Reactivation of Epstein-Barr virus (EBV) infection and posttransplant lymphoproliferative disorder (PTLD) pose a significant risk after T-cell-depleted (TCD) allogeneic hematopoietic stem-cell transplantation (HSCT). The pattern of EBV reactivation in patients receiving allogeneic HSCT, incorporating in vivo or in vitro alemtuzumab as the method of TCD, is not known. METHODS: Monitoring for EBV DNA was performed by quantitative polymerase chain reaction on whole blood in 111 consecutive adults undergoing HSCT using alemtuzumab-based TCD. Patients with more than 40,000 copies/mL were screened for PTLD, followed by the withdrawal of immunosuppression and a single infusion of rituximab. RESULTS: The 2-year cumulative incidence of EBV DNAemia was 40.3%. In vivo alemtuzumab was associated with earlier EBV reactivation than in vitro alemtuzumab (100-day incidence 22.7% vs. 2.8%, P=0.006). Eighteen patients (16%) had EBV DNAemia of more than 40,000 copies/mL. In evaluable patients, the initial rate of increase in EBV DNA levels was significantly faster in those who went on to treatment with rituximab than in patients who were left untreated (mean doubling time 3.5 days vs. 4.2 days, P=0.003). Rituximab treatment induced rapid declines in EBV DNA with an average half-life of 1.2+/-0.7 days. Only one patient (0.9%) had histologic confirmation of PTLD and subsequently attained a complete remission with rituximab that persists at 18 months. CONCLUSIONS: Alemtuzumab-based TCD is associated with a high frequency of EBV reactivation but a low (<1%) risk of PTLD using a strategy of preemptive rituximab therapy.

HLA-mismatched unrelated donors are a viable alternate graft source for allogeneic transplantation following alemtuzumab-based reduced-intensity conditioning.

The impact of human leukocyte antigen (HLA) mismatch after reduced-intensity conditioning allogeneic hematopoietic stem cell transplantation (RIT) using unrelated donors (UD) is unclear, and may be modulated by T-cell depletion. We therefore examined outcomes of 157 consecutive patients undergoing RIT after uniform conditioning with fludarabine, melphalan, and alemtuzumab (FMC). Donors were 10/10 HLA-matched (MUDs, n = 107) and 6 to 9/10 HLA-matched (MMUDs, n = 50), with no significant differences in baseline characteristics other than increased cytomegalovirus seropositivity in MMUDs. Rates of durable engraftment were high. Graft failure rates (persistent cytopenias with donor chimerism) were similar (8% vs 3%, P = .21), though rejection (recipient chimerism) was more frequent in MMUDs (8% vs 0%, P < .01). There were no significant differences between donors in the incidences of acute graft-versus-host disease (GVHD; 20% vs 22% grade 2-4, respectively, P = .83), chronic extensive GVHD (3-year cumulative incidence [CI] 23% vs 24%, P = .56), or treatment-related mortality (1-year CI 27% vs 27%, P = .96). Furthermore, there was no difference in 3-year overall survival (OS; 53% vs 49%, P = .44). Mismatch occurred at the antigenic level in 40 cases. The outcome in these cases did not differ significantly from the rest of the cohort. We conclude that RIT using HLA-mismatched grafts is a viable option using FMC conditioning.

Prognostic role of PET scanning before and after reduced-intensity allogeneic stem cell transplantation for lymphoma.

Allogeneic stem cell transplantation (SCT) is an established therapy for patients with relapsed lymphoma, but the role of positron emission tomography (PET) scanning preallogeneic and postallogeneic SCT is uncertain. We investigated whether pretransplantation PET status predicted outcome after allogeneic SCT and whether PET surveillance after transplantation provided additional information compared with computed tomography (CT) scanning. Eighty consecutive patients with lymphoma who received a reduced-intensity allogeneic SCT were entered onto a prospective trial. PET and CT scans were performed before transplantation and up to 36 months after transplantation. Forty-two patients were PET-positive before transplantation. Pretransplantation PET status had no significant impact on either relapse rate or overall survival. Thirty-four relapses were observed, of which 17 were PET-positive with a normal CT scan at relapse. Donor lymphocyte infusion (DLI) was administered in 26 episodes of relapse and was guided by PET alone in 14 patients. These findings suggest that, in contrast to autologous SCT, pretransplantation PET status is not predictive of relapse and survival after allogeneic SCT for lymphoma. Posttransplantation surveillance by PET detected relapse before CT in half of episodes, often allowing earlier administration of DLI in patients with recurrent lymphoma, and permitted withholding of potentially harmful DLI in those with PET-negative masses on CT scans.

p53-mediated apoptosis of CLL cells: evidence for a transcription-independent mechanism.

The p53 protein plays a key role in securing the apoptotic response of chronic lymphocytic leukemia (CLL) cells to genotoxic agents. Transcriptional induction of proapoptotic proteins including Puma are thought to mediate p53-dependent apoptosis. In contrast, recent studies have identified a novel nontranscriptional mechanism, involving direct binding of p53 to antiapoptotic proteins including Bcl-2 at the mitochondrial surface. Here we show that the major fraction of p53 induced in CLL cells by chlorambucil, fludarabine, or nutlin 3a was stably associated with mitochondria, where it binds to Bcl-2. The Puma protein, which was constitutively expressed in a p53-independent manner, was modestly up-regulated following p53 induction. Pifithrin alpha, an inhibitor of p53-mediated transcription, blocked the up-regulation of Puma and also of p21(CIP1). Surprisingly, pifithrin alpha dramatically augmented apoptosis induction by p53-elevating agents and also accelerated the proapoptotic conformation change of the Bax protein. These data suggest that direct interaction of p53 with mitochondrial antiapoptotic proteins including Bcl-2 is the major route for apoptosis induction in CLL cells and that p53's transcriptional targets include proteins that impede this nontranscriptional pathway. Therefore, strategies that block up-regulation of p53-mediated transcription may be of value in enhancing apoptosis induction of CLL cells by p53-elevating drugs.

Acute myeloid leukaemia blast cells with a tyrosine kinase domain mutation of FLT3 are less sensitive to lestaurtinib than those with a FLT3 internal tandem duplication.

FLT3 tyrosine kinase domain mutations (FLT3/TKDs) are associated with a favourable prognosis in acute myeloid leukaemia (AML), unlike FLT3 internal tandem duplications (FLT3/ITDs) that have a poor prognosis. Whilst FLT3/ITD+ cells are more susceptible to the cytotoxic effects of FLT3 inhibitors than wild type (WT) cells, the sensitivity of FLT3/TKD+ cells to therapeutic agents is unclear, as is the importance of the mutant level. We therefore studied the effect of cytarabine and the FLT3 inhibitor lestaurtinib, either alone or in combination, on in vitro survival of blast cells from 36 cases of AML (14 FLT3/WT, 11 FLT3/ITD+ and 11 FLT3/TKD+). All three groups showed similar sensitivity to the cytotoxic effects of cytarabine but FLT3/ITD mutant level was inversely correlated with cytarabine cytotoxicity (P = 0.04) whereas FLT3/TKD mutant level had no impact. FLT3/TKD+ cells showed a similar response to lestaurtinib as FLT3/WT cells, whereas FLT3/ITD+ cells were more sensitive (P = 0.004). There was no correlation between mutant level and lestaurtinib sensitivity for either FLT3/ITD+ or FLT3/TKD+ cells. Synergistic cytotoxicity of lestaurtinib plus cytarabine was demonstrated in all three groups. These results suggest that FLT3/TKD+ and FLT3/WT cases should not be differentiated when considering patients for treatment with FLT3 inhibitors.

Relationship between FLT3 mutation status, biologic characteristics, and response to targeted therapy in acute promyelocytic leukemia.

The prognostic significance of FLT3 mutations in acute promyelocytic leukemia (APL) is not firmly established and is of particular interest given the opportunities for targeted therapies using FLT3 inhibitors. We studied 203 patients with PML-RARA-positive APL; 43% of the patients had an FLT3 mutation (65 internal tandem duplications [ITDs], 19 D835/I836, 4 ITD+D835/I836). Both mutations were associated with higher white blood cell (WBC) count at presentation; 75% of the patients with WBC counts of 10 x 10(9)/L or greater had mutant FLT3. FLT3/ITDs were correlated with M3v subtype (P < .001), bcr3 PML breakpoint (P < .001), and expression of reciprocal RARA-PML transcripts (P = .01). Microarray analysis revealed differences in expression profiles among patients with FLT3/ITD, D835/I836, and wild-type FLT3. Patients with mutant FLT3 had a higher rate of induction death (19% vs 9%; P = .04, but no significant difference in relapse risk (28% vs 23%; P = .5) or overall survival (59% vs 67%; P = .2) at 5 years. In in vitro differentiation assays using primary APL blasts (n = 6), the FLT3 inhibitor CEP-701 had a greater effect on cell survival/proliferation in FLT3/ITD+ cells, but this inhibition was reduced in the presence of ATRA. Furthermore, in the presence of CEP-701, ATRA-induced differentiation was reduced in FLT3/ITD+ cells. These data carry implications for the use of FLT3 inhibitors as frontline therapy for APL.

No evidence that FLT3 status should be considered as an indicator for transplantation in acute myeloid leukemia (AML): an analysis of 1135 patients, excluding acute promyelocytic leukemia, from the UK MRC AML10 and 12 trials.

Fetal liver tyrosine kinase 3 (FLT3) internal tandem duplications (ITDs) are powerful adverse prognostic indicators for relapse in acute myeloid leukemia (AML) but the most efficacious therapy for FLT3/ITD(+) patients is currently unknown. We evaluated outcome according to FLT3/ITD status in 1135 adult patients treated according to United Kingdom Medical Research Council (UK MRC) AML protocols: 141 received an autograft, and 170 received a matched sibling allograft in first complete remission (CR). An FLT3/ITD was detected in 25% of patients and was an independent predictor for relapse (P < .001). It remained prognostic for increased relapse in patients who received a transplant (odds ratio [OR] = 1.91; 95% confidence intervals [CIs] = 1.13-3.21; P = .02), with no evidence of a difference in effect between patients who received an autograft (OR = 2.39; CIs = 1.24-4.62) and patients who received an allograft (OR = 1.31; CIs = 0.56-3.06) (test for interaction, P = .3) or between patients who did or did not receive a transplant (P = .4). These results were confirmed in an analysis of 186 patients randomized to receive or not receive an autograft in first CR and in a donor-versus-no donor analysis of 683 patients to assess the role of allograft (for latter, FLT3/ITD(-) OR = 0.70, CIs = 0.53-0.92; FLT3/ITD(+) OR = 0.59, CIs = 0.40-0.87; test for interaction, P = .5). These results suggest that at present there is no strong evidence that FLT3 status should influence the decision to proceed to transplantation.

RAS mutation in acute myeloid leukemia is associated with distinct cytogenetic subgroups but does not influence outcome in patients younger than 60 years.

The pathogenesis of acute myeloid leukemia (AML) involves the cooperation of mutations promoting proliferation/survival and those impairing differentiation. The RAS pathway has been implicated as a key component of the proliferative drive in AML. We have screened AML patients, predominantly younger than 60 years and treated within 2 clinical trials, for NRAS (n = 1106), KRAS (n = 739), and HRAS (n = 200) hot-spot mutations using denaturing high-performance liquid chromatography or restriction fragment length polymorphism (RFLP) analysis. NRAS mutations were confirmed in 11% of patients (126/1106) and KRAS mutations in 5% (39/739). No HRAS mutations were detected in 200 randomly selected samples. Codons most frequently mutated were N12 (43%), N13 (21%), and K12 (21%). KRAS mutations were relatively overrepresented in French-American-British (FAB) type M4 (P < .001). NRAS mutation was over-represented in the t(3;5)(q21 approximately 25;q31 approximately q35) subgroup (P < .001) and underrepresented in t(15;17)(q22;q21) (P < .001). KRAS mutation was overrepresented in inv(16)(p13q22) (P = .004). Twenty-three percent of KRAS mutations were within the inv(16) subgroup. RAS mutation and FLT3 ITD were rarely coexistent (14/768; P < .001). Median percentage of RAS mutant allele assayed by quantitative RFLP analysis was 28% (N12), 19% (N13), 25% (N61), and 21% (K12). RAS mutation did not influence clinical outcome (overall/disease-free survival, complete remission, relapse rate) either for the entire cohort or within cytogenetic risk groups.