Postinduction molecular MRD identifies patients with NPM1 AML who benefit from allogeneic transplant in first remission.

ABSTRACT: Selection of patients with NPM1-mutated acute myeloid leukemia (AML) for allogeneic transplant in first complete remission (CR1-allo) remains controversial because of a lack of robust data. Consequently, some centers consider baseline FLT3-internal tandem duplication (ITD) an indication for transplant, and others rely on measurable residual disease (MRD) status. Using prospective data from the United Kingdom National Cancer Research Institute AML17 and AML19 studies, we examined the impact of CR1-allo according to peripheral blood NPM1 MRD status measured by quantitative reverse transcription polymerase chain reaction after 2 courses of induction chemotherapy. Of 737 patients achieving remission, MRD was positive in 19%. CR1-allo was performed in 46% of MRD+ and 17% of MRD- patients. We observed significant heterogeneity of overall survival (OS) benefit from CR1-allo according to MRD status, with substantial OS advantage for MRD+ patients (3-year OS with CR1-allo vs without: 61% vs 24%; hazard ratio [HR], 0.39; 95% confidence interval [CI], 0.24-0.64; P < .001) but no benefit for MRD- patients (3-year OS with CR1-allo vs without: 79% vs 82%; HR, 0.82; 95% CI, 0.50-1.33; P = .4). Restricting analysis to patients with coexisting FLT3-ITD, again CR1-allo only improved OS for MRD+ patients (3-year OS, 45% vs 18%; compared with 83% vs 76% if MRD-); no interaction with FLT3 allelic ratio was observed. Postinduction molecular MRD reliably identifies those patients who benefit from allogeneic transplant in first remission. The AML17 and AML19 trials were registered at www.isrctn.com as #ISRCTN55675535 and #ISRCTN78449203, respectively.

Molecular, clinical and therapeutic determinants of outcome in NPM1 mutated AML.

Although NPM1-mutated acute myeloid leukemia (AML) carries a generally favorable prognosis, many patients still relapse and die. Previous studies identified several molecular and clinical features associated with poor outcome, however only FLT3-ITD mutation and adverse karyotype are currently used for risk stratification due to inconsistent results and uncertainty around how other factors should influence treatment, particularly given the strong prognostic impact of post-induction measurable residual disease (MRD). Here we analyzed a large group of patients with NPM1mut AML enrolled in prospective trials (NCRI AML17 and AML19, n=1357) to delineate the impact of baseline molecular and clinical features, post induction MRD status and treatment intensity on outcome. FLT3-ITD (HR 1.28, 95%CI 1.01-1.63), DNMT3A (HR 1.65, 95%CI 1.32-2.05), WT1 (HR 1.74, 95%CI 1272-2.38) and non-ABD NPM1 mutations (HR 1.64, 95%CI 1.22-2.21) were independently associated with poorer overall survival (OS). These factors were also strongly associated with MRD positivity. For patients achieving MRD negativity, these mutations (except FLT3-ITD) were associated with an increased cumulative incidence of relapse (CIR) and poorer OS. However, apart from the few patients with adverse cytogenetics, we could not identify any group of MRD negative patients with a CIR >40% or with benefit from allograft in first remission. Intensified chemotherapy with the FLAG-Ida regimen was associated with improved outcomes in all subgroups, with greater benefits observed in the highest risk molecular subgroups.

Successful outcome following allogeneic hematopoietic stem cell transplantation in adults with primary immunodeficiency.

The primary immunodeficiencies (PIDs), rare inherited diseases characterized by severe dysfunction of immunity, have been successfully treated by allogeneic hematopoietic stem cell transplantation (Allo-HSCT) in childhood. Controversy exists regarding optimal timing and use of Allo-HSCT in adults, due to lack of experience and previous poor outcomes. Twenty-nine consecutive adult patients, with a mean age at transplant of 24 years (range, 17-50 years), underwent Allo-HSCT. Reduced-intensity conditioning (RIC) included fludarabine (Flu)/melphalan/alemtuzumab (n = 20), Flu/busulfan (Bu)/alemtuzumab (n = 8), and Flu/Bu/antithymocyte globulin (n = 1). Stem cell donors were matched unrelated donors or mismatched unrelated donors (n = 18) and matched related donors (n = 11). Overall survival (OS), event-free survival, transplant-related mortality (TRM), acute and chronic graft-versus-host disease incidence and severity, time to engraftment, lineage-specific chimerism, immune reconstitution, and discontinuation of immunoglobulin replacement therapy were recorded. OS at 3 years for the whole cohort was 85.2%. The rarer PID patients without chronic granulomatous disease (CGD) achieved an OS at 3 years of 88.9% (n = 18), compared with 81.8% for CGD patients (n = 11). TRM was low with only 4 deaths observed at a median follow-up of 3.5 years. There were no cases of early or late rejection. In all surviving patients, either stable mixed chimerism or full donor chimerism were observed. At last follow-up, 87% of the surviving patients had no evidence of persistent or recurrent infections. Allo-HSCT is safe and effective in young adult patients with severe PID and should be considered the treatment of choice where an appropriate donor is available.

Predictors of recovery following allogeneic CD34+-selected cell infusion without conditioning to correct poor graft function.

Poor graft function is a serious complication following allogeneic hematopoietic stem cell transplantation. Infusion of CD34+-selected stem cells without pre-conditioning has been used to correct poor graft function, but predictors of recovery are unclear. We report the outcome of 62 consecutive patients who had primary or secondary poor graft function who underwent a CD34+-selected stem cell infusion from the same donor without further conditioning. Forty-seven of 62 patients showed hematological improvement and became permanently transfusion and growth factor-independent. In multivariate analysis, parameters significantly associated with recovery were shared CMV seronegative status for recipient/donor, the absence of active infection and matched recipient/donor sex. Recovery was similar in patients with mixed and full donor chimerism. Five -year overall survival was 74.4% (95% CI 59-89) in patients demonstrating complete recovery, 16.7% (95% CI 3-46) in patients with partial recovery and 22.2% (CI 95% 5-47) in patients with no response. In patients with count recovery, those with poor graft function in 1-2 lineages had superior 5-year overall survival (93.8%, 95% CI 82-99) than those with tri-lineage failure (53%, 95% CI 34-88). New strategies including cytokine or agonist support, or second transplant need to be investigated in patients who do not recover.

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.

Wilms' Tumour 1 (WT1) peptide vaccination in patients with acute myeloid leukaemia induces short-lived WT1-specific immune responses.

Wilms' Tumour 1 (WT1) is a zinc finger transcription factor that is over-expressed in acute myeloid leukaemia (AML). Its restricted expression in normal tissues makes it a promising target for novel immunotherapies aiming to accentuate the cytotoxic T lymphocyte (CTL) response against AML. Here we report a phase I/II clinical trial of subcutaneous peptide vaccination with two separate HLA-A2-binding peptide epitopes derived from WT1, together with a pan-DR binding peptide epitope (PADRE), in Montanide adjuvant. Eight HLA-A2-positive patients with poor risk AML received five vaccination cycles at 3-weekly intervals. The three cohorts received 0·3, 0·6 and 1 mg of each peptide, respectively. In six patients, WT1-specific CTL responses were detected using enzyme-linked immunosorbent spot assays and pWT126/HLA-A*0201 tetramer staining, after ex vivo stimulation with the relevant WT1 peptides. However, re-stimulation of these WT1-specific T cells failed to elicit secondary expansion in all four patients tested, suggesting that the WT1-specific CD8(+) T cells generated following vaccination may be functionally impaired. No correlation was observed between peptide dose, cellular immune response, reduction in WT1 mRNA expression and clinical response. Larger studies are indicated to confirm these findings.

Fludarabine, Cytarabine, Granulocyte Colony-Stimulating Factor, and Idarubicin With Gemtuzumab Ozogamicin Improves Event-Free Survival in Younger Patients With Newly Diagnosed AML and Overall Survival in Patients With NPM1 and FLT3 Mutations.

PURPOSE: To determine the optimal induction chemotherapy regimen for younger adults with newly diagnosed AML without known adverse risk cytogenetics. PATIENTS AND METHODS: One thousand thirty-three patients were randomly assigned to intensified (fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin [FLAG-Ida]) or standard (daunorubicin and Ara-C [DA]) induction chemotherapy, with one or two doses of gemtuzumab ozogamicin (GO). The primary end point was overall survival (OS). RESULTS: There was no difference in remission rate after two courses between FLAG-Ida + GO and DA + GO (complete remission [CR] + CR with incomplete hematologic recovery 93% v 91%) or in day 60 mortality (4.3% v 4.6%). There was no difference in OS (66% v 63%; P = .41); however, the risk of relapse was lower with FLAG-Ida + GO (24% v 41%; P < .001) and 3-year event-free survival was higher (57% v 45%; P < .001). In patients with an NPM1 mutation (30%), 3-year OS was significantly higher with FLAG-Ida + GO (82% v 64%; P = .005). NPM1 measurable residual disease (MRD) clearance was also greater, with 88% versus 77% becoming MRD-negative in peripheral blood after cycle 2 (P = .02). Three-year OS was also higher in patients with a FLT3 mutation (64% v 54%; P = .047). Fewer transplants were performed in patients receiving FLAG-Ida + GO (238 v 278; P = .02). There was no difference in outcome according to the number of GO doses, although NPM1 MRD clearance was higher with two doses in the DA arm. Patients with core binding factor AML treated with DA and one dose of GO had a 3-year OS of 96% with no survival benefit from FLAG-Ida + GO. CONCLUSION: Overall, FLAG-Ida + GO significantly reduced relapse without improving OS. However, exploratory analyses show that patients with NPM1 and FLT3 mutations had substantial improvements in OS. By contrast, in patients with core binding factor AML, outcomes were excellent with DA + GO with no FLAG-Ida benefit.

Risk-stratified adoptive cellular therapy following allogeneic hematopoietic stem cell transplantation for advanced chronic lymphocytic leukaemia.

Following reduced intensity-conditioned allogeneic stem cell transplantation (RIC allo-SCT) for chronic lymphocytic leukaemia (CLL), there is an inverse relationship between relapse and extensive chronic graft-versus-host disease (GVHD). We evaluated outcomes in 50 consecutive patients with CLL using the approach of alemtuzumab-based RIC allo-SCT and pre-emptive donor lymphocyte infusions (DLI) for mixed chimerism or minimal residual disease (MRD), with the intention of reducing the risk of GVHD. Forty two patients had high-risk disease, including 30% with 17p deletion (17p-). Of patients who were not in complete remission (CR) entering transplant, 83% subsequently achieved MRD-negative CR. Both MRD detection and uncorrected mixed chimerism were associated with greater risks of treatment failure. Nine of sixteen patients receiving DLI for persistent or relapsed disease subsequently attained MRD-negative CR. With a median follow-up of 4.3 years, 4-year current progression-free survival was 65% and overall survival was 75% (60% and 61% in respectively, patients with 17p-). DLI was associated with a 29% cumulative incidence of severe GVHD and mortality of 6.4%. At last follow-up, 83% of patients in CR were off all immunosuppressive treatment. In conclusion, the directed delivery of allogeneic cellular therapy has the potential to induce durable remissions in high-risk CLL without incurring excessive GVHD.

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.

A randomized comparison of CPX-351 and FLAG-Ida in adverse karyotype AML and high-risk MDS: the UK NCRI AML19 trial.

Liposomal daunorubicin and cytarabine (CPX-351) improved overall survival (OS) compared with 7+3 chemotherapy in older patients with secondary acute myeloid leukemia (AML); to date, there have been no randomized studies in younger patients. The high-risk cohort of the UK NCRI AML19 trial (ISRCTN78449203) compared CPX-351 with FLAG-Ida in younger adults with newly diagnosed adverse cytogenetic AML or high-risk myelodysplastic syndromes (MDS). A total of 189 patients were randomized (median age, 56 years). Per clinical criteria, 49% of patients had de novo AML, 20% had secondary AML, and 30% had high-risk MDS. MDS-related cytogenetics were present in 73% of the patients, with a complex karyotype in 49%. TP53 was the most common mutated gene, in 43%. Myelodysplasia-related gene mutations were present in 75 (44%) patients. The overall response rate (CR + CRi) after course 2 was 64% and 76% for CPX-351 and FLAG-Ida, respectively. There was no difference in OS (13.3 months vs 11.4 months) or event-free survival in multivariable analysis. However, relapse-free survival was significantly longer with CPX-351 (median 22.1 vs 8.35 months). There was no difference between the treatment arms in patients with clinically defined secondary AML or those with MDS-related cytogenetic abnormalities; however, an exploratory subgroup of patients with MDS-related gene mutations had significantly longer OS with CPX-351 (median 38.4 vs 16.3 months). In conclusion, the OS of younger patients with adverse risk AML/MDS was not significantly different between CPX-351 and FLAG-Ida.

FLT3 inhibitors as MRD-guided salvage treatment for molecular failure in FLT3 mutated AML.

Patients with FLT3-mutated AML have a high relapse rate and suboptimal outcomes. Many have co-mutations suitable for measurable residual disease (MRD) monitoring by RT-qPCR and those destined to relapse can be identified by high or rising levels of MRD, called molecular failure.  This provides a window for pre-emptive intervention, but there is little evidence to guide treatment. The use of FLT3 inhibitors (FLT3i) appears attractive but their use has not yet been evaluated.  We identified 56 patients treated with FLT3i at molecular failure.  The FLT3 mutation was an ITD in 52, TKD in 7 and both in 3. Over half of patients had previously received midostaurin. Molecular failure occurred at a median 9.2 months from diagnosis and was treated with gilteritinib (n = 38), quizartinib (n = 7) or sorafenib (n = 11). 60% achieved a molecular response, with 45% reaching MRD negativity. Haematological toxicity was low, and 22 patients were bridged directly to allogeneic transplant with another 6 to donor lymphocyte infusion. 2-year overall survival was 80% (95%CI 69-93) and molecular event-free survival 56% (95%CI 44-72). High-sensitivity next-generation sequencing for FLT3-ITD at molecular failure identified patients more likely to benefit. FLT3i monotherapy for molecular failure is a promising strategy which merits evaluation in prospective studies.

The role of Vδ2-negative γδ T cells during cytomegalovirus reactivation in recipients of allogeneic stem cell transplantation.

Reactivation of cytomegalovirus (CMV) remains a serious complication after allogeneic stem cell transplantation, but the role of γδ T cells is undefined. We have studied the immune reconstitution of Vδ2negative (Vδ2neg) γδ T cells, including Vδ1 and Vδ3 subsets and Vδ2positive (Vδ2pos) γδ T cells in 40 patients during the first 24 months after stem cell transplantation. Significant long-term expansions of Vδ2neg but not Vδ2pos γδ T cells were observed during CMV reactivation early after transplantation, suggesting direct involvement of γδ T cells in anti-CMV immune responses. Similarly, significantly higher numbers of Vδ2neg γδ T cells were detected in CMV-seropositive healthy persons compared with seronegative donors; the absolute numbers of Vδ2pos cells were not significantly different. The expansion of Vδ2neg γδ T cells appeared to be CMV-related because it was absent in CMV-negative/Epstein-Barr virus-positive patients. T-cell receptor-δ chain determining region 3 spectratyping of Vδ2neg γδ T cells in healthy subjects and patients showed restricted clonality. Polyclonal Vδ2neg cell lines generated from CMV-seropositive healthy donors and from a recipient of a graft from a CMV-positive donor lysed CMV-infected targets in all cases. Our study shows new evidence for role of γδ T cells in the immune response to CMV reactivation in transplantation recipients.

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.

Impact of in vivo alemtuzumab dose before reduced intensity conditioning and HLA-identical sibling stem cell transplantation: pharmacokinetics, GVHD, and immune reconstitution.

In vivo alemtuzumab reduces the risk of graft-versus-host disease (GVHD) and nonrelapse mortality after reduced intensity allogeneic transplantation. However, it also delays immune reconstitution, leading to frequent infections and potential loss of graft-versus-tumor responses. Here, we tested the feasibility of alemtuzumab dose deescalation in the context of fludarabine-melphalan conditioning and human leukocyte antigen (HLA)-identical sibling transplantation. Alemtuzumab was given 1-2 days before graft infusion, and dose reduced from 60 mg to 20 mg in 4 sequential cohorts (total n = 106). Pharmacokinetic studies were fitted to a linear, 2-compartment model in which dose reduction led to incomplete saturation of CD52 binding sites and greater antibody clearance. Increased elimination was particularly evident in the 20-mg group in patients who had CD52-expressing tumors at time of transplantation. The 20-mg dose was also associated with greater risk of severe GVHD (acute grade III-IV or chronic extensive) compared with > 20 mg (hazard ratio, 6.7; 95% CI, 2.5-18.3). In contrast, dose reduction to 30 mg on day -1 was associated with equivalent clinical outcomes to higher doses but better lymphocyte recovery at 12 months. In conclusion, alemtuzumab dose reduction to 30 mg is safe in the context of reduced intensity conditioning and HLA-identical sibling transplantation. This trial was registered at http://www.ncrn.org.uk as UKCRN study 1415.

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.

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.