Acute Myeloid Leukemia In Complete Remission Research Articles

Higher TIGIT+ γδ TCM cells may predict poor prognosis in younger adult patients with non-acute promyelocytic AML.

γδ T cells recognize and exert cytotoxicity against tumor cells. They are also considered potential immune cells for immunotherapy. Our previous study revealed that the altered expression of immune checkpoint T-cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) on γδ T cells may result in immunosuppression and is possibly associated with a poor overall survival in acute myeloid leukemia (AML). However, whether γδ T-cell memory subsets are predominantly involved and whether they have a relationship with clinical outcomes in patients with AML under the age of 65 remain unclear. In this study, we developed a multicolor flow cytometry-based assay to monitor the frequency and distribution of γδ T-cell subsets, including central memory γδ T cells (TCM γδ), effector memory γδ T cells (TEM γδ), and TEM expressing CD45RA (TEMRA γδ), in peripheral blood from 30 young (≤65 years old) patients with newly diagnosed non-acute promyelocytic leukemia (also known as M3) AML (AMLy-DN), 14 young patients with AML in complete remission (AMLy-CR), and 30 healthy individuals (HIs). Compared with HIs, patients with AMLy-DN exhibited a significantly higher differentiation of γδ T cells, which was characterized by decreased TCM γδ cells and increased TEMRA γδ cells. A generally higher TIGIT expression was observed in γδ T cells and relative subsets in patients with AMLy-DN, which was partially recovered in patients with AMLy-CR. Furthermore, 17 paired bone marrow from patients with AMLy-DN contained higher percentages of γδ and TIGIT+ γδ T cells and a lower percentage of TCM γδ T cells. Multivariate logistic regression analyses revealed the association of high percentage of TIGIT+ TCM γδ T cells with an increased risk of poor induction chemotherapy response. In this study, we investigated the distribution of γδ T cells and their memory subsets in patients with non-M3 AML and suggested TIGIT+ TCM γδ T cells as potential predictive markers of induction chemotherapy response.

Different Epidemiology and Characteristics of Invasive Pulmonary Aspergillosis in Acute Lymphoid Leukemia in Comparison with AML: Results of a Prospective Multicentric Observational Study of the Rete Ematologica Lombarda (REL)

Introduction. Invasive pulmonary aspergillosis (IPA) still affects the outcome of acute leukemia (AL) patients (pts). Although this aspect is better defined in acute myeloid leukemia (AML) ( Candoni et al, Mycoses 2020), some reports show a dismal impact also in acute lymphoid leukemia (ALL) ( Cattaneo et al, Leuk Lymphoma 2019). However, data concerning the epidemiology of IPA in adult ALL patients, as well as the best antifungal prophylaxis strategy, are still lacking. Therefore, to describe the incidence of IPA in AL pts and their clinical characteristics, as well as the predisposing factors, we carried out a prospective multicentric observational study within the Rete Ematologica Lombarda (REL). Methods. All consecutive AL patients diagnosed during a 3-year period and treated with curative intent were recorded. Diagnosis of IPA was defined according to EORTC/MSG criteria ( Donnelly et al, Clin Infect Dis 2020). Data concerning age, gender, AL type, chemotherapy (cht) received, complete remission (CR) achievement, antifungal prophylaxis, duration of neutropenia and 30-day mortality were collected. Patient's characteristics were analysed by standard descriptive statistics. The Student's t-test was used to compare continuous values; the Fisher's exact test was used to compare differences in percentage. P values below 0.05 were considered statistically significant. Results. Between 2018 and 2020, 207 AL pts (AML: 165, ALL: 42) were evaluated in five Hospitals participating to the REL. Median age of the whole cohort was 58y, and M/F ratio 112/95. After induction treatment, CR was achieved in 153 of the 200 evaluable pts (76.5%), more frequently in ALL (38/42, 90.5%) than in AML pts (115/158, 72.8%) (p=0.0146). During induction cht, proven/probable (p/p) and possible (poss) IPA were diagnosed in 32/207 pts (15.4%), equally divided in p/p and poss (16 each, 7.7%). P/p IPA were more frequent in ALL than in AML (ALL: 7/42, 16.6% vs AML: 9/165, 5.4%; p= 0.0235); similarly, considering also poss IPA, incidence was higher, even if not significantly (9/42, 21.4% vs 23/165, 13.9%; p=0.2374). Among ALL pts, IPA was more frequent in pts not achieving CR (2/4, 50%) than CR pts (7/38, 18%), whereas in AML CR achievement did not influence IPA frequency (CR group: 15/115, 13%; no CR group: 7/43: 16.5%). ALL pts were uniformly treated with pediatric-like protocols; in AML pts no statistically significant differences in IPA incidence were observed according to cht received (3+7/like: 13/81, 16%; 3+7+midostaurin: 2/27, 7.4%; FLAG-Ida: 3/21, 14.3%; CPX-351: 4/19, 21%). Antimould prophylaxis was protective against IPA in 202 evaluable pts (Yes: 22/171, 12.9%; No: 10/31, 32.3%, p=0.0134). Among ALL pts only 1 out of 15 (6.7%) receiving antimould prophylaxis developed IPA, as compared to 8/27 (29.6%) of those not receiving antimould prophylaxis (p=0.1235). All but two AML pts developing IPA were on posaconazole prophylaxis. Both mean age and duration of neutropenia were significantly lower in ALL than in AML pts (46.02y vs 57.75y, p<0.0001 and 16.66d vs27.96d, p<0.0001); however, an impact of mean duration of neutropenia on IPA incidence was observed only in ALL pts (IPA group: 24.38d; no IPA group: 14.79d, p=0.0359). Only 2 AML pts died at 30d: 1 (3.1%) out of 32 pts with IPA and 1 (0.6%) out of 175 pts without. No early death was observed in ALL pts. Table 1 summarizes the clinical characteristics of AL pts in relation to IPA. Conclusions. Despite younger age, shorter duration of neutropenia and higher percentage of CR achievement, IPA, particularly p/p, was more frequently observed in ALL pts than AML during induction cht. High dose induction cht seems to have no impact in IPA incidence in AML pts. On the other hand, intensive cht ALL protocols, including high doses of dexamethasone, could have a role in IPA development, as well as the lack of antimould prophylaxis, which is confirmed as a predictive factor for IPA. Therefore, studies with antimould agents as prophylaxis against aspergillosis in ALL pts during inductions would be desirable.

Identifying the Optimal Conditioning Intensity of Hematopoietic Stem Cell Transplantation in Patients with Acute Myeloid Leukemia in Complete Remission

Background Allogeneic hematopoietic stem cell transplantation (HSCT) as consolidation therapy is considered a curative option for acute myeloid leukemia (AML) in complete remission (CR) to prevent relapse. Many studies have investigated the optimal conditioning intensity, comparing reduced-intensity conditioning regimen (RIC) and myeloablative conditioning regimen (MAC) for patients with AML in CR. However, inconsistent results have been observed, possibly due to selection biases and significant heterogeneous treatment effects when focusing on average treatment effect. Some studies have attempted to assess treatment effects in specific subgroups, highlighting the heterogeneity conditioning intensity's impact. However, these analyses typically examined one factor at a time, overlooking potential multiple-factor combinations that might contribute to such heterogeneity.To address these limitations and surpass conventional comparison studies and subgroup analyses, machine-learning methods for group identification have been developed. In this study, we aimed to identify patient groups in which MAC or RIC had a superior PFS in patients with AML in CR using a machine-learning approach. Methods Our study included 3273 patients aged 40-69 years with AML in CR from the TRUMP2. These patients were divided into training (N=2020) and validation cohorts (N=1253). In the training cohort, we employed a group identification model to identify patient subsets. Preprocessing of variables was performed to ensure the ease of use in clinical practice. We modeled the magnitude of interaction between treatment and covariates without the main effect of covariates, thus leading to the construction of a scoring system that measures the individualized-treatment effect. The objective variable was set as 1-year PFS, and the interaction effects between treatment and covariates were estimated using a regression model with propensity score weight and LASSO regularization. The sum of the interaction term between treatment and covariates was defined as the “Conditioning Intensity Score”. Patients were categorized into high-score and low-score groups using a cut-off level of median in patients who received RIC. Subsequently, in the validation cohort, we estimated the average treatment effect of RIC compared to MAC for each group with hazard ratios (HR) using inverse treatment probability weight analysis. Results In the training cohort, the median age was 55 years (IQR, 48-61 years), with 1110 (55%) male patients. Among them, 666 (33%) received RIC, while the others underwent MAC. Conditioning Intensity Model was developed and consistent with followings; age over 49 (1.4 points), age over 49 and male (0.8 points), age over 49 and poor karyotype (0.5 point), age over 49 and cord blood transplantation (0.9 points), poor karyotype (-1.6 points), poor karyotype and primary induction failure (8.0 points), poor karyotype and bone marrow transplantation (-5.3 points), cord blood transplantation and hematopoietic cell transplant comorbidity index over 2 (2.1 points) and cord blood transplantation and extra bone marrow involvement (-8.6 points). The cut-off level was set as 2.2. The weighted HR of RIC on 1-year PFS was 0.56 (95%CI; 0.40-0.79, p < 0.001) in the high-score group and 1.23 (95%CI: 0.97- 1.57, p=0.094) in the low-score group. In the entire validation cohort, there were no significant differences in PFS between the RIC and MAC (67.7% [95% CI: 62.1-70.3%] in the RIC group vs. 70.3% [95% CI: 67.2-73.1%] in the MAC group, P=0.318). Patients were divided into two groups based on Conditioning Intensity Score. In the high-score group, the 1-year PFS was 73.9% (95% CI: 65.0-80.9%) in the RIC group vs. 66.2% (95% CI: 58.3-73.0%) in the MAC group ( P=0.181), with a weighted HR of 0.69 (95% CI: 0.43-1.11) for PFS in the RIC group compared to that the MAC group ( P=0.125) (Figure left). Conversely, in the low-score group, the 1-year PFS was 63.7% (95% CI: 56.3-70.2%) in the RIC group vs. 71.2% (95% CI: 67.8-74.2%) in the MAC group ( P=0.031), with a weighted HR of 1.35 (95% CI: 0.96-1.89) in the RIC group compared to the MAC group (log-rank P=0.086) (Figure right). Conclusion In conclusion, we have demonstrated the feasibility of utilizing a machine learning method for subgroup identification of conditioning regimens in patients with AML in CR. This established model holds promise for designing patient-specific regimens.

Comparable Survival for Fludarabine and Treosulfan Vs. Fludarabine and TBI As Conditioning Regimens in Patients with AML and MDS Undergoing Allogeneic Stem Cell Transplantation

Introduction Allogeneic stem cell transplantation (alloSCT) is a standard treatment option for patients (pts) with acute myeloid leukemia (AML) and myelodysplastic neoplasia (MDS) with relapse remaining the main cause of treatment failure. For AML pts transplanted in complete remission (CR) or MDS pts, the combination of fludarabin with fractionated total body irradiation (8 GyTBI, “FluTBI”) is an established conditioning regimen. Based on the favorable outcome data reported for fludarabine/treosulfan (“FluTreo”), especially for older and/or comorbid pts, this regimen has become a reliable dose reduced conditioning therapy prior to alloSCT and a valuable alternative to other TBI or chemotherapy-based regimens. Methods We conducted a retrospective analysis of 215 pts with AML in CR and 96 pts with MDS who underwent first alloSCT between 2010 and 2022 with a median follow-up (FU) of survivors of 34 months (range: 1 - 124 months). After conditioning therapy with FluTreo (150mg/m 2 fludarabin, 30g/m 2 treosulfan) or FluTBI (120mg/m 2 fludarabin, 4x2Gy TBI) pts were allografted from matched related (MRD; n=65), 10/10 human leukocyte antigen (HLA)-matched unrelated (MUD; n=200), or 9/10 HLA-matched unrelated donors (MMUD; n=46). For AML pts, any detectable molecular/cytogenetic alteration prior to alloSCT was classified as potentially measurable residual disease. Propensity score matching allowed us to identify 55 pair-matched FluTBI and FluTreo pts (nearest matching for age at alloSCT, sex, disease, HCT-CI score, ECOG, with a caliper distance of 0.5). Results Before matching, pts characteristics differed significantly between FluTreo and FluTBI in terms of age at alloSCT (median 64 vs. 47 yr), underlying disease (AML: 59% vs. 88%), ECOG (≥2 15% vs. 6%) and HCT-CI-Score (≥3 50% vs. 25%). In the AML subgroup, pts with measurable residual disease prior to alloSCT were significantly more frequent in the FluTreo group (68% vs. 48%, p<.001). GvHD prophylaxis consisted of calcineurin inhibitor and methotrexate/MMF in all pts. In addition, pts transplanted from unrelated donors received ATLG (Neovii). In the FluTreo group, more patients were transplanted from MRDs (24% vs. 14%, p.013). After matching, patient characteristics were well balanced with respect to age (median 57 vs. 55 yr, p.11), disease (AML/MDS) ( p .8), HCT-CI score ( p .24), sex ( p .85), donor types (MRD: 25.5% vs. 14.5%, MUD: 58.2% vs. 67.3%, MMUD: 16.4% vs. 18.2%, p .36) and measurable residual disease prior to alloSCT ( p .58). Risk groups for AML and MDS pts at diagnosis were comparable across both groups (ELN for AML and IPSS-R for MDS). Relapse incidences (RI) at 3 yrs were similar for FluTreo and FluTBI pts in the unmatched (25% vs. 25%, p.8) and matched cohorts (28% vs. 28% p .7). The non-relapse mortality (NRM) rate was <10% trending towards a lower NRM rate for the FluTBI group in the unmatched cohorts (at 1 yr: 8% vs. 5%, p .07) and a significant difference after matching (at 1 year 2% vs. 9% p .03). Overall survival (OS) for the whole cohort showed a trend towards an inferior outcome for FluTreo (3 yr: 71% vs. 79% p .06), after matching, no difference could be observed (3 yr: 80% vs. 71% p .54). Leukemia-free survival (LFS) was comparable across conditionings in the unmatched (59% vs. 66% p .42) and the matched cohorts (70% vs. 59% p .42). Irrespective of conditioning therapy, pts with measurable residual disease prior to alloSCT had comparable OS and LFS. However, for the matched cohort, pts with measurable disease treated with FluTreo showed a trend towards an inferior OS and LFS after 3 yr in contrast to FluTBI pts (OS: 66% vs. 80%, p .13; PFS: 58% vs. 67%, p .3). No significant differences could be observed in the incidence of acute onset graft versus host disease (GvHD) grade 2-4 at day 100 after alloSCT. Similarly, occurrence of chronic GvHD at 3 yr did not differ in the matched cohorts (32% vs. 43%, p .4). Conclusion With a meaningful median follow up of 34 months, our data for dose-reduced conditioning with either FluTreo or FluTBI highlight favorable and comparable survival rates of >70%. RI and NRM were low for both regimens. For pts with AML, who received FluTBI, measurable residual disease prior to alloSCT seemed to have no effect on outcome, while for the FluTreo cohort measurable residual disease prior to alloSCT showed a trend towards lower survival rates. This finding should be evaluated in a prospective trial comparing TBI and treosulfan based conditioning.

Influence of conditioning regimen intensity on outcomes post-allogeneic hematopoietic cell transplantation for acute myeloid leukemia in complete morphological remission.

The literature comparing outcomes between myeloablative (MAC) and reduced intensity conditioning (RIC) for acute myeloid leukemia (AML) is conflicting. We retrospectively analyzed 451 patients who underwent allogenic hematopoietic cell transplantation (alloHCT) for AML in complete remission (CR) with either RIC (n = 331) or MAC (n = 120) with the use of dual T-cell depletion as graft-versus-host disease (GVHD) prophylaxis. Univariate analysis demonstrated nonrelapse mortality (NRM) at 2 years was 19.1% for MAC and 22.5% for RIC (p = .44). Two-year cumulative incidence of relapse (CIR) was 19.8% for MAC and 24.5% for RIC (p = .15). Two-year overall survival (OS) was 61% and 53% for MAC and RIC, respectively (p = .02). Two-year graft-versus-host disease relapse-free survival (GRFS) was 40.8% for MAC and 33.7% for RIC (p = .30). A propensity score-matched analysis was done matching patients for age, HLA match, in vivo T-cell depletion, and Disease Risk Index (DRI). Two-year OS was 67% for MAC, 66% for RIC (p = .95). A subgroup analysis identified that matched related donor transplants benefit from MAC with OS at 2 years 82.6% versus 57.3% for RIC (p = .006). In the matched-related donor setting, MAC regimens may offer superior survival. Overall, for our cohort of predominantly in vivo T-cell depleted patients the outcomes of MAC and RIC were similar.

HLA-mismatched micro-transplantation as post-remission treatment compared to autologous hematopoietic stem cell transplantation or consolidation with single agent cytarabine for favorable-or intermediate-risk acute myeloid leukemia

ABSTRACTObjectivesOptimal post-remission treatment for individual favorable and intermediate risk acute myeloid leukemia (AML) patients has not yet been established. Human leukocyte antigen (HLA)-mismatched stem cell microtransplantation (MST), may improve outcomes and avoid graft-versus-host disease in patients with first complete remission of AML.MethodsWe retrospectively analyzed the efficacy, safety, and survival of 63 patients with favorable- or intermediate-risk AML who received MST, autologous stem cell transplantation (ASCT), or cytarabine single agent (CSA) as post-remission treatment from January 2014 to August 2021.ResultsThe neutrophil recovery time was shorter in the MST group than in the CSA group. The 2-year cumulative incidences of relapse in the MST, ASCT, and CSA groups were 27.27%, 29.41%, and 41.67%, respectively. During follow-up, 21 patients (33.30%) died of relapse, including six (9.52%), five (7.94%), and 10 (15.84%) in the MST, ASCT, and CSA groups, respectively. The estimated 2-year overall survival (OS) and relapse-free survival (RFS) were 62.20% vs. 50.00% (P = 0.101) and 57.10% vs. 50.00% (P = 0.136), in the >60 years MST and CSA groups (P = 0.101). The estimated 2-year OS was 100%, 66.20%, and 69.10% in the MST, ASCT, and CSA groups (MST vs CSA, P = 0.044), meanwhile, the estimated 2-year RFS was 100%, 65.40%, and 59.80% in patients ≤60 years.ConclusionMST, ASCT, and CSA are acceptable post-remission treatments for patients with favorable- and intermediate-risk AML and may not only improve the prognosis of the elderly but also prolong the OS and RFS of favorable- or intermediate-risk patients ≤60 years.

Hereditary angioedema with an acute attack resolved after bone marrow transplantation for acute myeloid leukemia: a case report

BackgroundHereditary angioedema (HAE), which is caused by C1-inhibitor (C1-INH) deficiency or dysfunction, is a rare and potentially life-threatening disease. In patients with HAE, excess production of bradykinin causes acute unpredictable recurrent attacks of angioedema in localized regions, including the larynx and intestines. Given the fact that HAE is an autosomal dominant disease, C1-INH produced in patients with HAE is 50% of that produced in healthy individuals. However, most patients with HAE present plasma C1-INH function of < 25% owing to the chronic consumption of C1-INH by kallikrein–kinin, contact, complement, coagulation, and fibrinolysis cascades. Recently, several therapeutic options have been developed for acute attacks and prophylaxis in the treatment of HAE; however, currently, there is no curative therapy for HAE.Case presentationHere we report the case of a 48-year-old male patient who presented with a long-standing history of HAE and underwent bone marrow transplantation (BMT) for acute myeloid leukemia (AML) at the age of 39 years and has been in complete remission of AML and HAE thereafter. Notably, after BMT, his C1-INH function gradually increased as follows: < 25%, 29%, 37%, and 45.6%. Since his 20 s, he intermittently presented with an acute attack of HAE once every 3 months from the initial attack. Further, after undergoing BMT, the number of acute attacks decreased to twice within 4 years until the age of 45 years, and subsequently, the patient has been free of acute attacks. C1-INH is mainly synthesized by hepatocytes, but it is known to be partially produced and secreted from peripheral blood monocytes, macrophages, endothelial cells, and fibroblasts. We speculate that the C1-INH function may be increased by extrahepatic production of C1-INH, possibly synthesized by differentiated cells derived from hematopoietic and mesenchymal stem cells after BMT.ConclusionsThis case report supports efforts to focus on extrahepatic production of C1-INH in the next strategy of new treatment development for HAE.

Primary Cancer Matters in Therapy-related Myeloid Neoplasm Patients Receiving Allogeneic Hematopoietic Cell Transplantation: A Study From the Chronic Malignancies Working Party of the EBMT

Primary Cancer Matters in Therapy-related Myeloid Neoplasm Patients Receiving Allogeneic Hematopoietic Cell Transplantation: A Study From the Chronic Malignancies Working Party of the EBMT

Loss of CD28 expression associates with severe T-cell exhaustion in acute myeloid leukemia.

Despite accumulated evidence in T-cell exhaustion in acute myeloid leukemia (AML), the immunotherapeutic targeting exhausted T cells such as programmed cell death protein 1 (PD-1) blockade in AML failed to achieve satisfying efficacy. Characteristics of exhausted T cells in AML remained to be explored. Phenotypic analysis of T cells in bone marrow (BM) using flow cytometry combining senescent and exhausted markers was performed in de novo AML patients and healthy donors as well as AML patients with complete remission (CR). Functional analysis of T-cell subsets was also performed in de novo AML patients using flow cytometry. T cells experienced a phenotypic shift to terminal differentiation characterized by increased loss of CD28 expression and decrease of naïve T cells. Additionally, lack of CD28 expression could help define a severely exhausted subset from generally exhausted T cells (PD-1+TIGIT+). Moreover, CD28- subsets rather than CD28+ subsets predominantly contributed to the significant accumulation of PD-1+TIGIT+ T cells in AML patients. Further comparison of de novo and CR AML patients showed that T-cell exhaustion status was improved after disease remission, especially in CD28+ subsets. Notably, higher frequency of CD28-TIGIT-CD4+ T cells correlated with the presence of minimal residual disease in AML-CR group. However, the correlation between CD28- exhausted T cells and cytogenetic risk or white blood cell count was not observed, except for that CD28- exhausted CD4+ T cells correlated with lymphocyte counts. Intriguingly, larger amount of CD28-TGITI+CD8+ T cells at diagnosis was associated with poor treatment response and shorter leukemia free survival. In summary, lack of CD28 expression defined a severely exhausted status from exhausted T cells. Accumulation of CD28- exhausted T cells was linked to occurrence of AML, and correlated to poor clinical outcome. Our data might facilitate the development of combinatory strategies to improve the efficacy of PD-1 blockade in AML.

Whole Genome Sequencing Comparison of Acute Myeloid Leukemia at Presentation and Remission Predicts Patient Outcome

Background: Acute myeloid leukemia (AML) is the most common leukemia in adults with more than 20,000 new cases in the U.S. each year. While about two-thirds of patients who receive standard induction chemotherapy reach complete remission, about half of those will relapse within two years. There are several known risk factors for relapse including age, cytogenetics, and a few specific mutations; however, there is still a need for better prognostic information to guide therapy and improve patient outcome. Measurement of residual disease has proven helpful in predicting patient outcome. Current approaches for measuring minimal residual disease (MRD) include multiparameter flow cytometry, which can detect abnormal myeloid blast populations based on the expression of cell-surface markers, and targeted molecular assays measuring variant frequency of common mutations. Molecular approaches have higher sensitivity than flow cytometry; however, many targeted assays follow only a single mutation, which does not capture the within patient genomic heterogeneity of AML. In this study, we applied whole genome sequencing (WGS) to study MRD at the time of complete remission in AML, revealing the full spectrum of genomic response to treatment and its correlation with patient outcome. Methods: Bone marrow biopsy or peripheral blood specimens were collected at the time of presentation and remission from 18 patients with AML. All patients were treated with standard-of-care induction chemotherapy. Among the 18 patients, 8 relapsed and 10 did not relapse. Corresponding clinical and outcome data (median follow-up of 26 months) were collected as well as matched flow cytometry data at remission on 11 of 18 cases. Deep whole genome sequencing (104x median depth) was performed on DNA from paired presentation and remission specimens. Single nucleotide variants were called from both samples, with a mutation caller capable of identifying low frequency variants. Quality filters and population frequency filters were applied to the variant set. Remaining variants were then classified into five classes based on their variant frequency pattern. Results: Figure 1A shows variant frequencies for two representative patients. Variants can be broadly classified into five classes: (A) likely germline variants, clustered at the expected variant frequency of 0.5 (grey); (B) leukemia-specific variants which are dramatically reduced in frequency upon treatment (dark red); (C) leukemia-enriched variants which are incompletely reduced at remission (bright red); (D) remission-specific variants that were not detectable at presentation but are prevalent at remission (dark blue); and (E) remission-enriched variants which have increased in frequency at remission (light blue). The proportion of variants belonging to classes B, C, D, and E correlates with both MRD status and patient outcome, as shown in Figure 1B. Patients with a higher ratio of leukemia-specific to leukemia-enriched variants are more likely to be alive at one year. Patients who relapsed tend to have a higher number of leukemia-enriched variants, which may represent variants in pre-leukemic cells or leukemic stem cell populations. Patients with a poor outcome also tend to have a higher number of new and increasing remission-specific and remission-enriched variants. This suggests that their remission state has not returned to normal, but is re-populated with clonal populations of cells containing a significant number of somatic variants. The few discrepancies between WGS and MRD flow cytometry suggest that the WGS data could be more predictive of patient outcome. For example, there are two MRD positive cases in which the WGS profiles suggest a good outcome, consistent with the actual patient outcome. In two other cases, neither flow cytometry nor WGS profiles predicted the patients’ relapses. We find that DNMT3A, TET2, ASXL1 mutations have high frequencies at both time points and cluster outside of the labeled classes of variants, whereas other driver gene mutations such as NPM1, NRAS, and IDH1 cluster with the leukemia-specific variants. Conclusion: Our study shows promising evidence for the utility of WGS to predict patient outcome in AML in the setting of intensive chemotherapy. Combining WGS with targeted deep sequencing approaches may further increase the predictive power of genomic approaches. Collection and analysis of a larger case series is ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immuno-AML, a Novel Immunogenomic Classifier Predicting Chemotherapy Response in Pediatric Patients with AML

Introduction Acute myeloid leukemia (AML) represents 20% of pediatric leukemias. Despite therapeutic improvements, ∼30% of children die due to therapy-related toxicity and relapse. In solid tumors, the density and functional orientation of the tumor microenvironment (TME) influence prognosis and response to chemotherapy and immunotherapy. Defining immunologic features associated with unfavorable response to standard treatment in pediatric AML might improve stratification systems that can be used for experimental interventions and personalized approaches. In this study, we investigated the potential mechanisms and effect of immune response and evasion in bone marrow (BM) of AML pediatric patients through an integrative gene expression analysis. Methods Thirty-two BM aspirates were collected from pediatric patients with non-promyelocytic AML at diagnosis in 3 different hospitals in Qatar, Pakistan, and Italy. Patients were assigned to 2 prognosis-related groups: patients who maintained a complete remission (CR) after first line of chemotherapy (sustained CR) vs patients who progressed/relapsed and/or obtained CR after hematopoietic stem cells transplantation (HSCT) (non-sustained CR). BM mRNA samples were analyzed with RNA sequencing (RNA-Seq) and/or NanoString's PanCancer IO 360 Gene Expression research panel. In particular, mRNA of 25 samples (Cohort 1, discovery set) were analyzed by NanoString's PanCancer IO 360. Subsequently, a subset of samples from Cohort 1 (n=19), was analyzed by RNA-Seq for technical validation. Cohort 1 results were then used to build a predictive classifier that was tested on Cohort 2 samples (n=7, validation set), analyzed with RNA-Seq. Results Gene expression analysis of 25 samples (Cohort 1, sustained CR, n=6; non-sustained CR, n=19) using NanoStringassay revealed 67 significant differentially expressed genes (DEGs; p < 0.05, LIMMA) among sustained CR vs non-sustained CR groups. Patients with sustained CR displayed coherent overexpression of immune genes related to IFN activation (IFIT3, IFITM1, IFI27, STAT1, GBP1), cytotoxic function (PRF1, GNLY), antigen presentation and B cell functions (TAP1, CD19), and T cell infiltration (CD3D, CD8A), consistent with a favorable T helper 1 (Th1)-cytotoxic polarization of the TME. This pattern was not observed in non-sustained CR patients, which overexpressed genes consistent with macrophage-myeloid suppression (CD68, TREM1) and intrinsic oncogene signaling linked with T cell exclusion and immune suppression (WNT5A, WNT-β- catenin pathway, and EFGR) (Fig1 A,B). This 67-gene signature (67GenSig) was technically validated using an orthogonal platform (RNA-Seq) on a subset of 19 patients (sustained CR, n=5; non-sustained CR, n=14) for which sufficient RNA-Seq data was available (65 out of 67 transcripts were available for analysis, 2 transcripts were filtered out after QC). Unsupervised PCA (Fig1 C) based on 67GenSig clearly segregated samples according to outcome, confirming the previous NanoString results. Next, we sought to identify a predictive classifier to precisely discriminate sustained CR from non-sustained CR AML pediatric patients using a minimal number of genes. By applying a Random Forest-based feature selection on 67GenSig transcripts of RNA-Seq data from Cohort 1 samples, we developed a 13-gene classifier (Immuno-AML) that was tested in a new independent dataset (Fig1 D; Cohort 2, n=7: sustained CR, n=4; non-sustained CR, n=3). Overall, genes included in the Immuno-AML predictor substantiated a positive Th1/cytotoxic vs myeloid polarization. The Immuno-AML predictor was able to classify patients in the validation cohort (Cohort 2) in "sustained CR" and "non-sustained CR" prognostic groups with a very good performance achieving 85.71% accuracy, 100% specificity, and 75% sensitivity. Conclusion Our data suggest that response to conventional therapy in AML pediatric patients is conditional to the presence of a Th1 polarization of the TME while is prevented by myeloid-mediated immune suppression and intrinsic oncogenic signaling. The optimized predictive signature (Immuno-AML) might be tested and/or refined in larger cohort to develop a clinical-grade predictive biomarker. It might help to provide a rational guidance for risk stratification criteria and personalized treatment decision paths, implying the possibility of combining immunotherapy drugs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A uniform conditioning regimen of busulfan, fludarabine, and antithymocyte globulin for allogeneic haematopoietic cell transplantation from haploidentical family, matched sibling, or unrelated donors-A single-centre, prospective, explorative study.

In a prospective, explorative study, the donor-source difference of haploidentical family (HF), matched sibling (MS), and unrelated donors (UD) was evaluated for the outcome of haematopoietic cell transplantations (HCT) in 101 patients with acute myeloid leukaemia (AML) in complete remission (CR). To eliminate compounding effects, a uniform conditioning regimen containing antithymocyte globulin (ATG) was used. After transplantation, there was a significantly higher cumulative incidence of acute graft-versus-host disease (GVHD) in HF-HCT patients (49%, 7%, and 16% for HF-, MS- and UD-HCT respectively; p< 0.001). A quarter of acute GVHD cases observed in HF-HCT patients occurred within three days of engraftment and were characterized by diffuse skin rash, fever, weight gain, and hypoalbuminaemia. This peri-engraftment acute GVHD was not observed in MS-HCT or UD-HCT patients. Additionally, a significantly higher proportion of HF-HCT patients achieved complete donor chimaerism in the peripheral mononuclear cells at one month (88%, 46%, and 69% for HF-, MS- and UD-HCT respectively; p = 0.001). There was no significant difference in engraftment, chronic GVHD, leukaemia recurrence, non-relapse mortality, and patient survival. In patients with AML in CR who received HCT using ATG-containing conditioning, stronger donor-patient alloreactivity was observed in HF-HCT, in terms of increased acute GVHD and higher likelihood of complete donor chimaerism.

Clinical implications and genetical insights of SOX6 expression in acute myeloid leukemia.

Transcription factor SOX6 belongs to Sry-related high-mobility-group box (SOX) family, has been reported to be downregulated and acts as a tumor-suppressor gene in various solid tumors, but in acute myeloid leukemia (AML) is incompletely understood. The SOX6 expression was analyzed between AML patients and normal controls from public data and our research cohort. Correlations between SOX6 expression and clinical, genetic features together with survival were further analyzed. In both public and our present datasets, we demonstrated that SOX6 expression is notably downregulated in AML patients compared with normal controls. Moreover, the expression level of SOX6 was dynamic, along withthediseasestatus. SOX6 was significantly decreased in relapsed/refractory AML compared with complete remission AML. Clinically, SOX6 underexpression was significantly correlated with bone marrow blasts, and WBC counts. Furthermore, decreased expression of SOX6 was more common in core binding factor AML (CBF-AML), rarely found in complex karyotype AML (CK-AML), and correlated with FLT3 mutations. By survival analyses, low-expression of SOX6 was associated with shorter overall survival (OS) and event-free survival (EFS) among cytogenetic normal AML (CN-AML) patients. Moreover, both univariate and multivariate analyses showed that low SOX6 expression was an independent unfavorable prognostic biomarker for CN-AML. Our findings indicated that SOX6 underexpression, as a frequent event in AML, was associated with genetic abnormalities and prognosis in AML. SOX6 might be a valuable biomarker for risk stratification, predictingprognosisand relapse of AML.

MicroRNA-181b-5p insufficiency predicts treatment response failure risk and unfavorable event-free survival as well as overall survival in acute myeloid leukemia patients.

The present study aimed to explore the correlation of microRNA (miR)-181b-5p expression with treatment response and long-term prognosis in acute myeloid leukemia (AML) patients. miR-181b-5p was detected in the bone marrow of 84 AML patients before therapy. After induction therapy, the patients exhibiting complete remission (CR) were recorded. Next, event-free survival (EFS) and overall survival (OS) were calculated. miR-181b-5p had excellent potential to discriminate AML patients from healthy donors [area under the curve (AUC): 0.922, 95% confidence interval (CI): 0.873-0.971)]. In addition, miR-181b-5p expression was decreased in AML patients with the FLT3-ITD mutation (P=0.032) or WT1 mutation (P=0.017) when compared to AML patients without these genetic mutations. Meanwhile, miR-181b-5p expression was negatively correlated with the National Comprehensive Cancer Network (NCCN) risk classification of AML (P=0.036). Furthermore, miR-181b-5p expression was elevated in CR AML patients compared to non-CR AML patients (P=0.030). Moreover, higher miR-181b-5p expression was associated with favorable accumulating EFS (P=0.001) and OS (P=0.024). In addition, higher miR-181b-5p expression was independently associated with better EFS (hazard ratio: 0.698, P=0.012). In conclusion, miR-181b-5p insufficiency is associated with induction therapy response failure, unfavorable accumulating EFS and OS in AML patients.

Post-remission outcomes in AML patients with high hyperleukocytosis and inaugural life-threatening complications.

Patients with hyperleukocytic (HL) acute myeloid leukemia (AML) are at higher risk of early death. Initial management of these patients is challenging, not fully codified and heterogenous. Retrospective studies showed that several symptomatic measures might decrease early death rate but long-term data are scarce. We aimed to analyze whether the therapeutic measures carried out urgently at diagnosis may influence the outcome among HL AML patients having achieved who survived inaugural complications. We retrospectively reviewed all medical charts from patients admitted to Saint-Louis Hospital between January, 1st 1997 and December, 31st 2018 with newly diagnosed AML and white blood cell (WBC) count above 50x109/L. Outcome measures were cumulative incidence of relapse (CIR), treatment-related mortality (TRM) defined as relapse-free death, and overall survival. Univariate and multivariate analyses were performed using Cox proportional hazards models. A total of 184 patients with HL AML in complete remission (CR) were included in this study. At 2 years after CR. 62.5% of patients were alive, at 5 years, cumulated incidence of relapse was 55.8%. We found that every therapeutic measure, including life-sustaining therapies carried out in the initial phase of the disease, did not increase the relapse risk. The use of hydroxyurea for more than 4 days was associated with a higher risk of relapse. At the end of the study, 94 patients (51.1%) were still alive including 23 patients out of 44 aged less than 60 yo that were able to return to work. We show that the use of emergency measures including life sustaining therapies does not come at the expense of a higher risk of relapse or mortality, except in the case of prolonged use of hydroxyurea. Patients with HL AML should be able to benefit from all available techniques, regardless of their initial severity.

Reduced intensity versus non-myeloablative conditioning regimen for haploidentical transplantation and post-transplantation cyclophosphamide in complete remission acute myeloid leukemia: a study from the ALWP of the EBMT.

The optimal conditioning regimen prior haploidentical stem cell transplantation (Haplo-SCT) with post transplantation cyclophosphamide (PT-Cy) for acute myeloid leukemia (AML) remains unknown. A non-myeloablative conditioning (NMAC) regimen (cyclophosphamide + fludarabine + TBI 2 Gy [CyFluTBI]) is a safe approach, but relapse incidence remains high in this setting. Alternatively, a reduced intensity conditioning (RIC) regimen combining thiotepa and reduced-dose busulfan with fludarabine (TBF) may decrease AML relapse. However, an excess of toxicity may counterbalance this potential benefit. We retrospectively compared CyFluTBI vs. TBF in CR AML patients who underwent Haplo-SCT with PT-Cy, in two different populations based on age. We analyzed 490 patients. In patients aged <60 years (n = 203), we observed a higher RI (HR = 3.59, 95% CI = 1.75-7.37, p < 0.01), lower LFS (HR = 1.98, 95% CI = 1.22-3.22, p < 0.01) and lower OS (HR = 1.73, 95% CI = 1.04-2.88, p = 0.04) in the CyFluTBI group, without significant difference in NRM. In older patients (n = 287), we observed that conditioning regimen did not significantly influence LFS (HR = 0.90, 95% CI = 0.56-1.44, p = 0.65), OS (HR = 0.81, 95% CI = 0.49-1.32, p = 0.39) and RI (HR = 1.78, 95% CI = 0.90-3.50, p = 0.10), but showed that CyFluTBI was associated with a significantly lower risk of NRM (HR = 0.48, 95% CI = 0.25-0.92, p = 0.03). Thus, younger patients seem to benefit from conditioning intensification from CyFluTBI to TBF regimens prior PT-Cy Haplo-SCT for CR AML, while older ones do not.

P583: PATIENTS AT HIGH RISK OF RELAPSE POST-TRANSPLANT: A PHASE 1 STUDY DESIGN WITH A NOVEL TREATMENT STRATEGY USING THE ESTIMAND FRAMEWORK

Background: The risk of disease relapse in patients (pts) allografted for acute myeloid leukemia (AML) is high and remains the main cause of transplant failure. The great majority of pts destined to relapse post-allogeneic stem cell transplant (SCT) will do so within the first 12 months and survival after early relapse is dismal. Therefore, strategies to prevent relapse are urgently required. A potent graft-versus-leukemia (GvL) effect is exerted after SCT, and strategies that may augment a GVL effect have the potential to reduce relapse risk post-SCT. Siremadlin (HDM201) is a novel investigational MDM2 inhibitor with single-agent (SA) anti-AML activity. MDM2 inhibitors, including siremadlin, possess potent immunomodulatory effects (IME) in murine solid tumor and AML models. The post-allogeneic SCT setting is ideal for investigating the IME of MDM2 inhibition on enhancing GvL effect. Here we present a phase Ib/II proof-of-concept study designed to assess safety and preliminary efficacy of a treatment strategy with siremadlin as monotherapy and, subsequently, in combination with donor lymphocyte infusions (DLIs) for AML in post-SCT setting using the estimand framework (ICH E9 (R1) addendum). Aims: The objective of the planned treatment strategy is to enhance GvL post-SCT by siremadlin as monotherapy and in combination with DLIs. Due to the risk of Graft-vs-Host Disease (GvHD) with DLI early after SCT, siremadlin will be given as priming monotherapy to prevent early relapse prior to starting siremadlin/DLI combination therapy. SA siremadlin maintenance after the combination therapy is intended to prolong and enhance the GvL reaction to eradicate residual leukemic blasts. Methods: The estimand framework mandates a careful definition of the experimental intervention according to the clinical question of interest. In this trial, the experimental intervention follows a treatment strategy: Patients (pts) with AML in complete remission (CR) post-SCT but at high risk for relapse based on pre-SCT risk factors will start with siremadlin priming monotherapy. Pts who tolerate the siremadlin dose with no evidence of GvHD are eligible to start siremadlin/DLI combination therapy. After combination therapy, pts may continue with SA siremadlin maintenance. Depending on the treatment journey, pts may receive only a part of the intervention’s sequence (e.g., pts in siremadlin priming monotherapy who are not eligible for DLI combination will continue the priming part). The clinical questions addressed in this trial are to determine the recommended dose (RD) of siremadlin across all parts of the treatment strategy and to assess preliminary efficacy regardless of the parts of the treatment strategy pts received. The doses of interest were preselected based on SA studies and in pts who received siremadlin after SCT. Results: The proposed approach is tailored to investigate safety and efficacy of the treatment strategy. The RD of siremadlin will be determined over 2 parts: A Bayesian logistic regression model will guide dose finding of siremadlin during priming (Cycle 1) and a Bayesian time-to-DLT model will assess the siremadlin dose for combination. Efficacy will be assessed by proportion of pts remaining in CR for at least 6 months. Image:Summary/Conclusion: In clinical practice, treatment decisions follow a strategy. The estimand framework guided the design of a clinical study in AML post SCT to conceptualize the assessment of a treatment strategy. Accordingly, safety and efficacy of the treatment strategy as a whole will be assessed taking into account different patient journeys.

Prognostic factors and impact of CMV reactivation on acute myeloid leukemia patients after HLA-matched myeloablative allogeneic stem cell transplantation in a high CMV prevalence country

IntroductionRelapse of acute myeloid leukemia (AML) after allogeneic stem cell transplantation (allo-SCT) leads to dismal outcomes. This study aimed to identify high-risk patients and explore the effects of cytomegalovirus (CMV) reactivation in a high CMV-seropositive population. MethodsThe study involved a single-center retrospective cohort in Thailand, analyzing clinical risk factors and CMV-mediated immune responses, correlated with transplant outcomes in AML patients. ResultsEighty-five patients with AML in complete remission (CR) undergoing HLA-matched myeloablative allo-SCT between 2011 and February 2021 were enrolled. The relapse rate was 27.1% with the median time of 7 months after transplantation. The 3-year relapse-free-survival (RFS) and overall-survival (OS) were 72.2% and 80.8%, respectively. The disease status (>CR1) and absence of chronic graft-versus-host disease (cGVHD) were independently significant adverse prognostic factors of RFS and OS. Ninety-two percent of recipient-donor pairs were both CMV seropositive. The CMV reactivation occurred in 54.1% of the patients. The clinically significant CMV infection rate was 49.4%. No CMV syndrome/disease or CMV-related mortality occurred. One-year cumulative incidence of relapse among CMV-reactivation and non-reactivation groups were 14.3% and 25.6%, respectively, without a statistically significant difference. Transplantation-related mortality was 11.1%. ConclusionsThe transplantation beyond CR1 and absence of cGVHD are powerful prognostic factors associated with inferior RFS and OS. In a high CMV prevalence country, there appears to be no impact of CMV reactivation on relapse in AML patients undergoing an allo-SCT.

Increased TOX expression concurrent with PD-1, Tim-3, and CD244 expression in T cells from patients with acute myeloid leukemia.

T cell dysregulation is a common event in leukemia. Recent findings have indicated that aberrant expression of immune checkpoint proteins may be associated with disease relapse and progression in acute myeloid leukemia (AML). TOX, a transcription factor in the HMG-box protein superfamily, was found to be a potential target for immunotherapy not only in solid tumors but also in hematological malignancies. However, little is known about TOX expression and co-expression with immune checkpoint proteins or the exhausted phenotype in the T cell subsets in AML. Thus, in this study, we analyzed TOX expression and co-expression with PD-1, Tim-3, and CD244 in T cells. TOX expression and co-expression with PD-1, Tim-3, and CD244 in CD3+, CD4+, regulatory T (Treg), and CD8+ T cells were analyzed by multi-color fluorescent flow cytometry in peripheral blood (PB) and bone marrow (BM) samples from patients with de novo AML and AML in complete remission (CR) and healthy individuals (HIs). A significantly increased percentage of TOX+CD3+, CD4+, and CD8+ T cells was found in PB from patients with de novo AML in comparison with HIs. Double-positive TOX+CD244+, TOX+PD-1+, and TOX+Tim-3+ T cells markedly increased in the CD3+, CD4+, and CD8+ T cell populations in de novo AML patients compared with HIs, and similar trends were demonstrated for TOX+Tim-3+CD3+/CD4+/CD8+ T cells in de novo AML compared with AML-CR patients. In addition, the number of TOX+, TOX+PD-1+, and TOX+Tim-3+Treg cells significantly increased in de novo AML patients compared with HIs, and TOX+PD-1+Treg cells were higher in de novo AML compared with AML-CR patients. Moreover, TOX positively correlated with Tim-3 expression in CD8+ and Treg cells, and a positive correlation between the expression of TOX+ CD4+ and CD244+CD4+ T cells was found. Furthermore, an increased percentage of TOX+Tim-3+ T cells in BM was also found in de novo AML patients compared with HIs. Increased TOX concurrent with PD-1, Tim-3, and CD244 in T cells may contribute to T cell exhaustion and impair their function in AML. Such exhausted T cells may be partially revised when AML patients achieve CR after chemotherapy. TOX may be considered a potential target for reversing T cell exhaustion and improving T cell function in AML.

Longitudinal Next Generation Sequencing Reveals the Clonal Hierarchy of IDH Mutated Clones and Impact on Survival in NPM1 Mutated AML

Longitudinal Next Generation Sequencing Reveals the Clonal Hierarchy of IDH Mutated Clones and Impact on Survival in NPM1 Mutated AML