Acute Myeloid Leukemia Patients In Remission Research Articles

A Phase I Trial of Activated Donor-Derived Marrow Infiltrating Lymphocytes to Treat Relapsed Hematologic Malignancies after Allogeneic Transplantation with Post-Transplantation Cyclophosphamide As Graft-Versus-Host Disease Prophylaxis

Background: Donor lymphocyte infusion (DLI) or a second allogeneic transplant (HCT) are the only curative treatments for hematologic malignancies relapsing after initial HCT. Both approaches lack significant efficacy and may cause considerable toxicity. Post-transplant cyclophosphamide (PTCy) used as graft-versus-host disease (GVHD) prophylaxis eliminates alloreactive T cells and enriches for tumor-specific T cells in the bone marrow. We hypothesized that expanded, donor-derived marrow infiltrating lymphocytes (alloMILs) collected post-HCT could restore antitumor immunity in relapsed recipients without the additional GVHD and toxicity seen with DLI or second HCT. Methods: We conducted a phase I dose-escalation trial (NCT02342613) of expanded alloMILs to treat bone marrow infiltrating hematologic malignancies relapsing after HCT with PTCy. Key inclusion criteria were age ≥ 18 years; relapsed hematologic malignancy ≥ 6 months after HLA matched or mismatched HCT with PTCy; donor CD3 + chimerism ≥ 30% in the peripheral blood; and lack of immunosuppressant use for 2 weeks prior to alloMILs harvest. Enrolled patients underwent salvage chemotherapy prior to or after alloMILs harvest to control the disease during the expansion process. Harvested bone marrow mononuclear cells were co-incubated with anti-CD3/CD28 beads and IL-2 for 7 to 14 days until the target CD3 + dose was reached. Given the differential risk of GVHD with matched versus mismatched DLI, dosing progressed from 1 x 10 7 to 5 x10 7 CD3 + cells/kg for matched HCT recipients and 1 x 10 6 to 5 x10 7 CD3 + cells/kg for mismatched HCT recipients. The primary endpoint was the feasibility of the product, defined as: successful harvest; expansion; infusion; and absence of grade III-IV acute GVHD for 90 days after infusion. Evaluation for HLA loss was performed using next-generation sequencing (NGS) of HLA loci. Single cell RNA sequencing (scRNAseq) was performed using 10x Genomics technology, Cell Ranger (v6.0.2), and Seurat (v4.3.0). Results: Thirty-two patients enrolled and underwent bone marrow harvest. Two patients were inaspirable, and five patients were not expanded due to low cell counts or high tumor volume. Twenty-five patients underwent alloMILs expansion, of which five failed to expand. Twenty patients received an infusion and six received a second infusion. Of the twenty patients receiving an infusion, eleven had multiple myeloma (MM) or plasma cell leukemia; five had acute myeloid leukemia (AML); one had chronic myeloid leukemia in blast phase; and three had chronic lymphocytic leukemia. Harvest, expansion, and infusion were well tolerated with minimal adverse events. No patient developed acute or chronic GVHD after infusion. Dosing advanced to the highest level of 5 x10 7 CD3 + cells/kg in both the mismatched and matched HCT cohorts. At day 60, the overall response rate (composite of complete (CR) and partial responses) was 40 percent (8/20) in the entire cohort; 33.3 percent (2/6) in the myeloid cohort (Figure 1A); 30 percent (3/10 evaluable patients) in the MM cohort; and 66.7 percent (2/3) in the lymphoid cohort. One MM patient achieved sustained > 2 years long CR and one AML patient is in ongoing CR, both of whom received alloMILs with no or minimal disease followed by maintenance therapy. HLA loss analysis was performed on pre-expansion samples from three AML patients receiving a mismatched HCT and alloMILs. Both non-responders in this cohort had loss of the host mismatched HLA haplotype. ScRNAseq on pre-expansion samples from ten AML patients, eleven MM patients, and four AML patients in remission after HCT (control) yielded 197 522 total cells and 32 587 CD8 + T cells. CD8 + T cells expressing a senescent-like gene signature previously associated with dysfunctional tumor killing in AML patients (Mazziotta et al ASH 2022) were associated with non-response primarily in AML patients (observed log2 fold change -2.48 compared with responders; FDR = 0.0017) (Figure 1B). Conclusions: AlloMILs demonstrated feasibility, a favorable safety profile, and early evidence of clinical activity in patients with measurable residual disease. The maximum dose of 5 x10 7 CD3 + cells/kg was reached, and no patient developed acute or chronic GVHD. HLA loss and CD8 + T cell senescence may be associated with non-response in AML patients. A clinical trial utilizing alloMILs as maintenance therapy for patients with measurable residual disease is in development.

Leukemia Blasts Induce NK-like Dysfunction in AML-Specific T Cells

Introduction Efforts to enhance T cell-mediated acute myeloid leukemia (AML) eradication face challenges possibly due to unique mechanisms of leukemia-induced T-cell dysfunction compared to exhaustion observed in solid tumors(Penter et al., Blood 2023). We previously showed the efficacy of Wilms' Tumor (WT) Antigen 1-specific T-cell receptor (TCR C4) inserted into CD8 + T cells (T TCR-C4) in preventing AML recurrence after hematopoietic cell transplantation (HCT) (Chapuis et al., Nat Med 2019). This led us to investigate the safety and efficacy of T TCR-C4 in post-HCT relapsed patients. Methods In this Phase I/II study conducted at Fred Hutchinson Cancer Center, 15 patients were enrolled and prospectively monitored for disease response. We manufactured T TCR-C4 using a lentiviral transduction system (Chapuis et al, Nat Med 2019) and performed comprehensive analyses of bone marrow (BM) and peripheral blood (PB) samples using single-cell RNA sequencing (scRNAseq) and TCRseq. Results T TCR-C4 treatment showed no survival advantage compared to matched controls (n =17). We hypothesized that active AML exacerbates T-cell dysfunction compared to patients who received prophylactic T TCR-C4 treatment. Identifying tumor-specific T cells poses a challenge in scRNAseq. By utilizing the WT1-transgene (WT1tg) sequence, we built a custom reference genome to dissect the transcriptional state of leukemia-specific cells. In PB scRNAseq profiles of 4 patients (24,472 CD8 + T cells) with post-infusion persistent blasts and T TCR-C4, we identified 5 main CD8 + T-cell signatures: Naïve, Stem-like, Activated-Exhausted, WT1tg positive, and NK-like (Figure 1). The NK-like signature has been associated with dysfunction in CAR-T cells (Good et al., Cell 2021). T TCR-C4 cells exhibited an NK-like skewing, although the expression of these markers was not as high as in endogenous NK-like CD8 + T cells. Trajectory inference revealed that NK-like endogenous CD8 + T cells represented the terminal state of differentiation, while T TCR-C4, despite expressing some NK-like markers, were in an intermediate state of differentiation along the trajectory (Figure 2). Combining scTCRseq data with proliferation signatures (Zheng et al., Science 2021) we found that WT1tg exhibited clonal expansion and proliferation, suggesting a functional intermediate state. We validated the 5 CD8 + T-cell differentiation states in BM samples (7,719 CD8 + T cells) from our cohort and in a publicly available large-scale BM CD8 + T-cell scRNAseq dataset (68,545 CD8+ T cells), with NK-like as the last differentiation state along the trajectory. Differential gene expression (DGE) of CD8 + T cells between active AML (AML+) and blasts-not-detectable (AML-) timepoints revealed reduced expression of stem-like and increased expression of NK-like markers in AML+ vs. AML- suggesting that leukemia drives NK-like terminal differentiation in T TCR-C4. To investigate if the NK-like derangement is AML-specific or a general dysfunction mechanism, we performed DGE between T TCR-C4 vs. TCR-T cells mesothelin-specific (T MSLN). The analysis revealed overexpression of exhaustion markers (CTLA4, PDCD1, TOX, CXCR6, NR4A2) in T MSLN and NK-like markers (NKG7, KLRD1, KLRG1, S1PR5) in T TCR-C4. To explore leukemia immune-escape mechanisms, we studied blasts gene expression. One patient who initially responded to T TCR-C4, exhibited monocytic blast differentiation upon relapse. We found overexpression of immune suppression genes (IL10, TNF, reactive oxygen species) in monocytic blasts at relapse vs. blasts before T TCR-C4 infusion. Published AML scRNAseq confirmed NK-like skewing in monocytic leukemia patients. Ongoing in-vitro assays will help to dissect the mechanisms of T TCR-C4 dysfunction. Discussion Our study reveals a novel AML-specific NK-like signature of T-cell dysfunction, exacerbated when AML blasts are present, potentially contributing to the restricted efficacy observed in TCR-T cell therapies. We observed that monocytic blast differentiation and the subsequent formation of an immune-suppressive environment imply the existence of intricate immune escape mechanisms. This study provides a critical foundation for the development of innovative interventions aimed at circumventing the dysfunction observed in cell-based therapies and fostering long-term remission in AML patients.

Dysregulated Activating and Inhibitory Receptors on Natural Killer Cells Predicts Immune Escape and Poor Outcomes in Acute Myeloid Leukemia

Background: Although improvements have been made in understanding the pathophysiology of Acute Myeloid Leukemia (AML), heading to development of emerging therapies targeting leukemic stem cells, relapse is still a major problem leading to poor long-term survival and low cure rates. Of particular interest, Natural Killer (NK) cells are regulated by opposing signals from receptors that activate and inhibit effector function and are known to mediate anti-leukemic immunosurveillance in AML, but the mechanisms underlying the control of hematopoietic neoplasms by NK cells remains unclear and not yet explored as therapeutical options. Aims: We hypothesized that functionally impaired NK cells due to imbalanced expression of inhibitory and activating receptors can predict poor outcomes in AML. Methods: NK cell phenotypes based on the expression of DNAM-1 and NKG2D (activating) and NKG2A and KIR2DL1 (inhibitory) receptors on CD56+, CD56bright CD16- and CD56dim CD16+ NK subsets were profiled by multiparametric flow cytometry in 100 de novo AML bone marrow (BM) samples (Table 1), followed up from February 2016 to June 2022 and 17 healthy bone marrows (HBM). Correlations between NK phenotype with ELN2017 risk stratification, complete remission (CR), relapse (R), measurable residual disease (MRD), overall survival (OS) and relapse-free survival (RFS) were verified. Six BM from AML patients in remission after induction chemotherapy (AML-CR) were used to evaluate NK cell frequency and receptors recovery. Co-culture of AML-exposed CD56+ NK cells and K562 cell line was used to assay for killing (1:1, 5:1, 10:1 E:T ratios) and degranulation (CD107a). Mann Whitney, Kruskal-Wallis and mixed effects tests were performed to determine associations, and Log Rank test and Kaplan Meir curves were used for survival analysis. Results: The frequency of all NK cell subsets were decreased in AML (P < .01). AML CD56+ NK cells showed decreased expression of NKG2D (P < .01), a trending to decreased DNAM-1 and increased NKG2A (P = .04) and KIR2DL1 (P < .01), while recovering to near healthy values in AML-CR. Expression of both NKG2D and DNAM-1 activating receptors was decreased (P < .01), while inhibitory NKG2A (P = .02) and KIR2DL1 (P < .01) were considerably increased in CD56dim NK, which is known to carry out the most cytotoxic activity. The expression of both activating receptors by CD56dim subset was found to be decreased in ELN2017 adverse versus low/intermediate risk (NKG2D, P = .05; DNAM-1, P < .01), in relapse (P < .01), and with similar trends in MRD+ patients after remission induction. The opposite was observed for inhibitory receptors, found to be increased in adverse risk (P < .01), relapse (NKG2A, P < .01; KIRDL1, P < .05) and MRD+ (NKG2A, P = .03) patients. Degranulation (P < .01) and specific target cell death (5:1, P = .01; 10:1, P = .002) were significantly decreased in AML. Patients with lower frequency of CD56dim NK expressing low NKG2D and DNAM-1 showed less degranulation and specific target cell death (P = .03), whereas patients with higher inhibitory receptors demonstrated lower degranulation (NKG2A, P=0.06) and cytotoxicity (KIR2DL1, P=.005). Of note, RFS was decreased in patients with lower frequency of CD56+ NK cells (P = .02) and both OS and RFS were decreased in patients with lower frequency of CD56dim NK cells (OS, P = .04; RFS, P = .03) and in patients with lower expression of NKG2D (OS, P = .03; RFS, P = .008) and DNAM-1 (OS, P = .02; RFS, P = .11) activating receptors, strongly suggesting an imbalance between activation and inhibition of NK cell activity. Conclusion: Our data indicate that CD56dim NK show an exhaustion immunophenotype with high expression of KIR2DL1 and NKG2A inhibitory receptors and low expression of NKG2D and DNAM-1 activating receptors and that this imbalance leads to impaired cytotoxicity, compromised anti-leukemic activity and can be predictive of poor outcomes in AML, such as response to treatment, relapse and MRD positivity. Additionally, this NK phenotype at diagnosis could be considered an adverse risk prognostic factor associated with lower survival rates. Closing these gaps in the knowledge of NK-mediated immune evasion in leukemia is of significant interest for targeting the leukemic microenvironment by NK cell-mediated immunotherapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bone Marrow WT1 mRNA Upregulation in Acute Myeloid Leukemia (AML) in Complete Molecular Remission (mCR): Understanding the Limitations and Advantages of WT1 As a Measurable Residual Disease (MRD) Target

Introduction WT1 mRNA levels assessed by q-PCR have been employed as a marker of remission in AML patients undergoing intensive chemotherapy (Nomdedeu J. et al. Leukemia 2013;27:2157) and hematopoietic cell transplantation (HCT) (Nomdedeu J et al. Biol Blood Marrow Transplant 2018;24:55). Patients with adjusted bone marrow WT1 mRNA levels greater than 100 copies showed a high probability of relapse. Despite the usefulness of WT1 quantification in this and other settings, this marker is scarcely employed as an MRD target because levels greater than >100 copies can also be observed in normal bone marrows. Furthermore, 10-20% of AML at diagnosis and most chronic myelomonocytic leukemia (CMML) cases lack WT1 upregulation. The high levels of bone marrow WT1 copy numbers observed in most AML, decline in responding patients, although the utility, if any, of WT1 levels in long-term survivors of AML is currently unknown. We investigated the frequency and biological findings of mCR AML samples showing WT1 upregulation(>100 copies) compared to that mCR AML with concordant low WT1 levels. Patients and methods The study included 717 bone marrow samples from 80 AML patients (23 patients with RUNX1-RUNX1T1, 18 pts with CBFb-MYH11, four pts with NPM1 mutations, and 35 pts with PML-RARa). Samples were obtained at diagnosis (58 samples) and the follow-up (659 samples, all in mCR and without relapses) from one month to 219 months after diagnosis. Chimeric transcripts and WT1 levels were investigated by standardized real-time methods (ELN). Hemoglobin and leukocyte counts were also recorded. Flow cytometry immunophenotyping was also performed using four-color flow cytometry. We analyzed the percentage of CD34+, CD117+, CD123+ cells, and the Myeloid/Lymphoid CD34 ratio. nCounter Stem cell panel (Nanostring) was used to investigate the differences between that AML in mCR, showing high (>100) and low (<100) WT1 levels. In an additional cohort (11 samples: 4 with WT1>100 and 7 with WT1 <100), we performed bulk RNAseq (Novogene) to investigate enriched genes in samples with high WT1 levels. Results Thirty-five patients had at least one sample with WT1 levels greater than 100 copies (44%) despite being in mCR. Twelve samples had WT1 levels greater than 300 copies. In one case, this level reached 770 copies. These WT1 "peak" samples were distributed as follows: 49/306 PML-RARa, 25/161 RUNX1-RUNX1T1, 10/174 CBFb-MYH11, and 1/18 NPM1. Patients showing WT1 peaks were older than those without elevations (47 years vs. 36 years, p:0.003) For PML-RARa samples, "peaking" was associated with low levels of hemoglobin (133 g/L for <100 copies vs. 125 g/L >100 copies, p:0.009). For RUNX1-RUNX1T1, "peaking" was linked to less platelets (132x109 vs. 197x109 p:0.05) , higher CD34 (1.5% vs. 1% p:0.05) and also higher CD117+ cells (1.6 vs.1%, p:0.01). Lastly, CBFb-MYH11+ samples with WT1 peaks had higher leukocyte counts that those showing low WT1 levels (4.5x109 vs. 2.9x109, p:0.006). Nanostring comparisons between mCR WT1 high vs. low samples showed upregulation of TOR1A, PIK3CB, PTPN6, and PGM1 with downregulation of HMGA1, HOXB2 and CDC6. RNA seq analysis detected an enrichment of SUZ12 targets in those samples with high WT1 levels. Conclusions Bone marrow mRNA WT1 levels commonly exceed the clinical relevant value of 100 copies in AML samples in mCR (13%). Peak samples were related to peripheral blood values and/or raised numbers of immature bone marrow cells (CD34, CD117). Bone marrow WT1 although unable to accurately predict relapses in long-term survivors of AML may be a non-invasive sensor of a yet undefined myeloid state.

Association of Platelet Count Detected by Novel Nano Probe with Prognosis After Initial Chemotherapy in Patients with Acute Myeloid Leukemia

Objective: To analyze the correlation between platelet count changing and the newly diagnosed patients with acute myeloid leukemia (AML) after initial induction of chemotherapy for the first time, and to explore the prognostic indicators affecting AML. Methods: 253 AML patients who received explicit diagnosis and treatment as AML. Platelet counts were detected by a near-infrared fluorescence nano probe based upon tirofiban, a clinically approved small-molecule glycoprotein IIb/IIIa inhibitor (GPIIb/IIIa). Results: Hemorrhage, platelet transfusion during chemotherapy and increased the platelet count after a course of treatment are risk factors for complete remission after chemotherapy, in which platelet count detected by nano probe after chemotherapy is an independent risk factor for complete remission in AML patients. Conclusion: Using nano probe to detect platelet by using small-molecule glycoprotein IIb/IIIa inhibitor (GPIIb/IIIa) can significantly improve descision making in AML patient in comparison to other traditional methods. In addition, we found that the prognosis of patients with newly diagnosed AML patients with increased platelet count after induction chemotherapy for the first time is better than no increase.

The Expression and Clinical Correlation of the CD68 Factor in Acute Myeloid Leukemia Patients

To analyze the expression and clinical characteristics of CD68 in bone marrow and peripheral blood of patients with acute myeloid leukemia (AML). The expression of CD68 in bone marrow blast cells was detected by four-color flow cytometry in 50 newly diagnosed AML patients and 23 controls. The expression of CD68 in peripheral blood of 85 newly diagnosed AML patients, 29 remission AML patients and 24 controls was detected by ELISA. The correlation between the expression rate of non-M3 AML bone marrow CD68, peripheral blood CD68 concentration and white blood cell count and other clinical data was compared respectively. The median CD68 expression rate in myeloid leukemia cells of non-M3 AML patients was 19.7%, significantly higher than control (0.2%) (P<0.001). The median concentration of non-M3 CD68 in peripheral blood was 67.97 pg/ml, significantly higher than in control (29.94 pg/ml)（P<0.01）. There was no statistically significant difference in the plasma CD68 concentration of the peripheral blood between the newly diagnosed (45.72 pg/ml) and the remission stage (55.12 pg/ml) of non-M3 AML patients by paired analysis (P>0.05). The results showed that the higher the expression rate of CD68 in bone marrow, the higher the count of white blood cells in peripheral blood, and the lower the count of hemoglobin and platelet in peripheral blood. The higher the plasma concentration of CD68 in peripheral blood, the higher the white blood cell count and the lower the complete remission rate. The expression of CD68 both in bone marrow and peripheral blood of patients with non-M3 AML is higher than that of control group. Patients with high expression of CD68 show a low rate of complete remission, suggesting that the expression level of CD68 is correlated with treatment response.

Harmony Alliance Provides a Machine Learning Researching Tool to Predict the Risk of Relapse after First Remission in AML Patients Treated without Allogeneic Haematopoietic Stem Cell Transplantation

Harmony Alliance Provides a Machine Learning Researching Tool to Predict the Risk of Relapse after First Remission in AML Patients Treated without Allogeneic Haematopoietic Stem Cell Transplantation

Maintenance therapies in acute myeloid leukemia: the renaissance of an old therapeutic concept.

Disease relapse remains the major cause of death in patients with acute myeloid leukemia (AML) and is driven by the persistence of leukemic cells following induction chemotherapy or allogeneic hematopoietic cell transplant (allo-HCT). Maintenance therapies to extend the duration of remission and to improve survival have been proposed for several years with mixed results but have experienced a renaissance recently. The oral hypomethylating agent CC-486 has been the first agent to show an overall survival (OS) benefit compared with observation in AML patients in remission following intensive chemotherapy who are not proceeding to allo-HCT. In the realm of maintenance therapy following allo-HCT, the FLT3 inhibitor sorafenib has yielded superior results in terms of OS and relapse-free survival in randomized trials compared with observation. Several open questions remain regarding patient selection, timing, duration and safety of maintenance therapies. Various targeted agents are currently tested in clinical trials and could potentially enable an even more individualized therapeutic approach. Maintenance therapies following intensive chemotherapy or allo-HCT offer a new therapeutic paradigm for an increasing number of AML patients and have been shown to result in an OS benefit in selected patients.

Results from Ongoing Phase 1/2 Trial of SL-401 As Consolidation Therapy in Patients with Acute Myeloid Leukemia (AML) in Remission with Minimal Residual Disease (MRD)

Results from Ongoing Phase 1/2 Trial of SL-401 As Consolidation Therapy in Patients with Acute Myeloid Leukemia (AML) in Remission with Minimal Residual Disease (MRD)

Treatment Patterns and Health Care Resources Use (HCRU) in Patients with Acute Myeloid Leukemia (AML): Real World Evidence (RWE) from 30 US Institutions

Background: AML is an aggressive hematological malignancy that is fatal if left untreated, it is therefore considered a medical emergency. Intensive chemotherapy (IC) is considered the standard of care in newly-diagnosed AML. The goal of treatment is to induce complete remission, with possible stem cell transplantation (SCT). Patients who are unfit for IC usually receive hypomethylating agents (HMA), azacitidine or decitabine. Many patients relapse and undergo salvage treatment. AML treatment is complex and requires significant healthcare resource use (HCRU) at each treatment phase. However, real world data on treatment patterns and HCRU in these patients are limited.Aims: The aim of this research was to compare treatment patterns and HCRU in AML patients who are newly-diagnosed and experiencing remission or relapse.Methods: This study used TriNetx syndicated electronic medical records network containing records for inpatient/outpatient services and procedures, diagnoses, adverse events, prescription drugs, and labs for over 30 million patients from 27 US hospital institutions. ICD-10 diagnosis disease codes were used to subgroup patients into three mutually exclusive cohorts: newly-diagnosed, in remission, and in relapse. Patient demographics and proportions of patients treated with IC, HMA, and SCT as well as the market shares for specific treatments were collected. HCRU of interest included hospitalizations, emergency room and physician visits, diagnostic radiology tests, and platelet and red blood cell transfusions. Rates of HCRU were calculated over the 6 months following treatment initiation in newly-diagnosed or relapsed patients and over the 6 months post-remission. The rates of HCRU across three cohorts were compared using Z-tests (two-tailed α=0.05).Results: Among 29,730 identified AML patients, mean age was 58 (SD 22), 54% were male, 61% were white, and 27% had prior myelodysplastic syndromes (MDS). Of these patients, 57% were newly-diagnosed, 27% were in remission, and 13% were in relapse. Newly-diagnosed patients were 62 (SD 21) years old on average, 55% were male, 57% were white, and 27% had prior MDS. Among treated newly-diagnosed AML patients, 47% received IC, and 23% received HMA. The most common IC combinations were cytarabine+daunorubicin (48%), cytarabine+fludarabine (36%), cytarabine+idarubicin (34%), and cytarabine+mitoxantrone (26%). Among patients treated with HMA, 65% were on azacytidine and 42% were on decitabine. Treatment patterns for patients in relapse were similar to those for newly-diagnosed patients, with slightly higher rates of IC (56%) and HMAs (28%). Among patients in remission who were previously treated with IC, 45% received SCT in addition to IC. HCRU varied significantly between patient cohorts. AML patients in remission demonstrated significantly lower rates of hospitalizations, diagnostic radiology tests, and red cell and platelet transfusions, while the rates of office visits were significantly higher and there was no significant difference in the rates of ER visits as compared to newly-diagnosed patients. The rates of all HCRU were significantly higher among patients in relapse as compared to newly-diagnosed patients (Table).Conclusions: This study offers real world evidence on the treatment of AML patients in the US. Most patients received IC, with cytarabine+daunorubicin as most common combination treatment. As expected, HCRU was significantly lower for patients in remission and significantly higher for patients in relapse, as compared to newly-diagnosed patients. Treatments that increase remission rates and decrease relapse rates may lead to significant improvements in the economic burden of AML.Table: Annual Health Care Resource use in patients with AML [Display omitted] DisclosuresBrandt:Novartis Pharmaceuticals Corporation: Employment. Manson:Novartis: Employment, Equity Ownership. Fuentes-Alburo:Novartis: Employment.

Chronic stress promotes acute myeloid leukemia progression through HMGB1/NLRP3/IL-1β signaling pathway.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor prognosis and overall survival. Clinical investigations show that chronic stress is commonly present in the course of AML and associated with adverse outcome. However, the underlying molecular mechanisms are elusive. In the present study, a chronic restraint stress mouse model was established to evaluate the effect of stress on AML. We found that mice under chronic stress exhibited significantly increased liver and spleen infiltration of leukemic cells and poorer overall survival. This was accompanied by elevated cellular NLR family pyrin domain containing 3 (NLRP3) and interleukin-1β (IL-1β) in the liver or bone marrow, and secreted IL-1β in the plasma, indicating the activation of inflammasomes under chronic restraint stress. High mobility group box 1 (HMGB1) expression was markedly increased in newly diagnosed AML patients, but reduced in complete remission AML patients. The expression level of HMGB1 was positively correlated with NLRP3 mRNA in AML patients. Knockdown of HMGB1 significantly decreased NLRP3 and IL-1β expression in AML cell lines, and secreted IL-1β in supernatant of AML cell culture, while HMGB1 stimulation caused contrary effects. These results implied that HMGB1 could be involved in the regulation of inflammasome activation in AML development. Mice model showed that chronic restraint stress-facilitated proliferation and infiltration of AML cells were largely abrogated by knocking down HMGB1. Knockdown of HMGB1 also ameliorated overall survival and remarkably neutralized NLRP3 and IL-1β expression under chronic restraint stress. These findings provide evidences that chronic stress promotes AML progression via HMGB1/NLRP3/IL-1β dependent mechanism, suggesting that HMGB1 is a potential therapeutic target for AML. KEY MESSAGES: • Chronic restraint stress promoted acute myeloid leukemia (AML) progression and mediated NLRP3 inflammasome activation in xenograft mice. • HMGB1 mediated NLRP3 inflammasome activation in AML cells. • Knockdown of HMGB1 inhibited AML progression under chronic stress in vivo.

Clinical Relevance of Morphologic and Molecular Complete Remission in AML Patients Undergoing Reduced Intensity or Non-Myeloablative Conditioning

Introduction: For most acute myeloid leukemia (AML) patients (pts) an allogeneic stem cell transplantation (HSCT) offers the best chance for relapse-free long-term survival. Evaluation of measurable residual disease (MRD) at HSCT allows risk stratification additionally to genetic risk at diagnosis. Pts with active AML or with positive MRD status (MRD+) pre-HSCT have similar and dismal outcomes following myeloablative conditioning. Reduced intensity (ric) or non-myeloablative (nma) conditioning regimes have been developed to allow HSCT also in AML pts with higher age or comorbidities. Here, we analyzed and compared the clinical relevance of morphologic and MRD-based remission status in AML pts prior to nma- or ric-HSCT. Methods: We analyzed 345 AML pts who received an allogeneic peripheral blood HSCT at a median age of 63 (range 21-77) years with active disease (34%), or in first (49%) or second (17%) complete remission (CR, 87% of pts in remission) or CR with incomplete peripheral recovery (CRi, 13% of pts in remission) after nma (75%) or ric (25%) conditioning. Donors were HLA matched related (11%), matched unrelated (67%), antigen mismatched unrelated (21%) or haploidentical related (1%). At diagnosis, cytogenetics and the mutation status of CEBPA, NPM1 and presence of FLT3-ITD were assessed. Using a next-generation targeted amplicon sequencing approach we analyzed a panel comprising 54 recurrently mutated genes in myeloid malignancies on the MiSeq platform (Illumina). Pre-HSCT morphologic remission status as well as MRD status in pts in morphologic CR/CRi based on NPM1 mutations, BAALC, MN1 and WT1 expression were evaluated. MRD+ pts were defined by being positive for any of the analyzed markers. Median follow up after HSCT was 2.2 years. Results: Pts transplanted with active disease differed from pts in remission with or without MRD pre-HSCT: they were less likely to have de novo AML (P=.02 &amp; P=.09, respectively) and had higher genetic risk including a higher frequency of an abnormal (P=.001 &amp; P&amp;lt;.001, respectively), a complex (P=.06 &amp; P=.04, respectively) or a monosomal karyotype (P&amp;lt;.001 &amp; P=.003, respectively), a lower frequency of NPM1 mutations (P&amp;lt;.001 &amp; P&amp;lt;.001, respectively) and worse ELN genetic risk (P&amp;lt;.001 &amp; P&amp;lt;.001, respectively). They were also more likely to receive ric-HSCT (P&amp;lt;.001 &amp; P&amp;lt;.001) because pts with active AML were frequently transplanted after FLAMSA conditioning. Pre-HSCT MRD- pts only differed from pre-HSCT MRD+ pts regarding a lower white blood count (P=.006) and lower circulating blasts at diagnosis (P=.05) while the proportion of pts transplanted in CR or CRi did not differ between MRD- and MRD+ AML pts. Also the number of applied chemotherapy cycles prior to HSCT did not differ between the three pts groups. Pre-HSCT MRD- pts had a significantly lower cumulative incidence of relapse/progression (CIR) compared to both MRD+ pts (P&amp;lt;.001) and pts transplanted with active disease (P&amp;lt;.001) while CIR did not differ between MRD+ pts and pts transplanted with active disease (P=.24, Figure 1A). Pre-HSCT MRD- pts had longer overall survival (OS) than pre-HSCT+ pts (P=.04) who again had longer OS than pts transplanted with active disease (P=.01, Figure 1B). In multivariate analyses, the MRD corrected remission status prior to HSCT remained a significant factor for CIR (Hazard Ratio 1.65, Confidence interval [CI] 1.31-2.06) after adjustment for ELN risk and for OS (Odds Ratio 0.63, CI 0.49-0.84) after adjustment for ELN risk, hemoglobin and platelet count at diagnosis. Conclusion: AML pts transplanted with active disease showed a variety of high-risk diagnostic parameters compared to pre-HSCT MRD- or MRD+ pts, as secondary disease and adverse genetic risk. In contrast, pre-HSCT MRD- and MRD+ pts could not be discriminated by high risk factors at diagnosis, underlining the importance of a dynamic risk stratification during remission. Both pts with active disease or a MRD+ status in CR/CRi prior to nma- or ric-HSCT showed dismal outcomes with higher CIR and shorter OS than MRD- pts. However, while CIR was comparable in pts with molecular or morphologic detectable disease, OS was worst in pts transplanted with active disease, indicating that MRD+ pts might still be salvageable after suffering relapse and able to achieve long-term outcomes. Figure Disclosures Jentzsch: JAZZ Pharmaceuticals: Honoraria; Novartis: Honoraria. Platzbecker:AbbVie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Amgen: Honoraria, Research Funding; Geron: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Schwind:Pfizer: Honoraria; JAZZ Pharmaceuticals: Honoraria; Novartis: Honoraria, Research Funding.

Next-generation sequencing for measurable residual disease detection in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is a blood cancer characterized by acquired genetic mutations. There is great interest in accurately establishing measurable residual disease (MRD) burden in AML patients in remission after treatment but at risk of relapse. However, inter- and intrapatient genetic diversity means that, unlike in the chronic myeloid and acute promyelocytic leukaemias, no single genetic abnormality is pathognomonic for all cases of AML MRD. Next-generation sequencing offers the opportunity to test broadly and deeply for potential genetic evidence of residual AML, and while not currently accepted for such use clinically, is likely to be increasingly used for AML MRD testing in the future.

Alteration of Serum Malondialdehyde Level As Biomarker of Oxidative Stress during Acute Myeloid Leukemia Treatment

Introduction Oxidative stress as a disruption of redox homeostasis is a state where reactive oxygen species (ROS) concentration is transiently/chronically increased or ROS-scavening capacity is declined. ROS have an important role in intracellular signaling process. On the other side increased ROS production could lead to oxidative damage of biomolecules as lipids, DNA and proteins. Many types of cancer cells have elevated levels of ROS for promoting cell proliferation and differentiation [1,2]. In the context of hematopoietic malignancy, in acute myeloid leukemia (AML) there is evidence of elevated ROS and/or accompanying oxidative stress [3], however little is known about the changes of oxidative status during AML treatment. The aim of this study was to estimate oxidative stress using the malondialdehyde (MDA) concentration as a marker of lipid peroxidation. The MDA concentrations were measured during treatment of AML patients in the initial stage (AML patients in diagnosis), after chemotherapy treatment (AML patients in remission), and after pretransplantation conditioning (AML patients before transplantation) in comparison with healthy controls. Methods Serum MDA concentration was analyzed in samples obtained at diagnosis, in remission, and before transplantation from patients (n=20) treated in the Institute of Hematology and Blood Transfusion, Prague, Czech Republic. As a control we used serum of healthy donors (n=13). Briefly, serum samples were prepared by alkaline hydrolysis, deproteinized and total MDA was derivatized by 2,4-dinitrophenylhydrazine (DNPH). MDA-DNPH derivates were separated by HPLC and determined using positive ion multiple reaction monitoring (MRM) mode by MS/MS. Results Using analysis of variance (ANOVA) we found significant differences in MDA concentrations in serum of AML patients and healthy controls (P &lt; 0.0001). Moreover, Tukey post-hoc test applied to comparison of all treatment periods of AML patients with healthy controls revealed significantly increased MDA levels in the period before transplantation (P &lt; 0.001) with respect to healthy controls. The MDA levels in the initial stage was also elevated as compared with healthy controls, but non significantly (P = 0.062). Comparing only AML patients in the individual treatment periods, we found significantly increased concentrations of MDA in samples of AML patients before transplantation with respect to AML patients in diagnosis (P = 0.023) and also to the patients in remission (P = 0.012). Conclusion Overall, throughout treatment periods for AML patients compared to healthy controls significant changes in MDA concentrations were identified. It is in agreement with previous findings stated that cancer cells have elevated levels of ROS possibly playing an important role in the initiation and progression of cancer. The highest MDA concentration was detected in samples of AML patients before transplantation which could be related to conditioning regimens [4]. The monitoring of AML patients at different times of disease treatment revealed that oxidative stress is changing not only as a result of disease progression but also as a result of treatment. Acknowledgement This work was supported by the European Regional Development Fund and the state budget of the Czech Republic (project AIIHHP: CZ.02.1.01/0.0/0.0/16_025/0007428, OP RDE, Ministry of Education, Youth and Sports), by the project of the Ministry of Health, Czech Republic, 00023736, by Grant from the Czech Science Foundation, Czech Republic, 19-02739S, and by ERDF OPPK CZ.2.16/3.1.00/24001. [1] Boonstra J, Post JA. Gene 2004;337, 1-13. [2] Schafer FQ, Buettner GR. Free Radic Biol. Med. 2001;30, 1191-1212. [3] Hole PS, Zabkiewicz J, Munje C, Newton Z, Pearn L, White P, Marquez N, Hills RK, Burnett AK, Tonks A, Darley RL. Blood. 2013;122(19):3322-3330. [4] Trachootham D, Alexandre J, Huang P. Nat Rev Drug Discov. 2009;8(7):579-591. Disclosures No relevant conflicts of interest to declare.

Gal9/Tim-3 expression level is higher in AML patients who fail chemotherapy

Immune checkpoint pathways active in Acute Myeloid Leukemia (AML) patients, especially during the course of remission induction chemotherapy, have not been well studied. Although dominant in mediating T cell dysfunction in cancer, it is now well-accepted that interruption of PD-1/PD-L1 axes alone does not always completely restore T cell function, indicating the involvement of additional negative regulatory pathways, such as TIM-3/Gal-9, in promoting T cell exhaustion.Here, we characterized these pathways in AML patients enrolled in a phase I dose escalation trial that combined Selinexor, a Selective Inhibitor of Nuclear Export (SINE), with high-dose cytarabine (HiDAC) and mitoxantrone (Mito) (NCT02573363) as induction therapy.To monitor changes in expression of immune checkpoint receptors, multi-parameter flow cytometry was performed on peripheral blood and bone marrow biopsy specimens at diagnosis and following induction therapy in 26 AML patients. Expression of CD47, PD-L1, PD-L2 and Gal9 was assessed on CD34+ AML blasts, as well as on CD34− cell populations. In parallel, we evaluated expression of inhibitory (PD1, CTLA4, LAG3, TIM-3) and stimulatory (CD28, ICOS, CD137, OX40, CD40L, HLA-DR) co-receptors on CD4+ and CD8+ T cell subsets.Compared to baseline, the frequency of Gal9+ CD34− cells was significantly higher in patients with treatment failure (TF) than in those in complete remission (CR), and this finding correlated with increased TIM-3 expression on marrow-resident T cells in TF patients. Moreover, when we measured the expression level of PD-1 and TIM-3 in bone marrow samples compared to peripheral blood, TIM-3 was significantly higher in BM specimens.Our results suggest that targeting the Gal9/Tim-3 axis could be effective in combination with induction chemotherapy to increase the likelihood of complete remission in AML patients.

Emerging role of EPHX1 in chemoresistance of acute myeloid leukemia by regurlating drug-metabolizing enzymes and apoptotic signaling.

Microsomal epoxide hyrolase 1 (EPHX1) is a critical biotransformation enzyme and participants in both the detoxification and activation of potentially genotoxic epoxides. In this study, we firstly aimed to investigate the role of EPHX1 in the chemoresistance of acute myeloid leukemic cells to aclarubicin (ACM) and mitoxantrone (MIT). EPHX1 mRNA expression and prognosis were measured in acute myeloid leukemia (AML) patients, and the function of EPHX1 in leukemic cell viability and apoptosis induced by ACM and MIT was also measured. Our results found that EPHX1 expression is obviously associated with recurrence rate, overall survival and time of obtaining first complete remission in AML patients. EPHX1 silencing promoted ACM and MIT induced decrease in cell viability and cell apoptosis of HL-60, K562, and THP-1 that was inhibited by EPHX1 overexpression. EPHX1 reduced the susceptibility of leukemic cells to ACM and MIT by regulating drug-metabolizing enzymes (CYP1A1, GSTM1, and GSTT1) and apoptotic signaling (Bax, Bcl-2, Caspase-3, Caspase-9, and PARP1). Moreover, Nrf2 overexpression significantly increased EPHX1 expression and leukemic cell viability and decreased leukemic cell apoptosis. Taken together, we summarized the recent findings about the chemoresistance-promoting role of EPHX1, and the potential of targeting EPHX1 was proposed to counteract drug resistance in leukemia treatment.

Clinical Studies with Bismuth-213 and Actinium-225 for Hematologic Malignancies.

Due to the shorter range and higher linear energy transfer of α-particles compared to β-particles, targeted α-particle therapy may produce more efficient tumor killing while sparing neighboring healthy cells. We will review the clinical studies using α-particle therapy for Acute Myeloid Leukemia (AML). A series of clinical trials were conducted to assess the safety, feasibility, and anti-leukemic effects of lintuzumab, an anti-CD33 humanized monoclonal antibody, labeled with the α-emitters bismuth- 213 (213Bi) and actinium-225 (225Ac). An initial phase I study conducted in 18 patients with relapsed or refractory AML demonstrated the safety and antitumor effects of 213Bi-lintuzumab. Subsequently, 213Bi-lintuzumab produced remissions in AML patients after partial cytoreduction with cytarabine in phase I/II trial. The 46- minute half-life of 213Bi and need for an onsite generator has limited its utility. Therefore, a secondgeneration construct was developed using 225Ac, a radiometal that yields four α-particle emissions. A phase I trial demonstrated that a single infusion of 225Ac-lintuzumab could be given safely at doses up to 111 kBq/kg with anti-leukemic activity across all dose levels studied. In a second phase I study, 28% of older patients with untreated AML had objective responses after receiving fractionated-dose 225Aclintuzumab and low-dose cytarabine. Based upon the encouraging results seen in phase I trials of 225Ac-lintuzumab, a phase II study of 225Ac-lintuzumab monotherapy for older patients with untreated AML is now in progress and is also being studied in a subset of patients with CD33-positive multiple myeloma.

Interleukin-15-Cultured Dendritic Cells Enhance Anti-Tumor Gamma Delta T Cell Functions through IL-15 Secretion.

Dendritic cell (DC) vaccination can be an effective post-remission therapy for acute myeloid leukemia (AML). Yet, current DC vaccines do not encompass the ideal stimulatory triggers for innate gamma delta (γδ) T cell anti-tumor activity. Promoting type 1 cytotoxic γδ T cells in patients with AML is, however, most interesting, considering these unconventional T cells are primed for rapid function and exert meaningful control over AML. In this work, we demonstrate that interleukin (IL)-15 DCs have the capacity to enhance the anti-tumoral functions of γδ T cells. IL-15 DCs of healthy donors and of AML patients in remission induce the upregulation of cytotoxicity-associated and co-stimulatory molecules on the γδ T cell surface, but not of co-inhibitory molecules, incite γδ T cell proliferation and stimulate their interferon-γ production in the presence of blood cancer cells and phosphoantigens. Moreover, the innate cytotoxic capacity of γδ T cells is significantly enhanced upon interaction with IL-15 DCs, both towards leukemic cell lines and allogeneic primary AML blasts. Finally, we address soluble IL-15 secreted by IL-15 DCs as the main mechanism behind the IL-15 DC-mediated γδ T cell activation. These results indicate that the application of IL-15-secreting DC subsets could render DC-based anti-cancer vaccines more effective through, among others, the involvement of γδ T cells in the anti-leukemic immune response.

PIM-1 mRNA expression is a potential prognostic biomarker in acute myeloid leukemia

BackgroundHigh expression of proviral integration site for Moloney murine leukemia virus-1 (PIM-1), a serine/threonine kinase, is associated with many cancers. The main purpose of this study were to investigate that the correlation between PIM-1 mRNA levels and clinicopathologic features and its clinical significance in acute myeloid leukemia (AML).MethodsqRT-PCR was performed for 118 de novo AML and 20 AML complete remission patients and 15 normal individuals. All statistical analysis were performed using Graphpad Prism5 software.ResultsWe observed that expression of PIM-1 mRNA was higher in AML patients than in healthy individuals and in complete remission AML patients (P = 0.0177). Further, high PIM-1 mRNA levels were more associated with high-risk FLT3+ AML patients than the FLT3− group (P = 0.0001) and were also associated with clinical factors such as risk stratification (P = 0.0029) and vital status (P = 0.0322). Kaplan–Meier survival analysis indicated that PIM-1 mRNA expression correlated with overall survival (OS), disease free survival (DFS), and relapse rate (RR) in AML patients. Most importantly, the high PIM-1-expressing patients took longer to achieve complete remission than the low expression group (P = 0.001). In addition, the complete remission rate was significantly lower in the high PIM-1 group (P = 0.0277) after induction therapy.ConclusionsAbove results suggest that PIM-1 mRNA levels may be an independent prognostic factor in AML.

CD123 target validation and preclinical evaluation of ADCC activity of anti-CD123 antibody CSL362 in combination with NKs from AML patients in remission

Despite the heterogeneity of acute myeloid leukemia (AML), overexpression of the interleukin-3 receptor-α (CD123) on both the more differentiated leukemic blast and leukemic stem cells (LSCs) provides a therapeutic target for antibody treatment. Here we present data on the potential clinical activity of the monoclonal antibody CSL362, which binds to CD123 with high affinity. We first validated the expression of CD123 by 100% (52/52) of patient samples and the correlation of NPM1 and FLT3-ITD mutations with the high frequency of CD123 in AML. In vitro studies demonstrated that CSL362 potently induced antibody-dependent cell cytotoxicity (ADCC) of AML blasts including CD34+CD38−CD123+ LSCs by natural killer cells (NKs). Importantly, compared with healthy donor (HD) NKs, NKs drawn from AML patients in remission had a comparable ADCC activity against leukemic cells; of note, during remission, immature NKs were five times higher in AML patients than that in HDs. Significantly, we report a case where leukemic cells were resistant to autologous ADCC; however, the blasts were effectively lysed by CSL362 together with donor-derived NKs after allogeneic hematopoietic stem cell transplantation. These studies highlight CSL362 as a promising therapeutic option following chemotherapy and transplant so as to improve the outcome of AML patients.