Factor In Acute Myeloid Leukemia Patients Research Articles

Bioinformatics Analysis Identifies EPAS1 as a Novel Prognostic Marker Correlated with Immune Infiltration in Acute Myeloid Leukemia

EPAS1 plays an important role in the development and progression of multiple tumor types by interacting with a series of other molecules. However, the prognostic and diagnostic values of EPAS1 in acute myeloid leukemia (AML) remain unknown. Here, we systematically explored and clarified the potential functions of EPAS1 in AML using data from Xena Browser and TCGA database. The expression of EPAS1 was significantly lower in AML patients than that in healthy people. The GO, KEGG, GSEA, and GSVA were performed to explore the potential functions and signaling pathways. The survival analysis was conducted using Cox regression analysis and the Kaplan-Meier method. Immune cell infiltration was evaluated via single-sample GSEA (ssGSEA). The results of enrichment analyses suggested that low-EPAS1 expression was related to the initiation, development, and prognosis of AML. The immune microenvironment landscape in AML was described by ssGSEA. ROC analysis of EPAS1 showed high discrimination ability between AML patients and healthy people. Kaplan-Meier method indicated that low-EPAS1 expression correlated significantly with a poor overall survival. Multivariate Cox regression analysis revealed that both age and EPAS1 expression were independent prognostic factors in AML patients. Furthermore, the nomogram based on these two variables performed well in discrimination and calibration. In summary, our study may provide new insights into the molecular mechanisms underlying AML and demonstrate the diagnostic and prognostic value of EPAS1 in AML for the first time.

The Expression of mi-RNAs Involved in the Hematopoietic Niche Microenvironment in Newly Diagnosed AML Patients

Introduction Acute myeloid leukemia (AML) is a genetically heterogeneous disease and the most common type of acute leukemia in adults. Despite the progress made in the last years in AML therapy, induction treatment failure and disease relapse still remain the main challenges in clinical practice. miRNAs are involved in the pathogenesis of AML as well as influence the differentiation and proliferation of normal hematopoietic cells. The expressions of specific miRNAs have been identified as potential biomarkers for predicting prognosis both in solid tumors and hematological malignancies. Methods The expression of 12 miRNAs involved in hematopoiesis and the microenvironment of the hematopoietic niche: miR-15a-5p, miR-34a-5p, miR-125b-5p, miR-139-5p, miR-146-5p, miR-181a-5p, miR-199b-5p, miR-204-5p, miR-218-5p, miR-299-5p, miR-424-3p, miR-452-5p was determined in duplicates using qPCR in bone marrow samples of newly diagnosed AML patients. The miRCURY LNA SYBR® Green PCR Kit (Qiagen) was used to carry out qPCR. miRNAs expression was normalized using the mean expression of all miRNAs in a given sample. The normalization was performed using the formula ∆Ct = Ct(reference) − Ct(miRNA of interest). This approach results in higher values for higher miRNA expression. Differential expression analysis was carried out using a t-test. Cox proportional hazard regression analyses of clinical factors and miRNAs for predicting overall survival in AML patients were performed. Results To the study group we enrolled 45 (23 women and 22 men) newly diagnosed AML adults with the mean age 45.1 ± 12.8 years. Among them, 13.9% patients (pts) had a low-risk, 44.2% intermediate-risk and 41.9% high-risk AML according to EuropeanLeukemiaNet (ELN) 2017 risk stratification (Döhner et al. 2017). In the study group, 16.7% of pts had NPM1 mutation, whereas 30.2% of pts had FLT3-ITD or FLT3-TKD mutation. The majority of patients (88.9%) received intensive treatment: DA-60 (daunorubicin 60mg/m2 + cytarabine 200mg/m2) regimen (50%), followed by DAC (DA-60 + cladribine 5mg/m2) (40%) and DA-90 (daunorubicin 90mg/m2 + cytarabine 100mg/m2) (10%). Azacitidine in standard dose was used as the low-intensity regimen. There were no statistically significant differences in miRNAs expression according to complete remission (CR) achievement. Patients with high-risk AML had higher expression of miR-125b-5p (fold change- FC=2.3, p=0.017) and lower expression of miR-34a-5p (FC=0.3, p=0.029), miR-199b-5p (FC=0.5, p=0.039) and miR-15a-5p (FC=0.6, p=0.043) as compared to low- and intermediate-risk pts. In patients with NPM1 mutation, miR-34a-5p, miR-146a-5p and miR-15a-5p were significantly upregulated, whereas miR-139-5p, miR-424-3p and miR-452-5p were downregulated. FLT-3-mutated patients had significantly lower expression of miR-424-3p (FC=0.2, p=0.019) comparing to FLT-3-unmutated controls. In patients with unfavorable karyotype, upregulation of miR-125b-5p (FC=2.8, p=0.002) and miR-139-5p (FC=3.0, p=0.027) was observed meanwhile lower expression of miR-34a-5p (FC=0.3, p=0.035) in these patients was detected. The median overall survival (OS) in the study cohort was 12.3 months. In univariate Cox regression analysis, hematopoietic cell transplantation-comorbidity index (HCTCI) score >0 (HR 2.86, 95%CI: 1.15-7.12, p=0.024), white blood cell (WBC) count at diagnosis (HR 1.01, 95%CI: 1.00-1.01, p=0.033), and higher expression (>median) of miR-199b-5p (HR 0.31, 95%CI: 0.12-0.81, p=0.027) (Fig.1) were factors influencing the outcome. In multivariate model, higher expression of miR-199-5p (HR 0.27, 95%CI: 0.08-0.91, p=0.036) retained their significant protective effect in the context of established prognostic factors. Conclusions Our preliminary results demonstrate that the expression of miRNAs involved in the microenvironment niche is deregulated in AML patients. The upregulation of miR-199-5p at the diagnosis is associated with longer overall survival of AML patients. Significantly lower expression of miR-199-5p was reported in ELN high-risk group. These data indicate that miR-199-5p may be a potential prognostic factor in AML patients. Larger studies are needed to confirm our observation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pan-cancer analysis of oncogenic TNFAIP2 identifying its prognostic value and immunological function in acute myeloid leukemia

BackgroundTumor necrosis factor alpha-induced protein 2 (TNFAIP2), a TNFα-inducible gene, appears to participate in inflammation, immune response, hematopoiesis, and carcinogenesis. However, the potential role of TNFAIP2 in the development of acute myeloid leukemia (AML) remains unknow yet. Therefore, we aimed to study the biological role of TNFAIP2 in leukemogenesis.MethodsTNFAIP2 mRNA level, prognostic value, co-expressed genes, differentially expressed genes, DNA methylation, and functional enrichment analysis in AML patients were explored via multiple public databases, including UALCAN, GTEx portal, Timer 2.0, LinkedOmics, SMART, MethSurv, Metascape, GSEA and String databases. Data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and Beat AML database were used to determine the associations between TNFAIP2 expression and various clinical or genetic parameters of AML patients. Moreover, the biological functions of TNFAIP2 in AML were investigated through in vitro experiments.ResultsBy large-scale data mining, our study indicated that TNFAIP2 was differentially expressed across different normal and tumor tissues. TNFAIP2 expression was significantly increased in AML, particularly in French–American–British (FAB) classification M4/M5 patients, compared with corresponding control tissues. Overexpression of TNFAIP2 was an independent poor prognostic factor of overall survival (OS) and was associated with unfavorable cytogenetic risk and gene mutations in AML patients. DNA hypermethylation of TNFAIP2 at gene body linked to upregulation of TNFAIP2 and inferior OS in AML. Functional enrichment analysis indicated immunomodulation function and inflammation response of TNFAIP2 in leukemogenesis. Finally, the suppression of TNFAIP resulted in inhibition of proliferation by altering cell-cycle progression and increase of cell death by promoting early and late apoptosis in THP-1 and U937AML cells.ConclusionCollectively, the oncogenic TNFAIP2 can function as a novel biomarker and prognostic factor in AML patients. The immunoregulation function of TNFAIP2 warrants further validation in AML.

PARP-1 inhibitors enhance the chemosensitivity of leukemia cells by attenuating NF-кB pathway activity and DNA damage response induced by Idarubicin

Idarubicin (IDA), an anthracycline antineoplastic drug, is commonly used in the treatment of acute myeloid leukemia (AML) with reasonable response rates and clinical benefits. However, some patients still relapse, or do not respond, and suffer high fatality rates. Recent studies have shown that overexpression of PARP-1 may represent an important risk factor in AML patients. The aim of the present study was to determine the underlying molecular mechanisms by which the PARP-1 inhibitor Olaparib enhances the chemosensitivity of the leukemia cell line K562 and THP1 to IDA. Our data demonstrated that PARP-1 is upregulated in AML patients as well as in K562 and THP1 cells, and that the suppression of PARP-1 activity by Olaparib enhances the inhibitory effect of IDA. A mechanistic study revealed that Olaparib decreases the expressions of p-ATM, p-IκBα, XIAP and p65, and upregulates Bax, cleaved-Caspase-3 and γ-H2AX. Olaparib can enhance the induction of DNA damage by IDA, probably mediated by the inhibition of the ATM-related DNA damage response. Moreover, we also found that the nuclear translocation of p65 and the nuclear export of NEMO are inhibited when IDA and Olaparib are combined. Our results suggest that Olaparib attenuates the activity of the NF-κB pathway and decreases the DNA damage response induced by IDA. Therefore, we conclude that Olaparib is a potentially valuable chemosensitizer for leukemia patients.

IER3 (IEX-1) dysregulation serves as a potential prognostic factor in acute myeloid leukemia patients.

IntroductionImmediate early response 3 (IER3) has association with hematological malignancies’ risk and prognosis, such as myelodysplastic syndrome, while its relation to acute myeloid leukemia (AML) is not clear. This study aimed to explore the correlation of IER3 with AML risk, clinical characteristics, complete remission (CR), event‐free survival (EFS), and overall survival (OS).MethodsA total of 93 de novo AML patients were included in this study. In addition, 30 patients with non‐hyperplasia hematologic malignancies requiring bone marrow testing (as disease controls) and 30 health donors (as health controls) were also recruited. Bone morrow samples of AML patients (before treatment), disease controls (before treatment), and health controls (at donation) were collected. IER3 in bone marrow mononuclear cells was detected by reverse transcription‐quantitative polymerase chain reaction.ResultsIER3 was increased in AML patients compared with disease controls and health donors (both P < .001), and receiver operating characteristic (ROC) curve showed that IER3 had certain capability of distinguishing AML patients from disease controls (area under curve (AUC): 0.735, 95% confidence interval (CI): 0.650‐0.820), and health donors (AUC: 0.789, 95% CI: 0.712‐0.866). Meanwhile, IER3 was correlated with FLT3‐ITD mutation (P = .030) and poor NCCN risk stratification (P = .031) in AML patients. Moreover, IER3 had negative association with CR in AML patients (P = .022), and showed certain potential in discriminating CR patients from non‐CR patients (AUC: 0.655, 95% CI: 0.533‐0.777). Besides, IER3 was negatively associated with EFS (P = .033), but not OS (P = .083) in AML patients.ConclusionIER3 dysregulation serves as a potential prognostic factor in AML patients.

Characteristics, clinical outcomes, and risk factors of SARS-COV-2 infection in adult acute myeloid leukemia patients: experience of the PETHEMA group

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection produces higher morbidity and mortality in hematological malignancies, but evidence in acute myeloid leukemia (AML) is scarce. A multicenter observational study was conducted to determine the clinical outcomes and assess the impact of therapeutic approaches in adult AML patients with SARS-CoV-2 infection in the first wave (March–May 2020). Overall, 108 patients were included: 51.9% with active leukemia and 70.4% under therapeutic schedules for AML. Signs and symptoms of SARS-CoV-2 were present in 96.3% of patients and 82.4% received specific treatment for SARS-CoV-2. The mortality rate was 43.5% and was correlated with age, gender, active leukemia, dyspnea, severe SARS-CoV-2, intensive care measures, neutrophil count, and D-dimer levels. A protective effect was found with azithromycin, lopinavir/ritonavir, and normal liver enzyme levels. During the SARS-CoV-2 first wave, our findings suggested an increased mortality in AML in a short period. SARS-CoV-2 management could be guided by risk factors in AML patients.

Effect of long non-coding RNA MBNL1-AS1 expression on prognosis of acute myeloid leukemia patients

Objective: To explore the prognosis effect of the expression of long-chain non-coding RNA (lncRNA) MBNL1-AS1 on acute myeloid leukemia (AML) patients. Methods: One hundred and twenty-five AML patients of the Cancer Genome Atlas (TCGA) from November 2001 to March 2010 were involved, including 70 patients who received chemotherapy only and other 55 patients treated with allogeneic hematopoietic stem cell transplantation (allo-HSCT) in addition to chemotherapy. According to the median expression of lncRNA MBNL1-AS1, patients of chemotherapy group were divided into high expression sub-group(n=35) and low expression sub-group (n=35), and patients of allo-HSCT group were also divided into high expression sub-group (n=28) and low expression sub-group (n=27) for prognosis analysis. Clinical characteristics at diagnosis, including peripheral white blood cell counts (WBC), blast percentages in peripheral blood and bone marrow (BM), French-American-British (FAB) subtypes and the frequencies of common genetic mutations in AML were described. The event-free survival (EFS) rate and overall survival (OS) rate of patients in different groups were analyzed, and the influence of the clinical characteristics of patients on the prognosis of AML was analyzed by COX multivariate analysis. Results: In the chemotherapy group, patients with low lncRNA-MBNL1-AS1 expression had significantly lower EFS and OS (60.0%, 8.6%) than patients with high lncRNA-MBNL1-AS1 expression (68.6%, 34.3%) (χ²=7.817, 10.880, all P<0.01). However, in the alloHSCT group, no significant differences were observed in EFS and OS of patients between high and low expression groups of lncRNA-MBNL1-AS1 (all P>0.05). COX multivariate analysis confirmed that age≥60 years old (EFS: HR (95%CI): 6.934 (1.918-25.075),P=0.003;OS: HR (95%CI): 4.119 (1.812-9.364), P=0.001), and low expression of lncRNA MBNL1-AS1 (EFS: HR (95%CI): 0.354 (0.126-0.941), P=0.038; OS: HR (95%CI): 0.424 (0.231-0.778), P=0.006)were independent risk factors for EFS and OS in the chemotherapy group. Conclusion: The long-chain non-coding RNA MBNL1-AS1 is related to the prognosis of AML, and its low expression is an independent poor prognostic factor in AML patients.

The Impact of Induction Treatment Response on the Prognosis of Pediatric Core Binding Factor-Acute Myeloid Leukemia Patients

To explore the impact of induction treatment response on the prognosis of pediatric core binding factor-acute myeloid leukemia (CBF-AML). The result of induce reaction and survival data of 157 pediatric CBF-AML patients in our hospital from September 2008 to December 2018 were retrospectively analyzed．The survival rate of the patients with different degrees of morphological remission after induction chemotherapy was comparative analyzed. Among the 157 children with CBF-AML, 113 (72.4%) patients achieved morphologic leukemia-free state (MLFS) after the first course of induction chemotherapy, 153 (98.1%) patients achieved MLFS after the second course of induction chemotherapy. The 5-year event-free survival (EFS) rate and 5-year overall survival (OS) rate of patients with non-remission (NR) status after the first course of induction of chemotherapy was significantly lower than the patients achieved MLFS and the patients achieved partial remission (PR). The 5-year EFS rate and 5-year OS rate of the patients with PR status after the second course of induction chemotherapy were lower than the patients achieved MLFS, but the difference was not statistically significant. Multivariable analyze showed that NR after the first course of induction chemotherapy and myeloid sarcoma were the independent risk factors affecting EFS of the patients. There were six patients with NR status after the first course of induction chemotherapy, in which all of them harbored t(8;21), three of them with sex chromosome deletion, two of them with myeloid sarcoma. NR status after the first course of induction chemotherapy was the independent risk factor affecting EFS and OS of CBF-AML patients, it can be taken as an indicator for higher risk stratification. PR status after the first course of induction chemotherapy may not be used as a diagnostic criterion for primary drug resistance.

WT-1, BAALC, and ERG Expressions in Iranian Patients with Acute Myeloid Leukemia Pre- and Post-chemotherapy.

Purpose: Acute myeloid leukemia (AML) is the most prevalent acute leukemia in adults. It possesses different cytogenetic and molecular features. The expression of Wilms tumor-1 (WT1), brain and acute leukemia, cytoplasmic (BAALC) and ETS-related gene (ERG) might be considered as prognostic factors in AML patients. The aim of this study was to determine the mRNA expressions of WT-1, BAALC and ERG genes in bone marrow of mononuclear cells and their effects on complete remission in the Iranian AML patients, pre- and post- chemotherapy. Methods: Forty AML patients with normal karyotype were evaluated. The mRNA gene expressions were measured with quantitative real-time PCR in bone marrow of mononuclear cells of AML patients at the baseline and after chemotherapy. The subtypes of AML and flow cytometry panel were also assessed. Complete remission (CR) after the treatment was addressed for all patients. Results: The mRNA expressions of WT-1, BAALC and ERG were significantly decreased after the treatment (p = 0.001, 0.017, 0.036). WT-1 mRNA expression was inversely correlated with CR after chemotherapy (P =0.024). There was also significant correlation between baseline expression of BAALC and CR (P =0.046). No significant correlation was observed between ERG and CR pre- and post- chemotherapy (P =0.464 and 0.781). There was also significant correlation between BAALC mRNA expression and CD34+ (P <0.001). Conclusion: The present study showed that WT-1 decreased significantly after standard chemotherapy which could have favorable effects on CR. Also, the high expression of BAALC could have a poor prognostic role in AML patients. The identification of these gene expressions can be an efficient approach in targeted therapy among AML patients.

Identification and Validation of STAT6 as a Prognostic and Predictive Biomarker in Acute Myeloid Leukemia.

BackgroundAcute myeloid leukemia (AML) is one of the most common hematological diseases in adults. The overall survival rate remains unsatisfactory. It is urgent to identify potential prognostic biomarkers and develop new molecular therapeutic strategies for AML. Signal transducer and activator of transcription (STAT) is a family of genes that encode intracellular transcription factors. STATs are associated with leukemogenesis, cellular transformation, and cell cycle in AML.MethodsWe used sequencing data and clinical data from The Cancer Genome Atlas (TCGA) and ONCOMINE to identify expression difference, gene variability and correlation as well as prognostic effects of STAT genes in AML patients. Then, we verified the expression difference of STAT6 between healthy control and AML patients and its prognostic impact in Gene Expression Omnibus (GEO) database and our own recruited cohort.ResultsThe mRNA level of STAT6 was increased in AML patients among TCGA, GEO and ONCOMINE public datasets and was found to be an independent risk factor of overall survival in all AML patients and patients who only received chemotherapy by multivariate analysis. In our study, STAT6 mRNA level was markedly up-regulated in AML patients (n=105) compared to healthy donor (n=39) (P=0.0435) as a validated cohort. Patients that only received chemotherapy in high STAT6 group showed significantly lower overall survival (OS) (P=0.0055).ConclusionSTAT6 expression was increased in AML patients. STAT6 was found to be an adverse prognosis factor in AML patients, especially those who only received chemotherapy treatments.

Prognostic Value of Interleukin-17 A Gene Polymorphism and Serum IL-17 Levels in Adult Acute Myeloid Leukaemia Patients

Background: T helper 17 (Th17) cells play a role in the pathogenesis and prognosis of acute myeloid leukemia (AML). Th17 cells produce interleukin (IL)-17A and IL-17F, that share strong surface expression and homology of the IL-23 receptor (IL-23R). Objective: To evaluate the expression of IL-17A gene polymorphism and IL-17 serum level in adult AML patients and to clarify its prognostic significance. Patients and methods: In this prospective study, 48 patients with de novo AML and 48 healthy matched controls were enrolled. Cases were diagnosed at clinical pathology and managed in Medical Oncology Departments. We evaluated both serum IL-17 levels, measured by ELISA, and IL-17A (rs2275913) gene polymorphism was assessed using realtime polymerase chain reaction (PCR) in all patients and control. All patients received “3+7” as induction, responders received 3-4 courses of high dose cytarabine as consolidation, while non-responders received salvage therapy. Results: Serum levels of IL-17 were higher in AML patients (median 27 pg/ml) vs 17 pg/ml in the control group (P < 0.001). Also, serum IL 17 level was higher in non-responders with median 37.5 (pg/ml) vs 23 (pg/ml) in responders (P < 0.001). while IL-17A mutant genotypes and alleles showed no significant relation between expression of IL-17 A gene polymorphism and response to treatment or outcome of studied AML cases. Conclusion: Serum IL-17 levels can be considered a useful diagnostic and prognostic factor in AML patients, unlike IL-17 A gene polymorphisms.

Prognostic Value of Interleukin-17 A Gene Polymorphism and Serum IL-17 Levels in Adult Acute Myeloid Leukaemia Patients

Background: T helper 17 (Th17) cells play a role in the pathogenesis and prognosis of acute myeloid leukemia (AML). Th17 cells produce interleukin (IL)-17A and IL-17F, that share strong surface expression and homology of the IL-23 receptor (IL-23R). Objective: To evaluate the expression of IL-17A gene polymorphism and IL-17 serum level in adult AML patients and to clarify its prognostic significance. Patients and methods: In this prospective study, 48 patients with de novo AML and 48 healthy matched controls were enrolled. Cases were diagnosed at clinical pathology and managed in Medical Oncology Departments. We evaluated both serum IL-17 levels, measured by ELISA, and IL-17A (rs2275913) gene polymorphism was assessed using realtime polymerase chain reaction (PCR) in all patients and control. All patients received “3+7” as induction, responders received 3-4 courses of high dose cytarabine as consolidation, while non-responders received salvage therapy. Results: Serum levels of IL-17 were higher in AML patients (median 27 pg/ml) vs 17 pg/ml in the control group (P < 0.001). Also, serum IL 17 level was higher in non-responders with median 37.5 (pg/ml) vs 23 (pg/ml) in responders (P < 0.001). while IL-17A mutant genotypes and alleles showed no significant relation between expression of IL-17 A gene polymorphism and response to treatment or outcome of studied AML cases. Conclusion: Serum IL-17 levels can be considered a useful diagnostic and prognostic factor in AML patients, unlike IL-17 A gene polymorphisms.

AML-134: Dysfunctions in Primary Hemostasis as Additional Risk Factors of Bleeding in Patients with Newly Diagnosed Acute Myeloid Leukemia (AML)

Background Bleeding events are common adverse events in newly diagnosed AML. The underlying mechanisms have not been fully investigated and need to be looked at in more depth. Objective The primary endpoints were qualitative and quantitative functions of plasma von Willebrand factor (vWF) by measuring of vWF activity (vWF:ristocetin cofactor [vWF:RCo]), vWF level (vWF:antigen [vWF:Ag]), factor VIII coagulation activity regarding vWF (vWF:FVIII), and FVIII activity (FVIII) as additional bleeding risk factors in AML patients since the diagnosis and during the first course of treatment. Patients and methods A total number of 10 patients with newly diagnosed AML (excluded APL) and 10 age-matched and sex-matched healthy individuals (control group). The severity of bleeding events was assessed according to Modified WHO Bleeding Scale. Results Four patients in the study group were experiencing bleeding events (G1-4) before and during the first course of treatment and six patients had no bleeding episodes (G0). There were no bleeding episodes in the control group (G0). Six patients with AML had levels of vWF:FVIII below normal ranges (median 29.85 %, range 20.25–40.55), four of them had different grades of spontaneous bleeding events before and during the first course of treatment. Thus, levels of vWF:FVIII below normal ranges in patients with AML was associated with higher risk of spontaneous bleeding events (p=0.019). We have monitored decreased levels of vWF:RCo in three patients with AML and similar in the control group. Three patients with vWF:RCo below normal ranges (median 26,6 %, range 22.5–72.85) in the study group had bleeding events (Grade 1-4), one of them died during the first course of chemotherapy due to gastrointestinal bleeding. Despite a similar decline in vWF:RCo below normal ranges in the control group, bleeding events have not emerged. Conclusions In our study group, decreased level of vWF:FVIII was associated with spontaneous bleeding events and may play an etiologic role in haemorrhagic complications in patients with newly diagnosed AML. The activity of vWF can be a promising risk factor of spontaneous bleeding combined with vWF:FVIII and vWF:Ag in newly diagnosed AML.

Signatures of Immune Dysfunction and Identification of Prognostic Immune-Related Risk Factors in Patients with Acute Myeloid Leukemia

There is growing appreciation that immune system plays an important role in anti-leukemia effect in patients with acute myeloid leukemia (AML). However, comprehensive illustration of phenotypic and functional immune signatures of AML at diagnosis is still lacking. Here, we conducted a prospective study to perform a comprehensive peripheral blood immune profiling through multiparameter flow cytometry and investigated prognostic immune-related risk factors in AML patients. In cohorts of 50 newly-diagnosed patients and 24 healthy controls, we performed phenotypic and functional analysis of circulating lymphocytes including CD4+ T, CD8+ T, natural killer (NK), natural killer-liked T (NKT), gamma delta T (γδ T) and B cells to elucidate the nature of immune defects in AML development. Our findings suggest that immune defects in AML patients are operative in T and NK cells that are important component of antitumor immunity while B cell function remains unaffected. Both CD8+ T and CD4+ T cells from AML patients exhibit features of senescence and exhaustion at diagnosis. These terminally senescent and exhausted T cells are phenotypically characterized by increased expression of CD57 and PD-1 and decreased expression of CD28. In addition, lessened frequency of NK, NKT and γδ T cells indicate slack innate immunity in AML. One important parameter involved in NK cell function is the maturation stage, and we found similarly increased NK cell maturation in AML. NK cell dysfunction was further supported by downregulation of activating receptor NKG2D and NKP30, which may weaken the recognition and interaction between NK cells and AML blasts. Furthermore, diseased γδ T cells demonstrate a highly-activated or even exhausted state through a profound PD-1 upregulation and NKG2D downregulation. We next performed immunologic studies on paired pre- and post-chemotherapy samples to analyze whether immune dysfunction was recovered during the course of induction chemotherapy. Immune reconstitution occurs only in patients with therapeutic response. T and NK cell function restoration is principally reflected in recovered proportions, decreased extent of terminal differentiation and partly improved T cell senescence and exhaustion. Moreover, we extended studies to analyze the difference in immune signatures between CR group (achieved stable complete remission after chemotherapy, n = 24) and RR group (not achieved complete remission after chemotherapy or suffered a relapse, n = 20). Patients in RR group exhibit selectively more senescent and exhausted CD8+ T (CD28lo/CD57hi/PD-1hi) and CD4+ T (PD-1hi) cells, lower percentages of γδ T cells and excessive NK maturation state. More importantly, we demonstrated that specific immune phenotypes apparently correlate with overall survival (OS) and event-free survival (EFS, the "event" refers to progression, relapse and death) in AML patients. Median values of the complete cohort were used for grouping the patients. Poor OS correlated with select pre-chemotherapy circulating PD-1+CD8+ and CD28-CD57+CD8+ T subsets (Figure 1). Elevated day +30 post induction chemotherapy PD-1+CD8+ T frequency and increased NK cell maturation correlated with unfavorable OS and EFS after induction chemotherapy. Moreover, decreased day +30 post induction chemotherapy CD28-CD57+CD8+ T and increased γδ T cell proportion were associated with improved EFS, but not with OS (Figure 2 and Figure 3). This is the first study to provide insights into longitudinal immunological landscape at diagnosis and after chemotherapy in patients with AML. Select immune phenotypes correlate with EFS and OS, implying that non-invasive immune testing of blood samples can be applied to identify high risk for relapse and unfavorable prognosis. Personized immunotherapies, aimed at reactivating senescent T cells, reverting exhausted T cells or improving innate NK and γδ T immunity, may contribute to better therapeutic effects and reduced early relapse. Disclosures No relevant conflicts of interest to declare.

The Proportional Association between WT1 mRNA Level in Peripheral Blood before Allogeneic Hematopoietic Cell Transplantation and Risk of Mortality in Acute Myeloid Leukemia Not in Remission

Abstract INTRODUCTION The prognosis for allogeneic hematopoietic cell transplantation (allo-HCT) is relatively poor in patients with acute myeloid leukemia (AML) who are not in remission (non-CR). Wilms' tumor-1 gene messenger ribonucleic acid (WT1 mRNA), which is overexpressed in almost all AML, has been used as an indicator of monitoring minimal residual disease (MRD) and has been regarded as one of the prognostic factors in AML patients with complete remission (CR). However, the association between the expression levels of WT1 mRNA in peripheral blood (WT1) before allo-HCT and risk of mortality in AML patients in non-CR remains quite elusive. We therefore assessed the effect of the pre-transplant WT1 level on survival after allo-HCT in AML patients in non-CR. METHODS We retrospectively analyzed data from consecutive patients undergoing allo-HCT at our institution between November 2007 and January 2017. We excluded patients who did not have an available WT1 value within 28 days before the start of conditioning. RESULTS A total of 125 AML patients, including 46 non-CR patients (36.8 %), were eligible for this study. The median age was 48 years (range: 17-68 years) and the number of males was 69 (55.2%). Forty-two patients (33.6%) had AML with myelodysplasia-related changes. This study included 34 patients (27.2%) who had undergone multiple transplantation. Myeloablative and reduced-intensity conditioning were used in 73 (58.4%) and 52 (41.6%) patients, respectively. In 63 patients who received transplants from either a related peripheral blood stem cell or bone marrow (BM) donor, four pairs (6.3%) were serologically mismatched for one antigen of HLA and 45 pairs (71.4%) received transplants from HLA haploidentical donors. In 32 patients who received transplants from an unrelated BM donor, seven pairs (21.9%) were serologically mismatched for one HLA antigen. Twenty-nine patients (23.2%) received unrelated cord blood transplantation. Among 46 non-CR patients, the median follow-up period among survivors was 1,092 days (range: 688-2,607 days) following allo-HCT. Receiver operating characteristic analysis for prediction of composite events including any cause of death or subsequent transplantation indicated that the best cutoff value for WT1 was 5,000 copies/ μg RNA in non-CR AML patients. Estimated two-year, event-free survival of non-CR patients with WT1 levels of less than 5,000 copies/ μg RNA was significantly higher than that of those with WT1 levels of 5,000 copies/ μg RNA or more (41.2% [95% confidence interval (CI), 18.6-62.6] vs 10.3% [95% CI, 2.6-24.3]; P=0.006). On multivariate analysis of the non-CR patients, WT1 5,000 copies/ μg RNA or more was significantly associated with increased risk of mortality (hazard ratio (HR), 2.7; 95% CI, 1.3-5.5; P=0.008). Log10-transformed WT1 (continuous scale) was also significantly related to increased risk of mortality (HR, 1.5; 95% CI, 1.0-2.1; P=0.037). In the entire cohort, log10-transformed WT1 (divided into four categories) before allo-HCT was found to be the most powerful prognostic factor of survival among remission status, cytogenetic risk group, and revised disease risk index, applying Akaike's information criterion. Moreover, under the assumption of a non-linear model, as the log10-transformed WT1 level rose, the HR of mortality increased monotonically; a non-linear fit was obtained using a restricted cubic spline function (three knots; Figure 1). CONCLUSION Our data showed that WT1 levels obtained before allo-HCT monotonically increased HR of mortality in a non-linear manner regardless of CR status. Of importance is the finding that WT1 levels before allo-HCT were proportionally correlated with probability of survival after allo-HCT in non-CR AML patients. This suggests that the level of expression of WT1 mRNA in peripheral blood might reflect tumor burden and that there may be a WT1 threshold in non-CR patients for benefiting from allo-HCT. Further prospective studies of WT1 are necessary to determine acceptable or feasible levels of disease activity for receiving allo-HCT in relapsed or refractory AML patients. Disclosures Nakamae: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Research Funding. Hino:Otsuka Pharmaceutical Co., Ltd.: Research Funding; Novartis: Research Funding. Nakamae:Otsuka Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Research Funding.

FLT3-ITD compared with DNMT3A R882 mutation is a more powerful independent inferior prognostic factor in adult AML patients after Allogeneic Hematopoietic Stem Cell Transplantation: A retrospective cohort study

ObjectiveThis study aimed to evaluate DNMT3A exon 23 mutations and their prognostic impacts in the presence of NPM1 and FLT3 mutations in acute myeloid leukemia (AML) patients who underwent allogeneic hematopoietic stem cell transplantation (HSCT).Materials and MethodsThis study comprised 128 adult AML patients referred to the Hematology-Oncology and Stem Cell Research Center of Shariati Hospital. NPM1 and FLT3-ITD mutations were detected by fragment analysis. For DNMT3A exon 23 mutation analysis, we used Sanger sequencing. Overall survival (OS) and relapse-free survival (RFS) curves were estimated by the Kaplan-Meier method and the log-rank test was used to calculate differences between groups.ResultsThe prevalence of DNMT3A exon 23 mutations was 15.6% and hotspot region R882 mutations were prominent. RFS and OS were compared in patients with and without DNMT3A exon 23 mutations using univariate analysis and there was no significant difference between these groups of patients. On the contrary, the FLT3-ITD mutation significantly reduced the OS (p=0.009) and RFS (p=0.006) in AML patients after allogeneic HSCT. In the next step, patients with AML were divided into four groups regarding FLT3-ITD and DNMT3A mutations. Patients with DNMT3A R882mut/FLT3-ITDpos had the worst OS and RFS. These results indicate that DNMT3A mutations alone do not affect the clinical outcomes of AML patients undergoing allogeneic HSCT, but when accompanied by FLT3-ITD mutations, the OS was significantly reduced (5-year OS 0% for DNMT3A R882mut/FLT3-ITDpos patients vs. 62% DNMT3A R882wt/FLT3-ITDneg, p=0.025) and the relapse rate increased.ConclusionIt can be deduced that DNMT3A R882mut/FLT3-ITDpos is an unfavorable prognostic factor in AML patients even after allogeneic HSCT.

Methylation-associated DOK1 and DOK2 down-regulation: Potential biomarkers for predicting adverse prognosis in acute myeloid leukemia.

DOK-1 and DOK-2 (DOK1/2) are closely related members of downstream of tyrosine kinase (DOK) family genes, which are found to be frequently rearranged in several hematopoietic cancers. However, the clinical implications of DOK1/2 in acute myeloid leukemia (AML) remain largely unknown. To investigate the clinical significance, real-time quantitative PCR (RQ-PCR) was carried out to detect DOK1/2 expressions in 125 de novo AML patients and 28 healthy controls. Real-time quantitative methylation-specific PCR (RQ-MSP) and bisulfite sequencing PCR (BSP) were applied to detect DOK1/2 methylation level and density. DOK1/2 expressions were significantly down-regulated in AML patients. The promoters of DOK1/2 were highly hypermethylated and negatively correlated with DOK1/2 expressions in AML patients. In addition, we also confirmed that DOK1/2 expressions could be restored by DOK1/2 demethylation using 5-aza-2'-deoxycytidine in leukemia cell line THP-1. Survival analyses showed that low-expressed DOK1/2 were associated with markedly shorter overall survival and leukemia free survival in both whole-cohort AML and non-M3 AML patients. Multivariate analyses further revealed that DOK1/2 were act as independent prognostic factors in AML patients. These findings indicate that decreased DOK1/2 expressions associated with their promoter hypermethylations predict adverse prognosis in AML.

Genome-wide genotype-based risk model for survival in core binding factor acute myeloid leukemia patients.

The present study attempted to build a single nucleotide polymorphism (SNP)-based risk model for predicting overall survival (OS) and event-free survival (EFS) in patients with core binding factor acute myeloid leukemia (CBF-AML). Adopting genome-wide SNP array using Affymetrix SNP array 6.0, we analyzed 868,157 SNPs with respect to OS and EFS in 104 patients with CBF-AML. Significant SNPs were identified from single SNP analysis. The risk model was constructed with incorporation of six SNPs and three clinical factors (age, c-kit exon 17 mutation, and LDH) for OS and six SNPs and three clinical factors (age, WBC, and LDH) for EFS. The model was further defined into low- and high-risk groups based on risk scores. The median age was 39years, and the subgroup of t(8;21) and inv(16) or t(16;16) was assessed in 68 (65.4%) and 36 patients (34.6%). Finally, six SNPs per each OS (rs4353685, rs4908185, rs7709207, rs12034, rs1554844, and rs17241868) and EFS (rs13385610, rs11210617, rs11169282, rs7709207, rs4438401, and rs16894846) were incorporated into the risk model. OS was significantly different in favor of the low risk group (80.4 ± 8.4%) compared to the high-risk group (22.0 ± 7.3% at 3years; p = 8.75 × 10- 13; HR 8.67). For EFS, there was also a significant difference between the low- (75.0 ± 5.8%) versus high-risk group (17.1 ± 6.3% at 3years; p = 5.95 × 10- 13; HR 7.67). A genome-wide SNP-based risk model can stratify CBF-AML patients according to their OS and EFS in 104 patients.

The prognostic implication of SRSF2 mutations in Chinese patients with acute myeloid leukemia.

Recently, somatic mutations in SRSF2 gene have been discovered in a proportion of hematologic malignancies including acute myeloid leukemia (AML). This study was aimed to investigate SRSF2 mutations in Chinese AML patients. High-resolution melting analysis (HRMA) was developed to screen SRSF2 mutations in 249 cases with AML, and then direct DNA sequencing was used to verify the results of HRMA. In this study, 3.6% (9/249) of Chinese AML patients were found with heterozygous SRSF2 mutations. Patients with SRSF2 mutations were older than those with wild-type SRSF2 (P = 0.014). No differences in the sex, blood parameters, French-American-British classification (FAB) subtypes, and karyotypes were observed between AML patients with and without SRSF2 mutations. Although the overall survival (OS) of SRSF2-mutated patients was inferior to those without mutations in both whole AML patients (median 4 vs. 11months, respectively; P = 0.006) and cytogenetically normal patients (median 2 vs. 12months, respectively; P = 0.008), multiple analysis disclosed that SRSF2 mutation was not an independent prognostic factor in AML patients. These results suggest that SRSF2 mutation occurs at a low frequency in aged AML patients and might not be associated with adverse prognosis in Chinese AML patients.

Examination of the FLT3 and NPM1 mutational status in patients with acute myeloid leukemia from southeastern Poland.

IntroductionAcute myeloid leukemia (AML) is a genetically heterogeneous disease at both the cytogenetic and molecular levels. In AML cells many chromosomal aberrations are observed, some of them being characteristic of a particular subtype of patients, and others being less significant. Besides chromosomal abnormalities, the leukemic cells can have a variety of mutations involving individual genes. The aim of this work was to investigate the frequencies of molecular alterations with the focus on FLT3-ITD and NPM1 mutations in AML patients of different age groups living in a southeastern region of Poland.Material and methodsThe study group comprised 50 consecutive AML patients. We analyzed bone marrow samples by conventional cytogenetics. Cytogenetic evaluation in selected cases was complemented by the FISH technique. The internal tandem mutation in the FLT3 gene was identified using polymerase chain reaction (PCR), and the NPM1 mutation was assessed by direct nucleotide sequencing.ResultsThe studies using classical cytogenetics showed chromosomal aberrations in 32 (64%) patients. In 18 cases no changes in the karyotype were found by conventional karyotyping. FLT3-ITD mutation was detected in 4 (8%) patients and mutation of NPM1 in 3 patients with AML (6%).ConclusionsThe incidence of both mutations in our study group was lower than described elsewhere. We have confirmed that FLT3-ITD occurred more commonly in older patients and it was associated with shorter overall survival. By contrast, mutation of exon 12 of the NPM1 gene seems to be a good prognostic factor in AML patients with normal karyotype.