Mutational analysis of DNMT3A improves the prognostic stratification of patients with acute myeloid leukemia.

Nucleophosmin1 (NPM1) mutations are the most frequently detected gene mutations in acute myeloid leukemia (AML) and are considered a favorable prognostic factor. We retrospectively analyzed the prognosis of 605 Japanese patients with de novo AML, including 174 patients with NPM1-mutated AML. Although patients with NPM1-mutated AML showed a high remission rate, this was not a favorable prognostic factor for overall survival (OS); this is contrary to generally accepted guidelines. Comprehensive gene mutation analysis showed that mutations in codon R882 of DNA methyltransferase 3A (DNMT3AR882 mutations) were a strong predicative factor indicating poor prognosis in all AML (p < 0.0001) and NPM1-mutated AML cases (p = 0.0020). Furthermore, multivariate analysis of all AML cases showed that DNMT3AR882 mutations and the co-occurrence of internal tandem duplication in FMS-like tyrosine kinase 3 (FLT3-ITD), NPM1 mutations, and DNMT3AR882 mutations (triple mutations) were independent factors predicting a poor prognosis related to OS, with NPM1 mutations being an independent factor for a favorable prognosis (hazard ratios: DNMT3AR882 mutations, 1.946; triple mutations, 1.992, NPM1 mutations, 0.548). Considering the effects of DNMT3AR882 mutations and triple mutations on prognosis and according to the classification of NPM1-mutated AML into three risk groups based on DNMT3AR882 /FLT3-ITD genotypes, we achieved the improved stratification of prognosis (p < 0.0001). We showed that DNMT3AR882 mutations are an independent factor for poor prognosis; moreover, when confounding factors that include DNMT3AR882 mutations were excluded, NPM1 mutations were a favorable prognostic factor. This revealed that ethnological prognostic discrepancies in NPM1 mutations might be corrected through prognostic stratification based on the DNMT3A status.

Immunogenicity of three versus four doses of 13-valent pneumococcal conjugate vaccine followed by 23-valent pneumococcal polysaccharide vaccine in allogeneic haematopoietic stem cell transplantation recipients: a multicentre, randomized controlled trial.

OBJECTIVE: This multicentre, phase 2, randomized, controlled study of allogeneic haematopoietic stem cell transplantation (allo-HSCT) recipients compared the immunogenicity of two anti-pneumococcal vaccine regimens: four doses of 13-valent pneumococcal conjugate vaccine (PCV13) followed by 23-valent pneumococcal polysaccharide vaccine (PPSV23) (3+1+1 experimental group), and three doses of PCV13 followed by PPSV23 (3+0+1 group). METHODS: Allo-HSCT recipients without active graft-versus-host disease at enrolment were eligible. The primary endpoint was the IgG response rate (≥0.20 mg/mL) for all eight measured serotypes at 5 months after the PPSV23 booster. RESULTS: Seventy-two recipients were randomized, and seventy recipients who received over one PCV13 dose were analysed. The mean ages were 47.2 years (standard deviation, 14.4) in the 3+1+1 group (n = 35) and 49.0 years (standard deviation, 14.3) in the 3+0+1 group (n = 35). There was no significant difference in the overall IgG response rate at 5 months after the PPSV23 booster between the 3+1+1 and 3+0+1 groups (100% (26/26) vs. 93% (27/29), respectively, relative risk (RR): 1.07; 95% confidence interval (CI): 0.97-1.19). This rate was high immediately before the PPSV23 booster in the 3+1+1 group (100% (26/26) compared with 81% (21/26), respectively, RR: 1.24; 95% CI: 1.03-1.49), but this difference disappeared 1 month after the PPSV23 booster (100% (26/26) vs. 97% (28/29), respectively, RR: 1.04; 95% CI; 0.97-1.11). No serious adverse events leading to study dropout occurred. DISCUSSION: We were not able to determine the efficacy of the experimental arm based on the IgG response rate at 5 months after the PPSV23 booster in allo-HSCT recipients.

Safety and efficacy of high-dose cytarabine MEAM therapy and other treatments for auto-peripheral blood stem cell transplantation: A retrospective comparative study.

AIM: The MEAM regimen consisting of ranimustine (MCNU), etoposide (ETP), cytarabine (Ara-C), and melphalan (MEL) is widely used before auto-peripheral blood stem cell transplantation (auto-PBSCT) for malignant lymphoma in Japan. The MEAM regimen generally consists of 200-400 mg/m2 for 4 days, but we decided to increase the dosage of Ara-C from the standard to 2 g/m2 for 2 days with the aim of increasing drug transferability to the central nervous system. We evaluate the safety and therapeutic efficacy of high-dose Ara-C MEAM therapy. METHODS: The high-dose Ara-C MEAM protocol consisted of MCNU 300 mg/m2 on day -7, ETP 200 mg/m2 on days -6, -5, -4, -3 and Ara-C 2 g/m2 on day -4 -3, and MEL 140 mg/m2 on day -2. We retrospectively analyzed 37 cases of malignant lymphoma at our institution between May 2014 and July 2020. RESULTS: All patients got engraftment and there were no cases of treatment-related mortality. In all cases, the 3-year overall survival (OS) and progression-free survival (PFS) after transplantation were 80.6% and 65.7%, respectively. Twenty-one cases of diffuse large B-cell lymphoma recurrence, for which there is proven usefulness of auto-PBSCT, showed good results after transplantation, with the 3-year OS and PFS after transplantation being 100% and 74.3%, respectively. CONCLUSION: The safety and efficacy of high-dose Ara-C MEAM therapy were demonstrated, but the expected therapeutic effect on central nervous system lesions could not be fully evaluated owing to the small number of cases.

Simultaneous detection of JAK2, CALR, and MPL mutations and quantitation of JAK2 V617F allele burden in myeloproliferative neoplasms using the quenching probe-Tm method in i-densy IS-5320.

INTRODUCTION: Accurate detection of myeloproliferative neoplasms (MPN)-associated gene mutations is necessary to correctly diagnose MPN. However, conventional gene testing has various limitations, including the requirement of skilled technicians, cumbersome experimental procedures, and turnaround time of several days. The gene analyzer i-densy IS-5320 allows gene testing using the quenching probe-Tm method. Specifically, pretreatment of samples including DNA extraction, amplification and detection of genes, and analysis of results are performed in a fully automatic manner after samples and test reagents are added into this system, which is compact and can be easily installed in a laboratory. The aim of this study is to investigate the sensitivity and specificity associated with the simultaneous detection of MPN-associated gene mutations. METHODS: We conducted an analysis of MPN-associated genes using i-densy IS-5320. We analyzed 384 samples (171 JAK2 V617F mutations, 10 JAK2 exon12 mutations, 104 CALR mutations, and 26 MPL mutations) that had been examined using conventional approaches such as allele-specific polymerase chain reaction (PCR), droplet digital PCR, and the direct sequencing method. RESULTS: The detection accuracy of JAK2 V617F, JAK2 exon 12, CALR, and MPL was 100.0% (383/383), 99.7% (383/384), 100.0% (370/370), and 99.7% (377/378), respectively. There was a strong positive correlation between the JAK2 V617F allele burden measured using conventional methods and i-densy IS-5320 (r = .989). CONCLUSION: Overall, i-densy IS-5320 exhibited good accuracy in terms of analyzing MPN-associated genes; thus, it can serve as a replacement for conventional methods of MPN-associated gene testing.

Outcomes of Patients with Early Hyperbilirubinemia after Allogeneic Hematopoietic Stem Cell Transplantation.

BACKGROUND: Because the cause of liver dysfunction after allogeneic hematopoietic stem cell transplantation (HSCT) is difficult to identify in the early stages, treatment may be delayed. Therefore, early factors associated with unfavorable outcomes of liver dysfunction must be identified. The objective of this study was to identify unfavorable prognostic factors for liver dysfunction during the early period after transplantation. METHODS: We defined liver dysfunction as elevated liver or biliary enzyme levels (corresponding to Grade 2 in the Common Terminology Criteria for Adverse Events version 4.0) within 30 days of transplantation and retrospectively investigated data from 82 patients who had undergone allogeneic HSCT at our center. RESULTS: Elevated liver or biliary enzyme levels were observed in almost half of the patients studied (n=40, 48.7%). Elevated total bilirubin (T-Bil) level was the most frequently observed unfavorable prognostic factor and had the greatest effect on overall survival (OS), progression-free survival (PFS), and non-relapse mortality (NRM) (probability of unfavorable outcome in patients without and with elevated T-Bil level: OS, 58.9% vs. 15.4%, p < 0.001; PFS, 46.4% vs. 15.4%, p < 0.001; NRM, 10.7% vs. 53.8%, p < 0.001). Moreover, the probability of an unfavorable outcome increased in relation to the degree of T-Bil elevation and absence of improvement over time in T-Bil level. CONCLUSION: Elevated T-Bil level was an important marker of outcomes for liver dysfunction after allogeneic HSCT.

Risk Factors for Acute Kidney Injury and Chronic Kidney Disease following Allogeneic Hematopoietic Stem Cell Transplantation for Hematopoietic Malignancies.

BACKGROUND: Acute kidney injury (AKI) and chronic kidney disease (CKD) are considered common complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). OBJECTIVES AND METHOD: In this study, 114 patients who had undergone allo-HSCT were retrospectively analyzed to investigate the risk factors for onset of posttransplant AKI and CKD as defined by the new Kidney Disease Improving Global Outcomes criteria. RESULTS: Seventy-four patients (64.9%) developed AKI and 25 (21.9%) developed CKD. The multivariate analysis showed that the risk factors for developing stage 1 or higher AKI were age ≥46 years at the time of transplant (p = 0.001) and use of ≥3 nephrotoxic drugs (p = 0.036). For CKD, the associated risk factors were disease status other than complete remission at the time of transplantation (p = 0.018) and onset of AKI after transplant (p = 0.035). The 5-year overall survival (OS) was significantly reduced by development of AKI (p < 0.001), but not CKD. Posttransplant AKI significantly increased the 5-year nonrelapse mortality (p < 0.001), whereas posttransplant CKD showed an increasing tendency, but the difference was not significant. CONCLUSIONS: Posttransplant AKI impacts OS, significantly increases the risk of CKD, and is significantly associated with disseminated intravascular coagulation and use of ˃3 nephrotoxic drugs.

Importance of prognostic stratification via gene mutation analysis in elderly patients with acute myelogenous leukemia.

INTRODUCTION: Acute myelogenous leukemia (AML) in elderly patients is associated with an increased incidence of complications and treatment-related toxicity because of the frequency of comorbid disease and age-related deterioration in organ function. Despite advances in AML treatment in recent years, elderly patients have experienced limited benefit, and their outcomes remain poor. This study aimed to perform a comprehensive gene mutation analysis in elderly AML patients and identify gene mutations that could serve as prognostic factors. METHODS: An analysis of gene mutations was performed for 281 AML patients, including 98 elderly patients aged 65 years or above. RESULTS: Compared to younger AML patients, elderly patients showed a higher frequency of the following gene mutations: TP53 (P = 0.026), PTPN11 (P = 0.006), RUNX1 (P = 0.024), TET2 (P = 0.002), and ASXL1 (P = 0.023). The complete remission rate was significantly lower in DNMT3A mutation-positive cases (4.26%, P = 0.011) and TP53 mutation-positive cases (2.13%, P = 0.031) than in negative cases. The overall survival rate was significantly poorer in cases with FLT3-ITD (P = 0.003), DNMT3A (P = 0.033), or TP53 mutation (P < 0.001). Conversely, cases with PTPN11 mutation (P = 0.014) had a significantly more favorable prognosis. In multivariate analysis, FLT3-ITD (P = 0.011) and TP53 mutation positivity (P = 0.002) were independent poor prognostic factors, as were a performance status of 3 or above (P < 0.001) and poor cytogenetic prognosis (P = 0.001). In contrast, PTPN11 mutation positivity (P = 0.023) was an independent favorable prognosis factor. CONCLUSION: Analysis of gene mutations in elderly AML patients is very important, not only for establishing prognosis, but also for introducing appropriate molecular-targeted treatments.

Influence of GST polymorphisms on busulfan pharmacokinetics in Japanese children.

BACKGROUND: Fatal adverse effects or relapse can occur with excessive or insufficient busulfan exposure in hematopoietic stem cell transplantation. Given that busulfan is mainly metabolized by glutathione S-transferase (GST), we investigated the influence of GST polymorphisms on busulfan pharmacokinetics in Japanese pediatric patients. METHODS: Blood samples were taken from patients receiving high-dose i.v. busulfan as the first dose. Plasma busulfan concentration was measured using high-performance liquid chromatography. The area under the plasma busulfan concentration-time curve (AUC) was calculated. The genotype of GSTA1 was determined on polymerase chain reaction (PCR)-restriction fragment length polymorphism. Multiplex PCR was used to detect the presence or absence of GSTM1 and GSTT1 in the genomic DNA samples. RESULTS: Twenty patients were consecutively enrolled. Phenotype prediction was defined as follows: poor metabolizer (n = 4), one or more GSTA1*B haplotype or GSTM1/GSTT1 double-null genotypes; and extensive metabolizer (n = 16), other genotypes. GSTA1, M1, and T1 independently had no significant differences in AUC0-∞ , clearance or elimination rate constant. For the infant with unexpectedly high AUC0-∞ (2,591 µmol/L min), the GSTA1, M1, and T1 polymorphisms were wild type. On further analysis, the poor metabolizer group had lower clearance and higher AUC0-∞, except for the aforementioned patient, compared with the extensive metabolizer group (1,531 vs 1,010 µmol/L min; P < 0.01). CONCLUSIONS: GST polymorphisms may have affected busulfan pharmacokinetics, but these effects were obscured by other factors, such as underlying disease, systemic conditions, treatment history, and race.

Therapeutic effects of tyrosine kinase inhibitors and subtypes of BCR-ABL1 transcripts in Japanese chronic myeloid leukemia patients with three-way chromosomal translocations.

We analyzed the clinical responses to thyrosine kinase inhibitors (TKIs) and the molecular and cytogenetic characteristics of 18 chronic myeloid leukemia (CML) patients with 3-way chromosomal translocations. The patients were 14 men and 4 women, aged 23-75 years (median 57 years). The Sokal risk was low in 12 patients, intermediate in 4 patients, and high in 2 patients. Newly identified translocation breakpoints were seen in 7 of the 18 patients. Three patients had the same breakpoints of t(9;22;11)(q34;q11.2;q23). The best responses to TKIs were partial cytogenic response (PCyR) in 2 patients, complete cytogenic response (CCyR) in 3 patients, molecular response (MR) 3.0 in 7 patients, MR 4.0 in 3 patients, and MR 4.5 or higher in 3 patients. A total of 66.7% of patients did not achieve MR 4.0 or higher. In 3 patients in whom TKIs resulted in MR 4.5 or higher for more than 2 years, TKI treatment was discontinued. However, all of them exhibited a loss of MR3.0, at 2, 6, and 20 months after the discontinuation of treatment, respectively, and TKI treatment needed to be restarted. According to Kaplan-Meier survival curve analysis, the overall survival (OS) was 100 months in 56% of the patients. The 60-months cumulative incidences of CCyR, MR3.0, MR4.0 and MR4.5 were 88.9%, 72.2%, 33.3%, and 16.7%, respectively. In the 11 analyzable patients, the BCR-ABL1 mRNA subtype was e14a2 type in 4 patients and e13a2 type in 7 patients.

Amlexanox Downregulates S100A6 to Sensitize KMT2A/AFF1-Positive Acute Lymphoblastic Leukemia to TNFα Treatment.

Acute lymphoblastic leukemias (ALL) positive for KMT2A/AFF1 (MLL/AF4) translocation, which constitute 60% of all infant ALL cases, have a poor prognosis even after allogeneic hematopoietic stem cell transplantation (allo-HSCT). This poor prognosis is due to one of two factors, either resistance to TNFα, which mediates a graft-versus-leukemia (GVL) response after allo-HSCT, or immune resistance due to upregulated expression of the immune escape factor S100A6. Here, we report an immune stimulatory effect against KMT2A/AFF1-positive ALL cells by treatment with the anti-allergy drug amlexanox, which we found to inhibit S100A6 expression in the presence of TNF-α. In KMT2A/AFF1-positive transgenic (Tg) mice, amlexanox enhanced tumor immunity and lowered the penetrance of leukemia development. Similarly, in a NOD/SCID mouse model of human KMT2A/AFF1-positive ALL, amlexanox broadened GVL responses and extended survival. Our findings show how amlexanox degrades the resistance of KMT2A/AFF1-positive ALL to TNFα by downregulating S100A6 expression, with immediate potential implications for improving clinical management of KMT2A/AFF1-positive ALL. Cancer Res; 77(16); 4426-33. ©2017 AACR.

Adult T-cell leukemia-lymphoma complicated by Takotsubo cardiomyopathy and HTLV-1-associated myelopathy after treatment with the anti-CCR4 antibody mogamulizumab.

Adult T-cell leukemia-lymphoma (ATL) caused by human T-lymphotropic virus type 1 (HTLV-1) is generally associated with poor prognosis. The anti-CCR4 antibody mogamulizumab is one of the options for refractory or relapsed ATL. Mogamulizumab is intravenously administered as a single agent at a dose of 1.0 mg/kg once a week for 8 weeks. The overall response rate is reported to be 50%. Reported common adverse events after administration of mogamulizumab are leukocytopenia, infusion reaction, and skin rash. Although rare, mogamulizumab-associated cardiomyopathy has also been reported. HTLV-1 is also associated with a number of diseases, such as HTLV-1-associated myelopathy (HAM). Approximately 0.25% of infected individuals are estimated to develop HAM. HAM is an inflammatory disease of the central nervous system, which is characterized by slowly progressive spastic paresis of bilateral lower limbs and bladder-rectal disorder. Coexistence of ATL and HAM has rarely been reported. Here we present the case of a patient with ATL in whom Takotsubo cardiomyopathy and HAM developed after mogamulizumab administration.

Angioimmunoblastic T-cell Lymphoma Associated with IgA Nephropathy.

Few cases of IgA nephropathy with angioimmunoblastic T-cell lymphoma (AITL) have been reported. We herein present the case of a 79-year-old Japanese man with AITL and IgA nephropathy. The patient presented with generalized edema, fatigue, and fever. Laboratory investigations revealed polyclonal gammopathy with a high level of IgA, microscopic hematuria, proteinuria, and some other immunological abnormalities. Computed tomography revealed generalized lymphadenopathy. A diagnosis of AITL and IgA nephropathy was made based on inguinal lymph node and renal biopsies. Following chemotherapy for AITL, the patient's edema, microscopic hematuria, and proteinuria were alleviated. These findings indicate that IgA nephropathy may occur in AITL patients.

Clinical characteristics and prognosis of acute myeloid leukemia associated with DNA-methylation regulatory gene mutations.

In recent years, it has been reported that the frequency of DNA-methylation regulatory gene mutations - mutations of the genes that regulate gene expression through DNA methylation - is high in acute myeloid leukemia. The objective of the present study was to elucidate the clinical characteristics and prognosis of acute myeloid leukemia with associated DNA-methylation regulatory gene mutation. We studied 308 patients with acute myeloid leukemia. DNA-methylation regulatory gene mutations were observed in 135 of the 308 cases (43.8%). Acute myeloid leukemia associated with a DNA-methylation regulatory gene mutation was more frequent in older patients (P<0.0001) and in patients with intermediate cytogenetic risk (P<0.0001) accompanied by a high white blood cell count (P=0.0032). DNA-methylation regulatory gene mutation was an unfavorable prognostic factor for overall survival in the whole cohort (P=0.0018), in patients aged ≤70 years, in patients with intermediate cytogenetic risk, and in FLT3-ITD-negative patients (P=0.0409). Among the patients with DNA-methylation regulatory gene mutations, 26.7% were found to have two or more such mutations and prognosis worsened with increasing number of mutations. In multivariate analysis DNA-methylation regulatory gene mutation was an independent unfavorable prognostic factor for overall survival (P=0.0424). However, patients with a DNA-methylation regulatory gene mutation who underwent allogeneic stem cell transplantation in first remission had a significantly better prognosis than those who did not undergo such transplantation (P=0.0254). Our study establishes that DNA-methylation regulatory gene mutation is an important unfavorable prognostic factor in acute myeloid leukemia.

The Therapeutic Outcomes of Mechanical Ventilation in Hematological Malignancy Patients with Respiratory Failure.

Objective In hematological malignancy patients, the complication of acute respiratory failure often reaches a degree of severity that necessitates mechanical ventilation. The objective of the present study was to investigate the therapeutic outcomes of mechanical ventilation in hematological malignancy patients with respiratory failure and to analyze the factors that are associated with successful treatment in order to identify the issues that should be addressed in the future. Methods The present study was a retrospective analysis of 71 hematological malignancy patients with non-cardiogenic acute respiratory failure who were treated with mechanical ventilation at Nippon Medical School Hospital between 2003 and 2014. Results Twenty-six patients (36.6%) were treated with mechanical ventilation in an intensive care unit (ICU). Non-invasive positive pressure ventilation (NPPV) was applied in 29 cases (40.8%). The rate of successful mechanical ventilation treatment with NPPV alone was 13.8%. The rate of endotracheal extubation was 17.7%. A univariate analysis revealed that the following factors were associated with the successful extubation of patients who received invasive mechanical ventilation: respiratory management in an ICU (p=0.012); remission of the hematological disease (p=0.011); female gender (p=0.048); low levels of accompanying non-respiratory organ failure (p=0.041); and the non-use of extracorporeal circulation (p=0.005). A subsequent multivariate analysis revealed that respiratory management in an ICU was the only variable associated with successful extubation (p=0.030). Conclusion The outcomes of hematological malignancy patients who receive mechanical ventilation treatment for respiratory failure are very poor. Respiratory management in an ICU environment may be useful in improving the therapeutic outcomes of such patients.

Epstein-Barr Virus-positive T-cell Lymphoproliferative Disease Following Umbilical Cord Blood Transplantation for Acute Myeloid Leukemia.

We report a case of the extremely rare condition Epstein-Barr virus (EBV)-positive T-cell lymphoproliferative disease (LPD) which occurred after umbilical cord blood transplantation. A 25-year-old Japanese man underwent cord blood transplantation from a male human leukocyte antigen 4/6-matched donor due to acute myeloid leukemia with trisomy 8. Bone marrow examination on day 30 showed chimerism with at least 90% donor cells and complete hematological response. Chronic symptoms of graft-versus-host disease appeared only on the skin and were successfully treated with cyclosporine alone. Three years later, however, the patient experienced repeated cold-like symptoms and was hospitalized with liver dysfunction. A high fever developed and was followed by significant edema of the right side of the face. The EBV DNA copy number in whole peripheral blood was 2×10(4)/mL. Liver biopsy showed invasion of EBV-infected CD8-positive T cells. Southern blotting analysis of the whole peripheral blood showed that the T-cell receptor Cß1 rearrangement was positive. On the basis of these results, EBV-positive T-cell LPD was diagnosed and treated with prednisolone, cyclosporine, and etoposide, followed by cyclophosphamide, doxorubicin, vincristine, and prednisone. However, the patient died of cardiac function failure, pneumonia, and pulmonary hemorrhage, all of unidentified cause. Most cases of EBV-related LPD after hematopoietic stem cell transplantation consist of EBV-positive B-cell LPD, and, to our knowledge, de novo EBV-positive T-cell LPD subsequent to transplantation has not been previously reported.