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.

NPM1-mutation-based measurable residual disease assessment after completion of two courses of post-remission therapy is a valuable clinical predictor of the prognosis of acute myeloid leukemia.

Recent studies have reported that measurable residual disease (MRD) analysis using NPM1 mutations helps determine whether allogeneic hematopoietic stem cell transplantation (allo-HSCT) is indicated in acute myeloid leukemia (AML) patients. However, the optimal timing and cutoff value for measuring MRD using genomic DNA remain undetermined. This study aimed to investigate the optimal timing and cutoff value to ascertain the value of NPM1 mutation in MRD assessment. NPM1-mutated MRD was quantified by real-time polymerase chain reaction of bone marrow samples from 56 patients with NPM1-positive AML who achieved hematological remission. The area under the receiver-operating characteristic curve was greatest when MRD was assessed after two courses of post-remission therapy with a cutoff value of 0.010% (specificity, 68.4%; sensitivity, 87.0%). Patients whose MRD was below the cutoff value throughout the course of treatment had significantly better overall survival and relapse-free survival rates. Of the 33 patients who did not undergo transplantation during the first remission, all of the 11 who were never MRD-negative at any point experienced a relapse. Evaluating MRD with a cutoff value of 0.010% after two courses of post-remission therapy helps predict prognosis and determine the indication for allo-HSCT.

Prognostic impact of CEBPA bZIP domain mutation in acute myeloid leukemia.

Mutations of CCAAT/enhancer-binding protein alpha (CEBPAmu) are found in 10% to 15% of de novo acute myeloid leukemia (AML) cases. Double-mutated CEBPA (CEBPAdm) is associated with a favorable prognosis; however, single-mutated CEBPA (CEBPAsm) does not seem to improve prognosis. We investigated CEBPAmu for prognosis in 1028 patients with AML, registered in the Multi-center Collaborative Program for Gene Sequencing of Japanese AML. It was found that CEBPAmu in the basic leucine zipper domain (bZIP) was strongly associated with a favorable prognosis, but CEBPAmu out of the bZIP domain was not. The presence of CEBPAmu in bZIP was a strong indicator of a higher chance of achieving complete remission (P < .001), better overall survival (OS; P < .001) and a lower risk of relapse (P < .001). The prognostic significance of CEBPAmu in bZIP was also observed in the subgroup with CEBPAsm (all patients: OS, P = .008; the cumulative incidence of relapse, P = .063; patients aged ≤70 years and with intermediate-risk karyotype: OS, P = .008; cumulative incidence of relapse, P = .026). Multivariate analysis of 744 patients aged ≤70 years showed that CEBPAmu in bZIP was the most potent predictor of OS (hazard ratio, 0.3287; P < .001). CEBPAdm was validated as a cofounding factor, which was overlapping with CEBPAmu in bZIP. In summary, these findings indicate that CEBPAmu in bZIP is a potent marker for AML prognosis. It holds potential in the refinement of treatment stratification and the development of targeted therapeutic approaches in CEBPA-mutated AML.

Pharmacokinetics of intravenous busulfan as condition for hematopoietic stem cell transplantation: comparison between combinations with cyclophosphamide and fludarabine.

Busulfan (Bu) has been used in combination with fludarabine (Flu; BuFlu) or cyclophosphamide (Cy; BuCy) as conditioning for allogeneic hematopoietic stem cell transplantation (HSCT). This multi-institutional prospective study compared pharmacokinetic (PK) parameters of Bu between BuFlu and BuCy. Plasma Bu concentrations were measured by high-performance liquid chromatography at the first dose of the first and fourth days of intravenous Bu administrations (total of 16 doses of 0.8 mg/kg). Thirty-seven patients were evaluable (BuFlu, N = 18; BuCy, N = 19). The median age was significantly higher in BuFlu. In BuFlu, the median area under the blood concentration-time curve of Bu on the fourth day was 1183 µmol min/L (range 808-1509), which was significantly higher than that on the first day [1095 µmol min/L (range 822-1453), P < 0.01]. In contrast, such differences were not observed in BuCy. Consistently, there was a significant decrease in the clearance of Bu on the fourth day as compared with the first day in BuFlu. These results suggest that the PK of Bu was altered during the co-administration of Flu, which was not the case with Cy. A large-scale study is required to evaluate the significance of the differences in the PK of Bu between the conditionings on HSCT outcomes.

Genetic and transcriptional landscape of plasma cells in POEMS syndrome.

POEMS syndrome is a rare paraneoplastic disease associated with monoclonal plasma cells; however, the pathogenic importance of plasma cells remains unclear. We performed comprehensive genetic analyses of plasma cells in 20 patients with POEMS syndrome. Whole exome sequencing was performed in 11 cases and found a total of 308 somatic mutations in 285 genes. Targeted sequencing was performed in all 20 cases and identified 20 mutations in 7 recurrently mutated genes, namely KLHL6, LTB, EHD1, EML4, HEPHL1, HIPK1, and PCDH10. None of the driver gene mutations frequently found in multiple myeloma (MM) such as NRAS, KRAS, BRAF, and TP53 was detected. Copy number analysis showed chromosomal abnormalities shared with monoclonal gammopathy of undetermined significance (MGUS), suggesting a partial overlap in the early development of MGUS and POEMS syndrome. RNA sequencing revealed a transcription profile specific to POEMS syndrome when compared with normal plasma cells, MGUS and MM. Unexpectedly, disease-specific VEGFA expression was not increased in POEMS syndrome. Our study illustrates that the genetic and transcriptional profiles of plasma cells in POEMS syndrome are distinct from MM and MGUS, indicating unique function of clonal plasma cells in its pathogenesis.

Clonal immunoglobulin λ light-chain gene rearrangements detected by next generation sequencing in POEMS syndrome.

Polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes (POEMS) syndrome is a rare plasma cell dyscrasia characterized by polyneuropathy, organomegaly, endocrinopathy, extravascular fluid overload, M protein, and a myriad of skin changes. The pathogenesis is poorly understood, but monoclonal plasma cells are λ-restricted and these immunoglobulin λ light chain variable (IGLV) region genes are derived from only two germlines, either IGLV1-44 or 1-40. Here we analyzed the clonal IGLV gene rearrangements of genomic DNA samples of bone marrow mononuclear cells using next-generation sequencing (NGS) to understand the clonal composition of IGLV genes in patients with POEMS syndrome (n = 30). The dominant IGLV gene rearrangement of POEMS syndrome-specific germline sequences were significantly increased in 11 POEMS patients (36.7%; IGLV1-44: n = 9, IGLV1-40: n = 2). In some cases, IGLV gene rearrangement clone was not detected as significant increase but was detected using cDNA samples by heteroduplex (HD) analysis and Sanger sequencing, suggesting that the quite small number of monoclonal plasma cells may produce large quantity of mRNA of monoclonal proteins. However, significant increase of dominant clone sizes was not directly linked to the initial disease status. On the other hand, in cases with significantly increased dominant clones, they decreased and increased accompanying with disease remission and relapse. These data demonstrate that monoclonal plasma cells are related to the pathogenesis of POEMS syndrome.

Long-term complete remission following tandem autologous stem cell transplantation and consolidative radiotherapy for refractory mediastinal gray-zone lymphoma.

Mediastinal gray zone lymphoma (MGZL) is a provisional entity with intermediate features between classical Hodgkin lymphoma (cHL) and diffuse large B-cell lymphoma. Outcomes for patients with MGZL are reportedly poorer than those for patients with cHL or primary mediastinal large B-cell lymphoma. Additionally, no standard management guidelines for patients with MGZL are available, primarily due to its recent identification, rarity, and challenges in diagnosis. Although recent several studies have suggested dose-adjusted EPOCH-R (etoposide, doxorubicin, vincristine, cyclophosphamide, prednisolone, and rituximab) may improve outcomes in patients with MGZL, numerous patients still suffer from relapsed/refractory MGZL, and the optimal management for such patients remains uncertain. Here, we report the first case of successful treatment of refractory MGZL by tandem high-dose chemotherapy supported by autologous stem cell transplantations (auto-SCTs) and consolidative radiotherapy (RT). To date, the patient remains in CR 33 months after the completion of RT, with no significant complications. This case suggests that tandem auto-SCTs may be a promising therapeutic option for relapsed/refractory MGZL.

Long-term efficacy of partial splenic embolization for the treatment of steroid-resistant chronic immune thrombocytopenia.

Thrombopoietin-receptor agonists have been recently introduced for a second-line treatment of immune thrombocytopenia (ITP). Splenectomy has tended to be avoided because of its complications, but the response rate of splenectomy is 60-80% and it has still been considered for steroid-refractory ITP. We performed partial splenic embolization (PSE) as an alternative to splenectomy. Between 1988 and 2013, 91 patients with steroid-resistant ITP underwent PSE at our hospital, and we retrospectively analyzed the efficacy and long-term outcomes of PSE. The complete response rate (CR, platelets > 100 × 109/L) was 51% (n = 46), and the overall response rate (CR plus response (R), > 30 × 109/L) was 84% (n = 76). One year after PSE, 70% of patients remained CR and R. The group with peak platelet count after PSE ≥ 300 × 109/L (n = 29) exhibited a significantly higher platelet count than the group with platelet count < 300 × 109/L (n = 40) at any time point after PSE. The failure-free survival (FFS) rates at 1, 5, and 10 years were 78, 56, and 52%, respectively. Second PSE was performed in 20 patients who relapsed (n = 14) or had no response to the initial PSE (n = 6), and the overall response was achieved in 63% patients. There were no PSE-related deaths. These results indicate that PSE is a safe and effective alternative therapy to splenectomy for patients with steroid-resistant ITP as it generates long-term, durable responses.

[MALT lymphoma accompanied by elevated serum IgM levels mimicking Waldenström's macroglobulinemia].

A 60-year-old man with chronic hepatitis C was referred to our hospital with significantly elevated total protein and serum IgM (9,500 mg/dl) levels identified via a routine checkup. Blood examination revealed increased serum IgM-monoclonal protein and serum-soluble IL-2 receptor (sIL2R) levels. Computed tomography and fluorodeoxyglucose positron emission tomography revealed pulmonary masses, abnormal soft tissue masses surrounding the bilateral kidneys, and thickened mucous membrane of the bladder with high fluorodeoxyglucose uptake. Pathological examination of the pulmonary mass revealed infiltration of medium-sized lymphocytes and plasma cells. Immunohistochemical analysis revealed tumor cells positive for CD138 and IgM, with a low positive rate of Ki-67 expression. Notably, the tumor cell-surrounding lymphocytes were positive for CD20. Although the patient was initially regarded as having Waldenström's macroglobulinemia owing to the significantly increased serum IgM levels, based on positive IgH-MALT1 translocation and negative MYD88 L265P mutation findings, he was further diagnosed with extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). Complete remission was achieved following six cycles of rituximab + CHOP therapy. This study data suggest that analysis of the MYD88 L265P mutation in tumor cells is suitable for accurately diagnosing hematopoietic malignancies with increased IgM monoclonal protein.

The Successful Treatment of a Cord Blood Transplant Recipient with Varicella Zoster Virus Meningitis, Radiculitis and Myelitis with Foscarnet.

Infections of the central nervous system (CNS) with varicella zoster virus (VZV) is a rare occurrence after allogeneic hematopoietic stem cell transplantation. We herein report a case of VZV meningitis, radiculitis and myelitis that developed 8 months after cord blood transplantation, shortly after the cessation of cyclosporine and low-dose acyclovir. Although treatment with acyclovir did not achieve a satisfactory response, the patient was successfully treated with foscarnet. Our report indicates that VZV infection should be considered in allo-hematopoietic stem cell transplantation (HSCT) patients with CNS symptoms and that foscarnet may be effective for the treatment of acyclovir-resistant VZV infections of the CNS. The development of optimal prophylactic strategies and vaccination schedules may eradicate post-transplant VZV disease.

Successful Treatment of Hepatocellular Carcinoma Complicated by Fanconi Anemia.

A 42-year-old woman with liver tumors was referred to our hospital. Her condition was complicated by Fanconi anemia, and she had undergone total laryngectomy 8 years ago. On admission, contrast-enhanced computed tomography revealed hypervascular tumors in the right hepatic lobe. Ultrasound-guided tumor biopsy revealed that the tumor comprised moderately differentiated hepatocellular carcinoma. Although the patient exhibited preserved liver function (Child-Pugh A), complete blood count revealed severe pancytopenia. Eventually, the tumor was successfully treated by transcatheter arterial embolization (TAE). Both platelet transfusion and systemic administration of antibiotics were performed. She was discharged 35 days after TAE.

Safety and Efficacy of Granulocyte Colony-Stimulating Factor Monotherapy for Peripheral Blood Stem Cell Collection in POEMS Syndrome.

Although autologous stem cell transplantation can achieve excellent responses in patients with POEMS syndrome, the optimal regimen for peripheral blood stem cell (PBSC) collection is still controversial. We retrospectively investigated the safety and efficacy of 41 PBSC collecting procedures in 37 patients with POEMS syndrome. PBSC mobilization was performed using cyclophosphamide + granulocyte colony-stimulating factor (G-CSF) (CG, n = 14) or G-CSF alone (G, n = 27). Twelve (85.7%) patients in the CG group and all (100%) patients in the G group received induction chemotherapy before PBSC collection. The proportions of good mobilizers (≥2.0 × 106 CD34+ cells/kg) were comparable between the 2 groups (CG versus G: 78.6% versus 70.4%, P = .71). Two (14.3%) patients in the CG group developed severe capillary leak symptoms during the PBSC mobilization period, whereas no patient in the G group experienced severe adverse events. Appropriate induction therapies followed by the G-CSF monotherapy compose an optimal strategy for PBSC collection.

Possible role of intragenic DNA hypermethylation in gene silencing of the tumor suppressor gene NR4A3 in acute myeloid leukemia.

Expression of the tumor suppressor gene NR4A3 is silenced in the blasts of acute myeloid leukemia (AML), irrespective of the karyotype. Although the transcriptional reactivation of NR4A3 is considered to have a broad-spectrum anti-leukemic effect, the therapeutic modalities targeting this gene have been hindered by our minimal understanding of the transcriptional mechanisms regulating its expression, particularly in human AML. Here we show the role of intragenic DNA hypermethylation in reducing the expression of NR4A3 in AML. Bisulfite sequencing analysis revealed that CpG sites at the intragenic region encompassing exon 3 of NR4A3, but not the promoter region, are hypermethylated in AML cell lines and primary AML cells. A DNA methyltransferase inhibitor restored the expression of NR4A3 following a reduction in DNA methylation levels at intragenic CpG sites. The in silico data revealed an enrichment of H3K4me1 and H2A.Z at exon 3 of NR4A3 in human non-malignant cells but that was excluded specifically in leukemia cells with CpG hypermethylation. This suggests that exon 3 represents a functional regulatory element involved in the transcriptional regulation of NR4A3. Our findings improve the current understanding of the mechanism underlying NR4A3 silencing and facilitate the development of NR4A3-targeted therapy.

Chronic active Epstein-Barr virus infection with marked pericardial effusion successfully treated with allogeneic peripheral blood stem cell transplantation.

A 23-year-old woman presented with a persistent fever and shortness of breath. Computed tomography showed marked pericardial effusion, hepatosplenomegaly, and cervical and mediastinal lymph node swelling. Epstein-Barr virus (EBV) antibody titers were abnormally elevated, and the copy number of EBV-DNA was increased in peripheral blood. Based on these observations, she was diagnosed with chronic active EBV infection (CAEBV). The EBV-infected cells in her peripheral blood were CD4(+)T lymphocytes. Fever and pericardial effusion improved following treatment with a combination of prednisolone, etoposide, and cyclosporine; however, peripheral blood EBV-DNA levels remained high. The patient underwent allogeneic peripheral blood stem cell transplantation from an EBV-seronegative, HLA-matched sibling donor, with fludarabine and melphalan conditioning. The post-transplantation course was uneventful, except for mild skin acute graft-versus-host disease (grade 2). EBV-DNA became undetectable in peripheral blood 98 days post transplantation. She has since been in good health without disease recurrence. CAEBV is a potentially fatal disease caused by persistent EBV infection of T lymphocytes or natural killer cells, thus requiring prompt treatment and allogeneic transplantation. Pericardial effusion is rarely observed in CAEBV and can impede its diagnosis. Therefore, we should be aware that patients may present with marked pericardial effusion as an initial manifestation of CAEBV.

Cessation of In-line Filters in Central Venous Catheters Does Not Significantly Influence the Incidence of Bloodstream Infections and Mortality in a Hospital Hematological Ward.

Objective The use of intravenous in-line filters is effective for the mechanical removal of large particles, precipitates, bacteria, fungi, large lipid globules, and air. However, the routine use of in-line filters remains controversial. Many patients with hematological diseases frequently suffer from bloodstream infections (BSIs) with fatal outcomes. Methods The year before cessation of an in-line filter was defined as the "filter period" and the year after its cessation was defined as the "non-filter period." The number of central line-associated bloodstream infections (CLABSIs), which are defined through surveillance, the catheter utilization rate, the number of patient deaths within 7 days after removal of the central venous catheters (CVCs), and the overall survival rate following CVC insertion were measured. Results During both periods, 84 patients had a total of 140 CVCs with a total number of catheter days of 3,407. There were 10 CVCs with CLABSIs, and the overall CLABSI rate was 2.9/1,000 catheter days, including 4 CVCs with CLABSIs (2.5/1,000 catheter days) during the filter period and 6 CVCs with CLABSIs (3.3/1,000 catheter days) during the non-filter period. The CLABSI rate, catheter utilization rate, and mortality did not differ significantly between the two periods. The only independent variable that was found to be significantly associated with the development of CLABSIs was a neutrophil count of <500×10(6)/L (p<0.05). Conclusion Our study revealed that the cessation of in-line filters from CVCs does not significantly influence the incidence of BSIs and mortality in patients with hematological disease. To confirm our results, however, a large-scale randomized controlled study is warranted.

Successful Allogeneic Stem Cell Transplantation for Severe Aplastic Anemia after Treatment of Lymphoproliferative Disorder Caused by Rabbit Antithymocyte Globulin.

Immunosuppressive therapy (IST) with a combination of antithymocyte globulin (ATG) and cyclosporine (CsA) is an effective therapeutic modality for patients with aplastic anemia (AA) who are not eligible for allogeneic stem cell transplantation (Allo-SCT) from a human leukocyte antigen-identical sibling donor. However, there have been reports of some patients developing lymphoproliferative disorder (LPD) after IST for AA. We herein report a case of a 26-year-old man with severe AA (SAA) complicated by LPD after a single course of IST, who was successfully treated with Allo-SCT from an unrelated donor. Two months after starting IST for SAA, he developed LPD in the stomach. CsA was reduced, however, his neutrophil counts decreased, and CsA could not be discontinued. The patient was treated with rituximab monotherapy, and LPD resulted in complete remission. However, he failed IST for SAA and underwent Allo-SCT with reduced-intensity conditioning to recover his hematopoiesis. The patient has achieved complete hematopoietic recovery without the recurrence of LPD for five years after transplantation. This is the first report of successful Allo-SCT for SAA after the treatment of LPD caused by the use of rabbit ATG. This case provides useful information for the management of SAA with the development of LPD after IST.

Early-Onset Severe Diffuse Alveolar Hemorrhage after Bortezomib Administration Suggestive of Pulmonary Involvement of Myeloma Cells.

Severe acute lung injury is a rare but life-threatening complication associated with bortezomib. We report a patient with multiple myeloma who developed a severe diffuse alveolar hemorrhage (DAH) immediately after the first bortezomib administration. The patient was suspected to have pulmonary involvement of myeloma, which caused DAH after rapidly eradicating myeloma cells in the lungs with bortezomib. Rechallenge with bortezomib was performed without recurrent DAH. In patients with multiple myeloma who manifest abnormal pulmonary shadow, we should be aware of early-onset severe DAH after bortezomib administration, which might be due to pulmonary involvement of myeloma cells.

Successful diagnosis of type II enteropathy-associated T-cell lymphoma using flow cytometry and the cell block technique of celomic fluid manifesting as massive pyoid ascites that could not be diagnosed via emergency laparotomy.

Enteropathy-associated T-cell lymphoma (EATL), an intestinal tumor of intraepithelial T lymphocytes, is a rare and highly aggressive disease. We herein describe a case of type II EATL with massive pyoid ascites in which a histological examination could not be performed despite emergency laparotomy that was successfully diagnosed using flow cytometry and the cell block technique to analyze the celomic fluid. This case suggests that EATL should be included in the differential diagnosis of pyoid ascites of unknown origin and that flow cytometry and the cell block technique of assessing celomic fluid are useful procedures for diagnosing EATL, especially in cases in which conducting a histological examination is impossible.

Posterior reversible encephalopathy syndrome in an adult patient with acute lymphoblastic leukemia after remission induction chemotherapy.

Posterior reversible encephalopathy syndrome (PRES) has been reported in childhood leukemia patients increasingly frequently. However, the development of PRES in adult leukemia patients during chemotherapy is very rare. We present a case of PRES in an adult patient with acute lymphoblastic leukemia (ALL) after remission induction chemotherapy. A 28-year-old woman with ALL was administered remission induction chemotherapy consisting of cyclophosphamide, daunorubicin, vincristine, prednisone, and L-asparaginase. After initiation of chemotherapy, the patient developed paralytic ileus and hypertension, and on day 30, she suddenly developed generalized convulsions, loss of visual acuity, and muscle weakness in the legs. Magnetic resonance imaging findings and her signs and symptoms were typical of PRES. The symptoms gradually improved following treatment with an anticonvulsant and an antihypertensive agent, and the patient underwent allogeneic bone marrow transplantation. She has completely recovered from PRES and has been asymptomatic without leukemia relapse. During remission induction chemotherapy for ALL, PRES may be caused by multiple drugs, such as L-asparaginase, vincristine, and corticosteroids, with different mechanisms of action. PRES should be recognized as an important complication, which will occur more frequently with the increased intensity of chemotherapy for adult ALL patients.