[Evaluation of the correlation between JAK2V617F allele burden by Quenching Probe-Tm method and allele-specific quantitative PCR method].

The JAK2V617F mutation is a driver mutation of myeloproliferative neoplasms (MPNs). V617F allele burden is considered a risk factor for complications associated with MPNs and is a predictor of prognosis. In Japan, V617F allele burden has been measured in laboratory settings using the i-densyTM IS-5320 genetic analyzer with the quenching probe-Tm (QP-Tm) method. However, since 2020, allele-specific quantitative PCR (AS-qPCR) is being performed in clinical settings for measuring V617F allele burden. To investigate the clinical usefulness of the QP-Tm method in patients with MPNs, we evaluated the V617F allele burden measured by both the methods. A good correlation was observed between the V617F allele burden determined using QP-Tm and that determined using AS-qPCR (P<0.001, rs=0.952). The median mutant allele burden, as determined using the QP-Tm method, was significantly higher in patients with polycythemia vera than in those with essential thrombocythemia. The results of this study suggested that the QP-Tm method will continue to be useful clinical ancillary test for measuring V617F allele burden.

Successful treatment with ABL tyrosine kinase inhibitor for patients with acute myeloid leukemia with BCR-ABL1.

Acute myeloid leukemia (AML) with BCR-ABL1 is rare and has a poor prognosis with conventional chemotherapy or ABL tyrosine kinase inhibitors (TKIs) alone. We reported a case of AML with BCR-ABL1 patient who was successfully treated with dasatinib alone; additionally, we previously reported another case of long-term remission maintained with imatinib monotherapy. These results suggested that a treatment with a novel and significantly potent TKI may be effective in AML with BCR-ABL1 patients with low tumor burden and without additional chromosome aberrations and ABL kinase domain mutations.

Jumping translocations of 1q in donor cell-derived myelodysplastic syndrome after cord blood transplantation: Case report and review of the literature.

Donor cell-derived leukemia and myelodysplastic syndrome (DCL) is a rare complication in patients after allogenic stem cell transplantation (SCT). Since 1971, numerous cases of DCL have been reported, but the detailed mechanisms of DCL are still unclear. A patient with jumping translocations (JTs) of 1q in umbilical cord blood donor cell-derived myelodysplastic syndrome (MDS), which likely occurred due to genetic alterations of TET2 and ASXL1 after cord blood transplantation (CBT), was examined in this study. Previously reported DCL cases after CBT that focused on the cytogenetic and molecular characteristics of these patients and patient outcome were reviewed. A total of 30 cases of DCL after CBT were identified between 2005 and 2018. The median time from CBT to the development of DCL was 16 months. The number of patients with DCL who were diagnosed with acute myeloid leukemia (AML) and MDS was 19 and 8, respectively. JTs were frequently observed in 5 of 27 DCL patients who had cytogenetic abnormalities, including our patient. Molecular abnormalities were described in 7 of the cases, and the most frequent abnormality was an NPM1 mutation. Other gene mutations that were usually found in de novo MDS or AML were observed in JT-DCL after CBT. From these results, chromosomal abnormalities such as JTs that occur subsequent to genetic alterations were seemed an important mechanisms underlying DCL onset in patients after CBT. Further molecular analyses regarding the genetic alterations of JTs are required to understand the pathogenesis of umbilical cord blood-derived JT-DCL.

High-grade trichoblastic carcinoma arising through malignant transformation of trichoblastoma: Immunohistochemical analysis and the expression of p53 and phosphorylated AKT.

Trichoblastoma (TB) is a benign cutaneous adnexal neoplasm. The lesion typically presents as a slow-growing, solitary, well-circumscribed nodule measuring up to 3 cm in diameter. On rare occasions, TB causes malignant transformation into an aggressive form described as high-grade trichoblastic carcinoma. Four such cases have been reported to date; all were described as high-grade trichoblastic carcinomas. Here, we describe the case of a 72-year-old Japanese male patient with a rapidly enlarging subcutaneous tumor on his lower back, which was diagnosed as high-grade trichoblastic carcinoma. Histopathologically, the tumor featured both benign and malignant components, and a transition zone between these states was clearly evident. In the immunohistochemical analysis, a malignant component was positive for p53 and showed stronger staining of phospho-RAC-α serine/threonine-protein kinase (AKT) Ser473 in comparison with a benign component. These results suggest that loss of p53 function and activation of phosphatidylinositol 3-kinase-AKT signaling pathways played important pathogenic roles in malignant transformation of the present case.

STAT5A regulates DNMT3A in CD34(+)/CD38(-) AML cells.

Signal transducer and activator of transcription 5 (STAT5) is activated in CD34(+)/CD38(-) acute myelogenous leukemia (AML) cells. Inhibition of STAT5 induced apoptosis and sensitized these cells to the growth inhibition mediated by conventional chemotherapeutic agents. The present study attempted to identify molecules that are regulated by STAT5 in CD34(+)/CD38(-) AML cells by utilizing cDNA microarrays, comparing the gene expression profiles of control and STAT5A shRNA-transduced CD34(+)/CD38(-) AML cells. Interestingly, DNA methyltransferase (DNMT) 3A was downregulated after depletion of STAT5A in CD34(+)/CD38(-) AML cells. Reporter gene assays found that an increase in activity of DNMT3A occurred in response to activation of STAT5A in leukemia cells. On the other hand, dephosphorylation of STAT5A by AZ960 decreased this transcriptional activity. Further studies utilizing a chromatin immunoprecipitation assay identified a STAT5A-binding site on the promoter region of DNMT3A gene. Forced expression of STAT5A in leukemia cells caused hypermethylation on the promoter region of the tumor suppressor gene, PTEN, and downregulated its mRNA levels, as measured by methylation-specific and real-time polymerase chain reaction, respectively. Taken together, these data suggest that STAT5A positively regulates levels of DNMT3A, resulting in inactivation of tumor suppressor genes by epigenetic mechanisms in AML cells.

[Successful eculizumab treatment for multiple coronary thrombosis complicated in paroxysmal nocturnal hemoglobinuria].

We herein report a rare case of paroxysmal nocturnal hemoglobinuria (PNH) who repeatedly developed coronary arterial thromboembolism. Anticoagulant therapies including heparin, aspirin as an antiplatelet agent and even drug-eluting stent placement in the coronary artery failed to prevent the recurrence of ischemic heart disease. Of note, initiating the administration of a humanized anti-C5 antibody, eculizumab, achieved prompt thrombolysis and maintenance treatment with eculizumab prevented the recurrence of thromboembolic disease in this patient. Taking these observations together, we suggest that the use of eculizumab be considered for treatment or prevention of arterial thrombosis complicated by PNH, although arterial thrombosis is an extremely rare event in the Japanese population.

Recombinant human soluble thrombomodulin is active against hemophagocytic lymphohistiocytosis associated with acquired immunodeficiency syndrome.

A 39-year-old man was admitted to our hospital to initiate highly active anti-retroviral therapy (HAART) for documented acquired immune deficiency syndrome. The HIV load was 2.5 million copies/mL and the CD4-positive lymphocyte count was only 52 cells/µL at presentation. The HAART regimen consisted of lamivudine and abacavir as the backbone, plus raltegravir and lopinavir/ritonavir as the base. The day after initiating HAART, his body temperature rose to 102.4 °F (39.1 °C), accompanied by elevated levels of liver enzymes, neutropenia, coagulopathies, and an extremely high serum ferritin level, prompting us to suspect hemophagocytic lymphohistiocytosis (HLH) and disseminated intravascular coagulation (DIC). To correct the coagulation abnormalities, recombinant thrombomodulin (rTM) was initiated at 375 U/kg. Surprisingly, fever resolved almost immediately, in parallel with dramatic decreases in serum levels of ferritin and liver enzymes and prompt normalization of coagulopathy with only two doses of rTM. The patient subsequently developed amebiasis, which was successfully treated using metronidazole. In summary, the use of rTM dramatically improved not only DIC, but also HLH, suggesting potent anti-inflammatory effects of the agent. Although further clinical reports and trials are needed, rTM appears to provide an additional therapeutic option in the management of HLH.

Effect of recombinant human soluble thrombomodulin on clinical outcomes of patients with coagulopathy after hematopoietic stem cell transplantation.

From 2001 to 2012, 71 individuals with hematological diseases received HSCT in our institution. Of these, 41 developed disseminated intravascular coagulation (DIC) in association with various underlying conditions. The patients who developed DIC after 2008 (n = 23) were treated by recombinant human soluble thrombomodulin (rTM), and the others (n = 11) were treated by either heparin and/or antithrombin III concentrate. Seven patients did not receive any anticoagulant therapy. Of note, treatment for coagulopathy by rTM significantly improved clinical outcomes of patients at day 100 and dramatically prolonged their overall survival (P = 0.044). Taken together, rTM is useful to improve clinical outcomes of transplant recipients with coagulopathy.

Recombinant human soluble thrombomodulin safely and effectively rescues acute promyelocytic leukemia patients from disseminated intravascular coagulation.

We treated individuals for disseminated intravascular coagulation (DIC) caused by acute promyelocytic leukemia (APL) (n=9) using human soluble thrombomodulin (rTM) in combination with all-trans retinoic acid (ATRA) and chemotherapy, and compared the clinical outcomes with historical control patients (n=8) treated with ATRA and/or chemotherapy. Two control patients developed intracranial vascular incidents. On the other hand, no bleeding related mortality was noted in rTM-treated patients. Notably, treatment with rTM rescued patients from DIC earlier than historical controls (log rank test, p=0.019). These results suggest that administration of rTM should be considered for the treatment of individuals with DIC associated with APL.

Expression of p-JAK2 predicts clinical outcome and is a potential molecular target of acute myelogenous leukemia.

Our study determined if Janus kinase 2 (JAK2) was activated in acute myelogenous leukemia (AML; n = 77, excluding acute promyelocytic leukemia) by immunohistochemistry (IHC) using a phosphor-specific antibody against JAK2. p-JAK2 was detectable in all cases, although its levels varied between patient samples (high levels, n = 31; low levels, n = 46). The quantification of levels of p-JAK2 by IHC was well correlated with that assessed by Western blot analyses and fluorescence-activated cell sorting (FACS). Levels of p-JAK2 were directly correlated with high white blood cell count (52.3 × 10(3) /L in patients with high p-JAK2 vs. 28.3 × 10(3) /L in patients with low p-JAK2, p < 0.01) and were inversely correlated with complete remission rates (45% in patients with high p-JAK2 vs. 78% in patients with low p-JAK2, p < 0.003). In addition, multivariate analysis confirmed that high levels of p-JAK2 remained a significant factor for overall survival (hazard ratio = 2.213; 95% confidence interval, 1.212-4.041, p = 0.023). Moreover, we found that AZ960, a novel and specific inhibitor of the JAK2 kinase, potently inhibited the clonogenic growth and induced apoptosis of freshly isolated AML cells from patients in association with cleavage of caspase 3 and downregulation of anti-apoptotic Bcl-xL proteins. Taken together, JAK2 may be a promising molecular target for treatment of AML.

Inhibition of signal transducer and activator of transcription 5 by the inhibitor of janus kinases stimulates dormant human leukemia CD34+ /CD38- cells and sensitizes them to antileukemia agents.

To verify molecular mechanisms by which leukemia stem cells (LSCs) maintain a dormant state, we explored the activity of the major prosurvival signal pathways in CD34(+) /CD38(-) compartment, supposed to contain LSCs, and CD34(+) /CD38(+) counterparts from patients with acute myelogenous leukemia (AML, n = 11) by fluorescence-activated cell sorting (FACS). CD34(+) /CD38(-) cells expressed a greater amount of p-janus kinase 2 (JAK2) and p-signal transducer and activator of transcription 5 (STAT5) than CD34(+) /CD38(+) counterparts in all patients except for one case. In addition, we found that CD34(+) /CD38(-) cells were relatively resistant to cytarabine- and the inhibitor of the fms-like tyrosine kinase 3 (FLT3)-mediated growth inhibition, as measured by the clonogenic assay. Interestingly, blockade of JAK2/STAT5 signaling by the specific JAK2 inhibitor AZ960 stimulated cell cycling in CD34(+) /CD38(-) cells in conjunction with downregulation of cyclin-dependent kinase inhibitor p21(waf1) and sensitized these cells to the growth inhibition mediated by cytarabine and the FLT3 kinase inhibitor. Moreover, exposure of CD34(+) /CD38(-) cells to AZ960 potently induced apoptosis in parallel with downregulation of antiapoptotic protein Bcl-xL, as measured by Western blot analysis. Taken together, JAK2/STAT5 signaling may be a promising molecular target to eradicate CD34(+) /CD38(-) leukemia cells in individuals with AML.

Effects of the dipeptidyl peptidase-IV inhibitor ASP8497 on glucose tolerance in animal models of secondary failure.

UNLABELLED: Sulfonylureas promote insulin secretion and potently lower blood glucose levels, however, they induce hypoglycemia and undergo a reduction in efficacy when administered long-term (secondary failure). The dipeptidyl peptidase (DPP)-IV inhibitor ASP8497, (2S,4S)-4-fluoro-1-({[4-methyl-1-(methylsulfonyl)piperidin-4-yl]amino}acetyl)pyrrolidine-2-carbonitrile monofumarate, inhibits the degradation of glucagon-like peptide-1 (GLP-1), an incretin hormone, and promotes insulin secretion in a glucose-dependent manner. ASP8497 is therefore less likely to induce hypoglycemia and less likely to show reduced efficacy even after repeated administration. Here, to determine whether or not ASP8497 improves glucose tolerance in Zucker fatty rats, we examined the effects of ASP8497 and gliclazide, a sulfonylurea, on glucose tolerance after repeated administration. We also developed an animal model of secondary failure using streptozotocin-nicotinamide-induced diabetic mice. RESULTS: ASP8497 (3mg/kg) improved glucose intolerance in Zucker fatty rat without any attenuation (blood glucose AUC: P=0.034 vs. vehicle) while gliclazide (10mg/kg) did not (P=0.916 vs. vehicle). Furthermore, ASP8497 (3, 10mg/kg) was found to effect glucose tolerance dose-dependently (3mg/kg: P=0.230, 10mg/kg: P=0.003 vs. glibenclamide (10mg/kg)) by enhancing insulin secretion in mice inadequately controlled with glibenclamide. Our results suggest that ASP8497 may be effective even in patients with secondary failure who are unable to maintain satisfactory glycemic control using sulfonylureas.

Effects of serotonin 5-HT(3) receptor antagonists on CRF-induced abnormal colonic water transport and defecation in rats.

The effects of corticotropin releasing factor (CRF) and serotonin (5-HT)(3) receptor antagonists on intestinal water transport are not well understood. Hence, we established a CRF-induced abnormal water transport model in rat colon, and evaluated the effects of 5-HT(3) receptor antagonists including ramosetron, alosetron, and cilansetron, and the antidiarrheal agent loperamide, in this model. In addition, the effects of 5-HT(3) receptor antagonists and loperamide on abnormal defecation induced by CRF in rats were examined. Colonic water transport was measured in colonic loops in conscious rats. Centrally administered CRF (3-30 microg/kg) markedly decreased colonic fluid loss, whereas oral administration of ramosetron (3, 30 microg/kg), alosetron (300 microg/kg), cilansetron (300 microg/kg), or loperamide (3 mg/kg) significantly inhibited it. Ramosetron (1-10 microg/kg), alosetron (10-100 microg/kg), cilansetron (10-100 microg/kg), or loperamide (0.3-3 mg/kg) also showed dose-dependent inhibition of CRF-induced defecation in rats. These results suggest that 5-HT(3) receptors are involved in both abnormal colonic water transport and defecation induced by CRF, and that the inhibitory effects of 5-HT(3) receptor antagonists on CRF-induced abnormal defecation partly result from their ameliorating action on colonic water transport.

Effect of ramosetron on conditioned emotional stress-induced colonic dysfunction as a model of irritable bowel syndrome in rats.

The aim of this study was to establish a pathophysiologic model of irritable bowel syndrome, and then to evaluate the pharmaceutical efficacy of ramosetron, a potent serotonin 3 (5-HT(3)) receptor antagonist, and other anti-irritable bowel syndrome agents in this model. Rats stressed by a conditioned stress procedure exhibited marked prolongation of freezing time, an index of fear level, and an increase in the frequency of defecation (P<0.01). A corticotropin-releasing factor (CRF) antagonist, alpha-helical CRF, inhibited both defecation and freezing behavior, while the antidiarrheal loperamide inhibited defecation only. The 5-HT(3) receptor antagonists ramosetron, cilansetron and alosetron also inhibited defecation (ED(50) values: 0.012, 0.094, 0.078 mg/kg p.o., respectively) without affecting freezing behavior. Ramosetron showed longer-lasting effect on defecation than cilansetron. Stress also resulted in increases in both proximal and distal colonic transit rates. Ramosetron and other 5-HT(3) receptor antagonists at doses inhibiting stress-induced defecation also ameliorated both stress-stimulated colonic transit rates. These results suggest that ramosetron, as well as agents used for the treatment of irritable bowel syndrome with diarrhea, has beneficial effects against emotional stress-induced colonic dysfunction. Furthermore, this emotional stress model may be useful in evaluation of drugs to treat irritable bowel syndrome presenting with diarrhea.