Enhanced ALOX12 Gene Expression Predicts Therapeutic Susceptibility to 5-Azacytidine in Patients with Myelodysplastic Syndromes.

5-azacytidine (AZA), a representative DNA-demethylating drug, has been widely used to treat myelodysplastic syndromes (MDS). However, it remains unclear whether AZA's DNA demethylation of any specific gene is correlated with clinical responses to AZA. In this study, we investigated genes that could contribute to the development of evidence-based epigenetic therapeutics with AZA. A DNA microarray identified that AZA specifically upregulated the expression of 438 genes in AZA-sensitive MDS-L cells but not in AZA-resistant counterpart MDS-L/CDA cells. Of these 438 genes, the ALOX12 gene was hypermethylated in MDS-L cells but not in MDS-L/CDA cells. In addition, we further found that (1) the ALOX12 gene was hypermethylated in patients with MDS compared to healthy controls; (2) MDS classes with excess blasts showed a relatively lower expression of ALOX12 than other classes; (3) a lower expression of ALOX12 correlated with higher bone marrow blasts and a shorter survival in patients with MDS; and (4) an increased ALOX12 expression after AZA treatment was associated with a favorable response to AZA treatment. Taking these factors together, an enhanced expression of the ALOX12 gene may predict favorable therapeutic responses to AZA therapy in MDS.

[Successful immunosuppressive therapy in female hemophilia A developing inhibitor after perioperative administration of factor VIII products].

A 62-year-old woman was diagnosed as a hemophilia A carrier (factor VIII activity 35%) on preoperative examination of an ovarian tumor. A total of 35,600 units of recombinant factor VIII products was administered perioperatively. On postoperative day 95, a subcutaneous hematoma formed and immunosuppressive therapy with prednisolone was started based on an APTT of 66 seconds, factor VIII (FVIII) activity of 3%, and FVIII inhibitor of 1 BU/ml. During this treatment, the patient was hospitalized due to ankle joint bleeds and required hemostatic treatment, but the inhibitor disappeared and FVIII activity recovered to 30% after postoperative day 438 with cyclophosphamide. F8 analysis revealed the patient carried a heterozygosity of p.Arg391Cys, which has previously been categorized as cross-reacting material (CRM)-positive severe hemophilia A. No high-risk mutations for inhibitor development were found. We also report the results of a desmopressin acetate hydrate test administered to the patient to prepare for future treatment in case of hemorrhage, since high-dose FVIII administration may have been a factor in inhibitor development.

Successful correction of factor V deficiency of patient-derived iPSCs by CRISPR/Cas9-mediated gene editing.

BACKGROUND: Factor V (FV) deficiency is a monogenic inherited coagulation disorder considered to be an ideal indication for gene therapy. To investigate the possibility of therapeutic application of genome editing, we generated induced pluripotent stem cells (iPSCs) from a FV-deficient patient and repaired the mutation of factor V gene (F5) using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9). METHODS: The patient's peripheral blood mononuclear cells were reprogrammed for iPSCs. The targeting vector was designed with homology arms against F5 containing the corrected sequence. Cas9 ribonucleoprotein (RNP) complex and targeting vector were electroporated into iPSCs. Gene-edited iPSCs were differentiated into hepatocyte-like cells (HLCs). RESULTS: The mutation of F5 in patient-derived iPSCs was repaired by CRISPR/Cas9. In concentrated culture supernatants of patient-derived iPS-HLCs, neither FV antigen nor activity was detected, while in those of gene-corrected iPS-HLCs, FV antigen and specific activity were 67.0 ± 13.1 ng/mL and 173.2 ± 41.1 U/mg, respectively. CONCLUSIONS: We successfully repaired the mutation of F5 using the CRISPR/Cas9 and confirmed the recovery of FV activity with gene-corrected iPS-HLCs. Gene-edited iPSCs are promising for elucidating the pathophysiology as well as for a modality of gene therapy.

Transducin ß-like 1, X-linked and nuclear receptor co-repressor cooperatively augment the ligand-independent stimulation of TRH and TSHß gene promoters by thyroid hormone receptors.

Mutations in TBL1X, a component of the nuclear receptor co-repressor (N-CoR) and silencing mediator of retinoic acid and thyroid hormone receptor co-repressor complexes, have recently been implicated in isolated central hypothyroidism (CeH). However, the mechanisms by which TBL1X mutations affect negative feedback regulation in the hypothalamus-pituitary-thyroid axis remain unclear. N-CoR was previously reported to paradoxically enhance the ligand-independent stimulation of TRH and TSHß gene promoters by thyroid hormone receptors (TR) in cell culture systems. We herein investigated whether TBL1X affects the unliganded TR-mediated stimulation of the promoter activities of genes negatively regulated by T3 in cooperation with N-CoR. In a hypothalamic neuronal cell line, the unliganded TR-mediated stimulation of the TRH gene promoter was significantly enhanced by co-transfected TBL1X, and the co-transfection of TBL1X with N-CoR further enhanced promoter activity. In contrast, the knockdown of endogenous Tbl1x using short interfering RNA significantly attenuated the N-CoR-mediated enhancement of promoter activity in the presence of unliganded TR. The co-transfection of N365Y or Y458C, TBL1X mutants identified in CeH patients, showed impaired co-activation with N-CoR for the ligand-independent stimulation of the TRH promoter by TR. In the absence of T3, similar or impaired enhancement of the TSHß gene promoter by the wild type or TBL1X mutants, respectively, was observed in the presence of co-transfected TR and N-CoR in CV-1 cells. These results suggest that TBL1X is needed for the full activation of TRH and TSHß gene promoters by unliganded TR. Mutations in TBL1X may cause CeH due to the impaired up-regulation of TRH and/or TSHß gene transcription despite low T3 levels.

Expression of programmed cell death ligand 1 is associated with poor overall survival in patients with diffuse large B-cell lymphoma.

Programmed cell death ligand 1 (PD-L1) is expressed on both select diffuse large B-cell lymphoma (DLBCL) tumor cells and on tumor-infiltrating nonmalignant cells. The programmed cell death 1 (PD-1)/PD-L1 pathway inhibits host antitumor responses; however, little is known about how this pathway functions in the tumor microenvironment. The aim of this study was to determine the clinicopathological impact of PD-L1(+) DLBCL. We performed PD-L1/PAX5 double immunostaining in 1253 DLBCL biopsy samples and established a new definition of PD-L1(+) DLBCL. We also defined the criteria for microenvironmental PD-L1(+) (mPD-L1(+)) DLBCL (ie, PD-L1(-) DLBCL in which PD-L1(+) nonmalignant cells are abundant in the tumor microenvironment). Of the 273 patients whose clinical information was available, quantitative analysis of PD-1(+) tumor-infiltrating lymphocytes (TILs) was performed. The prevalence rates of PD-L1(+) and mPD-L1(+) DLBCL were 11% and 15.3%, respectively. Both PD-L1(+) and mPD-L1(+) DLBCL were significantly associated with non-germinal center B-cell (GCB) type and Epstein-Barr virus positivity. The number of PD-1(+) TILs was significantly higher in GCB-type tumors and lower in mPD-L1(-) and PD-L1(+) DLBCL. Patients with PD-L1(+) DLBCL had inferior overall survival (OS) compared with that in patients with PD-L1(-) DLBCL (P = .0009). In contrast, there was no significant difference in OS between mPD-L1(+) and mPD-L1(-) DLBCL (P = .31). The expression of PD-L1 maintained prognostic value for OS in multivariate analysis (P = .0323). This is the first report describing the clinicopathological features and outcomes of PD-L1(+) DLBCL. Immunotherapy targeting the PD-1/PD-L1 pathway should be considered in this distinct DLBCL subgroup.

HBA1C AND MEAN GLUCOSE DERIVED FROM SHORT-TERM CONTINUOUS GLUCOSE MONITORING ASSESSMENT DO NOT CORRELATE IN PATIENTS WITH HBA1C >8.

OBJECTIVE: Optimum therapy for patients with diabetes depends on both acute and long-term changes in plasma glucose, generally assessed by glycated hemoglobin (HbA1c) levels. However, the correlation between HbA1c and circulating glucose has not been fully determined. Therefore, we carefully examined this correlation when glucose levels were assessed by continuous glucose monitoring (CGM). METHODS: Fifty-one patients (70% female, 30% male) were examined; among them were 28 with type 1 diabetes and 23 with type 2 diabetes. Clinically determined HbA1c levels were compared with blood glucose determined by CGM during a short time period. RESULTS: Changes in HbA1c levels up to 8.0% showed a clear and statistically strong correlation (R = 0.6713; P<.0001) with mean blood glucose levels measured by CGM, similar to that observed in the A1c-derived Average Glucose study in which patients were monitored for a longer period. However, we found no statistical correlation (R = 0.0498; P = .83) between HbA1c and CGM-assessed glucose levels in our patient population when HbA1c was >8.0%. CONCLUSION: Short-term CGM appears to be a good clinical indicator of long-term glucose control (HbA1c levels); however, cautions should be taken while interpreting CGM data from patients with HbA1c levels >8.0%. Over- or underestimation of the actual mean glucose from CGM data could potentially increase the risks of inappropriate treatment. As such, our results indicate that a more accurate analysis of CGM data might be useful to adequately tailor clinical treatments. ABBREVIATIONS: ADAG = A1c-Derived Average Glucose CGM = continuous glucose monitoring %CV = percent coefficient of variation HbA1c = glycated hemoglobin.

Development of immersion vaccine for bacterial cold-water disease in ayu Plecoglossus altivelis.

Flavobacterium psychrophilum (F. psychrophilum) is the causative agent of bacterial cold-water disease (BCWD) that occurs in ayu Plecoglossus altivelis. Formalin-killed cell of F. psychrophilum has long been studied as an immersion vaccine for BCWD. In this study, we explored the possibility of F. psychrophilum collagenase (fpcol) for use as the immersion vaccine. BCWD convalescent ayu sera contained specific IgM antibodies against somatic F. psychrophilum and fpcol, meaning that fpcol is a promising antigen for the vaccine development. The recombinant fpcol was successfully expressed in Escherichia coli and Brevibacillus chosinensis (B. chosinensis). The culture supernatant of the B. chosinensis was used as an immersion vaccine solution. The vaccinated ayu were then challenged by soaking into F. psychrophilum culture. In two experimental groups, the relative percentages of survivals were 63 and 38%, respectively, suggesting that fpcol is promising as the immersion vaccine for ayu-BCWD.

Characteristics of Japanese aldosterone-producing adenomas with KCNJ5 mutations.

Somatic mutations in KCNJ5 gene have been identified in patients with adrenal aldosterone-producing adenomas (APAs). We previously reported that Japanese patients with APAs had distinct characteristics from patients in Western countries; i.e. they had a high frequency of KCNJ5 mutations and exhibited a frequent association with cortisol co-secretion. Therefore, APAs among Japanese patients may have different features from those in Western countries. We added recent cases, examined 47 cases (43% male) of APAs, including clinicopathological features, KCNJ5 mutations, and the mRNA levels of several steroidogenic enzymes, and compared the results obtained to those reported in other countries. While the prevalence of KCNJ5 mutations is approximately 40% in Western countries, 37 APA cases (78.7%) showed mutations: 26 with p.G151R and 11 with p.L168R. Although a significant gender difference has been reported in the frequency of KCNJ5 mutations in Europe, we did not find any gender difference. However, the phenotypes of Japanese patients with mutations were similar to those of patients in Western countries; patients were younger and had higher plasma aldosterone levels, lower potassium levels, and higher diastolic blood pressure. Reflecting these phenotypes, APAs with mutations had higher CYP11B2 mRNA levels. However, in contrast to APAs in Western countries, Japanese APAs with mutations showed lower CYP11B1, CYP17A1, and CYP11A1 mRNA levels. These findings demonstrated that Japanese APA patients may have distinct features including a higher prevalence of KCNJ5 mutations, no gender difference in the frequency of these mutations, and characteristics similar to the zona glomerulosa.

GNAS mutations in adrenal aldosterone-producing adenomas.

Mutations in GNAS, which encodes Gsα, have been documented in detail, particularly in human pituitary GH-secreting adenomas. Mutations have also recently been reported in adrenal cortisol-producing adenomas (CPAs), in addition to those in the PRKACA gene. However, mutations have not yet been examined in aldosterone-producing adenomas (APAs). Therefore, we herein investigated mutations in the GNAS gene in APAs. Two of the 15 (13%) CPAs with overt Cushing's syndrome and one of the 9 (11%) CPAs with subclinical Cushing's syndrome examined had the somatic mutations, p.R201S and p.R201C in the GNAS gene. We identified mutations in the GNAS gene (p.R201C) in 2 out of the 33 (6%) APAs tested, both of which showed autonomous cortisol secretion, while 24 APAs had mutations in the KCNJ5 gene (18 with p.G151R and 6 with p.L168R). These GNAS and KCNJ5 mutations were mutually exclusive in these adenomas. We herein demonstrated for the first time the presence of GNAS mutations in APAs, as well as in some cortisol-secreting adenomas. Our results suggest that these mutations, in addition to mutations in the KCNJ5 gene and other genes such as ATP1A1, ATP2B3 and CACNA1D, may be responsible for the tumorigenesis of APAs and CPAs with subclinical Cushing's syndrome.

GPIbα regulates platelet size by controlling the subcellular localization of filamin.

Interaction between the cytoplasmic domain of GPIbα with its cytoskeletal binding partner, filamin, is a major determinant of platelet size, and deficiency of either protein results in macrothrombocytopenia. To clarify the mechanism by which GPIbα-filamin interactions regulate platelet production, we manipulated the expression levels of filamin and GPIb in cultured embryonic stem cells (ESCs) that were subsequently differentiated into platelets. Knocking down filamins A and B resulted in the production of ESC-derived proplatelets with abnormally large swellings and proplatelet shafts that generated giant platelets in culture. Large platelets could also be generated by overexpressing GPIbα in ESCs, or by overexpressing in vivo a transgene encoding a chimeric protein containing the cytoplasmic domain of GPIbα. To identify the mechanism by which the GPIb:filamin ratio regulates platelet size, we manipulated filamin and GPIbα levels in HEK293T cells and examined the effects of overexpressing either protein on their ability to traffic to the cell periphery. Accumulation of either protein within the endoplasmic reticulum resulted in trapping of the other. Taken together, these data demonstrate that coordinated expression of GPIbα and filamin is required for efficient trafficking of either protein to the cell surface, and for production of normal-sized platelets.

Decreased serum levels of immunoglobulin A, immunoglobulin M and immunoglobulin G in a patient with primary biliary cirrhosis: A case report.

Primary biliary cirrhosis (PBC) is a cholestatic liver disease with an elevated serum immunoglobulin (Ig)M level. Patients with PBC may develop extrahepatic manifestations, including hypogammaglobulinemia. However, hypogammaglobulinemia seldom occurs, and the associated changes of lymphocytes remain unknown. Furthermore, the impact of Ig on the progression of PBC is still unclear. Here, we describe a case of hypogammaglobulinemia developed in a female patient with PBC. The patient was diagnosed with PBC at the age of 46 years and treated with ursodeoxycholic acid and bezafibrate. At the age of 50 years, the patient developed bronchitis, and laboratory test results indicated a marked decrease in serum levels of IgA, IgM and IgG. Then, the patient was diagnosed as having idiopathic hypogammaglobulinemia and treated with Ig replacement therapy; however, respiratory infections recurred frequently, leading to the patient's death at the age of 53 years. An autopsy revealed hyperplastic bone marrow with CD3, CD20 and IgG positive lymphocytes. However, no CD79a, CD138, IgA and IgM positive lymphocytes were observed. Moreover, the severity of PBC progressed even after the onset of hypogammaglobulinemia. In addition, CD3 positive cells were seen around chronic non-suppurative destructive cholangitis in the autopsy specimen of the liver. Thus, the present case demonstrated changes of lymphocytes in hypogammaglobulinemia developed in patients with PBC. Furthermore, the clinical course of the present case of PBC may indicate that the Ig-mediated mechanisms may be non-essential for the progression of PBC.

CD34(+) cell therapy is safe and effective in slowing the decline of hepatic reserve function in patients with decompensated liver cirrhosis.

BACKGROUND AND AIM: Preclinical studies in rodent models of chronic liver fibrosis have shown that transplantation of peripheral blood (PB) CD34(+) cells leads to hepatic regeneration and a reduction of liver fibrosis by suppressing hepatic stellate cell activity and increasing matrix metalloproteinase activity. The aim of this study was to examine the safety and clinical efficacy of intrahepatic transplantation of autologous granulocyte colony-stimulating factor (G-CSF)-mobilized PB-CD34(+) cells in patients with decompensated liver cirrhosis. METHODS: PB-CD34(+) cells were isolated from G-CSF-mobilized apheresis products. Ten patients were treated with G-CSF-mobilized PB-CD34(+) cells (treatment group) and seven patients were treated with standard medical therapy. For mobilization, patients in the treatment group received subcutaneous injections of 10 µg G-CSF/kg/day for 5 days. The cells were then injected at three different doses (5 × 10(5) , 1 × 10(6) and 2 × 10(6) cells/kg) through the hepatic artery. Thereafter, all patients were followed up for 24 months. RESULTS: G-CSF treatment and leukapheresis were well tolerated, and no serious adverse events were observed. Patients in the treatment group had a significant but transient splenomegaly. After 24 weeks, serum albumin was significantly increased in patients who had received middle or high doses of CD34(+) cells compared with baseline. Doppler ultrasound showed a significant increase in hepatic blood flow velocity and blood flow volume after CD34(+) cell therapy. The hepatic vein pressure gradient decreased in two patients who received high-dose CD34(+) cells at week 16. CONCLUSIONS: CD34(+) cell therapy is feasible, safe and effective in slowing the decline of hepatic reserve function.

Somatic mutations of the catalytic subunit of cyclic AMP-dependent protein kinase (PRKACA) gene in Japanese patients with several adrenal adenomas secreting cortisol [Rapid Communication].

Somatic mutations of the catalytic subunit of the cyclic AMP-dependent protein kinase (PRKACA) gene have recently been identified in about 35% of cortisol-producing adenomas (CPAs), with the affected patients showing overt Cushing's syndrome. Since we recently reported higher prevalence of mutations of the KCNJ5 gene and associations with autonomous cortisol secretion in Japanese aldosterone-producing adenomas than in Western countries, there might be different features of CPAs between Japan and the West. We therefore investigated mutations of the PRKACA gene in Japanese patients with several adrenal tumors secreting cortisol, including overt Cushing's syndrome, subclinical Cushing's syndrome, and aldosterone-producing adenomas (APAs) co-secreting cortisol operated on at Gunma University Hospital. Of the 13 patients with CPA who showed overt Cushing's syndrome, 3 (23%) had recurrent somatic mutations of the PRKACA gene, p.L206R (c.617 T>G), and there were no mutations in subclinical Cushing's syndrome. Among 33 APAs, 24 had somatic mutations of the KCNJ5 gene, either G151R or L168R, 11 (33%) had autonomous cortisol secretion, but there were no mutations of the PRKACA gene. We established a PCR-restriction fragment length polymorphism assay and revealed that the mutated allele was expressed at a similar level to the wild-type allele. These findings demonstrated that 1) the prevalence of Japanese patients with CPA who showed overt Cushing's syndrome and whose somatic mutations in the PRKACA gene was similar to that in Western countries, 2) the mutation might be specific for CPAs causing overt Cushing's syndrome, and 3) the mutant PRKACA allele was expressed appropriately in CPAs.

Hypermetabolic pulmonary and bone marrow lesions in a patient with chronic adult T-cell leukemia.

A 69 years old woman with adult T-cell leukemia (ATL) (chronic type) was referred for a fluorine-18 fluorodeoxyglucose positron emission computed tomography ((18)F-FDG PET/CT). Multiple hypermetabolic pulmonary and bone lesions were evidence. The patient underwent chemotherapy, but did not respond, and she died approximately 8 months from the onset of symptoms. Autopsy showed ATL cells infiltrating the lung parenchyma and the pulmonary hilum. In conclusion, we present a case of hypermetabolic pulmonary lesions associated with thoracic CT findings on a (18)F-FDG PET/CT scan in a patient with a chronic adult T-cell leukemia.

Pigment epithelium-derived factor (PEDF) inhibits survival and proliferation of VEGF-exposed multiple myeloma cells through its anti-oxidative properties.

Vascular endothelial growth factor (VEGF) has been reported not only to induce angiogenesis within the bone marrow, but also directly stimulate the proliferation and survival of multiple myeloma cells, thus being involved in the development and progression of this second most common hematological malignancy. We, along with others, have found that pigment epithelium-derived factor (PEDF) has anti-angiogenic and anti-vasopermeability properties both in cell culture and animal models by counteracting the biological actions of VEGF. However, effects of PEDF on VEGF-exposed myeloma cells remain unknown. In this study, we examined whether and how PEDF could inhibit the VEGF-induced proliferation and survival of myeloma cells. PEDF, a glutathione peroxidase mimetic, ebselen, or an inhibitor of NADPH oxidase, diphenylene iodonium significantly inhibited the VEGF-induced reactive oxygen species (ROS) generation, increase in anti-apoptotic and growth-promoting factor, myeloid cell leukemia 1 (Mcl-1) expression, and proliferation in U266 myeloma cells. VEGF blocked apoptosis of multiple myeloma cells isolated from patients, which was prevented by PEDF. PEDF also reduced p22phox levels in VEGF-exposed U266 cells. Furthermore, overexpression of dominant-negative human Rac-1 mutant mimicked the effects of PEDF on ROS generation and Mcl-1 expression in U266 cells. Our present study suggests that PEDF could block the VEGF-induced proliferation and survival of multiple myeloma U266 cells through its anti-oxidative properties via suppression of p22phox, one of the membrane components of NADPH oxidase. Suppression of VEGF signaling by PEDF may be a novel therapeutic target for multiple myeloma.

A case of 8p11 myeloproliferative syndrome with BCR-FGFR1 gene fusion presenting with trilineage acute leukemia/lymphoma, successfully treated by cord blood transplantation.

The 8p11 myeloproliferative syndrome is a rare neoplasm associated with chromosomal translocations involving the fibroblast growth factor receptor 1 (FGFR1) gene located at chromosome 8p11-12. FGFR1 encodes a transmembrane receptor tyrosine kinase. The resultant fusion proteins are constitutively active tyrosine kinases that drive the proliferation of hematopoietic cells, whose uncontrolled growth can present as a myeloproliferative neoplasm. We report here the case of a 50-year-old man harboring the t(8;22)(p12;q11) chromosomal translocation in cells from both bone marrow and lymph nodes. He presented with acute leukemia and lymphoma with trilineage features. A novel mRNA in-frame fusion between exon 4 of the breakpoint cluster region (BCR) gene at chromosome 22q11 and exon 9 of FGFR1 gene on chromosome 8p11-12 was identified by reverse transcription polymerase chain reaction analysis and was confirmed by DNA sequencing. Because the patient was refractory to chemotherapy, cord blood transplantation was performed in progressive disease. It resulted in a successful outcome in which cytogenetic complete remission has been maintained for 2 years till date.

[Fatal zygomycosis caused by Mucor indicus after haplo-identical stem cell transplantation].

A 62-year-old woman with acute lymphoblastic leukemia in first complete remission was treated with unrelated cord blood transplantation, but exhibited primary graft failure. She then underwent HLA-haploidentical peripheral blood stem cell transplantation from her daughter. The conditioning regimen consisted of fludarabine 30 mg/m(2)/day for 6 days, intravenous busulfan 3.2 mg/kg/day for 2 days, and thymoglobulin 1 mg/kg/day for 2 days. Voriconazole was administered to prevent fungal infections. The patient achieved prompt hematopoietic recovery. Fever was observed 21 days after the second transplant, followed by sigmoid colon perforation and a liver space occupying lesion (SOL). A filamentous fungus was detected in a percutaneous biopsy of the liver SOL. In spite of changing the antifungal drug from voriconazole to liposomal amphotericin B, the patient died on day 41. The fungus was identified as Mucor indicus, a type of zygomycete. Although Mucor indicus inhabits soil, an infectious disease is extremely rare, and breakthrough infection after voriconazole prophylaxis had not been reported until now. It is mandatory to consider preventive antifungal treatment for drug-resistant fungal infectious diseases in patients after neutrophilic recovery with a strongly immunocompromised state after a HLA-haploidentical transplant.

A prospective, multicenter, open-label phase III study of emicizumab prophylaxis in patients with acquired hemophilia A.

BACKGROUND: Emicizumab is a bispecific antibody that mimics the cofactor function of activated factor (F) VIII. It prevents bleeds in patients with congenital hemophilia A regardless of the inhibitor status; however, no prospective clinical studies have been conducted for emicizumab in patients with acquired hemophilia A (PwAHA). OBJECTIVES: To describe the primary analysis results from a prospective, multicenter, open-label phase III study evaluating the efficacy, safety, and pharmacokinetics of emicizumab in PwAHA (AGEHA; JapicCTI-205151). METHODS: Emicizumab was administered subcutaneously at 6 mg/kg on day 1 and 3 mg/kg on day 2, followed by 1.5 mg/kg once weekly from day 8 onward. Predefined criteria for the completion of dosing included FVIII activity of >50 IU/dL. RESULTS: By the cutoff date (April 23, 2021), 12 patients on immunosuppressive therapy were enrolled, and 11 of them (91.7%) completed emicizumab treatment. The mean trough plasma emicizumab concentration rapidly reached a steady state (1 week), achieving the efficacious level that was established in patients with congenital hemophilia A (>30 µg/mL). Before first emicizumab administration, 7 patients (58.3%) experienced 77 major bleeds. During emicizumab treatment, no major bleeds occurred in any patient. Neither death due to bleeding or infection nor any study treatment-related serious adverse event was reported. One asymptomatic, nonserious deep vein thrombosis was discovered with no laboratory findings indicating any trend toward hypercoagulation. CONCLUSION: These results suggest that emicizumab prophylaxis with the tested dosing regimen and completion criteria may have a favorable benefit-risk profile in PwAHA.

KCNJ5 mutations in aldosterone- and cortisol-co-secreting adrenal adenomas.

Adrenal aldosterone-producing adenomas (APA) are rarely associated with the clear co-secretion of cortisol. Somatic mutations of the potassium channel KCNJ5 gene, with the hotspots G151R and L168R, have been recently identified in patients with APA. However, whether APAs that secrete cortisol have these mutations remains unclear. We examined three patients with APAs showing clear autonomous secretion of cortisol who possessed a 1 mg dexamethasone suppression test (DST) with a failure of the serum cortisol level to drop below 3.0 µg/dL, a morning plasma ACTH level of less than 10 pg/mL, and suppressed accumulation in the intact adrenal on (131)I- adosterol scintigraphy, or postoperative adrenal insufficiency. Laparoscopic adrenectomy revealed all tumors to be golden yellow, and histological examination confirmed them to be adrenocortical adenomas. All these patients required replacement therapy with hydrocortisone after surgery. Sequencing demonstrated that 2 of three cases showed a mutation of the KCNJ5 gene, one with c.451G>A, p.G151R and one with c.503T>G, p.L168R. Furthermore, the mRNA levels of steroidogenic enzymes including CYP11B1, CYP11B2, HSD3B2, CYP17A1, CYP11A1 and KCNJ5 in the 3 cases did not differ from those in 8 pure APAs not showing any of the above conditions for autonomous cortisol secretion. In addition, all 8 pure APAs harbored mutations of the KCNJ5 gene. These findings suggested that at least some aldosterone- and cortisol-co-secreting adrenal tumors have mutations of the KCNJ5 gene, suggesting the origin to be APA, and pure APAs may show a high incidence of KCNJ5 mutations.