Clonal evolution process from essential thrombocythemia to acute myeloid leukemia in the original patient from whom the CALR-mutated Marimo cell line was established.

We previously reported the Marimo cell line, which was established from the bone marrow cells of a patient with essential thrombocythemia (ET) at the last stage after transformation to acute myeloid leukemia (AML). This cell line is widely used for the biological analysis of ET because it harbors CALR mutation. However, genetic processes during disease progression in the original patient were not analyzed. We sequentially analyzed the genetic status in the original patient samples during disease progression. The ET clone had already acquired CALR and MPL mutations, and TP53 and NRAS mutations affected the disease progression from ET to AML in this patient. Particularly, the variant allele frequency of the NRAS mutation increased along with the disease progression after transformation, and the NRAS-mutated clone selectively proliferated in vitro, resulting in the establishment of the Marimo cell line. Although CALR and MPL mutations co-existed, MPL was not expressed in Marimo cells or any clinical samples. Furthermore, mitogen-activated protein kinase (MAPK) but not the JAK2-STAT pathway was activated. These results collectively indicate that MAPK activation is mainly associated with the proliferation ability of Marimo cells.

Initiating-clone analysis in patients with acute myeloid leukemia secondary to essential thrombocythemia.

Most of essential thrombocythemia (ET) patients have the clone harboring a mutation in one of the JAK2, CALR, or MPL gene, and these clones generally acquire additional mutations at transformation to acute myeloid leukemia (AML). However, the proliferation of triple-negative clones has sometimes been observed at AML transformation. To clarify the clonal evolution of ET to AML, we analyzed paired samples at ET and AML transformation in eight patients. We identified that JAK2-unmutated AML clones proliferated at AML transformation in three patients in whom the JAK2-mutated clone was dominant at ET. In two patients, TET2-mutated, but not JAK2-mutated, clones might be common initiating clones for ET and transformed AML. In a patient with JAK2-mutated ET, SMARCC2, UBR4, and ZNF143, but not JAK2, -mutated clones proliferated at AML transformation. Precise analysis using single-cell sorted CD34+/CD38- fractions suggested that ET clone with JAK2-mutated and AML clone with TP53 mutation was derived from the common clone with these mutations. Although further study is required to clarify the biological significance of SMARCC2, UBR4, and ZNF143 mutations during disease progression of ET and AML transformation, the present results demonstrate the possibility of a common initial clone involved in both ET and transformed AML.

Paroxysmal nocturnal hemoglobinuria-related thrombosis in the era of novel therapies: a 2043-patient-year analysis.

ABSTRACT: Thrombophilia is one of the principal features of paroxysmal nocturnal hemoglobinuria (PNH) and constitutes the main cause of disease morbidity/mortality. Anticomplement treatment has revolutionized the natural history of PNH, with control of the hemolytic process and abolition of thrombotic events (TEs). However, no guidelines exist for the management of thromboembolic complications in this setting, with type and duration of anticoagulation depending on individual practices. Besides, a scarcity of data is present on the efficacy of direct oral anticoagulants (DOACs). Herein, we accrued a large real-world cohort of patients with PNH from 4 US centers to explore features, predictors of TE, and anticoagulation strategies. Among 267 patients followed up for a total of 2043 patient-years, 56 (21%) developed TEs. These occurred at disease onset in 43% of cases, involving more frequently the venous system, typically as Budd-Chiari syndrome. Rate of TEs was halved in patients receiving complement inhibitors (21 vs 40 TEs per 1000 patient-years in untreated cases, with a 2-year cumulative incidence of thrombosis of 3.9% vs 18.3%, respectively), and varied according to PNH granulocytes and erythrocytes clone size, type, disease activity parameters, as well as number (≥2 mutations, or less) and variant allelic frequency of PIGA mutations. Anticoagulation with warfarin (39%), DOACs (37%), and low-molecular weight heparin (16%) was administered for a median of 29 months (interquartile range [IQR], 9-61.8). No thrombotic recurrence was observed in 19 patients treated with DOACs at a median observation of 17.1 months (IQR, 8.9-45) whereas 14 cases discontinued anticoagulation without TE recurrence at a median time of 51.4 months (IQR, 29.9-86.8).

Molecular and clinical analyses of PHF6 mutant myeloid neoplasia provide their pathogenesis and therapeutic targeting.

PHF6 mutations (PHF6MT) are identified in various myeloid neoplasms (MN). However, little is known about the precise function and consequences of PHF6 in MN. Here we show three main findings in our comprehensive genomic and proteomic study. Firstly, we show a different pattern of genes correlating with PHF6MT in male and female cases. When analyzing male and female cases separately, in only male cases, RUNX1 and U2AF1 are co-mutated with PHF6. In contrast, female cases reveal co-occurrence of ASXL1 mutations and X-chromosome deletions with PHF6MT. Next, proteomics analysis reveals a direct interaction between PHF6 and RUNX1. Both proteins co-localize in active enhancer regions that define the context of lineage differentiation. Finally, we demonstrate a negative prognostic role of PHF6MT, especially in association with RUNX1. The negative effects on survival are additive as PHF6MT cases with RUNX1 mutations have worse outcomes when compared to cases carrying single mutation or wild-type.

Comparison of clonal architecture between primary and immunodeficient mouse-engrafted acute myeloid leukemia cells.

Patient-derived xenografts (PDX) are widely used as human cancer models. Previous studies demonstrated clonal discordance between PDX and primary cells. However, in acute myeloid leukemia (AML)-PDX models, the significance of the clonal dynamics occurring in PDX remains unclear. By evaluating changes in the variant allele frequencies (VAF) of somatic mutations in serial samples of paired primary AML and their PDX bone marrow cells, we identify the skewing engraftment of relapsed or refractory (R/R) AML clones in 57% of PDX models generated from multiclonal AML cells at diagnosis, even if R/R clones are minor at <5% of VAF in patients. The event-free survival rate of patients whose AML cells successfully engraft in PDX models is consistently lower than that of patients with engraftment failure. We herein demonstrate that primary AML cells including potentially chemotherapy-resistant clones dominantly engraft in AML-PDX models and they enrich pre-existing treatment-resistant subclones.

[FLT3 inhibitors in the treatment of FLT3-mutated acute myeloid leukemia].

Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene represent the most common genetic alteration in acute myeloid leukemia (AML), identified in approximately one third of patients newly diagnosed with AML. FLT3 internal tandem duplication mutations (FLT3-ITD) are associated with increased relapse and inferior overall survival. Multiple inhibitors of FLT3 signaling have been developed in the last few years with variable kinase-inhibitory properties, pharmacokinetics, and toxicity profiles. At present, two FLT3 inhibitors (gilteritinib and quizartinib) have been approved as monotherapies for relapsed/refractory FLT3-mutated AML in Japan, and many more drugs are currently being researched in clinical trials as monotherapies or in combination with conventional chemotherapy or hypomethylating agents and in various settings, including front line, relapsed/refractory disease, and maintenance therapy after consolidation chemotherapy or allogeneic stem cell transplantation. Despite significant advances, some issues need to be overcome, including the resistance to FLT3 inhibitors and controversies regarding the role of FLT3 inhibitors in maintenance therapies and the role of allogeneic stem cell transplantation in FLT3-mutated AML.

[Bacillus cereus bacteremia in patients with hematological disorders].

Bacillus cereus bacteremia is an infectious disease that may sometimes be fatal with a rapid clinical course. We performed a retrospective analysis on 12 patients with Bacillus cereus bacteremia recruited from January 2010 to March 2015. The primary diseases were acute leukemia (n=5), myelodysplastic syndromes (n=3), malignant lymphoma (n=3), and hemophagocytic syndrome (n=1). Neutrophil count at the onset of this bacteremia was less than 500 cells/µl in 9 patients. At the onset of bacteremia, we observed neurological symptoms (n=7), gastrointestinal symptoms (n=6), and findings suspected of infection at the venous catheter insertion site (n=6). Vancomycin was administered to all the patients; 10 patients showed improvement whereas 2 died early after allogeneic hematopoietic stem cell transplantation owing to bacteremia. Three patients had sequelae of central nervous system disorders. Neurological and gastrointestinal symptoms with fever may be predictors for this bacteremia, and early administration of appropriate antibacterial drugs may improve the prognosis. Future research should be aimed toward the identification of the clinical features of poor prognosis and establishment of remedies for Bacillus cereus bacteremia.

Allogeneic hematopoietic stem cell transplantation at the first remission for younger adults with FLT3-internal tandem duplication AML: The JALSG AML209-FLT3-SCT study.

In this phase II multicenter study (JALSG AML209-FLT3-SCT), we aimed to prospectively elucidate the role of allogeneic hematopoietic stem cell transplantation (allo-HSCT) at first complete remission (CR1) for FLT3-internal tandem duplication (ITD)-positive AML. Newly diagnosed de novo AML patients with FLT3-ITD were enrolled at the achievement of CR1 and received allo-HSCT as soon as possible after the first consolidation therapy. Mutations of 57 genes in AML cells at diagnosis were also analyzed. Among 48 eligible patients with a median age of 38.5 (17-49) years, 36 (75%) received allo-HSCT at a median of 108 days after CR1. The median follow-up was 1726 days. The primary end-point, 3-year disease-free survival (DFS) based on an intent to treat analysis, was 43.8% (95% confidence interval [CI], 30%-57%), suggesting the efficacy of this treatment because the lower limit of the 95% CI exceeded the threshold response rate of 20%. The 3-year overall survival, post-transplant DFS, and non-relapse mortality rates were 54.2% (95% CI, 39%-67%), 58.3% (95% CI, 41%-72%), and 25.0% (95% CI, 12%-40%), respectively. The median ITD allelic ratio (AR) was 0.344 (0.006-4.099). Neither FLT3-ITD AR nor cooccurring genetic alterations was associated with a poor DFS. This prospective study indicated the efficacy and safety of allo-HSCT for FLT3-ITD AML patients in CR1. This study was registered at: www.umin.ac.jp/ctr/ as #UMIN000003433.

Prospective evaluation of prognostic impact of KIT mutations on acute myeloid leukemia with RUNX1-RUNX1T1 and CBFB-MYH11.

The prognostic impact of KIT mutation on core-binding factor acute myeloid leukemia (CBF-AML) remains controversial. We registered 199 newly diagnosed de novo CBF-AML patients, aged 16 to 64 years, who achieved complete remission. They received 3 courses of high-dose cytarabine therapy and no further treatment until hematological relapse. Mutations in exons 8, 10-11, and 17 of the KIT gene were analyzed. Furthermore, we analyzed mutations in 56 genes that are frequently identified in myeloid malignancies and evaluated minimal residual disease (MRD). The primary end point was relapse-free survival (RFS) according to KIT mutations. The RFS in KIT-mutated patients was inferior to that in unmutated patients (hazard ratio, 1.92; 95% confidence interval, 1.23-3.00; P = .003). Based on subgroup analysis, KIT mutations had a prognostic impact in patients with RUNX1-RUNX1T1, but not in those with CBFB-MYH11, and only exon 17 mutation had a significant prognostic impact. Multivariate Cox regression analysis with stepwise selection revealed that the KIT exon 17 mutation and the presence of extramedullary tumors in patients with RUNX1-RUNX1T1, and loss of chromosome X or Y and NRAS mutation in patients with CBFB-MYH11 were poor prognostic factors for RFS. MRD was evaluated in 112 patients, and it was associated with a poorer RFS in the patients with CBFB-MYH11, but not in those with RUNX1-RUNX1T1. These results suggested that it is necessary to separately evaluate AML with RUNX1-RUNX1T1 or CBFB-MYH11 according to appropriate prognostic factors. This study was registered at www.umin.ac.jp/ctr/ as #UMIN000003434.

Day 0 bone marrow pathology of allogeneic hematopoietic stem cell transplantation is a novel prognostic factor in myeloid malignancies.

Residual disease (RD) is one of the risk factors for relapse after hematopoietic stem cell transplantation (HSCT) in hematological malignancies. Although recent advances in the technology for detecting minimal/measurable RD, such as multiparameter flow cytometry and quantitative PCR, enable risk stratifications of disease relapse, these examinations still have limitations in routine clinical practice. In this study, we assessed RD in bone marrow (BM) specimens on day 0 of allogeneic HSCT by immunostaining of case-specific leukemic blast markers and analyzed the relationship between day 0 BM status and HSCT outcomes. We analyzed 82 adult HSCT recipients with myeloid malignancies. BM histology of day 0 revealed almost empty marrow with a small number of residual BM cells. However, residual blasts could be detected by immunostaining even for only a few cells. When patients were divided into two groups according to the existence of RD on day 0, those with positive RD showed significantly lower overall survival rate (27% vs. 73%, P<0.001) and higher cumulative incidence of relapse (46% vs. 9%, P=0.006) at one year compared to those with negative RD. Furthermore, even if they were not in remission at the point of the pre-conditioning evaluation, the patients who achieved negative RD on day 0 showed comparable prognosis with those who maintained remission before conditioning. This study shows the efficacy of day 0 BM pathology of allogeneic HSCT as a prognostic factor that can contribute to clinical decisions on post-transplant strategies.

FLT3 mutations in acute myeloid leukemia: Therapeutic paradigm beyond inhibitor development.

FMS-like tyrosine kinase 3 (FLT3) is a type III receptor tyrosine kinase that plays an important role in hematopoietic cell survival, proliferation and differentiation. The most clinically important point is that mutation of the FLT3 gene is the most frequent genetic alteration and a poor prognostic factor in acute myeloid leukemia (AML) patients. There are two major types of FLT3 mutations: internal tandem duplication mutations in the juxtamembrane domain (FLT3-ITD) and point mutations or deletion in the tyrosine kinase domain (FLT3-TKD). Both mutant FLT3 molecules are activated through ligand-independent dimerization and trans-phosphorylation. Mutant FLT3 induces the activation of multiple intracellular signaling pathways, mainly STAT5, MAPK and AKT signals, leading to cell proliferation and anti-apoptosis. Because high-dose chemotherapy and allogeneic hematopoietic stem cell transplantation cannot sufficiently improve the prognosis, clinical development of FLT3 kinase inhibitors expected. Although several FLT3 inhibitors have been developed, it takes more than 20 years from the first identification of FLT3 mutations until FLT3 inhibitors become clinically available for AML patients with FLT3 mutations. To date, three FLT3 inhibitors have been clinically approved as monotherapy or combination therapy with conventional chemotherapeutic agents in Japan and/or Europe and United states. However, several mechanisms of resistance to FLT3 inhibitors have already become apparent during their clinical trials. The resistance mechanisms are complex and emerging resistant clones are heterogenous. Further basic and clinical studies are required to establish the best therapeutic strategy for AML patients with FLT3 mutations.

Clinical significance of ASXL2 and ZBTB7A mutations and C-terminally truncated RUNX1-RUNX1T1 expression in AML patients with t(8;21) enrolled in the JALSG AML201 study.

We analyzed the clinical significance and genetic features of ASXL2 and ZBTB7A mutations, and the alternatively spliced isoform of the RUNX1-RUNX1T1 transcript, which is also called AML1-ETO9a (AE9a), in Japanese CBF-AML patients enrolled in the JALSG AML201 study. ASXL2 and ZBTB7A genes were sequenced using bone marrow samples of 41 AML patients with t(8;21) and 14 with inv(16). The relative expression levels of AE9a were quantified using the real-time PCR assay in 23 AML patients with t(8;21). We identified ASXL2 (34.1%) and ZBTB7A (9.8%) mutations in only AML patients with t(8;21). ASXL2-mutated patients had a significantly higher WBC count at diagnosis (P = 0.04) and a lower frequency of sex chromosome loss than wild-type patients (33 vs. 76%, respectively, P = 0.01). KIT mutations were the most frequently accompanied with both ASXL2 (36%) and ZBTB7A (75%) mutations. Neither ASXL2 nor ZBTB7A mutations had an impact on overall or event-free survival. Patients harboring cohesin complex gene mutations expressed significantly higher levels of AE9a than unmutated patients (P = 0.03). In conclusion, ASXL2 and ZBTB7A mutations were frequently identified in Japanese AML patients with t(8;21), but not in those with inv(16). Further analysis is required to clarify the detailed biological mechanism of AE9a regulation of the cohesin complex.

Identification of the novel deletion-type PML-RARA mutation associated with the retinoic acid resistance in acute promyelocytic leukemia.

All-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are essential for acute promyelocytic leukemia (APL) treatment. It has been reported that mutations in PML-RARA confer resistance to ATRA and ATO, and are associated with poor prognosis. Although most PML-RARA mutations were point mutations, we identified a novel seven amino acid deletion mutation (p.K227_T233del) in the RARA region of PML-RARA in a refractory APL patient. Here, we analyzed the evolution of the mutated clone and demonstrated the resistance of the mutated clone to retinoic acid (RA). Mutation analysis of PML-RARA was performed using samples from a chemotherapy- and ATRA-resistant APL patient, and the frequencies of mutated PML-RARA transcript were analyzed by targeted deep sequencing. To clarify the biological significance of the identified PML-RARA mutations, we analyzed the ATRA-induced differentiation and PML nuclear body formation in mutant PML-RARA-transduced HL-60 cells. At molecular relapse, the p.K227_T233del deletion and the p.R217S point-mutation in the RARA region of PML-RARA were identified, and their frequencies increased after re-induction therapy with another type of retinoiec acid (RA), tamibarotene. In deletion PML-RARA-transduced cells, the CD11b expression levels and NBT reducing ability were significantly decreased compared with control cells and the formation of PML nuclear bodies was rarely observed after RA treatment. These results indicate that this deletion mutation was closely associated with the disease progression during RA treatment.

Extracorporeal photopheresis with TC-V in Japanese patients with steroid-resistant chronic graft-versus-host disease.

There are few established therapies for chronic graft-versus-host disease (cGVHD) refractory to first-line treatment with steroids. We evaluated the efficacy and safety of extracorporeal photopheresis (ECP) with a third-generation TC-V device in Japanese patients with cGVHD. Fifteen patients with steroid-resistant or -intolerant cGVHD after allogeneic hematopoietic stem cell transplantation participated in this multicenter open-label study. Extracorporeal photopheresis was conducted on days 1-3, week 1; days 1-2, weeks 2-12; and days 1-2, weeks 16, 20, and 24. The composite primary endpoint consisted of evaluation of response and changes in steroid dose 24 weeks after ECP initiation. Secondary endpoints included response over time, concomitant drug dose, quality of life, and safety. Twelve patients completed scheduled ECP therapy; eight (66.7%) showed a response at week 24. In all 15 patients, the mean (± standard deviation) steroid dose decreased 0.115 ± 0.230 mg/kg/day from screening to week 24. Five serious, potentially treatment-related adverse events (heart failure, thrombosis in the device, pneumonia, edema, and wheezing) occurred; none were fatal. This study confirmed that ECP using the TC-V device was effective, with an acceptable toxicity profile. Further studies in larger cohorts are clearly warranted to determine its optimal use in Japanese patients with cGVHD.

Impact of hospital length of stay on the risk of readmission and overall survival after allogeneic stem cell transplantation.

Patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) are at high risk of readmission for complications. We sought to examine the association between HSCT hospital length of stay and the incidence of readmission and survival after discharge. We retrospectively reviewed the cases of 230 allo-HSCT recipients. The cumulative incidence of readmission with non-relapse transplant-related complications (including infections; acute and chronic GVHD; liver, lung, renal, or neurological complications; and haematological abnormalities) 2 years after the first discharge was 49.7% in patients with length of stay ≤ 100 days (n = 156), and 66.6% in patients with length of stay > 100 days (n = 74) (P = 0.02). The cumulative incidence of readmission with infections 2 years after first discharge was lower in the length of stay ≤ 100 days group than in the length of stay > 100 days patients (27.1 vs. 41.3%, P = 0.04). Length of stay > 100 days was the only risk factor identified that correlated positively with the rate of readmission for non-relapse transplant-related complications [relative risk (RR) 1.53; 95% confidence interval (CI) 1.08-2.18, P = 0.018] or infections [RR 1.64; CI 1.03-2.61; P = 0.038]. Close follow-up of patients with longer length of stay after allo-HSCT is advised.

Mutation analysis of therapy-related myeloid neoplasms.

We analyzed the genetic mutation status of 13 patients with therapy-related myeloid neoplasms (t-MN). Consistent with previous reports, t-MN cells preferentially acquired mutations in TP53 and epigenetic modifying genes, instead of mutations in tyrosine kinase and spliceosome genes. Furthermore, we compared the mutation status of three t-MN cells with each of the initial lymphoid malignant cells, and identified common mutations among t-MN and the initial malignant cells in two patients. In a patient who developed chronic myelomonocytic leukemia (CMML) after follicular lymphoma (FL), TET2 mutation was identified in both CMML and FL cells. Notably, the TET2 mutation was also identified in peripheral blood cells in the disease-free period with the same allelic frequency as CMML and FL cells, but not in a germ-line control, indicating that the TET2 mutation occurred somatically in the initiating clone for both malignant cells. On the other hand, a germ-line MYB mutation was identified in a patient who developed myelodysplastic syndromes (MDS) after FL. These results suggest that germ-line deposition and clonal hematopoiesis are closely associated with t-MN susceptibility; however, further analysis is necessary to clarify the mechanism required to provide the initiating clone with lineage commitment and clonal expansion.

Outcomes of strategic alternative donor selection or suspending donor search based on Japan Marrow Donor Program coordination status.

In allogeneic hematopoietic stem cell transplantation (allo-HSCT) from unrelated donors, delays in donor search are adversely associated with patient outcome. However, the optimal duration for either waiting for an unrelated donor or selecting alternative sources remains undetermined. Using data from the Japan Marrow Donor Program (JMDP) registry, we retrospectively analyzed 349 adult patients who had searched for unrelated donors. Two hundred and three patients received allo-HSCT from JMDP donors (Group A) with a median of 140 days required to identify a donor, 60 received allo-HSCT from alternative sources (Group B) after a median of 111.5 days at which point either all donor candidates had failed or the patient achieved a second or subsequent complete remission, and 77 suspended allo-HSCT (Group C) after a median of 310 days. The 5-year overall survival (OS) rate in Group A was superior to that of Group C (48.6 vs 38.5%, P = 0.001). Although Group B included more patients with high or very high disease risk index (DRI) at the time of allo-HSCT compared with Group A, the 5-year OS was not significantly different between Groups A and B (48.6 vs 40.9%, P = 0.07), indicating that switching to alternative donors may benefit patients with high DRI.

Immune-Mediated Hematopoietic Failure after Allogeneic Hematopoietic Stem Cell Transplantation: A Common Cause of Late Graft Failure in Patients with Complete Donor Chimerism.

Late graft failure (LGF) without evidence of residual recipient cells is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-SCT) and often requires stem cell infusion from the same donor when the patient fails to respond to conventional therapies. We screened the peripheral blood (PB) of 14 patients who developed donor-type LGF at 2 to 132 months after allo-SCT for the presence of the markers for immune-mediated bone marrow (BM) failure. Increased glycosylphosphatidyl inositol-anchored protein-deficient (GPI-AP-) leukocytes, which accounted for .009% to 0.147% of the total granulocytes, were detected in 5 patients (severe aplastic anemia, n = 2; follicular lymphoma, n = 1; acute lymphoblastic leukemia, n = 1; myelodysplastic syndromes; n = 1) and 4.7% to 81.2% HLA-allele-lacking leukocytes (HLA-LLs) were detected in 2 patients (acute myelogenous leukemia, n = 1; and myelodysplastic syndromes, n = 1). Three of the 5 patients with increased GPI-AP- leukocytes were treated with antithymocyte globulin (ATG), and 2 patients achieved transfusion independence. These results suggest that immune mechanisms that are similar to acquired aplastic anemia underlie condition of approximately one-half of the patients with donor-type LGF, and that in patients with increased GPI-AP- cells, donor-derived hematopoiesis may be restored by ATG therapy alone without donor stem cell infusion.