Proposal of criteria for dyserythropoiesis in the diagnosis of myelodysplastic syndromes.

The percentage manifesting dysplasia in bone marrow needed to qualify as significant is ≥10 % in each lineage. However, detailed analyses of this threshold have not been reported. Here, we analyzed dyserythropoiesis (dysE) in 109 myelodysplastic syndromes (MDS) patients with 21 immune thrombocytopenia (ITP)/12 hemolytic anemia (HA) patients as a control. In present study, mild megaloblastic erythroblasts were specifically named 'red cell with abnormal chromatin clumping (RCACC)'. RCACC ≥10 % in erythroblasts was observed in 29 % of ITP patients and 58 % of HA patients. The numbers of MDS patients with RCACC in erythroblasts <10, 10-19 and ≥20 % were 1, 3, and 105, respectively. We analyzed dysE criteria according to the WHO classification (original WHO dysE). Most of our MDS patients (98 %) had original WHO dysE ≥20 %. The ITP patients with original WHO dysE ≥10 % was 48 %, and there were no ITP patients had original WHO dysE ≥20 %. Sixty-seven percent of HA patients had original WHO dysE ≥10 %, and three patients (25 %) had original WHO dysE ≥20 %. Raising the threshold of the original WHO dysE from 10 to 20 or 30 % may provide more suitable criteria. If RCACC is not included in dysE criteria, we think that '10 %' is a suitable threshold for the determination of dyserythropoiesis.

Correlation between dysplastic lineage and type of cytopenia in myelodysplastic syndromes patients with refractory anemia according to the FAB classification.

OBJECTIVES: To analyze the correlation between dysplastic lineage and type of cytopenia in myelodysplastic syndromes. METHODS: We analyzed the correlation between dysplasia and cell count using the data set of our previous morphologic study. RESULTS: There were no correlations between dysgranulopoiesis of 10% or more and absolute neutrophil count (ANC). Similarly, hyposegmented mature neutrophils (Pelger) of 10% or more were not related to ANC. Interestingly, the platelet count of patients with dysmegakaryopoiesis (dys Mgk) was higher than that of patients without dys Mgk (dys Mgk ≥10% vs <10%, P = .08; dys Mgk ≥40% vs <40%, P = .02; micromegakaryocytes ≥10% vs <10%, P = .004). CONCLUSIONS: Since low cell counts did not correlate with the presence of dysplastic features, we suggest that dysplastic features do not directly relate to apoptosis.

[Aplastic anemia complicated with ulcerative colitis].

A 37-year-old female who presented with pancytopenia in April 2008 was diagnosed with aplastic anemia stage 2 with a normal karyotype. She had a PNH phenotype in her red blood cells (RBC) and granulocytes, and HLA DR15. Her aplastic anemia was deteriorated from stage 2 to stage 3, and she required periodic RBC transfusions. Four months after cyclosporine therapy, the pancytopenia improved and she did not need RBC transfusion. However, three months thereafter, she again required RBC transfusions after developing severe ulcerative colitis. Although mesalazine and steroid pulse therapy improved her ulcerative colitis, her transfusion dependency persisted. Eleven months after the diagnosis of aplastic anemia, equine anti-thymocyte globulin (ATG) and cyclosporine were administered, but no hematological improvement was obtained. Six months after the administration of ATG and cyclosporine, transformation to refractory cytopenia with multilineage dysplasia (RCMD) with 7-monosomy was observed. An allogeneic bone marrow transplant (BMT) from a HLA-identical sibling was performed 23 months after the diagnosis of aplastic anemia. Complete remission of both the aplastic anemia and ulcerative colitis was obtained without medication. Although the relationship between aplastic anemia and ulcerative colitis remains unclear, immunological abnormalities might be involved in the pathogenesis of both disorders because she had PNH phenotype in RBC and HLA DR15 and because allogeneic BMT improved both disorders.

Morphologic analysis in myelodysplastic syndromes with del(5q) treated with lenalidomide. A Japanese multiinstitutional study.

Lenalidomide is known to be effective in myelodysplastic syndromes (MDS) with del(5q) in improving anemia and suppressing del(5q) cells. MDS with del(5q) shows increase of nonlobulated megakaryocytes. However, histopathology of MDS with del(5q) treated with lenalidomide has not been fully studied. We investigated the morphologic changes in lenalidomide treated low- or intermediate-1-risk MDS with del(5q). All of evaluable patients showed high proportion of nonlobulated megakaryocytes. The nonlobulated megakaryocytes were markedly decreased in 6 patients during therapy in parallel with suppression of del(5q) cells. Our analysis suggests that single allele deletion of common deleted region inhibits nuclear lobulation of megakaryocytes.

Intensified consolidation therapy with dose-escalated doxorubicin did not improve the prognosis of adults with acute lymphoblastic leukemia: the JALSG-ALL97 study.

We designed a treatment protocol for newly diagnosed adult acute lymphoblastic leukemia (ALL) in the pre-imatinib era, employing intensified consolidation therapy with a total of 330 mg/m² doxorubicin and adopting slightly modified induction and maintenance regimen of the CALGB 8811 study. Of 404 eligible patients (median age 38 years, range 15-64 years), 298 (74%) achieved complete remission (CR). The 5-year overall survival (OS) rate was 32%, and the 5-year disease-free survival (DFS) rate was 33%. Of 256 Philadelphia chromosome (Ph)-negative patients, 208 (81%) achieved CR and the 5-year OS rate was 39%, and 60 of them underwent allogeneic-hematopoietic stem cell transplantation (allo-HSCT) from related or unrelated donors during the first CR, resulting in 63% 5-year OS. Of 116 Ph-positive patients, 65 (56%) achieved CR and the 5-year OS rate was 15%, and 22 of them underwent allo-HSCT from related or unrelated donors during the first CR, resulting in 47% 5-year OS. In Ph-negative patients, multivariate analysis showed that older age, advanced performance status and unfavorable karyotypes were significant poor prognostic factors for OS and higher WBC counts for DFS. The present treatment regimen could not show a better outcome than that of our previous JALSG-ALL93 study for adult ALL.

Differences in the distribution of subtypes according to the WHO classification 2008 between Japanese and German patients with refractory anemia according to the FAB classification in myelodysplastic syndromes.

We reported the different clinical features between Japanese and German refractory anemia (RA) patients in FAB classification. We re-analyzed the clinical features by WHO classification revised in 2008. The frequencies of refractory cytopenia with unilineage dysplasia (RCUD) and myelodysplastic syndrome-unclassified (MDS-U) with pancytopenia in Japanese patients were higher than in German patients (p<0.001). Refractory cytopenia with multilineage dysplasia patients showed the most unfavorable prognosis in both countries. The higher frequencies of MDS-U with pancytopenia and RCUD in Japanese patients may influence the different clinical characteristics between Japanese and German FAB-RA patients.

A Phase I/II study of nilotinib in Japanese patients with imatinib-resistant or -intolerant Ph+ CML or relapsed/refractory Ph+ ALL.

Nilotinib is a second-generation BCR-ABL kinase inhibitor with improved potency and selectivity compared to imatinib. A Phase I/II dose-escalation study was designed to evaluate the efficacy, safety, and pharmacokinetics of nilotinib in Japanese patients with imatinib-resistant or -intolerant Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML) or relapsed/refractory Ph+ acute lymphoblastic leukemia (ALL). A total of 34 patients were evaluated in this analysis and had a median duration of drug exposure of 293 (range 13-615) days. All 6 CML-CP patients without complete hematologic response (CHR) at baseline rapidly achieved CHR. A major cytogenetic response was achieved in 94% of patients with CML-CP, including a complete cytogenetic response in 69%. A major molecular response was achieved by 56%. These responses were also observed in patients with CML in advanced stages and Ph+ ALL. Non-hematologic adverse events were mostly mild to moderate. Grade 3 or 4 neutropenia and thrombocytopenia occurred in 50 and 28% of patients, respectively. Overall, the results of this study suggest that nilotinib induced significant responses in imatinib-resistant or -intolerant patients with CML-CP and CML in advanced stages and Ph+ ALL. The results of this study confirmed the efficacy and safety of nilotinib in Japanese patients.

Phase 1/2 clinical study of dasatinib in Japanese patients with chronic myeloid leukemia or Philadelphia chromosome-positive acute lymphoblastic leukemia.

A phase 1/2 study was conducted to assess the safety and efficacy of dasatinib in Japanese patients with chronic myelogenous leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) resistant or intolerant to imatinib. In phase 1, 18 patients with chronic phase (CP) CML were treated with dasatinib 50, 70, or 90 mg twice daily to evaluate safety. Dasatinib

Long-term efficacy of imatinib in a practical setting is correlated with imatinib trough concentration that is influenced by body size: a report by the Nagasaki CML Study Group.

Imatinib has dramatically improved long-term survival of chronic myelogenous leukemia (CML) patients. To analyze its efficacy in a practical setting, we registered most of CML patients in Nagasaki Prefecture of Japan. Of these, 73 patients received imatinib as an initial therapy. The overall survival rate of these patients was 88.7% at 6 years, and the cumulative complete cytogenetic response rate was 82.5% at 18 months. These results are comparable with the data of other reports including the IRIS study; however, the administered imatinib dose was smaller in our study than that in other reports. To address these discrepancies, we measured the trough concentration of imatinib among 35 patients. Although 39% of the patients were administered less than 400 mg/day, the trough level was comparable to those of previous reports. The trough level of imatinib showed a significant relationship with its efficacy, and was clearly related to dose of imatinib administrated and dose of imatinib divided by body surface area (BSA). Considering the smaller BSA of Japanese patients as compared to those of foreign origin, the results suggest that a lower dose of imatinib could maintain enough trough level and provided excellent results for the treatment of CML in our registry.

Adhesion-dependent growth of primary adult T cell leukemia cells with down-regulation of HTLV-I p40Tax protein: a novel in vitro model of the growth of acute ATL cells.

In order to better understand the biology of adult T cell leukemia (ATL), we aimed to establish a novel method, which allows the primary growth of ATL cells using a co-culture system with murine bone marrow-derived stromal cells, MS-5. ATL cells grew in close contact with MS-5 layers and formed so-called "cobblestone areas" (CAs) without the addition of IL-2. In clinical samples, eight of ten (80.0%) cases of acute or lymphoma type ATL cells formed CAs. The frequency of CA forming cells in ATL cells ranged from 0.03 to 1.04%. The morphology, immunophenotyping, and DNA analysis indicated that cells composing CA were compatible with ATL cells, and clonally identical to primary CD4-positive ATL cells. Furthermore, in ATL cells composing CA, the expression of p40Tax was down-regulated in transcriptional and translational level, while that of HTLV-I basic leucine zipper factor (HBZ) gene was comparable to the level of primary ATL cells, resembling expression pattern of proviral genes in in vivo ATL cells. By microarray analysis, several genes which coded products involved in cell-cell interaction, and cellular survival and proliferation, were differentially expressed in ATL cells composing CA compared with primary samples. In conclusion, our co-culture system allows for the first time the growth of primary ATL cells in vitro, and might be useful as an in vitro assay for biological and clinical studies to develop molecular targeting drugs against ATL.

Prospective monitoring of BCR-ABL1 transcript levels in patients with Philadelphia chromosome-positive acute lymphoblastic leukaemia undergoing imatinib-combined chemotherapy.

The clinical significance of minimal residual disease (MRD) is uncertain in patients with Philadelphia chromosome-positive acute lymphoblastic leukaemia (Ph+ ALL) treated with imatinib-combined chemotherapy. Here we report the results of prospective MRD monitoring in 100 adult patients. Three hundred and sixty-seven follow-up bone marrow samples, collected at predefined time points during a uniform treatment protocol, were analysed for BCR-ABL1 transcripts by quantitative reverse transcription polymerase chain reaction. Ninety-seven patients (97%) achieved complete remission (CR), and the relapse-free survival (RFS) rate was 46% at 3 years. Negative MRD at the end of induction therapy was not associated with longer RFS or a lower relapse rate (P = 0.800 and P = 0.964 respectively). Twenty-nine patients showed MRD elevation during haematological CR. Of these, 10 of the 16 who had undergone allogeneic haematopoietic stem cell transplantation (HSCT) in first CR were alive without relapse at a median of 2.9 years after transplantation, whereas 12 of the 13 who had not undergone allogeneic HSCT experienced a relapse. These results demonstrate that, in Ph+ ALL patients treated with imatinib-combined chemotherapy, rapid molecular response is not associated with a favourable prognosis, and that a single observation of elevated MRD is predictive of subsequent relapse, but allogeneic HSCT can override its adverse effect.

Diagnosis and classification of myelodysplastic syndrome: International Working Group on Morphology of myelodysplastic syndrome (IWGM-MDS) consensus proposals for the definition and enumeration of myeloblasts and ring sideroblasts.

The classification of myelodysplastic syndromes is based on the morphological criteria proposed by the French-American-British (FAB) and World Health Organization (WHO) groups. Accurate enumeration of blast cells, although essential for diagnosis of myelodysplastic syndrome and for assignment to prognostic groups, is often difficult, due to imprecise criteria for the morphological definition of blasts and promyelocytes. An International Working Group on Morphology of Myelodysplastic Syndrome (IWGM-MDS) of hematopathologists and hematologists expert in the field of myelodysplastic syndrome reviewed the morphological features of bone marrows from all subtypes of myelodysplastic syndrome and agreed on a set of recommendations, including recommendations for the definition and enumeration of blast cells and ring sideroblasts. It is recommended that (1) agranular or granular blast cells be defined (replacing the previous type I, II and III blasts), (2) dysplastic promyelocytes be distinguished from cytologically normal promyelocytes and from granular blast cells, (3) sufficient cells be counted to give a precise blast percentage, particularly at thresholds that are important for diagnosis or prognosis and (4) ring sideroblasts be defined as erythroblasts in which there are a minimum of 5 siderotic granules covering at least a third of the nuclear circumference. Clear definitions and a differential count of a sufficient number of cells is likely to improve precision in the diagnosis and classification of myelodysplastic syndrome. Recommendations should be applied in the context of the WHO classification.

The GAS5 (growth arrest-specific transcript 5) gene fuses to BCL6 as a result of t(1;3)(q25;q27) in a patient with B-cell lymphoma.

The BCL6 gene is frequently disrupted at its 5' noncoding region by 3q27 chromosomal translocations in B-cell lymphoma. As a result of translocation, BCL6 is juxtaposed to reciprocal partners, such as the immunoglobulin (Ig) gene family. Besides the Ig loci, multiple non-Ig partners of the BCL6 translocation have been reported. Here we describe the identification of the GAS5 (growth arrest-specific transcript 5) gene as a novel partner of the BCL6 in a patient with diffuse large B-cell lymphoma, harboring the t(1;3)(q25;q27). In this case, the chromosome 1 breakpoint was located within the intronic small nucleolar RNA (snoRNA) sequence of GAS5 and the chromosome 3 breakpoint at 4 kb upstream of BCL6 exon 1a. As the result of chromosomal translocation, the GAS5-BCL6 chimeric transcripts were expressed, in which the 5'-terminal oligopyrimidine (5'TOP) sequence of GAS5 was fused to the whole coding sequence of BCL6. The GAS5 gene on chromosome 1q25 is the second BCL6 partner, to the SNHG5 on 6q15, which is classified as a non-protein-coding multiple snoRNA host and 5'-TOP class gene.

Karyotype at diagnosis is the major prognostic factor predicting relapse-free survival for patients with Philadelphia chromosome-positive acute lymphoblastic leukemia treated with imatinib-combined chemotherapy.

To identify factors associated with relapse-free survival (RFS), 80 patients with newly diagnosed Philadelphia chromosome-positive acute lymphoblastic leukemia, enrolled in a phase II study of imatinib-combined chemotherapy, were analyzed. The median follow-up of surviving patients was 26.7 months (maximum, 52.5 months). Twenty-eight out of 77 patients who had achieved CR relapsed. The probability of RFS was 50.5% at 2 years. Multivariate analysis revealed that the presence of secondary chromosome aberrations in addition to t(9;22) at diagnosis constitute an independent predictive value for RFS (p=0.027), and increase the risk of treatment failure by 2.8-fold.

A novel gene, ANKRD28 on 3p25, is fused with NUP98 on 11p15 in a cryptic 3-way translocation of t(3;5;11)(p25;q35;p15) in an adult patient with myelodysplastic syndrome/acute myelogenous leukemia.

We identified a novel gene fusion of ANKRD28 (ankyrin repeat domain 28) on 3p25 to NUP98 on 11p15 in a patient with adult myelodysplastic syndrome/acute myelogenous leukemia. A partially cryptic 3-way translocation, t(3;5;11)(p25;q35;p15), that had initially been supposed to be t(3;5)(p25;q35) was revealed by precise breakpoint mapping via fluorescence in situ hybridization analysis with bacterial artificial chromosome clones. This translocation produces the expression of 2 in-frame fusion transcripts, the novel ANKRD28-NUP98 and NUP98-NSD1, and 1 out-of-frame NSD1-ANKRD28 transcript. Transient overexpression of ANKRD28-NUP98 in NIH/3T3 cells, but not the C-terminal deletion mutant of ANKRD28 (DeltaC-ANKRD28), caused significantly increased focus formation compared with mock-transfectant controls. ANKRD28-NUP98 was localized in the nucleolus and cytoplasm, whereas ANKRD28 and DeltaC-ANKRD28 were found exclusively in the cytoplasm. Alteration of the subcellular localization of ANKRD28 might have contributed to the leukemogenesis in this case. This report is the first of ANKRD28 as an NUP98 fusion partner, and this case implies that this fusion may be responsible for hematologic malignancies.

[POEMS syndrome presenting with transient immunoglobulin isotype switching after successful treatment with autologous peripheral blood stem cell transplantation].

A 60-year-old man was admitted with muscle weakness and numbness in the extremities. Based on the existence of monoclonal gammopathy of the IgG-lamda type, a slight increase of plasma cells in the bone marrow, and an elevated level of serum vascular endothelial growth factor (VEGF), the diagnosis of POEMS syndrome was made. After peripheral blood stem cell collection by etoposide and G-CSF, the patient received high dose melphalan (200 mg/m2) therapy supported by autologous peripheral blood stem cell transplantation (autoPBSCT). After high-dose chemotherapy with autoPBSCT, the serum VEGF level normalized and the monoclonal IgG-lamda, disappeared. The patient gradually recovered from a bedridden state and at the time of writing has no impairment in his activities of daily life. After the autoPBSCT, monoclonal IgG-kappa, protein was detected transiently in serum. The new monoclonal immunoglobulin was considered to be due to normal immune reconstitution after myeloablation rather than alteration of the abnormal plasma cell clone, similarly as oligoclonal immunoglobulins occur in multiple myeloma after autoPBSCT. AutoPBSCT with high-dose chemotherapy should be considered among the treatments of choice for POEMS syndrome.

Imatinib provides durable molecular and cytogenetic responses in a practical setting for both newly diagnosed and previously treated chronic myelogenous leukemia: a study in nagasaki prefecture, Japan.

To evaluate the efficacy of imatinib in a practical setting, we registered 43 patients with newly diagnosed chronic myelogenous leukemia (CML) (group I) and 56 patients with previously diagnosed CML (group II) at 11 hematology centers in Nagasaki prefecture, Japan, from December 2001 to July 2005 and analyzed the molecular responses. Cytopenia, fluid retention, and skin rash were major adverse events, along with elevation in creatine phosphokinase levels. With a follow-up of approximately 3.5 years, imatinib treatment led to 88.7% overall survival (OS) and 85.2% progression-free survival (PFS) rates for group I, and 79.8% OS and 76.6% PFS rates for group II; the rates were not significantly different despite a lower average imatinib dose in group II. The rates of complete cytogenetic response at 30 months and major molecular response at 24 months were 86.1% and 62.5%, respectively, in group I, and 77.9% and 58.3% in group II; the rates were not significantly different. As has been reported by other groups, these results demonstrate that imatinib treatment can provide excellent clinical and molecular effects for not only newly diagnosed but also previously treated CML patients in practical settings that cover a wider variety of patients than clinical trials.