Keiko Ando

Telomeric length and telomerase activity vary with age in peripheral blood cells obtained from normal individuals

The telomerase activity and length of telomeres of peripheral blood mononuclear cells obtained from 124 healthy individuals aged 4–95years was measured. Telomerase activity level was semiquantitatively assessed by a fluorescent-telomeric repeat amplification protocol (fluorescent-TRAP) using an internal telomerase assay standard, fluorescent primers and an automated laser fluorescent DNA sequencer. Telomeric length, measured by assay of terminal restriction fragments (TRFs), was determined in HinfI-digested DNA by Southern blot analysis using a (TTAGGG)4 probe. TRF length was determined in 80 individuals and age-related progressive reduction of size was observed. TRF length in peripheral blood mononuclear cells obtained from normal individuals (aged 4–39years) decreased by approximately 84bp per year, while in individuals aged ≥40years it decreased by 41bp per year. In contrast, telomerase activity showed an apparent biphasic pattern with aging. Individuals aged 4–39years showed a progressive decrease in telomerase activity, whereas 65% of those aged ≥40years showed relatively stable but very low telomerase activity, and the remaining individuals aged ≥40years had no detectable telomerase activity. These data obtained from normal individuals might in the future be of value to help risk stratify and manage the care of patients with leukemia.

Can eradication therapy for Helicobacter pylori really improve the thrombocytopenia in idiopathic thrombocytopenic purpura? Our experience and a literature review

Helicobacter pylori has recently been postulated to play a role in the pathogenesis of autoimmune diseases, including idiopathic thrombocytopenic purpura (ITP). We investigated the prevalence ofH pylori infection and the effects of its eradication in 61 patients with ITP.H pylori infection was found in 50 patients (83%), an incidence significantly higher than not only healthy volunteers in Japan (60%) but also subjects in other reported ITP series (approximately 43%–71%). In our study, the mean age ofH pylori-positive ITP patients (58.0 years) was significantly higher than that ofH pylori-negative ITP patients (40.5 years). Bacterium eradication efforts were performed in 29 infected ITP patients and succeeded in 27 patients (93%). The 29 patients with eradicatedH pylori infections showed significant increases in platelet counts compared with patients with uneradicated infections or who wereH pylori-negative. During the follow-up period (median, 11.0 months), 16 (55%) of 29 patients achieved a major or a minor response. The patients who achieved a major response had not received previous prednisolone therapy, suggesting a relationship between prednisolone therapy and the response to eradication efforts.The assessment ofH pylori infection and its eradication should be attempted in cases of ITP, because this approach may be a good new strategy for treating some ITP patients, especially elderly Japanese patients. Some regional factors have been suggested as causes ofH pylori-associated ITP.

Successful treatment with cyclosporin A for myelodysplastic syndrome with erythroid hypoplasia associated with T‐cell receptor gene rearrangements

Myelodysplastic syndrome (MDS) with erythroid hypoplasia, a rare form of MDS, has not yet been clearly defined. We report four patients with MDS with erythroid hypoplasia who received immunosuppressive therapy. All were elderly, had severe transfusion‐dependent anaemia, morphological evidence of myelodysplasia and a low percentage (3·2–13·6%) of erythroid precursors. Administration of cyclosporin A (CsA) improved their anaemia; all transfusion‐dependent patients achieved transfusion‐independence. An inverted CD4/8 ratio was seen in three patients who also demonstrated T‐cell receptor (TCR)‐β and ‐γ gene rearrangements by Southern blotting and clonality by polymerase chain reaction. Treatment with CsA can be an attractive alternative treatment for patients with MDS with erythroid hypoplasia, which may be associated with a clonal abnormality in T cells.

Telomere Dynamics and Genetic Instability in Disease Progression of Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is characterized by a Philadelphia (Ph) translocation creating a novel BCR-ABL oncoprotein, and CML patients have a chronic phase for several years followed by an intractable blast cell proliferation, called blast transformation. In the blast phase, more than 60% of patients show additional cytogenetic changes, e. g., double Ph, +8, i(17q). In this review, we would like to address genetic changes, including genome instability, cytogenetic changes, and telomere dynamics that relate to karyotypic instability. In the chronic phase, approximately 60% of CML patients show reduced telomere length without highly elevated telomerase activity or microsatellite alterations, indicating that telomere reduction may be linked to cell replication. Therefore, the Ph translocation might be a first event to immortalize cell proliferation. In the blast phase, 50% of CML patients have high levels of elevated telomerase activity and the same number of patients had microsatellite changes. Of note is that most patients with telomerase up-regulation in the blast phase had additional cytogenetic changes and >60% of them showed microsatellite changes at least at one locus. In contrast, most patients without telomerase activity did not show microsatellite changes. These findings may indicate that telomerase up-regulation in the blast phase of CML patients is closely associated with microsatellite changes (representative of genome instability), while blast cells in the remaining patients (30%) maintain their proliferative capability without microsatellite changes and telomerase up-regulation. This further suggests that there is also an unknown mechanism for genome stability without the process of telomerase up-regulation in some patients with CML in blast crisis.

Hemophagocytic syndrome associated with CD8 positive T-cell chronic lymphocytic leukemia.

We describe a case of hemophagocytic syndrome (HPS) associated with CD8-positive T-cell chronic lymphocytic leukemia (CD8 + T-CLL). A 68-year-old man with CD8 + T-CLL presented with fever, progressive pancytopenia, and lymphadenopathy. Laboratory findings showed a hyper-ferritinemia, abnormalities of coagulation tests, and liver and renal dysfunction with hypoproteinemia. He did not respond to any treatments and died of respiratory failure 10 days after the admission and 14 months after the onset of CD8 + T-CLL. Pathological findings of the autopsy demonstrated infiltration of CD8 + T-CLL cells in multiple organs along with the increase of histiocytes with prominent hemophagocytosis. Serum concentration levels of IL-6, soluble IL-2 receptor and VEGF were all elevated at admission. These findings revealed that he had a secondary HPS. It was suggested that HPS should be considered in patients with an unexplained cytopenia and a fever during the clinical course of CD8 + T-CLL.

Anaplastic large-cell lymphoma which showed severe inflammatory status and myelodysplasia with increased VEGF and IL-6 serum levels after long-term immunosuppressive therapy.

We report a patient with anaplastic large‐cell lymphoma (ALCL) who has been given immunosuppressive therapy for Evans syndrome for 10 years. He was admitted with spike fever, intra‐abdominal lymphadenopathy, and multiple liver masses. Examination of biopsy specimens obtained by para‐aortic lymph nodes and liver masses resulted in a diagnosis of ALCL. Immunohistochemically, these cells were reactive to anti‐CD30 antibody but were not of B‐ or T‐lineage. Bone marrow aspiration demonstrated the invasion of giant neoplastic cells and trilineage myelodysplasia. Because the patient showed severe inflammatory symptoms, we examined serum levels of various cytokines. Pretreatment levels of IL‐6 and VEGF in this patient were significantly elevated compared to those of normal controls. He was treated with combination chemotherapy (ABVD regimen), achieving complete remmesion. Myelodysplasia and serum IL‐6 and VEGF also normalized after treatment. We assumed that ALCL resulted from long‐term immunosuppressive therapy and that the up‐regulation of IL‐6 and VEGF played a role in pathogenesis of this type of lymphoma. Am. J. Hematol. 66:49–52, 2001.

PNH-phenotype cells in patients with idiopathic cytopenia of undetermined significance (ICUS) with megakaryocytic hypoplasia and thrombocytopenia.

The recent World Health Organization classification has identified a condition marked by having fewer than 10% of dysplastic cells and fewer than 5% of blasts in the bone marrow (BM) as idiopathic cytopenia of undetermined significance (ICUS) (Wimazal et al, 2007; Brunning et al, 2008). We recently reported that ICUS patients may be heterogeneous (Ando et al, 2010), and the pathophysiology of some ICUS patients may be different from that of myelodysplastic syndromes (MDS). During the last decade, the immunological background of MDS patients, especially in those with low-grade dysplasia, has been postulated as significant, and immunosuppressive therapy is widely proposed in the consensus guidelines for MDS treatment (Bowen et al, 2003). Candidates for immunosuppressive therapy (IST) are those with hypocellular bone marrow or the presence of DRB1*1501. The correlation between the presence of paroxysmal nocturnal haemoglobinuria (PNH)-phenotype cells (a minor PNH clone: <1% of CD55and CD59-deficient cells) and a good response to IST has also been reported. It has been reported that patients with aplastic anaemia (AA) or MDS displaying >0Æ003% of PNH-phenotype cells showed significantly favourable responses to IST (Wang et al, 2002; Nakao et al, 2006). However, the prevalence of increased PNH-type cells in patients with ICUS remained unclear. We analysed a total of 29 patients, including 11 patients with morphologically identified ICUS, nine with refractory cytopenia with unilineage dysplasia (RCUD), and nine patients with refractory cytopenia with multilineage dysplasia (RCMD): the haematological and clinical data of some patients were reported previously (Ando et al, 2010). Patients with fewer than 25 analysable megakaryocytes were defined as undetermined, even if the percentage of dysplastic megakaryocytes was more than 10%. MDS was diagnosed according to morphological criteria defined by the FrenchAmerican-British classification and International Working Group on Morphology of myelodysplastic syndromes (Valent et al, 2007; Mufti et al, 2008). To detect a minor PNH clone, peripheral blood obtained from patients was transferred to Kanazawa University (transported overnight at 4 C), and analysed using a highly sensitive flow cytometric assay, as reported previously (Sugimori et al, 2006). We considered a patient to be positive for PNH-phenotype cells when the presence of CD55and CD-59 deficient granulocytes were >0Æ003%, and when CD55and CD59-deficient erythrocytes were >0Æ005%. Of the 11 ICUS patients, two showed an increase in the percentage of PNH-type cells, whereas none of 9 RCUD or 9 RCMD patients did. Of the two patients with ICUS showing increased PNH-type cells, one showed clonal monosomy 8 and the other had a normal karyotype. Both patients who were positive for PNH–type cells had markedly low platelet counts, although there was no significant difference in the platelet count (P = 0Æ2125 by unpaired t-test) (Fig 1) between the ICUS patients and those negative for PNH-type cells. The BM of the two patients (unique patient numbers 54 and 09–01) showed markedly megakaryocytic hypoplasia and was therefore difficult to assess for the morphology of megakaryocytes (Table SI). It has been reported that approximately 20% of MDSrefractory anaemia and RCMD cases, and 50% of AA patients showed an increase in the percentage of PNH-type cells, and a common immunological background was postulated between the low-risk MDS and AA patient groups (Nakao et al, 2006; Sugimori et al, 2006). In the present study, PNH– type cells were detectable in only two patients with ICUS, both of whom showed prominent thrombocytopenia and megakaryocytic hypoplasia in the BM. This indicates that some ICUS patients with predominant thrombocytopenia may share immune pathophysiology characterized by the presence of increased PNH-type cells with patients with AA. Recent research by Wang et al (2009) demonstrated that nine refractory anaemia (RA) patients had a minor population of PNH-phenotype granulocytes (<1% of granulocytes), and six of them showed neither ‡10% dysgranulopoiesis nor ‡10% dysmegakaryopoiesis; five of them lacked clonal cytogenetic abnormalities, suggesting the possibility that these patients could be given diagnoses of ICUS. Wang et al (2002) reported that 21 RA patients with increased PNH-type cells showed significantly lower percentages of abnormal karyotypes and pseudo-Pelger neutrophil anomalies, and significant lower platelet counts than RA patients negative for increased PNHtype cells. These findings, in combination with our results, indicate that an increase in the percentage of PNH-type cells may be characteristic of BM failure with few signs of dysplasia. Our findings further suggest that ICUS to be heterogeneous; some patients present with predominant neutropenia (Ando et al, 2010) and others show predominant thrombocytopenia with increased PNH-phenotype cells. Another important issue is the limitation of diagnosing ICUS with the current criteria. Patients given diagnoses of Correspondence

Idiopathic neutropenia with fewer than 5% dysplasia may be a distinct entity of idiopathic cytopenia of undetermined significance

Dear Editor, A condition marked by fewer than 10% of dysplastic cells and fewer than 5% of blasts in the bone marrow (BM) is now categorized as idiopathic cytopenia of undetermined significance (ICUS); if clonal cytogenetic changes are detectable in ICUS patients, the diagnosis can be changed to myelodysplastic syndrome (MDS) [1]. This categorization is very practical and clear-cut in separating MDS from those with low-grade dysplasia [1], and it thus became possible to analyze the clinical and hematologic features to differentiate refractory cytopenia with unilineage dysplasia (RCUD) from ICUS. However, only a single report dealing with possible ICUS with dysplastic features in each cell lineage appears to exist, that by Wimazal et al. [2]. We therefore focused on cytopenia patients with fewer than 5% of BM blasts and reassessed the dysplastic features, in combination with the cytogenetic results, to shed light on low-grade dysplasia. From 1994 to 2008, we performed BM examinations with cytogenetic studies in 445 patients with cytopenia, 237 of whom were given diagnoses of MDS or suspected MDS. As well as we could, we used the initial BM examination to rule out the possibility of other underlying disorders inducing cytopenia, and as a result, 137 patients with fewer than 5% marrow blasts were enrolled in this study. Of these 137 patients, 56 who were followed for more than 6 months and for whom specimens were available for reanalyzable marrow films (200 cells being examined in each cell lineage) were used in this study [3, 4]; two patients with hypoplastic BM without cytogenetic changes were excluded from this study since we could not completely rule out the possibility of low-grade aplastic anemia. In this study, we reassessed the bone marrow films for 16 patients with ICUS (Electronic supplementary materials, File 1), 16 with RCUD (Electronic supplementary materials, File 2), and 22 patients with refractory cytopenia with multilineage dysplasia (RCMD; Electronic supplementary materials, File 3). No particular difference in the peripheral blood data was found among patients with ICUS, RCUD, and RCMD. RCUD patients had more dysgranulopoietic cells than those with ICUS (16.7± 19.4% vs 3.5±3.3%, P=0.0116) because of the presence of hypogranular neutrophils or pseudo-Pelger anomaly, while no significant difference in percentages of dyserythropoietic cells was noted (P=0.1809; Electronic supplementary materials, File 4). This indicates that ICUS patients can usually be diagnosed from the absence of prominent dysgranulopoiesis. We then separated the ICUS patients into two groups according to the percentages of dysplastic cells (Table 1). ICUS patients with fewer than 5% dysplastic cells in at least one cell lineage had a significantly lower absolute neutrophil count than those with 5% to 9% of dysplastic Electronic supplementary material The online version of this article (doi:10.1007/s00277-009-0845-0) contains supplementary material, which is available to authorized users. K. Ando : T. Iwabuchi :K. Ohyashiki (*) The First Department of Internal Medicine (Hematology Division), Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan e-mail: ohyashik@rr.iij4u.or.jp

Non-random chromosomal deletion clustering at 20q in Waldenström macroglobulinemia

Abstract Chromosome change at 20q11–q12, including del(20q), is sometimes reported in plasma cell dyscrasia, but most cases are found during or after chemotherapy. It is therefore still uncertain whether del(20q) is a primary change or therapy-related. We performed cytogenetic studies and fluorescent in situ hybridization (FISH) analysis using 20q12 and 20qter probes to ascertain the possible involvement of 20q in nine patients with Waldenström macroglobulinemia (WM). The FISH study demonstrated deletions of 20q12 and/or 20qter in four of nine patients (44%) with WM at diagnosis, and one of them had the del(20q) chromosome. Moreover, one patient had de novo appearance of the del(20q) chromosome with 20q12 deletion after chemotherapy, although this patient had neither the del(20q) chromosome nor 20q12 deletion at WM diagnosis. Based on the results of this study, we conclude that chromosomal breakage at 20q13 is a non-random genetic change which plays a role in the neoplastic process of WM.

Low body weight and body mass index may be associated with musculoskeletal pain following imatinib discontinuation in chronic myeloid leukemia

It is difficult to predict musculoskeletal pain as a withdrawal syndrome following the discontinuation of imatinib (IM) in patients with chronic myeloid leukemia. We investigated a link between physical size and musculoskeletal pain following IM discontinuation. In total, seven out of 24 patients developed musculoskeletal pain after discontinuing IM. Those with symptoms had a significantly lower body weight (BW) and body mass index (BMI) than those without symptoms. While previous reports indicated that physical size is associated with the pharmacokinetics of IM, our current study suggests that lower BW and BMI may be associated with musculoskeletal pain following IM discontinuation.