Eiji Usui

Characteristics of t(8;21) acute myeloid leukemia (AML) with additional chromosomal abnormality: concomitant trisomy 4 may constitute a distinctive subtype of t(8;21) AML.

t(8;21)(q22;q22) is the most frequently observed karyotypic abnormality associated with acute myeloid leukemia (AML), especially in FAB M2. Clinically, this type of AML often shows eosinophilia and has a high complete remission rate with conventional chemotherapy. t(8;21) AML is also frequently associated with additional karyotypic aberrations, such as a loss of the sex chromosome; however, it is unclear whether these aberrations change the biological and clinical characteristics of t(8;21) AML. To investigate this issue, 94 patients with t(8;21) AML were categorized according to their additional karyotypic aberrations, which were detected in more than three cases, and then morphologic features, phenotypes, expression of cytokine receptors, and clinical features were compared to t(8;21) AML without other additional aberrant karyotypes. t(8;21) AML with loss of the sex chromosome and abnormality of chromosome 9 were found in 27 cases (29.3%) and 10 cases (10.6%), respectively; however, no differences were observed from the t(8;21) AML without other additional karyotypes in terms of morphological and phenotypic features. There was also no significant difference in the clinical outcome among these three groups. On the other hand, trisomy 4 was found in three cases (3.2%) and these cells showed low expressions of CD19 (P=0.06) and IL-7 receptor (P=0.05), and high expressions of CD33 (P=0.13), CD18 (P=0.03), and CD56 (P=0.03) when compared to t(8;21) AML without additional karyotypes. Moreover, all three t(8;21) AML cases with trisomy 4 did not show eosinophilia in their bone marrow and died within 2.4 years. These observations suggest that additional karyotypic aberration, t(8;21) with trisomy 4 is rare, but it may constitute a distinctive subtype of t(8;21) AML.

Non-DNA-binding Ikaros isoform gene expressed in adult B-precursor acute lymphoblastic leukemia

Ikaros, a zinc finger transcription factor, is essential for lymphoid development. Mutant mice expressing dominant-negative Ikaros gene (Ikaros) isoforms develop an aggressive form of lymphoid malignancies. We examined the expression of Ikaros isoforms in 11 leukemic cell lines and adult acute lymphoblastic leukemia cells from 36 patients with B-precursor acute lymphoblastic leukemia (pre-B ALL) and nine with T-precursor acute lymphoblastic leukemia (pre-T ALL), using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. In one pre-B ALL cell line, INC cells, and primary leukemic cells from 16 patients with pre-B ALL, we found the predominant expression of a non-DNA-binding Ikaros isoform, Ik-6. However, Ik-6 was not detected in pre-T ALL cells. All of pre-B ALL cells expressing Ik-6 were CD10+, whereas CD10− pre-B ALL cells did not express Ik-6. The expression of Ik-6 was not related to karyotype abnormalities such as t(9;22) and t(4;11). Proteins from the cells that expressed Ik-6 alone failed to bind to the Ikaros protein-specific binding sequence in DNA. Ikaros proteins lacking the DNA binding sequences were detected in the cytoplasm but not in the nucleus of the cells. When INC and primary pre-B ALL cells that express Ik-6 alone were irradiated and cultured in the absence of serum, these cells produced functional Ikaros isoforms, Ik-1 and Ik-2. Purified CD19+ CD10− and CD19+ CD10+ cells from normal human bone marrow did not express Ik-6. The predominant expression of Ik-6, which is the result of post-transcription dysregulation, is characteristic of adult pre-B ALL, especially CD10+ pre-B ALL.

Additional t(11;17)(q23;q21) in a patient with Philadelphia-positive mixed lineage antigen-expressing leukemia

We describe very uncommon phenotypic and cytogenetic findings in a 40-year-old female with blast phase of Philadelphia chromosome (Ph)-positive CML. In addition to the t(9;22)(q34;q11) that was detected in all metaphases, a t(11;17)(q23;q21) was identified in 15 of 20 metaphases. Reverse transcription-polymerase chain reaction showed the major and minor bcr/abl fusion transcripts in the cells from a bone marrow (BM) sample. Fluorescence in situ hybridization (FISH) analysis also showed that fusion signals of the bcr and abl probes were found in 95% of blastic cells and in 64% of neutrophils. MLL gene rearrangement was also detected in some blastic cells but not in neutrophils by FISH analysis. Phenotypically, blastic cells expressed mixed lineage antigens such as CD34, CD33, CD13, CD19, CD7, and CD41. Immunogenotypically, some population of BM cells showed monoclonal rearrangements of immunoglobulin heavy chain and T-cell receptor gamma chain genes by Southern blot analysis. Clinical course was aggressive, and therapy was poorly tolerated. Such findings seem to support an association between Ph and an abnormality of 11q23 with poor prognosis, and suggest that the expression of both abnormal genes may be related to this mixed lineage antigen-expressing leukemia.

Successful treatment with low-dose splenic irradiation for massive splenomegaly in an elderly patient with hairy-cell leukemia

Abstract: The therapeutic approach to hairy‐cell leukemia (HCL) is in some instances still debated. Although management with α‐interferon and purine analogues is well established, there is an alternative role for therapeutic splenectomy in patients with massive splenomegaly who have failed to respond to systemic therapy. Most patients with HCL will not be suitable for treatment with splenectomy as their ages at diagnosis are high. Here, we report an elderly Japanese HCL patient whose refractory massive splenomegaly responded well to low‐dose splenic irradiation.

Fluorescent In Situ Hybridization Analysis of Philadelphia Chromosome-Negative Chronic Myeloid Leukemia with the bcr/abl Fusion Gene

This report describes a patient with Philadelphia chromosome-negative (Ph-) but bcr/abl fusion gene-positive chronic myeloid leukemia (CML) and a molecular analysis of the mechanisms behind the Ph- status. Spectral karyotyping-fluorescent in situ hybridization (SKY-FISH) analysis showed no abnormal translocation; however, a bcr/abl fusion gene was detected by reverse transcriptase-polymerase chain reaction analysis. FISH analysis showed that signals from the 9q and 22q subtelomere probes were detected on the der(9) and der(22) chromosomes, respectively. On the other hand, FISH analysis of the abl and bcr genes with dual fusion probes, which can detect the bcr/abl fusion gene on both the der(9) and der(22) chromosomes, showed the signal for bcr/abl fusion on the der(22) chromosome but not on the der(9) chromosome.These results indicate that insertion of the abl gene into the bcr region on the der(22) chromosome or retranslocation between the der(9) chromosome and the der(22) chromosome may have caused the Ph- CML in this case.

Molecular analysis of PDGFRα/β genes in core binding factor leukemia with eosinophilia

Abstract:  Eosinophilia sometimes occurs in acute myeloid leukemia (AML), especially in core binding factor (CBF) leukemia. However, the pathogenesis of the differentiation from leukemic progenitors to eosinophils is not well understood in this type of leukemia. Recent reports showed that a novel fusion tyrosine kinase, Fip1‐like1 (FIP1L1) platelet‐derived growth factor receptor alpha (PDGFRα), is found in idiopathic hypereosinophilic syndrome. The involvement of another chimeric gene, PDGFRβ, was also reported in myeloproliferative disorder with eosinophilia. These chimeric genes cause constitutive activation of PDGFR tyrosine kinases. On the other hand, a two‐hit model for the pathogenesis of AML, which seems to be caused by inactivating mutations in transcription factors and genetic lesions in tyrosine kinase resulting in constitutive activation, has been proposed. On the basis of these findings, we screened for the expression of the FIP1L1‐PDGFRα fusion gene and for mutations in the juxtamembrane and tyrosine kinase domains of PDGFRα/β genes in 22 cases of CBF leukemia with eosinophilia. Among these cases, no FIP1L1‐PDGFRα fusion gene was found. Although cDNA sequencing also detected three types of single‐nucleotide alterations at kinase domains in PDGFRα/β genes, all of them were silent changes and polymorphisms. Therefore, PDGFRα/β genes do not appear to play a significant pathogenetic role in eosinophilia or leukemogenesis of CBF leukemia.

High frequency of CD29high intermediate monocytes correlates with the activity of chronic graft-versus-host disease.

To the Editor: Chronic graft-versus-host disease (cGVHD) is a major cause of morbidity and mortality in patients after allogeneic hematopoietic stem cell transplantation (HSCT) (1, 2). ‘Trial-and-error system’ remains the only way to identify the effectiveness of immunosuppressive drug in the individual patient, and valid biomarkers for cGVHD are eagerly needed to identify the response to the drug (3, 4). Monocyte-derived interleukin-10 (IL-10) spot-forming cells (SFCs) can be used as a biomarker for evaluating the activity of cGVHD (5). Recently, monocytes have been classified into three subpopulations (6). Among them, CD14CD16 intermediate monocytes are found at low frequency, but they have unique features and expand with cytokine treatment and in inflammation (7). Now, we have demonstrated that CD29 intermediate monocytes increased in cGVHD. These results could be clinically relevant for treatment strategies. We have updated our published results (5) in this study by adding thirty additional samples from 57 previous and three new patients for flow cytometric analysis because a new standard of monocyte phenotypes was reported (6). Patients were subclassified into those with no, active, and inactive cGVHD as previously described (5, 8, 9). Peripheral blood mononuclear cells (PBMCs) were obtained from patients who underwent allogeneic HSCT between 2000 and 2012 with no evidence of infection. Fresh samples were obtained and assayed from patients between 6 and 139 months (mean: 47.9 months) after HSCT. The study was registered (UMIN-Clinical Trials Registry: 000006733). First, we have performed flow cytometric analysis of the expression of CD29, a ligand of fibronectin, and intracellular (IC) IL-10 in monocytes after stimulation with or without lipopolysaccharide (LPS) in patients after HSCT (Fig. 1A). The absolute number of intermediate monocytes, but neither classical nor nonclassical monocytes, increased significantly in patients with active cGVHD compared with those with no or inactive cGVHD (P < 0.05) (Fig. 1B). The expression of CD29 on intermediate monocytes was significantly higher (P < 0.05) in patients with active cGVHD than those with no or inactive cGVHD. CD29 expression of monocytes increased after reacting with fibronectin as we have reported (5). Fibronectin strongly deposited under the basal layer of skin in cutaneous cGVHD (5, 10). Moreover, the expressions of IC IL-10 on intermediate monocytes from patients with active cGVHD stimulated both with and without LPS were significantly higher (P < 0.05) than those with no or inactive cGVHD (Fig. 1B). These results suggest that CD29 intermediate monocytes were the main producer of IL-10 during active cGVHD. Secondly, we studied chemokine/homing receptors on monocytes. Classical monocytes represented with lower expression of CX3CR1 (P < 0.01) and higher expression of CCR2 (P < 0.01) and CD62L (P < 0.05) than each expression of nonclassical monocytes (Fig. 1C). Intermediate monocytes showed intermediate expressions for CX3CR1, CCR2, and CD62L between classical and non-classical monocytes. Namba et al. (11) suggested that CX3CR1 monocytes might be recruited from the circulation to the fractalkine epidermis in cGVHD. CCR2 is a receptor for monocyte chemoattractant protein-1 and it involved in migration of monocytes to inflammatory site. CD62L has a key role in adherence of monocytes to vascular endothelium from the blood (12). Thus, migration of blood monocytes into target organ of cGVHD might be controlled by chemokine/homing receptors. In summary, the findings described here have demonstrated that flow cytometric analysis of intermediate monocytes can be used, as a simple and useful biomarker, for

A case of acute myeloid leukemia with t(7;11)(p15;p15) mimicking myeloid crisis of chronic myelogenous leukemia

The chromosome aberration t(7;11)(p15;p15) is uncommon but recurrent in leukemia. We experienced a case of acute leukemia with t(7;11)(p15;p15), the hematological appearance of which mimicked myeloid crisis in chronic myeloid leukemia (CML). This case showed splenomegaly, a decreased neutrophil alkaline phosphatase (NAP) score, increased vitamin B12 value, and cells at all stages of neutrophilic maturation in both bone marrow and peripheral blood. We initially had difficulty differentiating acute myeloid leukemia (AML) M2 with marked myeloid differentiation from myeloid crisis of Philadelphia chromosome (Ph)-negative CML. Immature myeloid cells in the peripheral blood disappeared and cytogenetic analysis indicated that marrow cells changed to the normal karyotype after remission induction therapy. Therefore, this case was thought not to be myeloid crisis but AML M2 subtype. TheNUP98/HOXA9 fusion transcript was detected by reverse transcription-polymerase chain reaction (RT-PCR) at exon A but not exon B ofNUP98.

Bilateral Osteonecrosis of the Head of the Femur during Treatment with Retinoic Acid in a Young Patient with Acute Promyelocytic Leukemia

All-trans retinoic acid (ATRA) is the drug of choice for the treatment of acute promyelocytic leukemia (APL). In general, ATRA is well tolerated, but it does have side effects, the most severe of which is ATRA syndrome. We report the case of a young patient with APL treated with ATRA for induction and maintenance therapy who then developed avascular necrosis of both femoral heads. We also review cases of APL patients with osteonecrosis of the femoral head after ATRA therapy.

Aggressive neoplastic plasma cell growth with MLL gene rearrangement after high-dose therapy with autologous stem cell support for multiple myeloma

We report a case of a patient with IgA κ multiple myeloma (MM) mobilized with etoposide and subsequently receiving high-dose melphalan (HDM) with stem cell support. She relapsed rapidly post transplantation. Southern blot and fluorescent in situ hybridization analysis showed MLL gene rearrangement in the myeloma cells, which was not detected in the sample at diagnosis or in the PBSC harvested with etoposide plus G-CSF. These observations suggest that clonal rearrangement of the MLL gene is caused by etoposide. Patients with MM undergoing HDM with stem cell rescue may be at an increased risk of not only secondary leukemia, but also secondary genetic abnormalities in myeloma cells, especially those receiving priming with etoposide for peripheral blood stem cell collection. Bone Marrow Transplantation (2001) 27, 555–558.