Hideki Mitsui

Donor Cell-Derived Chronic Myeloproliferative Disease with t(7;11)(p15;p15) after Cord Blood Transplantation in a Patient with Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

We report a case of donor cell-derived chronic myeloproliferative disease with t(7;11)(p15;p15) occurring after cord blood transplantation (CBT). A 41-year-old man developed precursor B-cell acute lymphoblastic leukemia with a karyotype of 46, XY, t(9;22)(q34;q11) and inv(9)(p11;q13), for which he received CBT from a sex-mismatched donor at the first complete remission of the leukemia. Five months after CBT, gradual neutrophilia of unknown origin developed following the myeloid reconstitution after CBT. Karyotyping of bone marrow cells at 9 months after CBT showed 46,XX, t(7;11)(p15;p15) in 17/20 dividing cells, but neither Philadelphia chromosome (Ph) nor inv(9)(p11;q13) was present. This is the first report of chronic myeloproliferative disease with t(7;11)(p15;p15) that developed in donor cells after CBT. The donor was well-developed and healthy, at least at the time of follow-up, half a year after the birth.

Aberrant expression of immunoglobulin light chain isotype in B lymphocytes from patients with monoclonal gammopathies : isotypic discordance and clonal B-cell excess

A study was made of 29 patients with monoclonal gammopathies to detect aberrations in immunoglobulin (Ig) light chain isotype expression in lymphocytes at various levels of B‐cell differentiation, namely, circulating surface Ig positive (SIg +) cells, Ig‐secreting cells (plaque forming cells, PFC) and mitogen‐induced PFC. By using kappa‐lambda analysis, two major phenotypes of aberrant Ig light chain isotype expression were found in circulating B cells at these three levels of differentiation: an absolute increase in B cells bearing the same Ig light chain isotype as that of monoclonal protein (clonal B‐cell excess), and a relative decrease in those B cells (isotypic discordance). Isotypic discordance (ID) was found to be essentially negative in patients with monoclonal gammopathy of undetermined significance (MGUS) provided that they were in a stable condition. In myeloma patients, ID was found only in stage I, except for a remission case of stage III (4/7 in stage I, 0/8 in stage II, and 1/6 in stage III). ID was not restricted to a circulating SIg+ cell level but was also demonstrable at a spontaneous or pokeweed mitogen‐induced PFC level. However, ID was negative at a PFC level induced by Staphylococcus aureus Cowan I. Clonal B‐cell excess (CE) was frequently found in patients with active myeloma but not in stable patients (0/8 in MGUS, 1/7 in stage I, 8/8 in stage II, and 4/6 in stage III). CE was positive not only at a circulating SIg+ cell level but also at a circulating PFC level. Furthermore, patients with CE at a PFC level were found to have a higher proliferating capacity, defined as a percentage labelling index of marrow myeloma cells, than those without CE at a PFC level (P<0.02). ID and CE can therefore be considered as useful markers for discriminating between MGUS and myeloma, evaluating the clinical stability and predicting the clinical course.

New variant of acute promyelocytic leukemia with IRF2BP2-RARA fusion.

We present an acute promyelocytic leukemia (APL) patient with two subtypes of IRF2BP2–RARA, in which the IRF2BP2 gene showed completely new breakpoints. Bone marrow examination revealed morphologic features indicative of APL. However, promyelocytic leukemia–RARA fusion was not detected. A paired‐end mRNA sequencing followed by RT‐PCR and direct sequencing revealed two types of fusion transcripts between exon 1B of IRF2BP2 and exon 3 of RARA. The patient received all‐trans retinoic acid and conventional chemotherapy, but showed resistance. This is the second report of IRF2BP2 involvement in APL, and we describe various breakpoints for the IRF2BP2–RARA fusion gene.

Quantitative polymerase chain reaction analysis with allele-specific oligonucleotide primers for individual IgH VDJ regions to evaluate tumor burden in myeloma patients

Quantitative polymerase chain reaction (PCR) with patient-specific, allele-specific oligonucleotide (ASO) primers for individual immunoglobulin H VDJ region (ASO-PCR) amplification was performed using several sources of clinical material, including mRNA from peripheral blood cells (PBMNCs), whole bone marrow cells (BMMNCs), and the CD20+ CD38- B-cell population in bone marrow, as well as cell-free DNA from the sera of patients with multiple myeloma (MM). We designed the ASO primers and produced sufficient PCR fragments to evaluate tumor burden in 20 of 30 bone marrow samples at diagnosis. Polymerase chain reaction amplification efficiency depended on primer sequences because the production of ASO-PCR fragments did not correlate with serum M-protein levels. However, the ASO-PCR levels in BMMNCs showed statistically significant correlations with those in PBMNCs and CD20+ CD38- B-cells. The good association between the BMMNC and PBMNC data indicated that PBMNCs could be a suitable source for monitoring minimal residual disease (MRD). In the case of cell-free DNA, ASO-PCR levels showed a unique pattern and remained high even after treatment. Because the sequence information for each ASO-PCR product was identical to the original, the cell-free DNA might also be useful for evaluating MRD. Moreover, the ASO-PCR products were clearly detected in 17 of 22 mRNA samples from CD20+ CD38- populations, suggesting that MM clones might exist in relatively earlier stages of B cells than in plasma cells. Thus, ASO-PCR analysis using various clinical materials is useful for detecting MRD in MM patients as well as for clarifying MM pathogenesis.

High-affinity interleukin 2 receptors on B cell chronic lymphocytic leukemia cells are induced by phorbol myristate acetate but not by calcium ionophore

The role of phorbol myristate acetate (PMA: a protein kinase-C (PKC) activator) and calcium ionophore A23187 in the induction mechanism of the interleukin 2 receptor (IL2R) on B-cell chronic lymphocytic leukemia (B-CLL) cells was studied. B-CLL cells from five patients were cultured with PMA or A23187 for 72 h and used for the following experiments. Interleukin 2 (IL2) cross-linking assays showed that PMA induced the expression of IL2R subunits (p55 and p70/75) in all cases examined, but that A23187 induced neither subunit. Radiolabeled IL2 binding assays also demonstrated that PMA induced both high-affinity IL2R (HA-IL2R) and low-affinity IL2R (LA-IL2R) on B-CLL cells, but that A23187 did not. After treatment with PMA, three of five cases did not respond to IL2 even though they expressed HA-IL2R, suggesting impaired signal transduction. No cases responded to IL2 after treatment with A23187. In conclusion, PMA but not A23187 stimulates B-CLL cells to induce the expression of p55 and p70/75, indicating that the PKC pathway plays a more important role than the calcium pathway in the induction of IL2R subunits in B-CLL cells.

Early Trilineage Recovery by Granulocyte Colony-Stimulating Factor in a Patient with Aplastic Anemia

Dr. Yuzuru Kanakura, Second Department of Internal Medicine, Osaka University Medical School, Yamadaoka 2-2, Suita Osaka 565 (Japan) Among the cytokines active in hematopoiesis, granulocyte colony-stimulating factor (G-CSF) has functions mainly on cells of neutrophilic granulocyte lineage [1]. A large number of clinical trials have suggested that G-CSF can accelerate the recovery of neutrophils when used after high-dose cytotoxic chemotherapy or bone marrow transplantation [2, 3]. In addition, G-CSF have been shown to have beneficial effects in patients with aplastic anemia (AA) who suffer from severe neutropenia [4]. With the exception of rare instances, however, G-CSF is known to have little or no effect on erythrocyte or platelet counts [2, 4-7]. We report here a unique case with acquired AA who showed trilineage recovery shortly after administration of rhG-CSF. Case Report A 57-year-old man was referred to Osaka University Hospital because of pancytopenia in April 1993. He had a 3-month history of pancytopenia and had received a total of 800 ml of red blood cell (RBC) in a previous hospital. Laboratory data were as follows: hemoglobin (Hb), 11.2 g/dl (immediately after RBC transfusion); reticulo-cytes88 × 109/1, white blood cells (WBC), 3.2 × 109/1 with differentials of 35% neutrophils and 56% lymphocytes; and platelets (PLT) 73 × 109/1. Bone marrow aspirate and biopsy revealed uniform hypocellularity (nucleated cell count, 14 × 109/l)withnomegakaryo-cytes and no morphological abnormalities. Based on these findings, he was diagnosed as having mild AA [8]. Daily anabolic steroids were thus administered orally at a dose of 30 mg/kg body weight in April 1993. This treatment did not improve the hematologic parameters and hemoglobin decreased (fig. 1). Because RBC transfusions were required every 2 or 4 weeks, the patient was admitted to our hospital on September 21, 1993. Hemoglobin was 8.6 g/dl, reticulocytes 80 × 109/1, WBC 2.71 × 109/1 (47.8% neutrophils, 3.8% eosinophils, 39.9% lymphocytes, 8.1% monocytes), and PLT

A Novel Chromosomal Abnormality, t(6;10)(q27;q22), Found in a Polycythemic Potential Donor for Allogeneic Hematopoietic Stem Cell Transplantation

A 59-year-old man was a potential donor for allogeneic hematopoietic stem cell transplantation and was found to be healthy but slightly polycythemic. The bone marrow was morphologically normal, but karyotyping of bone marrow cells showed t(6;10)(q27;q22) in 7 of 20 metaphases analyzed by G-banding and only the t(6;10) abnormality in 3 of 5 metaphases analyzed by the spectral karyotyping method. G-banding analysis of peripheral blood mononuclear cells cultured with phyto-hemagglutinin for 72 hours showed a normal karyotype in all 20 metaphases analyzed. These findings suggest clonal expansion with t(6;10)(q27;q22) in the bone marrow of this individual. He was determined to be ineligible for donation. A coordinated nationwide work-up for older donors is necessary to ensure high-quality standards.