Norihiro Awaya

Serial quantification of minimal residual disease of t (8 ; 21) acute myelogenous leukaemia with RT-competitive PCR assay

The chromosomal translocation (8;21)(q22;q22) in the AML M2 subtype according to the FAB classification, results in the production of a novel fusion gene AML1/ETO. The chimaeric AML1/ETO transcript is useful for the detection of minimal residual disease (MRD). Recently, several studies on the detection of AML1/ETO transcripts in t(8;21) AML have been reported. However, the clinical significance of a small number of AML1/ETO transcripts by a reverse transcription–polymerase chain reaction (RT‐PCR) remains to be elucidated. We have developed a novel quantitative RT‐competitive PCR assay and evaluated the clinical usefulness of this method by the monitoring of MRD in eight patients with t(8;21) AML. In four patients in first continuous complete remission (CR) the value of MRD was always <0.1 fg of the competitor dose throughout their courses, whereas in four relapsed patients there was an increase in the value of MRD to <0.1 fg of the competitor dose before cytogenetic relapse. We conclude that the detection of the presence of cells with AML1/ETO fusion transcripts by our RT‐competitive PCR assay may be useful to monitor disease progression and to predict subsequent relapse.

Retinoid Resistance in Leukemic Cells

Recent studies have shown that a high proportion of patients with acute promyelocytic leukemia (APL) achieve complete remission after treatment with all-trans retinoic acid (RA). Nevertheless, despite an initial good response, most patients who received continuous treatment with all-trans RA relapsed and develop RA-resistant disease. The detailed mechanisms for this development of RA resistance by APL cells are still unclear. Several possible mechanisms have been considered to explain in vitro resistance to RA. One obvious explanation is the generation of new mutations in the retinoid receptors. However, UF-1 cells (the first permanent APL cell line with RA-resistant features) had no point mutations in the ligand-binding domain of the RAR-alpha gene. Another potential mechanism for clinical RA resistance is the pharmacologic alteration in the metabolism of all-trans RA. Continuous treatment with all-trans RA in APL is associated with a progressive reduction of the plasma concentrations of RA. Induction of cytochrome P-450, cellular RA-binding protein (CRABP) and P-glycoprotein resulted in lower plasma and cellular levels of active retinoids. Thus, acquired resistance to RA may be explained at least in part by drug metabolism in leukemic cells.

Novel variant isoform of G-CSF receptor involved in induction of proliferation of FDCP-2 cells: relevance to the pathogenesis of myelodysplastic syndrome.

Recent studies have shown that point mutations in granulocyte colony‐stimulating factor receptor (G‐CSFR) are involved in the pathogenesis of severe congenital neutropenia (SCN) and in the transformation of SCN to acute myelogenous leukemia (AML). It is reasonably speculated that the abnormalities in the signal transduction pathways for G‐CSF could be partly responsible for the pathogenesis and the development to AML in patients with myelodysplastic syndromes (MDS). Therefore, we investigated the structural and functional abnormalities of the G‐CSFR in 14 patients with MDS and 10 normal subjects. In in vitro colony forming assay, MDS samples showed reduced response to growth factors. However, G‐CSF, but not GM‐CSF and IL‐3, enhanced clonal growth in three cases of high risk patients with MDS (RAEB, RAEB‐t, and MDS having progressed to acute myeloid leukemia (AML)) and one low risk patient (RA). Eight out of 14 patients including above 4 patients demonstrated a common deletion of the G‐CSFR cDNA; a deletion of three nucleotides (2128–2130) in the juxtamembrane domain of the G‐CSFR, which resulted in a conversion of Asn630Arg631 to Lys630. To assess the functional activities of this deletion in the G‐CSFR isoform, a mutant with the same three‐nucleotide deletion was constructed by site‐directed mutagenesis. FDCP‐2 cells expressing the G‐CSFR isoform responded to G‐CSF, and exhibited proliferative responses than did those cells having wild‐type G‐CSFR. Moreover, these isoforms showed prolonged activation of STAT3 in response to G‐CSF than did the wild‐type. These results suggest that the deletion in the juxtamembrane domain of the G‐CSFR gives a growth advantage to abnormal MDS clones and may contribute to the pathogenesis of MDS.

Angioimmunoblastic T-Cell Lymphoma with Polyclonal Proliferation of Plasma Cells in Peripheral Blood and Marrow

phoadenopathy rapidly progressed after his first visit to our hospital in September, and a month later, WBC count was elevated to 27.3 ! 10 9 /l. The peripheral blood smear showed 31% of plasma cells, and the bone marrow smear disclosed diffuse infiltration of plasma cells (41.2% of all nucleated cells) ( fig. 1 ). No dysmorphic feature of plasma cells was observed. The patient was admitted to our hospital on October 8, 2004. On admission, he had fever, dyspnea and generalized lymphadenopathy. Chest X-ray showed consolidation in the right lower and left upper lung fields and mild bilateral pleural effusion. A computed tomographic scan revealed hepatosplenomegaly as well as mediastinal, pulmonary hilar, axillary, paraaortic and ilioinguinal lymphadenopathy. Laboratory findings showed leukocytosis (WBC 28.2 ! 10 9 /l with 30.5% plasma cells), mild anemia (hemoglobin 11.5 g/dl) and thrombocytopenia (platelets 65 ! 10 9 /l). Flow cytometric analysis showed that plasma cells in the peripheral blood and marrow were positive for CD19 and CD38, but lacking CD10, CD20 and CD56. No laterality of and -immunoglobulin light chain expression on the plasma cells was observed ( fig. 2 a, b). Gene rearrangement of immunoglobulin heavy chains (JH) and light chains (J ), using DNA extracted from the peripheral blood, was negative. The values of serum IgG, IgA and IgM were 6,690, 2,100 and 333 mg/dl, respectively. Immunoelectrophoresis of serum protein revealed a polyclonal increase in -globuAngioimmunoblastic T-cell lymphoma (AILT) is characterized by generalized lymphadenopathy, anemia, hepatosplenomegaly, skin eruption and polyclonal hypergammaglobulinemia. Polyclonal plasmacytosis in peripheral blood and bone marrow is not a recognized feature of AILT. Polyclonal plasmacytosis is a rare event, which has been observed in infectious diseases, drug reactions, malignant diseases and autoimmune diseases [1–5] . Most cases have an indolent or self-limited clinical course. However, for cases with lymphadenopathy and rapid polyclonal plasma cell proliferation, the underlying disease has not been well characterized to date, probably because immediate initiation of treatment is required without histological confirmation of lymph nodes. Lymph node biopsy has not been performed, even in the reported cases [6, 7] . Here, we report a case of AILT that presented with progressive polyclonal plasmacytosis in the peripheral blood and bone marrow. A 63-year-old man presented with cervical and axillary lymphadenopathy on September 6, 2004. The white blood cell (WBC) count and its differential were normal. His past medical history was unremarkable except that he received clarithromycin for his common cold for 4 days and olopatadine hydrochloride for subsequent skin rash from his general practitioner in July 2004. The patient reported immediate disappearance of the skin rash, which was deemed to be caused by clarithromycin. LymReceived: May 17, 2006 Accepted after revision: July 7, 2006 Published online: November 10, 2006

Establishment of a new human acute monocytic leukemia cell line TZ-1 with t(1;11)(p32;q23) and fusion gene MLL-EPS15

Human leukemia cell lines are of great value in investigating basic and applied aspects of cell biology and clinical medicine. There have been 37 leukemia cell lines carrying 11q23 translocation and MLL rearrangements; however, cell lines harboring with t(1;11)(p32;q23) have not been established. We report here for the first time a new acute monocytic leukemia (AMoL) cell line with t(1;11)(p32;q23), designated TZ-1, and herein describe its biological characteristics. Mononuclear cells isolated from the ascites from a patient with AMoL (French–American–British classification; acute myeloid leukemia M5a) were isolated and passaged by liquid culture medium for a year. TZ-1 cells revealed typical monocytic features in morphology and had a t(1;11)(p32;q23) translocation. The immunoprofiling as determined by flow cytometry showed that TZ-1 cells are positive for myeloid and monocytic markers with lymphoid-associated markers. Fluorescence in situ hybridization and reverse transcription-polymerase chain reaction analyses revealed MLL-EPS15 fusion transcript and protein. Taken together, these results suggest that TZ-1 is a new monocytic leukemia cell line with t(1;11) translocation and fusion gene MLL-EPS15. The established cell line, TZ-1, could provide a valuable model in the analysis of the pathogenesis of MLL-EPS15-positive leukemia and in the development of new agents for this type of leukemia.

Bone marrow metastasis of small cell carcinoma of the lung mimicking Burkitt lymphoma/leukaemia.

A 65-year-old Japanese man was admitted for the evaluation of anaemia and thrombocytopenia. Six months earlier, he had undergone lobectomy of the right middle lobe for small cell carcinoma of the lung (T2N0M0, clinical stage IB), and subsequently he had received two courses of chemotherapy consisting of cisplatinum and etoposide. The findings of physical examination were unremarkable. The complete blood count showed a normal white blood cell count of 8Æ6 · 10/l with 4% myelocytes, 4% metamyelocytes, 62% neutrophils, 19% lymphocytes, 10% monocytes, 1% eosinophils, a haemoglobin of 8Æ6 g/dl, and a platelet count of 17 · 10/l. Blood chemistry showed a high serum lactate dehydrogenase level (3655 IU/l, normal range 120–200 IU/l). Bone marrow aspiration showed a diffuse monotonous infiltration of medium sized cells with basophilic cytoplasm containing abundant vacuoles, which were suggestive of Burkitt lymphoma/leukaemia (top). The cells were myeloperoxidase-negative, and were also negative for B-cell markers, such as CD19, CD10 and CD20, by flow-cytometric analysis. The only positive surface marker of the cells was CD56 (neural cell adhesion molecule). After admission, interstitial pneumonitis of unknown aetiology rapidly progressed and the patient died with acute respiratory failure. Autopsy confirmed multiple metastases of small cell carcinoma of the lung with a diffuse infiltration of the cells into the bone marrow (bottom). Bone marrow metastasis in patients with small cell carcinoma of the lung has been frequently documented. However, the marrow is seldom the sole site of metastasis and metastases are usually identified as cohesive cell aggregates in the bone marrow smears. Our experience indicates that bone marrow metastasis of small cell carcinoma of the lung may simulate a haematological malignancy. In this case, the sole expression of CD56 was a diagnostic clue.

Multilineage Involvement of Light Microscopic Myeloperoxidase-Negative Acute Leukemia

The classification of acute leukemia has traditionally been based on a combination of morphology and cytochemical staining data, including myeloperoxidase (MPO) reaction; however, a recent World Health Organization (WHO) classification entails use of cytogenetic and molecular findings in addition to the classic morphological and immunophenotypic analyses. Nevertheless, there have been rare cases in which blastic cells show multilineage phenotypes. These cases may be classified as acute leukemia of ambiguous lineage in the recent WHO classification. We report the case of a 49-year-old man with acute leukemia with multilineage phenotypes. Morphological findings led to a diagnosis of acute myeloid leukemia M2 by the French-American-British classification, but at light microscopy the results of MPO staining were negative for blast cells. In contrast, results of reverse transcription polymerase chain reaction and fluorescence-activated cell sorter analyses were positive for expression of MPO messenger RNA and protein. The blast cells expressed CD4, CD19, CD22, CD33, CD38, CD79a, and HLA-DR and showed rearrangement of the immunoglobulin heavy chain and TCR-β genes. Results of immunoelectron microscopic analysis of the blast cells were positive for MPO, CD19, CD33, CD34, CD38 and glycophorin A but not for platelet peroxidase. According to these results, the blast cells had at least 4 lineage phenotypes. We concluded that the multiparameter analyses conducted in this case, including immunological and ultrastructural assays, were important in arriving at the appropriate diagnosis of acute leukemia of ambiguous lineage in the new WHO classification.