Eijiro Omoto

Two acute monocytic leukemia (AML-M5a) cell lines (MOLM-13 and MOLM-14) with interclonal phenotypic heterogeneity showing MLL-AF9 fusion resulting from an occult chromosome insertion, ins(11;9)(q23;p22p23)

We describe two new human leukemia cell lines, MOLM-13 and MOLM-14, established from the peripheral blood of a patient at relapse of acute monocytic leukemia, FAB M5a, which had evolved from myelodysplastic syndrome (MDS). Both cell lines express monocyte-specific esterase (MSE) and MLL-AF9 fusion mRNA. Gene fusion is associated with a minute chromosomal insertion, ins(11;9)(q23;p22p23). MOLM-13 and MOLM-14 are the first cell lines with, and represent the third reported case of, MLL gene rearrangement arising via chromosomal insertion. Both cell lines carry trisomy 8 which was also present during the MDS phase, as well as the most frequent trisomies associated with t(9;11), ie, +6, +13, +19 variously present in different subclones. Despite having these features in common, differences in antigen expression were noted between the two cell lines: that of MOLM-13 being CD34+, CD13−, CD14−, CD15+, CD33+; whereas MOLM-14 was CD4+, CD13+, CD14+, CD15+, CD33+. Differentiation to macrophage-like morphology could be induced in both cell lines after stimulation with INF-γ alone, or in combination with TNF-α, which treatment also induced or upregulated, expression of certain myelomonocyte-associated antigens, including CD13, CD14, CD15, CD64, CD65 and CD87. Together, these data confirm that both cell lines are likely to be novel in vitro models for studying monocytic differentiation and leukemogenesis.

The effects of a simplified method for cryopreservation and thawing procedures on peripheral blood stem cells

A simplified method for cryopreservation at −80°C of peripheral blood stem cells (PBSC) has been increasingly used for autologous PBSC transplantation in Japan. Although this method, using 6% hydroxyethyl starch (HES) and 5% dimethyl sulfoxide (DMSO) as a cryoprotectant without rate-controlled freezing, has several advantages over the conventional method using 10% DMSO with rate-controlled freezing, little is known about effects of long-term cryopreservation for years and thawing process on hematopoietic progenitors. We examined the recovery rates of BFU-E and CFU-GM in sample tubes cryopreserved by the simplified method under various conditions as follows: (1) long-term storage for 1–5 years; (2) DMSO exposure for 1 h after rapid thawing; and (3) thawing at a lower temperature other than 37°C. In our study, we found that the recovery rates of BFU-E and CFU-GM were not affected by the length of cryopreservation period; they remained at more than 70% on average for 16–61 months. In our hands, a 1-h exposure to DMSO after rapid thawing was not toxic for hematopoietic progenitors. Furthermore, there was no significant difference in the recovery rates of BFU-E and CFU-GM between thawing at 37°C and 20°C. These observations indicate that PBSC cryopreserved for at least 5 years by the simplified method can be used clinically without losing hematopoietic activity, and suggest that hematopoietic activity of the thawed PBSC may be unaffected when PBSC are infused slowly within 60 min or even when PBSC are thawed gradually at room temperature.

G-CSF reduces IFN-γ and IL-4 production by T cells after allogeneic stimulation by indirectly modulating monocyte function

Despite a 10-fold increase of T cell dose, the incidence and severity of acute GVHD following allogeneic transplantation of G-CSF-mobilized PBSC is not increased compared to BMT. Experimental murine studies demonstrate that G-CSF polarizes donor T cells toward a type 2 cytokine response. To determine whether G-CSF alters T cell cytokine responses, we investigated the effects of G-CSF administration on T cell proliferative and cytokine responses to alloantigen and Con A in nonadherent PBMC (NAC) and CD3+ T cells obtained from normal individuals before and after G-CSF administration (10 μg/kg × 4 days). Although T cell proliferative and cytokine (IFN-γ and IL-4) responses to alloantigen stimulation and Con A were significantly reduced in post-G-CSF NAC, they were restored by the removal of non-T cells from post-G-CSF NAC. Furthermore, there was less T cell alloreactivity in MLR in the presence of autologous post-G-CSF monocytes than in the presence of pre-G-CSF monocytes. This alteration was not replicated in vitro by culturing PBMC with G-CSF. These results suggest that G-CSF administration suppresses T cell proliferative and cytokine (IFN-γ and IL-4) responses to allogeneic stimulation by indirectly modulating monocyte function. Bone Marrow Transplantation (2000) 25, 1035–1040.

Successful treatment of advanced natural killer cell lymphoma with high-dose chemotherapy and syngeneic peripheral blood stem cell transplantation

CD56+ angiocentric lymphoma has currently been recognized as a distinct clinical entity which is the prototype of the putative NK cell lymphomas. A 16-year-old Japanese girl with advanced CD56+ angiocentric lymphoma received high-dose chemotherapy supported with syngeneic peripheral blood stem cell transplantation (PBSCT). Prior to syngeneic PBSCT, she received six cycles of conventional chemotherapy before transplantation, resulting in a partial response. PBSC were mobilized with granulocyte colony-stimulating factor (G-CSF) and collected from her identical twin. High-dose cyclophosphamide, MCNU, etoposide, and carboplatin were used for pretransplant conditioning. Syngeneic PBSCT was well tolerated. She achieved complete remission and is now surviving in continuous complete remission for more than 30 months after syngeneic PBSCT. Thus, marrow-ablative chemotherapy facilitated by autologous or allogeneic PBSCT should be considered as part of the primary therapy for poor prognosis NK cell lymphomas.

Relapsed Acute Promyelocytic Leukemia Previously Treated with All-Trans Retinoic Acid: Clinical Experience with a New Synthetic Retinoid, Am-80

All-trans retinoic acid (ATRA), a potent differentiating drug for acute promyelocytic leukemia (APL), induces a high incidence of complete remission (CR) in patients with APL and is now established as a first-line therapy. However, ATRA resistance has become a clinical problem. Patients who relapsed after ATRA-induced CR have had difficulty in obtaining a second CR with ATRA therapy. Although several mechanisms have been postulated, treatment strategies to overcome resistance have not been established. We used a new synthetic retinoid, Am-80, as reinduction therapy for APL relapse after from ATRA-induced CR. Am-80 was several times more potent than ATRA in inducing differentiation in vitro. At a 6 mg/m2 dose, there were 24 evaluable patients; 14 (58%) achieved CR between days 20 and 58 (median, 37 days). Clinical response correlated with the in vitro response to Am-80. Adverse effects included retinoic acid syndrome (n = 1), hyperleukocytosis (n = 1), xerosis (n = 9), cheilitis (n = 8), hypertriglyceridemia (n = 16), and hypercholesterolemia (n = 15). Am-80 is active in APL after relapse from ATRA-induced CR. Further clinical trials are needed to establish strategies to overcome ATRA resistance.

Bone marrow necrosis in a patient with acute myeloblastic leukemia during administration of G-CSF and rapid hematologic recovery after allotransplantation of peripheral blood stem cells.

Allogeneic peripheral blood stem cell transplantation from an HLA‐identical sibling was performed for a 38‐year‐old male with refractory acute myeloblastic leukemia. The patient was conditioned with total body irradiation (TBI) and high‐dose cytosine arabinoside (Ara‐C). G‐CSF (300 μg/body) was started for priming of residual leukemic cells 24 hrbefore the beginning of TBI (day −9). However, intolerable generalized bone pain appeared shortly after the start of first dose of G‐CSF, and persisted for 3 days in spite of the cessation of G‐CSF. Posttransplant hematopoietic engraftment was very rapid. Bone marrow biopsy specimens on day 14 and 30 showed typical bone marrow necrosis histologically. This is the first case of bone marrow necrosis during administration of G‐CSF, and our experience suggests that PBSC could repopulate hematopoiesis in spite of severe bone marrow necrosis. Am. J. Hematol. 57:238–240, 1998.

All-trans retinoic acid therapy for newly diagnosed acute promyelocytic leukemia: comparison with intensive chemotherapy

Abstract We analyzed the results of treating patients with newly diagnosed acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) in the JALSG AML-92 study and compared them with those of the AML-87 and AML-89 studies, which consisted of standard chemotherapy. In the AML-92 study, patients were scheduled to receive 45 mg/m2 oral ATRA daily until achievement of a complete remission (CR). If patients had initial leukocyte counts of >3.0×109/l, they received 40 mg/m2 daunorubicin (DNR) for 3 days and 200 mg/m2 behenoyl cytarabine (BHAC) for 5 days in addition to ATRA. During remission induction therapy, if the patients showed peripheral blood myeloblast and promyelocyte counts of >1.0×109/l, they received additional DNR and BHAC on the same schedule. After achievement of a CR, patients received three courses of consolidation and six courses of maintenance/intensification chemotherapy. Of 196 evaluable patients, 173 (88%) achieved a CR: 59 of 62 (95%) treated with ATRA alone, 41 of 49 (84%) treated with ATRA plus later chemotherapy, 63 of 73 (86%) treated with ATRA plus initial chemotherapy, and 10 of 12 (83%) treated with ATRA plus both initial and later chemotherapy. The CR rate in AML-92 was significantly higher than that in AML-89, but not than that achieved in AML-87. In addition, the early mortality and relapse rates in AML-92 were significantly lower than those in AML-89, but were not than those in AML-87. At a median follow-up of 36 months the predicted 4-year event-free survival (EFS) rate for 196 evaluable patients and the 4-year disease-free survival (DFS) rate for the CR cases were 54% and 62%, respectively. There was a significant difference in DFS between AML-92 and AML-87 (P = 0.0418) but not between AML-92 and AML-89 (P = 0.0687). In contrast, significant differences in EFS between AML-92 and both AML-87 (P = 0.0129) and AML-89 (P = 0.005) were observed. These results suggest that non-cross-resistant therapy combined with ATRA and intensive chemotherapy for APL contributes synergistically to the significant improvement in EFS.

Myelodysplastic syndrome with translocation (8; 21): a distinct myelodysplastic syndrome entity or M2-acute myeloid leukemia with extensive myeloid maturation?

Abstract We report a case of refractory anemia with excess of blasts in transformation with the translocation (8; 21), which is frequent in acute myeloid leukemia (AML) but not in myelodysplastic syndromes (MDS). Bone marrow blasts were 1.6% and an extensive myeloid differentiation was noted. Fluorescence in situ hybridization analysis revealed the presence of 21q22 translocations in mature neutrophils, indicating that clonogenic blast progenitors could actively undergo terminal differentiation to mature end cells in vivo. We consider that t(8; 21)+ MDS may represent a rare clinical manifestation of M2-AML, in which blast progenitors have an extensive differentiation potential to mature neutrophils without maturation arrests.

Re-induction of complete remission with a new synthetic retinoid, Am-80, for relapse of acute promyelocytic leukaemia previously treated with all-trans retinoic acid

Two patients with relapsed acute promyelocytic leukaemia previously treated with all‐trans retinoic acid (ATRA), were treated with a new synthetic retinoid, Am‐80. In both patients pancytopenia gradually resolved without an increase in leukaemic cells, and differentiation of leukaemic cells was observed morphologically in bone marrow. Without the use of anti‐leukaemic agents, both cases achieved complete remission (CR) on days 52 and 38 of treatment, respectively. On the day of CR, PML gene rearrangement and the t(15;17) translocation disappeared, though PML‐RAR α chimaeric messenger RNA was still detected by reverse transcriptase polymerase chain reaction. Both patients then received conventional chemotherapy for consolidation of CR. These clinical experiences suggest that Am‐80 may be an active agent for APL patients who have relapsed from ATRA‐induced remission.

Improvement with disodium cromoglycate of neutrophil phagocytosis and respiratory burst activity in a patient with hyperimmunoglobulin E syndrome.

We report a case of hyperimmunoglobulin E syndrome (HIE) complicated by neutrophil deficiency which was successfully treated with oral administration of disodium cromoglycate. A 48‐year‐old Japanese man with HIE developed Streptococcus pneumoniae meningitis. Laboratory tests after the meningitis revealed persistent neutropenia (300‐800/mm3) and defects of phagocytosis and bacterial killing by neutrophils. Administration of disodium cromoglycate was started, and neutrophil counts gradually increased to 1200‐1600/mm3 TTie impaired neutrophil activities returned to normal. The patient improved clinically; during the 2‐year treatment, he had only two brief episodes of the common cold. Disodium cromoglycate may have potential clinical use in the treatment of cases of HIE even with neutrophil deficiency.