Shinpei Nakazawa

The JAK2 inhibitor AG490 predominantly abrogates the growth of human B-precursor leukemic cells with 11q23 translocation or Philadelphia chromosome

The Janus kinase (JAK) family is one of intracellular protein tyrosine kinases (PTKs) present in hematopoietic and lymphoid cells and has been shown to play a crucial role in a variety of biological responses. It was reported that a human B-precursor leukemic cell line was potently inhibited in its proliferation by one of synthetic PTK inhibitors (tyrphostins), AG490, via anti-JAK2 activity. However, no extensive studies about it have been performed. In the present study, we tested 16 human lymphoid leukemic cell lines (B-precursor, 12; T cell, four) for their sensitivity to AG490 using 3H-thymidine incorporation and colony formation assays, and found that B-precursor cell lines with 11q23 translocation or Philadelphia chromosome (Ph1) whose JAK2 proved to be constitutively phosphorylated were predominantly sensitive to AG490 at a concentration that has few inhibitory effect on normal hematopoiesis. We first revealed the association of JAK2 with BCR-ABL in Ph1-positive cell lines and with Brutons tyrosine kinase (BTK) in cell lines with 11q23 translocation by coimmunoprecipitation experiments. Of interest, AG490 markedly down-regulated phosphorylation of JAK2, but rather transiently up-regulated phosphorylation of BCR-ABL and BTK, suggesting direct implication of AG490 in the process of the JAK2 dephosphorylation. These results indicate that AG490 exerts a potent inhibitory activity to B-precursor leukemia with specific chromosomal abnormalities, and a therapeutic approach using AG490 is expected.

Long-term follow-up of childhood acute lymphoblastic leukemia in Tokyo Children's Cancer Study Group 1981-1995.

The objectives were as follows: Firstly, to estimate the overall probability of event-free survival (EFS) and isolated CNS relapse in the studies for children with acute lymphoblastic leukemia (ALL) during the 1980s and 1990s. Secondly, to report the EFS according to presenting features and lineage. Thirdly, to evaluate the treatment results re-classified by the risks of NCI criteria. Four consecutive protocol studies were performed in the Tokyo Childrens Cancer Study Group: L81–10 protocol (1981–1984, 189 patients), L84–11 (1984–1989, 484 patents), L89–12 (1989–1992, 418 patients) and L92–13 (1992–1995, 347 patients). Overall EFS at 5 years in each protocol was 56.5 ± 3.8(1 s.e.)%, 71.0 ± 2.1%, 67.8 ± 2.3%, and 63.4 ± 2.7%, respectively. The cumulative isolated CNS relapse rate at 5 years was 8.1 ± 2.1%, 3.5 ± 0.9%, 3.6 ± 1.0%, 1.0 ± 0.6. The EFS in SR/HR (standard risk/high risk) according to the NCI criteria in B-precursor ALL at 5 years was 61.9 ± 4.3%/41.4 ± 7.4% (lineage was not confirmed.), 72.5 ± 2.6%/63.4 ± 5.0%, 77.4 ± 2.7%/56.3 ± 4.7%, and 67.8 ± 3.4%/56.7 ± 5.4% in each protocol. Also EFSs according to NCI SR/HR at 5 years of T-ALL in protocols L84–11, L89–12 and L92–13 were 55.6 ± 16.6%/60.9 ± 10.1%, 72.7 ± 13.4%/51.6 ± 9.1%, and 77.1 ± 14.4%/53.6/10.1%, respectively. The truncation of maintenance therapy to 6 months resulted in a decreased EFS in L92–13, particularly due to an increase of bone marrow relapse after cessation of therapy in SR and HR. The NCI risk criteria work properly even in the patients treated by different intensities, so that it makes the comparison possible among the patients in various groups. The overall EFSs in childhood ALL improved in 1980s, but it seemed stable or decreased in 1990s. The short maintenance therapy resulted in poor outcome in SR on the L92–13 protocol. Many of these late relapsers were effectively rescued and overall survival remained at a high level. The proportion of patients who received cranial irradiation reduced without any increase of the CNS events.

Clinical and cytogenetic characteristics of myelodysplastic syndromes developing myelofibrosis

Myelofibrosis occurs in various hematologic neoplasias, including myelodysplastic syndrome (MDS), with a relatively low incidence. To gain insight into the clinical and cytogenetic implications of MDS patients in whom myelofibrosis develops, statistical analysis was done on 82 primary MDS patients with successful cytogenetic results. Seven patients had myelofibrosis during the course of the disease (8.5%, Group I), 34 had abnormal karyotypes without myelofibrosis (41.5%, Group II), and the other 41 had normal karyotypes without myelofibrosis (50%, Group III). All of the MDS patients except one with myelofibrosis had cytogenetic abnormalities, and four of them had multiple chromosome abnormalities. In univariant analysis, MDS patients with myelofibrosis showed no significant differences in age, sex, or peripheral blood data. In contrast, patients with chromosome abnormalities evolved into myelofibrosis with a high incidence compared with those with normal karyotypes (14.6% versus 2.4%, P = 0.054). The occurrence of myelofibrosis was higher during the first 6 months after the diagnosis of MDS than in the next 6 months (6.1% versus 0%, P = 0.045). Most of the MDS patients survived for less than 10 months after myelofibrosis was evident. Furthermore, survival was significantly shorter in Group I compared with Groups II (P < 0.05) and III (P < 0.01). Among the MDS patients in whom myelofibrosis developed, some were associated with acute megakaryoblastic leukemia, indicating a heterogeneity of clinical features in MDS with myelofibrosis.

11;19 Translocation in a congenital leukemia with two cell populations of lymphoblasts and monoblasts

This report describes a case of a female infant of congenital leukemia with a chromosomal translocation t(11;19) (q23;p13) which presented initially with lymphoid features and at relapse with monocytic ones. The clinical course and the results of sequential cytochemical, cytogenetic and immunological studies are considered to be consistent with the interpretation of leukemogenesis of the myelo-monocytoid progenitor cell which still retains the capability of exhibiting lymphoid features to a limited extent. Although leukemia with t(11;19) has been classified as ANLL, most commonly M5 of FAB classification, the patient with this chromosomal abnormality may have a mixed leukemia in which cells with lymphoid features and those with monocytic ones exist.

Molecular studies of chronic myelogenous leukemia using the polymerase chain reaction

Thirty‐two cases of chronic myelogenous leukemia (CML) were studied to determine whether there was a correlation between the position of the chromosome breakpoint within the breakpoint cluster region (bcr) on chromosome 22 and the type of chimeric mRNA expression. One case with the chromosome breakpoint in zone 2 of the major bcr (Mbcr) and six cases with breakpoints in zone 3 expressed Mbcr exon 2‐abl (b2‐a) mRNA, and they were in distinguishable at the level of mRNA expression. The remaining ten cases with breakpoints in zone 3 and all ten cases with breakpoints in zone 4 expressed Mbcr exon 3‐abl (b3‐a) mRNA with or without b2‐a mRNA. Three cases with breakpoints in zone 5 expressed b3‐a mRNA, and none of these expressed Mbcr exon 4‐abl(b4‐a) mRNA. The cases with breakpoints in zones 4 or 5 had b3‐a mRNA expression indistinguishable from those with breakpoints in zone 3. In two patients, the breakpoint in the bcr could not be determined by Southern hybridization using the 3 bcr probe or the large bcr probe. However, when analyzed for chimeric mRNA expression, both of them exhibited b3‐a chimeric mRNA, suggesting the possibility that the entire Mbcr is deleted in the majority of leukemic cells in these patients. These studies indicate that Southern hybridization analysis combined with the polymerase chain reaction assay is a useful approach to understanding the pathologic role of bcr‐abl gene recombination and expression in the development of CML. 68:2426‐2430, 1991.

Clinical and biologic characteristics of CD7+ acute myeloid leukemia: Our experience and literature review

Six patients with acute myeloid leukemia (AML) expressing CD7 antigen (CD7+ AML) were studied. They consisted of five patients with M1 and one with an M2 morphology. Two cases expressed other lymphoid-associated antigens, in addition to CD7. The complete remission rate was 50%. One patient had central nervous system recurrence. Cytogenetic analysis demonstrated normal karyotypes in all the cases. All but one had germline configurations of the T-cell receptor (TCR) genes and immunoglobulin heavy chain gene. However, all did not have detectable recombinase activating gene-1 activity by the RT-PCR technique. We performed colony formation assay in two patients, and no enhancement of colony formation by granulocyte colony-stimulating factor was noted. The results presented here, together with those reported previously, suggest that CD7+ AML may demonstrate lineage infidelity.

Replacement of m-calpain by μ-calpain during maturation of megakaryocytes and possible involvement in platelet formation

Localization of calpains in human bone marrow cells was studied immunohistochemically employing monoclonal antibodies against the high-Ca(2+)-requiring form (m-calpain) and the low-Ca(2+)-requiring form (mu-calpain). Most cells were stained with anti-m-calpain more strongly than with anti-mu-calpain, and staining with anti-mu-calpain was prominent only in megakaryocytes. To confirm the result, megakaryoblastic cell line (T-33) cells were subjected to immunoblot analysis. However no immunoreactivity to mu-calpain was seen in T-33 cells. Bone marrow from a patient with idiopathic thrombocytopenic purpura showed immature megakaryocytes (stage II) strongly stained by anti-m-calpain antibody while mature cells (stage III) were strongly stained by anti-mu-calpain antibody. These results suggest that mu-calpain plays a crucial role in mature megakaryocytes, possibly in platelet production.

Ph1-positive CML-derived myeloid-monocytoid precursor cell line producing substance(s) that stimulates normal CFU-C

A new Ph1-chromosome positive cell line, KOPM-28. was established from a patient with chronic myelogenous leukemia (CML) in blast crisis. KOPM-28 cells were phenotypically immature: without azurophilic granules; negative for myeloperoxidase and positive for specific and nonspecific esterases. The nonspecific esterase reaction was intensified by TPA, and retinoic acid reinforced the specific esterase reaction without inducing morphological changes. KOPM-28 cells were not phagocytic. The cells expressed complement receptors, myeloid-monocytoid antigens, an Ia-like antigen and T4 antigen. CALLA, T-lymphocyte specific antigens, B-lymphocyte related antigen and platelet-megakaryocyte-megakaryoblast specific antigen were not detected. KOPM-28 cells formed colonies in semi-solid medium; this ability was augmented by GM-CSA. The addition of culture medium conditioned by KOPM-28 cells to normal bone marrow cells resulted in the increase of the CFU-C colonies. These findings indicate that KOPM-28 cells have features of myeloid and monocytoid precursor cells and that they are producing substance(s) which stimulates normal CFU-C.

Expression of thrombopoietin receptor and its functional role in human B-precursor leukemia cells with 11q23 translocation or Philadelphia chromosome.

Thrombopoietin (TPO) is a hematopoietic growth factor which plays a central role in normal megakaryocytopoiesis and thrombopoiesis. Although the interaction between TPO and its receptor c-Mpl encoded by the c-mpl gene is now known to be implicated in the proliferation and/or differentiation of abnormal myeloid cells and normal hematopoietic stem cells, little is known about a role of the TPO/c-Mpl system in lymphoid leukemia cells. In the present study, we first examined the expression of c-mpl/c-Mpl in 23 human lymphoid leukemic cell lines (T-lineage 4, B-lineage 19) using three distinct methods. The c-mpl mrna was detectable in as many as 20 cell lines (t-lineage 3, b-lineage 17) by reverse transcriptase-polymerase chain reaction, but its translated product, c-mpl, was demonstrable by western blot only in b-lineage cell lines. flow cytometric analysis revealed the surface c-mpl expression in 13 of 17 b-lineage cell lines, but its higher expression (>40%) was restricted in nine B-precursor cell lines, eight of which had 11q23 translocation or Philadelphia chromosome (Ph1). We also demonstrated that two of eight cell lines with 11q23 translocation or Ph1 exhibited a significant proliferative response to TPO in the 3H-thymidine uptake and colony-forming assays. Triggering of these cell lines by TPO transiently up-regulated tyrosine phosphorylation of JAK-2 and Shc, indicating that their receptor is functional. Primary leukemia cells separated from patients with B-precursor acute lymphoblastic leukemia with Ph1 or 11q23 translocation also showed the surface c-Mpl expression and a significant responsiveness to TPO. These results suggest that the TPO/c-Mpl interaction may play a physiological role in the growth regulation of B-precursor leukemia cells particularly with specific chromosomal abnormalities. Leukemia (2000) 14, 1598–1605.

Treatment of Childhood Acute Lymphoblastic Leukemia: the Results of the Tokyo Children’s Cancer Study Group L84-11 Treatment Protocol Study

The Tokyo Children’s Cancer Study Group protocol (TCCSG L84-11), a randomized trial, was designed to evaluate moderate dose methotrexate (MD-MTX) therapy plus late skull irradiation and intensified central nervous system (CNS) prophylaxis by triple intrathecal (IT) therapy. From 1984 to 1988, children with newly diagnosed acute lymphoblastic leukemia (ALL), except for B-ALL, were enrolled in this protocol. Patients at high risk had one or more of the following risk factors: age below 2 or above 7 years, white cell count of 20,000/μ1 or more. Patients at extremely high risk had the following risk factors: T-cell phenotype, white-cell count of 100,000/μ1 or more, or mediastinal mass. Other patients were in the standard risk group. The standard risk (S) regimen consisted of two arms (S-1, S-2). For the remission induction, conventional VPL therapy was carried out. After the remission, triple IT therapy and 18 Gy radiation were given to S-l, and MD-MTX, late radiation (24 weeks later) and maintenance therapy was the standard form. The therapy was terminated after 3.5 years. The high-risk (H) regimen consisted of two arms (H-l, H-2). The outline of this regimen was similar to that of the S regimen. In the H regimen, cyclophosphamide, anthracyclines, BH-AC, cytosine arabinoside, and 1-asparaginase were administered for cyclic intensification therapy. The extremely high risk regimen (Hex) was a modified protocol of BFM-ALL83. There were 471 eligible patients. The remission rates were 97–100%. The patients with relapsed disease were 9/93 (S-1), 8/92 (S-2), 21/127 (H-1), 20/113 (H-2), and 8/46 (Hex). Event = free survival rates (EFS) were 80%, 66-74% and 69% in the S-, H-, and Hex regimens, respectively. In each group, no significant difference in EFS was demonstrated between the two arms (S-l vs. S-2, and H-l vs. H-2). The most common site of relapse was the bone marrow (49/66, 74%). Patients with CNS relapse were 5/93 (S-1), 2/92 (S-2), 1/127 (H-1), 0/113 (H-2), and 2/46 (Hex). The unexpected sequela of this protocol was the severe neurotoxicity that accumulated in the H-2 subgroup. Although the follow-up period is short, these preliminary results indicate that this protocol apparently reduces the CNS relapse rate, especially in the high-risk group. However, further modification of CNS prophylaxis is necessary to avoid neurological sequela.