Toru Tezuka

p16/MTS1/INK4A gene is frequently inactivated by hypermethylation in childhood acute lymphoblastic leukemia with 11q23 translocation.

The p16 gene encoding a specific inhibitor of cyclin-dependent kinases 4 and 6 has been reported to be inactivated at a variety of rates in malignant tumors. We studied frequency and mechanism of inactivation of the p16 gene in various types of childhood acute lymphoblastic leukemia (ALL) using 36 leukemic cell lines established from children (B precursor-ALL, 28; B-ALL/Burkitt’s lymphoma, 3; and T-ALL, 5). On Southern blot, homozygous deletions or hemizygous deletions with rearrangement were detected in 14 cell lines. The expression of p16 protein was not observed on Western blot in 18 of 22 cell lines with intact p16 gene, but induced in 16 cell lines after treatment with the demethylating agent, indicating the silencing of the p16 gene by hypermethylation. Of note, the p16gene was inactivated by hypermethylation of the 5′ CpG island in nine of nine cell lines with 11q23 translocation, but was restored with the treatment of the demethylating agent. Partial methylation of the p16 gene was also demonstrated in three of eight primary leukemia samples with this translocation, suggesting that the p16 gene inactivation by hypermethylation might play a role in the leukemogenesis and disease progression of ALL with 11q23 translocation.

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.

Leukemic cells with 11q23 translocations express granulocyte colony-stimulating factor (G-CSF) receptor and their proliferation is stimulated with G-CSF

We report a 20-month-old boy with acute lymphoblastic leukemia with the 11q23 translocation whose blasts markedly increased in peripheral blood after intravenous granulocyte colony-stimulating factor (G-CSF) administration, but disappeared after stopping G-CSF. The in vitro study showed that the leukemic cells separated from this patient expressed G-CSF receptor (G-CSFR) and an addition of G-CSF stimulated their proliferation by 3H-thymidine incorporation assay (stimulation index, 4.9). To clarify whether or not leukemic cells with 11q23 translocations generally express G-CSFR and show proliferative response to G-CSF, we performed the similar in vitro experiments using eight leukemic cell lines with 11q23 translocations. We found that all cell lines examined expressed G-CSFR (20–98%) and proliferation of seven leukemic cell lines was significantly enhanced in response to G-CSF (stimulation index >1.5 in five cell lines), suggesting a possible participation of the G-CSF/G-CSFR interaction in the process of growth regulation of leukemic cells with 11q23 translocations.

The KOR-SA3544 antigen predominantly expressed on the surface of Philadelphia chromosome-positive acute lymphoblastic leukemia cells is nonspecific cross-reacting antigen-50/90 (CD66c) and invariably expressed in cytoplasm of human leukemia cells.

We previously reported a novel monoclonal antibody KOR-SA3544 which predominantly reacted with a surface antigen (sSA3544) expressed on Philadelphia chromosome (Ph1)-positive acute lymphoblastic leukemia (ALL). In the present study, we demonstrate that the antibody specifically recognized nonspecific cross-reacting antigen (NCA)-50/90 (CD66c), one of the carcinoembryonic antigen (CEA)-related glycoproteins encoded by a member of the CEA gene family. In addition, we show that the SA3544 antigen (NCA-50/90) was invariably expressed in cytoplasm of all of the human leukemic cell lines examined (sSA3544-positive B-lymphoid two, sSA3544-negative T or B-lymphoid and non-lymphoid 24) regardless of the presence or absence of surface expression of this antigen. Immunoelectromicroscopic examination revealed that the cytoplasmic antigen was mainly present in granules in sSA3544-positive leukemia cells, whereas it was diffusely present in cytosol in sSA3544-negative leukemia cells. Thus, among members of the CEA family, NCA-50/90 was first demonstrated to be expressed not only on the surface of some leukemia cells, but also in cytoplasm of various types of leukemia cells.

A novel 203 kD aberrant BCR-ABL product in a girl with Philadelphia chromosome positive acute lymphoblastic leultaemia

Summary We report a girl with Ph1‐positive ALL with the aberrant BCR‐ABL product. In this case. bcr exon 3 jointed not to ordinal abl exon 2 but to exon 3 resulting in the production of a 20 3 kD BCR‐ABL fusion protein with marked tyrosine kinase activity. To our knowledge, this is the first report of an aberrant BCR‐ABL product in childhood. This case was characterized with younger age and low leucocyte count at the onset, but relapsed early like the typical Phl‐positive ALL, suggesting the diversity in the clinicopathogenesis of Ph1‐positive ALL.

Participation of granulocyte colony-stimulating factor in the growth regulation of leukemia cells from Philadelphia chromosome-positive acute leukemia and blast crisis of chronic myeloid leukemia

Granulocyte colony-stimulating factor (G-CSF) has been shown to support the growth of multipotential hematopoietic stem cells in addition to the cells of neutrophilic lineage. Philadelphia chromosome (Ph1)-positive leukemia has its origin in the hematopoietic stem cell. In the present study, we demonstrated that the proliferation of leukemic cells from chronic myeloid leukemia in blast crisis (CML-BC) and Ph1-positive acute lymphoblastic leukemia (ALL) cases is frequently stimulated with G-CSF in vitro. We next studied a total of 12 leukemic cell lines established from CML-BC (n = 6) and Ph1-positive acute leukemia (n = 6): four ‘myeloid’, five ‘biphenotypic’, and three ‘lymphoid’ types. All cell lines expressed G-CSF receptor (G-CSFR) in flow cytometric analysis, but their proliferative response to G-CSF in 3H-thymidine incorporation assay varied. The ‘biphenotypic’ cell lines expressed G-CSFR at higher levels and showed the most pronounced response to G-CSF. The ‘lymphoid’ cell lines showed intermediate G-CSFR expression with the modest response to G-CSF. Unexpectedly, ‘myeloid’ cell lines showed lower G-CSFR expression and lower G-CSF response compared with ‘biphenotypic’ cell lines. In three of four ‘myeloid’ cell lines, proliferation was partially inhibited by an addition of anti-G-CSF neutralizing monoclonal antibody into culture medium. Further, the % inhibition of 3H-thymidine uptake of cell lines positively correlated with the amount of their intracellular G-CSF measured by enzyme immunoassay, suggesting an autocrine growth mechanism via the G-CSF/G-CSFR interaction. These results suggest that G-CSF play an important role in the growth regulation of leukemia cells from Ph1-positive acute leukemia and CML-BC.

Transient increase in CD45RO expression on T lymphocytes in infected newborns.

Although infections are a significant cause of mortality and long-term morbidity of newborns, the early diagnosis of neonatal infections remains difficult. It has been shown recently that up-regulation of CD45RO expression on umbilical cord lymphocytes might be a good indicator for intrauterine viral infections. To evaluate clinical relevance of changes in CD45 isoforms to the diagnosis of neonatal infections, we examined CD45RA and CD45RO expression on total, CD4+, and CD8+ lymphocytes from newborns. Lymphocytes from 55 newborns who were hospitalized in Kiyose Metropolitan Childrens Hospital or Yamanashi Medical University Hospital were analyzed. Newborns were classified into three groups according to the possibility of bacterial or viral infection; I (“no” infection, n = 24), II(“possible” infection, n = 17), and III(“definite” infection, n = 14). Results were compared between two of three groups. The CD45RO expression on total or CD4+ lymphocytes in group III (bacterial 12, viral 2) was significantly higher than that in group I or II. Particularly, the CD45RO expression on CD4+ lymphocytes was always greater than 10% in group III, and showed no overlap with the respective value in group I. After termination of infection, the increased CD45RO expression in group III gradually returned to normal levels. These results indicate that not only viral but also bacterial infections trigger transient and reversible changes in CD45 isoform expression by neonatal CD4+ T lymphocytes. This may be a novel marker for the early diagnosis of neonatal infections.

Expression of granulocyte colony-stimulating factor receptor on CD10-positive human B-cell precursors

We examined the expression of CD10 and G‐CSF receptor (G‐CSFR) on the lymphoid population of mononuclear cells obtained from bone marrow (BM) using two‐colour analysis. In the BM of children with ALL in remission, the CD10+ population was significantly increased (20.6 ± 5.1% compared with that of controls (2.5 ± 0.5%). More than half (61.3 ± 2.9%) of the CD10+ cells coexpressed G‐CSFR, but not CD13. These results indicate G‐CSFR+ B‐cell precursors are markedly increased in BM of ALL in remission, suggesting the probable involvement of G‐CSF in the human early B‐cell ontogeny.

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.

A successful case of allogeneic bone marrow transplantation for osteosarcoma with multiple metastases of lung and bone.

A successful case of allogeneic bone marrow transplantation for osteosarcoma with multiple metastases of lung and bone