An effective chemotherapeutic regimen for acute myeloid leukemia and myelodysplastic syndrome in children with Down's syndrome.

In recent pediatric collaborative studies of acute myeloid leukemia (AML), patients with Down's syndrome (DS) have better outcome than other patients when they were treated according to their intensive AML protocols. This may be attributed to enhanced sensitivity of DS AML cells to selected chemotherapeutic agents. We evaluated a less intensive chemotherapeutic regimen which was specifically designed for children with AML-DS. Remission induction chemotherapy consisted of daunorubicin (25 mg/m2/day for 2 days), cytosine arabinoside (100 mg/m2/day for 7 days), and etoposide (150 mg/m2/day for 3 days). Patients received one to seven courses of consolidation therapy of the same regimen. Thirty-three patients were enrolled on the study and their clinical, hematologic and immunophenotypic features were analyzed. Of the 33 patients, all were younger than 4 years and diagnosed as having acute megakaryoblastic leukemia or myelodysplastic syndrome. All patients achieved a complete remission and estimated 8 year event-free survival rate was 80+/-7%. Three patients relapsed and two died due to cardiac toxicity and one due to septic shock. The results of our study showed that patients with AML-DS constitute a unique biologic subgroup and should be treated according to a less intensive protocol designed for AML-DS.

Lineage switch in childhood leukemia with monosomy 7 and reverse of lineage switch in severe combined immunodeficient mice.

Morphophenotypic lineage switches occur in a small percentage of those with acute leukemia, and the underlying mechanisms are not clear. In this study, we attempted to induce a lineage switch in acute myelocytic leukemia (AML) with monosomy 7, whose lineage had switched from acute T-lymphocytic leukemia (T-ALL) during chemotherapy, in severe combined immunodeficient (SCID) mice. Although the transplanted myeloid cells were engrafted in SCID mice without cytokine administration, T-ALL developed in SCID mice treated with recombinant human granulocyte-macrophage colony-stimulating factor or recombinant human interleukin 3. Analysis of the nucleotide sequences of the rearranged T-cell receptor gamma-chain (TCR-gamma) gene revealed that this lineage switch resulted from the selection of the T-lineage subclone in SCID mice, which had expanded at onset. In addition, we found that the T-lineage and myeloid cells belonged to the distinct subclones, which were different in TCR-gamma gene rearrangements, but were derived from a common clone with an identical N-ras gene mutation for both subclones. In in vitro cultures, only the myeloid subclone grew; the T-lineage subclone failed to grow even in the presence of recombinant human granulocyte-macrophage colony-stimulating factor or recombinant human interleukin 3. These results suggested that the initial diagnostic T-lymphoid subclone, whose growth was dependent on these cytokines and the hematopoietic microenvironment, emerged from a bipotential T-lymphoid/myeloid leukemic stem cell, and further genetic event(s) induced the myeloid subclone, which grew independently of these cytokines and the microenvironment.

Successful engraftment of unrelated cord blood stem cells for familial erythrophagocytic lymphohistiocytosis. Kinki Cord Blood Bank.

Familial erythrophagocytic lymphohistiocytosis (FEL) is an autosomal recessive disorder that can only be corrected by stem cell transplantation. One of our patients with FEL in second complete remission underwent successful cord blood stem cell transplantation (CBSCT); the donor was an HLA one-locus mismatched and unrelated individual. The conditioning regimen consisted of BU/CY/VP-16. The transfused cell dose was 6.8 x 10(7)/kg, which contained 1.36 x 10(5)/kg of CD34 cells and 3.4 x 10(4)/kg of CFU-GM. After CBSCT, there were no major infectious complications. Acute grade I GVHD was well controlled. Neutrophil counts reached >0.5 x 10(9)/l by day 20 and platelet counts reached >50 x 10(9)/l by day 40. Deficient natural killer activity returned to normal after the transplant. The patient recovered well more than 7 months after receiving CBSCT, without showing evidence of chronic GVHD. We recommend CBSCT for FEL patients who have no HLA-matched siblings or unrelated donors.

Unrelated bone marrow transplantation for Epstein-Barr virus-associated T/NK-cell lymphoproliferative disease.

Epstein-Barr virus (EBV)-associated T/NK-cell lymphoproliferative disease (LPD) has been linked to several different disorders, including chronic active EBV infection, EBV-associated hemophagocytic syndrome, hypersensitivity to mosquito bites, hydroa vacciniforme, aggressive NK-cell leukemia, and nasal/nasal-type NK-cell lymphoma. In most instances, these disorders are refractory to conventional treatments and have a poor prognosis. Here, we report a new treatment strategy for EBV-associated T/NK-cell LPD, consisting of immunochemotherapy, intensive combination chemotherapy, and stem cell transplantation. The five patients studied, two with T-cell and three with NK-cell LPD, lacked a human leukocyte antigen-matched, related donor, and therefore received bone marrow grafts from HLA-matched, unrelated donors. The preconditioning regimen consisted of total-body irradiation (12 Gy), etoposide (900 mg/m(2)), and cyclophosphamide (120 mg/kg) or melphalan (210 mg/m(2)). All patients had residual LPD by a quantitative PCR technique prior to transplantation. After unrelated bone marrow transplantation (UBMT), four of the five patients remain in continuous complete remission at a median of 19 months, without detectable EBV-DNA in peripheral blood. Thus, UBMT appears to be a reasonable option for the treatment of patients with EBV-associated T/NK-cell LPD. Detection of EBV-DNA by PCR offers an important tool for assessing minimal residual disease in patients with EBV-associated T/NK-cell LPD.

CD33+ B-cell precursor acute lymphoblastic leukemia in children: a distinct subgroup of B-cell precursor acute lymphoblastic leukemia.

Clinical and laboratory features associated with CD33 expression were analysed in 123 children with B-precursor acute lymphoblastic leukemia (ALL), including 85 at onset, 34 at relapse and four in a refractory state to induction therapy. CD33 was demonstrated in 13 patients (15.3%) at onset, and it was associated with coexpression of T-cell and multipotential hematopoietic cell-associated antigens, i.e. CD2, CD4 and CD7, which were observed in four of 11 analysed patients (P < 0.01). Patients with CD33 expression were older than those without CD33 (P < 0.01). Although CD33 was the strongest predictor of a poor outcome (event-free survival, 44% for CD33+ and 75% for CD33-patients; P = 0.0041) in univariate analysis, multivariate analysis did not demonstrate significance (P = 0.0645). Fourteen of 38 patients (36.8%) at relapse or in a refractory state showed CD33 expression. Analysis of CD33 expression had also been performed at onset in 16 of these patients and showed acquisition of CD33 in six of 13 patients who had been negative for this antigen at onset. Thus, it seems that CD33+ B-precursor ALL is derived from undifferentiated cells minimally committed to B-cell lineage and more homogeneous than so-called My+ B-precursor ALL with regard to the clinical and biological features. The frequent expression of CD33 on the cells which acquired resistance to chemotherapy may have resulted from expansion of a CD33+ original minor clone or clonal evolution.

Intensification of chemotherapy using block therapies as consolidation and reinduction therapies for acute lymphoblastic leukemia during childhood.

Between April 1994 and March 1997, 143 children (age range, 1-15 years) with newly diagnosed acute lymphoblastic leukemia (ALL), except for those patients with t(9;22), were treated according to protocol-94 of the Osaka Childhood Leukemia Study Group. In this trial, the intensity of chemotherapy was enforced in the consolidation and reinduction phases by introducing AML-type block therapies consisting of concentrated administration of 4 to 6 drugs during 5 or 6 days. For patients in the higher risk groups, rotational combination chemotherapy was introduced following the early phase. A total of 124 children with B-cell precursor ALL (B-pre ALL) were classified into 3 groups, the ultrahigh-risk group (UHRG) (15 patients), the high-risk group (HRG) (61 patients), or the standard-risk group (SRG) (48 patients), based on age. leukocyte count, immunophenotype, central nervous system leukemia, response to treatment, and selected chromosomal abnormalities. The complete remission rate was 93%, and the 6-year event-free survival (EFS) rate was 79%+/-4%. EFS rates for the UHRG, HRG, and SRG groups were 67%+/-12%, 80%+/-6%, and 81%+/-6%, respectively. Nineteen patients with T-cell ALL were treated with the protocol for the UHRG. Thirteen patients (68%) attained complete remission, and the 6-year EFS rate was 55%+/-12%. Thus, intensification of chemotherapy improved the EFS rate and AML-type block therapies appeared to be effective as the consolidation and reinduction therapies for B-pre ALL.

Splenectomy in haemophagocytic lymphohistiocytosis: report of histopathological changes with CD19+ B-cell depletion and therapeutic results.

The pathogenesis of haemophagocytic lymphohistiocytosis (HLH) in children without a known familial pattern of inheritance is often difficult to establish. Splenic enlargement, one of the main clinical findings in this disorder, has led to the use of splenectomy for uncontrollable coagulopathy, persistent cytopenia or both. This procedure is also thought to be a useful tool in making a differential diagnosis in cases of the immunochemotherapy-resistant HLH. We report here five cases of splenectomized childhood HLH, in which subsets of mononuclear spleen cells were analysed either by flow cytometry or immunohistochemistry, and the results were compared with those from cases of hereditary spherocytosis (controls). There was a statistically significant depletion of CD19+ B cells in the HLH cases (3.8 +/- 3.2% vs. 52.6 +/- 4.5%, P < 0. 0001) associated with an increase of T cells in three cases and of natural killer cells in another. The histopathological findings included atrophic white pulps, B-cell depletion with fibrosis and haemosiderosis in all five cases. Despite temporary therapeutic benefits, three of the HLH patients had a rapidly deteriorating post-splenectomy course and all three eventually died. These results demonstrate striking depletion of B cells in the enlarged spleens of children with HLH, which may be an intrinsic feature of HLH pathogenesis. Further study is needed to establish the therapeutic value of splenectomy in this disease.

Low frequency of the p53 gene mutations in neuroblastoma.

BACKGROUND: The p53 gene frequently is affected by point mutations, rearrangements, or deletions that contribute to the genesis or progression of a wide variety of human adult solid tumors; however, to the authors' knowledge, this gene alteration has not been analyzed in neuroblastoma. METHODS: Genomic DNA samples from 20 children with neuroblastoma, including 16 patients with advanced disease, were screened for the presence of mutations in exons 5-9 of the p53 gene, where over 90% of mutations have been reported to be located in human cancer. The screening technique employed polymerase chain reaction/single-strand conformation polymorphism analysis followed by direct DNA sequencing. RESULTS: Heterozygous mutations were detected in 2 of the 20 cases. A silent mutation (T to G transversion) at codon 172 and a missense mutation (G to T transversion) at codon 259 were found in patients with Stage II and Stage IV disease, respectively. Thus, p53 mutations were found to occur in neuroblastoma, but at a low frequency (2 of 20). CONCLUSIONS: Our data suggest that in a minority of neuroblastomas, p53 gene mutations may play a contributing role in tumorigenesis, but other genes presumably play a major role in this tumor.

Hepatocyte growth factor is constitutively produced by human bone marrow stromal cells and indirectly promotes hematopoiesis.

Bone marrow (BM) stromal cells are required for normal hematopoiesis. A number of soluble factors secreted by these cells that mediate hematopoiesis have been characterized. However, the mechanism of hematopoiesis cannot be explained solely by these known factors, and the existence of other, still unknown stromal factors has been postulated. We showed that hepatocyte growth factor (HGF) is one such cytokine produced by human BM stromal cells. BM stromal cells were shown to constitutively produce HGF and also to express the c-MET/HGF receptor. The production of HGF was enhanced by addition of heparin and phorbol ester. Dexamethasone and tumor growth factor-beta (TGF-beta) inhibited the production of HGF. Interleukin-1 alpha (IL-1 alpha) tumor necrosis factor-alpha (TNF-alpha), and N6,2'-o-dibutyryl-adenosine-3':5'-cyclic monophosphate (dbc-AMP) showed no obvious influence on HGF production. Western blot analysis of HGF derived from BM stromal cells showed two bands at 85 and 28 kD corresponding to native and variant HGF, respectively. Addition of recombinant HGF significantly promoted the formation of burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte erythroid macrophage (CFU-GEM) by BM mononuclear cells in the presence of erythropoietin and granulocyte-macrophage colony-stimulating factor (GM-CSF), but the formation of CFU-GM was not modified. However, HGF had no effects on colony formation by purified CD34+ cells. Within BM mononuclear cells, c-MET was expressed on a proportion of cells (CD34-, CD33+, CD13+, CD14+, and CD15+), but was not found on CD34+ cells. We conclude that HGF is constitutively produced by BM stromal cells and that it enhances hematopoiesis. In addition, expression of c-MET on the stromal cells suggests the presence of an autocrine mechanism, operating through HGF, among stromal cells.

Cytokine production regulating Th1 and Th2 cytokines in hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is caused by the hyperactivation of T cells and macrophages. The clinical characteristics associated with this disease result from overproduction of Th1 cytokines including interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNF-alpha). In this study, we analyzed the production of IL-12 and IL-4, which determine Th1 and Th2 response, respectively, and IL-10, which antagonizes Th1 cytokines, in 11 patients with HLH. IL-12 was detected in plasma in all patients (mean peak value, 30.0 +/- 5.0 pg/mL), while IFN-gamma was massively produced in nine patients (mean peak value, 79.2 +/- 112.0 U/mL). IL-4 was not detected in any of the patients. Plasma IL-10 levels were elevated in all patients (mean peak value, 2,698.0 +/- 3,535.0 pg/mL). There was a positive correlation between the levels of IFN-gamma and IL-10 (P < .01). The plasma concentrations of these cytokines were initially high, before decreasing after the acute phase. However, the decrease in IL-10 levels was slower than that of IFN-gamma. Although the concentration of IL-12 was high at the acute phase, in some patients, a peak in the level was delayed until the chronic phase. Thus, in HLH, production of cytokines that promote development of Th1 cells appears to be predominant over that for Th2 cell development. Overproduction of IL-10 was also observed indicating that a mechanism suppressing hyperactivation of Th1 cells and monocytes/macrophages functions in patients with this disease.