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.

Allogeneic hematopoietic transplantation of CD34+ selected cells from an HLA haplo-identical related donor. A long-term follow-up of 135 patients and a comparison of stem cell source between the bone marrow and the peripheral blood.

We studied the outcome of allogeneic transplants in 135 patients who received selected BM and/or PBSC CD34+ cells from HLA haplo-identical related donors. Donor engraftment was achieved in 108 of 128 evaluable transplants. Engraftment failure occurred more often in non-malignant than in malignant diseases (10 of 25 vs 17 of 103, P = 0.010). The CD34+ cell dose was associated with the speed of neutrophil and platelet recovery, but the cell source was not. Acute GVHD (> or = grade II) developed in 21.0 +/- 3.7%. Chronic GVHD occurred more frequently in malignancies than in non-malignancies (44.1 +/- 7.6% vs 0.0%, P = 0.0075), and more in PBSC recipients than in BM recipients (53.6 +/- 9.4% vs 17.4 +/- 9.3%, P = 0.0054). Relapse rate was higher in high risk patients than in standard risk patients (78.7 +/- 7.1% vs 22.1 +/- 10.0%, P = 0.0001). Probabilities of disease-free survival (DFS) were 14.2 +/- 3.5% in malignancies and 25.7 +/- 9.2% in non-malignancies. Probabilities of DFS in standard and high risk patients were 39.4 +/- 9.2% and 5.7 +/- 2.8% (P = 0.0001). A high incidence of graft failure, infection and relapse was observed and resulted in high mortality.

Juvenile myelomonocytic leukemia relapsing after allogeneic bone marrow transplantation successfully treated with interferon-alpha.

We report a 5-year-old boy with juvenile myelomonocytic leukemia (JMML) which relapsed after an allogeneic bone marrow transplant who was successfully treated with interferon-alpha (IFN-alpha). One year after starting the therapy, he remains clinically well and in complete remission while continuing treatment with IFN-alpha and bestatin. Although the precise mechanism by which remission was induced is uncertain, a GVL effect combined with a direct antileukemia effect of IFN-alpha may be responsible. Further assessment of the role of IFN-alpha in relapsed JMLL patients is warranted.

Allogeneic peripheral stem cell transplantation using positively selected CD34+ cells from HLA-mismatched donors.

We examined five children who underwent allogeneic peripheral stem cell transplantation (PSCT) using positively selected CD34+ cells from three or two loci-mismatched donors. CD34+ cells mobilized from peripheral blood were separated by immunomagnetic beads. CD34+ cells at 2.2-6.2 x 10(6)/kg were transplanted into three patients with refractory leukemia, a patient with relapsed medulloblastoma and a patient with Fanconi's anemia following a conditioning regimen which included irradiation, alkylating agents and antithymocyte globulin treatment. The number of infused CD3+ cells included in grafts was 2.3-22.7 x 10(4)/kg. Four patients achieved engraftment and hematopoietic reconstitution (> 5 x 10(8)/l of neutrophils on day 10 or 11). Graft rejection was observed in the patient with Fanconi's anemia, but a rapid engraftment was obtained after second PSCT. Although no prophylactic agents other than ATG (included in the conditioning regimen) were used, greater than grade I acute GVHD was not observed, but limited chronic GVHD was observed in two patients. The two patients with leukemia relapsed on days 103 and 210, respectively, and the patient with medulloblastoma died of disease on day 159. The patient with Fanconi's anemia died of fungal infection. CMV and HHV-6 diseases developed in four and two patients, respectively. Thus, although SCT using positively selected peripheral CD34+ cells may be an alternative approach for overcoming graft rejection and GVHD from HLA- mismatched donors, persistent immune deficiency attributing to extremely low numbers of T cells in grafts can potentially lead to reactivation of herpes viruses.

Thrombotic microangiopathy associated with reactivation of human herpesvirus-6 following high-dose chemotherapy with autologous bone marrow transplantation in young children.

Thrombotic microangiopathy (TMA) is a serious complication of BMT. Several factors are important in the etiology of TMA, such as cyclosporin A, GVHD, irradiation, intensive conditioning chemotherapy and infection, which cause damage to vascular endothelial cells leading to activation of these cells. We describe two young children with TMA following high-dose chemotherapy with autologous BMT. Development of TMA was accompanied by reactivation of HHV-6, which was identified by both an increase in the copy number of HHV-6 DNA in the peripheral blood and a significant increase in antibody titers to HHV-6. Thus, it was suggested that reactivation of HHV-6 together with high-dose chemotherapy played an important role in the pathogenesis of TMA in these patients. Since HHV-6 is known to infect vascular endothelial cells, and CMV which is virologically closely related to HHV-6, has been reported to be a pathogen that causes TMA, infection with HHV-6 of vascular endothelial cells may induce TMA via damage and activation of these cells.

Double-conditioning regimens consisting of thiotepa, melphalan and busulfan with stem cell rescue for the treatment of pediatric solid tumors.

Major dose-limiting factors of high-dose thiotepa (TEPA) and melphalan are life-threatening mucositis and neurotoxicity. To administer a maximum dose of these drugs safely and to obtain a maximum anti-cancer effect, a double-conditioning regimen with a single grafting, two cycles of administration of a combination of TEPA (300-600 mg/m2) plus melphalan (70-150 mg/m2) with a 1-week interval was attempted in 20 patients with pediatric advanced or chemotherapy-resistant solid tumors (seven rhabdomyosarcoma, four hepatoblastoma, three neuroblastoma and four other malignancy). Combinations of TEPA plus melphalan/busulfan (Bu) (8-10 mg/kg) and TEPA plus Bu were given to four and two patients with brain tumors, respectively. In an additional two patients, three cycles of drug administration were performed. According to the results of the dose-escalating study, the maximum tolerable doses of TEPA and melphalan for children aged 2 years old or older were 1000 mg/m2 and 280 mg/m2, respectively. Mucositis was dose-limiting. Renal toxicity was also dose-limiting in young children (<2 years old). There were two treatment-related deaths (7%) (fungal pneumonia and renal tubular acidosis). Among 13 patients who received high-dose chemotherapy during CR, 10 are alive with no evidence of disease (15-59 months, median: 35 months) and in 13 evaluable patients without CR, six are alive without regrowth of the disease (14-59 months, median: 39 months). Thus, these novel conditioning regimens allowed us to increase the dose intensity to nearly the maximum for each drug and seemed to reduce adverse effects compared to previously reported regimens with these drugs. With regard to the effect on outcome, the results of this study seem to be encouraging, but a further study on a larger number of patients is required.

Serum levels of soluble adhesion molecules in stem cell transplantation-related complications.

To assess the involvement of vascular endothelial cell activation and damage in stem cell transplantation (SCT)-related complications, such as acute and chronic GVHD and thrombotic microangiopathy (TMA), we investigated the changes in serum levels of soluble forms of vascular cell adhesion molecule-1 (sVCAM-1) and E-selectin (sE-selectin) in SCT. The soluble form of intercellular adhesion molecule-1 (sICAM-1) was also analyzed. In patients with acute GVHD (grades II-IV), serum levels of sE-selectin and sICAM-1 increased around onset of GVHD (day 30). While the increase of sE-selectin levels was transient, sICAM-1 levels remained high until day 60. In patients with extensive chronic GVHD, sVCAM-1 as well as sE-selectin levels significantly increased. The appearance of clinical symptoms was preceded by elevations of sVCAM-1 and sE-selectin levels on day 60, and sICAM-1 levels on days 30 and 60. For the analysis of TMA, to exclude the influence of GVHD, serum levels were measured in auto-SCT patients. Around the onset of TMA, sVCAM-1 and sE-selectin levels significantly increased in patients with TMA without an increase of sICAM-1 levels. These findings support the notion that activation and injury of endothelium are commonly involved in the pathogenesis of acute and chronic GVHD and TMA.

Inverse relationship between human herpesvirus-6 and -7 detection after allogeneic and autologous stem cell transplantation.

Human herpesvirus-6 (HHV-6) and -7 were analyzed in 25 and 18 patients with allogeneic (allo) and autologous (auto) stem cell transplantation (SCT), respectively, by weekly examination of viral DNA in peripheral mononuclear cells using semiquantitative PCR and serologic tests up to 12 weeks after SCT. HHV-6 DNA was detected in 29.6% and 27.9% of samples after allo- and auto-SCT, respectively. The proportions of HHV-6-DNA-positive samples increased in week 3 and 4 after allo-SCT, and in week 1 to 3 after auto-SCT. The frequency of HHV-7 DNA detection, however, was higher after auto-SCT (24.7%) than allo-SCT (12.8%) (P 10(2) copies of HHV-6 DNA (/10(5) cells) on two consecutive occasions were allo-SCT recipients and three showed clinical episodes. Conversely, three of five patients with continuous reactivation of HHV-7 were auto-SCT recipients. Thus, the frequencies of HHV-6 and -7 DNA detection showed an inverse relationship comparing allo- and auto-SCT, suggesting a different mechanism may regulate HHV-6 and -7 reactivation.

Ganciclovir is effective for prophylaxis and treatment of human herpesvirus-6 in allogeneic stem cell transplantation.

Human herpesvirus 6 (HHV-6) infection and disease are serious complications of allogeneic hematopoietic stem cell transplantation (allo-SCT). Ganciclovir (GCV) is effective against HHV-6 in vitro but the antiviral susceptibility of HHV-6 has not been well characterized in vivo. We retrospectively compared the HHV-6 reactivation rate in pediatric allo-SCT recipients with and without GCV prophylaxis. The HHV-6 reactivation rate at 3 weeks after allo-SCT in patients without prophylactic GCV administration was significantly higher than that in those receiving prophylactic GCV (11/28 vs 0/13, P < 0.01). Five of 36 patients without prophylactic GCV showed clinical manifestations including skin rash, interstitial pneumonitis, persistent thrombocytopenia, enterocolitis and thrombotic microangiopathy, respectively. HHV-6-associated symptoms were observed in one of the 13 patients receiving prophylactic GCV. This patient showed fever, diarrhea and graft rejection concomitantly with a sudden increase of HHV-6 DNA copy number. Patients who received GCV for treatment of HHV-6 infection showed an improvement in symptoms and/or decrease of HHV-6 copy number. Thus, GCV is effective for treating HHV-6 disease after allo-SCT in vivo.

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.

Second transplantation with CD34+ blood cells from an HLA-mismatched related donor after engraftment failure of transplanted cord blood cells.

Unrelated cord blood transplantation (CBT) has been worldwide for bone marrow reconstitution. CBT is associated with a high frequency of engraftment failure and rejection due to a small dose of graft cells. In cases of engraftment failure or rejection following unrelated CBT, retransplantation from the original donors is impossible. We report a successful transplantation with CD34+ blood cells selected from a 2-loci HLA-mismatched mother to a child with acute monocytic leukemia after engraftment failure of the primary CBT. Selected CD34+ blood cell transplantation is a useful approach for retransplantation in the setting of engraftment failure.

p53 expression in various stages of 4-nitroquinoline 1-oxide induced carcinoma in the rat tongue.

p53 expression in rat tongue epithelium was investigated immunohistochemically following experimental carcinogenic induction by 4-nitroquinoline 1-oxide (4NQO). p53 assays were performed using PAb240 anti-p53 monoclonal antibody during various stages of experimental carcinogenesis. I found that p53-positive epithelial cells increased in direct proportion to development of the tumor. Significant increases were observed during the periods from control to 4 weeks and from 8 to 12 weeks after 4NQO administration. Although no histological changes were observed in the earlier period, hyperplasia and dysplasia were observed in later stages. Mutant type p53 was detected by immunoprecipitation at 16 and 20 weeks after administration of 4NQO. Results suggested that epithelial p53 detected prior to onset of histological changes was wild-type, while that in the hyperplastic or dysplastic epithelium was mutant.

Correlation of lining thickness and expression of alpha 2 and alpha 3 integrins within the epithelial lining of odontogenic cysts.

Using monoclonal antibodies, we performed immunohistochemical investigations of the expression of alpha 2, alpha 3 and beta 4 integrin subunits within the squamous epithelial linings of odontogenic cysts. Tissue samples consisted of both follicular cysts and odontogenic keratocysts from 15 patients. It was found that beta 4 integrin was expressed on the basement membrane regardless of the histological type of the cyst. The degree of immunostaining for alpha 2 and alpha 3 integrin expression corresponded to the thickness of the epithelial cyst wall. We found that the thickness of the epithelial lining of odontogenic cysts had a direct correlation with the expression of integrin molecules.

Human soluble IL-6 receptor: its detection and enhanced release by HIV infection.

By using a fluorescence sandwich ELISA for the quantification of soluble human IL-6R, normal human PBMC were found to be induced to release IL-6R into culture supernatant by stimulation with PHA. Furthermore, certain promonocyte cell lines and human T-cell leukemia virus I (HTLV-I)-positive cell lines produced sIL-6R into culture supernatants constitutively. However, this was not found with HTLV-I negative T cell lines and Burkitt's B cell line. In addition, generation of supernatant IL-6R of the promonocyte cell line was significantly increased 27-fold after PMA treatment and sevenfold after infection with HIV. The released IL-6R molecules were characterized as an apparent m.w. of 50 to 55 kDa by both size-exclusion HPLC and immunoprecipitation of the soluble protein with IL-6R-specific mAb followed by SDS-PAGE analysis. Furthermore, increased levels of serum IL-6R were detected in blood donors seropositive for HIV. Moreover, the released IL-6R could bind efficiently to purified rIL-6 on solid phase and suppressed the proliferative responses of PBMC. These results suggest that the release of soluble IL-6R might be linked to regulatory functions of immune responses induced by IL-6 stimulation during normal and human retrovirus-infected cell growth and differentiation.

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.

Age-related changes in surface antigens on peripheral lymphocytes of healthy children.

The age-related changes in proportion of various subsets within lymphocytes were investigated in cord blood and peripheral blood from healthy children and adults. The percentages of T and B cells did not show age-related changes, whereas natural killer (NK) cells increased significantly with age. Within lymphocytes or the CD3+ T cell population the proportion of CD45RAbright+ lymphocytes decreased and that of CD45RO+ cells increased, while that of CD45RAdim+ cells showed no age-related change. Within lymphocytes, the percentage of CD45RAbright+ CD4+ cells decreased, together with a decline of that of CD4+ cells. The proportions of CD45RAbright+ CD8+ cells and S6F1bright+ CD8+ cells increased with age, and the age-dependent increase of the proportion of CD8+ cells seems to be mainly attributable to the increases in these subsets. The CD45RAdim+ CD4+ and CD45RAdim+ CD8+ cells co-expressing CD45RO at a low level nevertheless showed no age-related changes. In gamma delta T cells, both delta TCS1+ and delta TCS1- T cells increased with age, but the delta TCS1- gamma delta T cells increased more than the delta TCS1+ subset. Among lymphocytes, the percentages of CD20+, CD21+ and CD22+ cells remained similar, with no age-related changes, but the proportion of CD5+ cells within lymphocytes or B cells decreased. The proportions of CD16+ NK cells among lymphocytes increased with age, and this change was attributable to the increase of CD56+ cells.

Mixed phenotype of blasts in acute megakaryocytic leukaemia and transient abnormal myelopoiesis in Down's syndrome.

Blasts from eight cases with acute megakaryoblastic leukaemia (AMKL) and seven with transient abnormal myelopoiesis in Down's syndrome (TAM) were investigated to clarify their phenotypic characteristics. CD41 and CD7 were the most frequently expressed in both disorders. CD41 was positive in six TAM and five AMKL cases, and CD7 was positive in five TAM and five AMKL cases, respectively. CD33 was detected in four TAM and five AMKL cases. Other myeloid-lineage associated antigens such as CD13 and CD11b could not be found in TAM but were expressed in five AMKL cases. Interestingly, CD56, a neural adhesion molecule, was expressed in three of four TAM and one of five AMKL cases. Cytoplasmic CD3 antigen was also noted in three of five examined cases. A short-term culture study was conducted on blasts from two TAM cases and five AMKL cases. In two cases in which CD41 was not expressed before culture, the expression of CD41 was enhanced after culture with or without 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The expression of CD7 remarkably was depressed, while that of CD13 was enhanced after culture with TPA. These findings suggest that blasts of TAM and AMKL originate from very immature cells and represent a mixed phenotype. In the present study, distinction of phenotypical differences between blast in TAM and AMKL was not possible.