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Most patients with acute myeloid leukemia (AML) enter complete remission (CR) after treatment with chemotherapy, but a large number of them experience relapse with resistant disease. To identify genes that are associated with their prognoses, we analyzed gene expression in 54 pediatric patients with AML using an oligonucleotide microarray that contained 12 566 probe sets. A supervised approach using the Student t test selected a prognostic set of 35 genes, some of which are associated with the regulation of cell cycle and apoptosis. Most of these genes had not previously been reported to be associated with prognosis and were not correlated with morphologically classified French-American-British (FAB) subtypes or with karyotypes. These results indicate the existence of prognosis-associated genes that are independent of cell lineage and cytogenetic abnormalities, and they can provide therapeutic direction for individual risk-adapted therapy for pediatric AML patients.

While studying Ikaros proteins in childhood acute myeloid leukemia (AML), Ikaros isoform 6 (Ik6) expression was detected in 7 of 10 cases of M4 and M5 leukemia, but in none of the remaining French-American-British subtypes (M2, 8 cases; M7, 6 cases). The spliced Ikaros isoforms 4 to 8 (Ik4-8) suppress the function of full-length Ik1 or Ik2 in a dominant-negative manner, owing to their reduced numbers of DNA binding sites. Thus, dominant-negative Ikaros isoforms may inhibit the normal transcriptional regulation of hematopoietic cell development. To clarify the function of Ik6 in developing blood cells, this isoform was transiently transfected into an Ik2(+), interleukin-3 (IL-3)-dependent 32D murine myeloid precursor cell line and studied the expression of Bcl-2 family proteins in relation to in vitro cell growth, using a tetracycline-inducible TREx system. The possibility of aberrant cell regulation due to Ikaros functional changes was examined by cotransfecting both Ik2 and Ik6 into Ikaros/Aiolos/Helios triple-negative Cos-7 cells. The results demonstrated IL-3-independent growth by Ik6-transfected 32D clones coincident with up-regulation of the antiapoptotic protein Bcl-XL. Up-regulation of Bcl-XL, but not of other Bcl-2 family proteins, was associated with the suppression of functional Ik2 by Ik6 in a dominant-negative fashion. Thus, the pathogenesis of myelomonocytic/monocytic AML may involve aberrant regulation of apoptosis due to unscheduled expression of the Ik6 isoform.

This study analyzed data on 35 infants with acute myeloid leukemia (AML) who were treated with intensive chemotherapy between 1995 and 1998 in Japan. The incidence of boys, younger age (< 6 months old), and hyperleukocytosis at onset was high in patients with the M4/M5 subtype (n = 23) in the French-American-British classification, compared with the non-M4/M5 subtype (n = 12). Thirteen (56%) and 16 (70%) patients with the M4/M5 subtype also showed 11q23 translocations and MLL gene rearrangements, respectively, whereas only one patient with the non-M4/M5 subtype had this rearrangement. All 35 patients were treated with the ANLL91 protocol consisting of etoposide, high-dose cytarabine, and anthracyclines. Overall survival and the event-free survival (EFS) rates at 3 years of all patients were 76% (95% confidence interval [CI], 61.3%-90.7%) and 72% (95% CI, 56.4%-87.9%), respectively. EFS showed no significant difference between 2 subgroups divided by age, gender, presence of the MLL gene rearrangements, and white blood cell count at onset; EFS in patients with the M4/M5 subtype tended to be better than those with the non-M4/M5 subtype. Although all 6 patients who underwent allogeneic stem cell transplantation (SCT) have been in complete remission, no benefit of SCT was confirmed. These findings suggest that the intensive chemotherapy with the ANLL91 protocol might have been responsible for the observed good outcome of infant AML, even without SCT. The presence of the MLL gene rearrangements or the age at onset had no impact on the outcome of infant AML.

The hemophagocytic lymphohistiocytoses (HLH) comprise a heterogeneous group of disorders characterized by dysregulated activation of T cells and macrophages. Although some patients with HLH harbor perforin gene mutations, the cause of the remaining cases is not known. The phenotype of HLH bears a strong resemblance to X-linked lymphoproliferative disease (XLP), an Epstein-Barr virus (EBV)-associated immunodeficiency resulting from defects in SH2D1A, a small SH2 domain-containing protein expressed in T lymphocytes and natural killer cells. Here it is shown that 4 of 25 male patients with HLH who were examined harbored germline SH2D1A mutations. Among these 4 patients, only 2 had family histories consistent with XLP. On the basis of these findings, it is suggested that all male patients with EBV-associated hemophagocytosis be screened for mutations in SH2D1A. Patients identified as having XLP should undergo genetic counseling, and be followed long-term for development of lymphoma and hypogammaglobulinemia.

A prospective multicenter trial of 119 children 1 to 18 years of age with newly diagnosed aplastic anemia (AA) was conducted, comparing treatment using antithymocyte globulin (ATG), cyclosporine (CyA), and danazol (DAN) with or without rhG-CSF (400 microg/m(2), day on days 1-90). All children with very severe AA received rhG-CSF (VSAA group, n = 50). The other children were randomized to receive ATG, CyA, DAN, and rhG-CSF (G-CSF+ group, n = 35) or ATG, CyA, and DAN without rhG-CSF (G-CSF- group, n = 34). After 6 months, the hematologic response rate was 71%, 55%, and 77% in the VSAA group, G-CSF+ group, and G-CSF- group, respectively. There was no difference in the incidence of febrile episodes and documented infections between the G-CSF+ and G-CSF- groups. Bone marrow transplantation (BMT) was attempted in 22 patients in whom initial immunosuppressive therapy (IST; n = 18) failed or in whom a relapse occurred after an initial response (n = 4). Nineteen of the 22 patients are alive and well after a median follow-up of 18 months (range, 3 to 66 months) since BMT. The probability of survival at 4 years was 83% +/- 7% in the VSAA group, 91% +/- 5% in the G-CSF+ group, and 93% +/- 6% in the G-CSF- group. Myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) developed in one patient in each of the three groups; the overall risk for MDS/AML was 3% +/- 2% at 4 years. Because the results of IST were encouraging, it is suggested that children with AA receive IST as first-line therapy if there is no human leukocyte antigen-matched sibling donor.

An attractive hypothesis is that in utero exposure of hematopoietic cells to oncogenic agents can induce molecular changes leading to overt acute lymphoblastic leukemia (ALL) in infants and perhaps older children as well. Although supported by studies of identical infant twins with concordant leukemia, and of nontwined patients with MLL gene rearrangements, this concept has not been extended to the larger population of B-lineage ALL patients who lack unique nonconstitutive mutations or abnormally rearranged genes. We therefore sought to demonstrate a prenatal origin for 7 cases of B-cell precursor ALL (either CD10(+) or CD10(-)) that had been diagnosed in infants and children 14 days to 9 years of age. Using a polymerase chain reaction-based assay, we identified the same clonotypic immunoglobulin heavy-chain complementarity determining region or T-cell receptor V(D)2-D(D)3 sequences in the neonatal blood spots (Guthrie card) and leukemic cell DNAs of 2 infants with CD10(-) ALL and 2 of the 5 older patients with CD10(+) ALL. Nucleotide sequencing showed a paucity of N or P regions and shortened D germ line and conserved J sequences, indicative of cells arising from fetal hematopoiesis. Our findings strongly suggest a prenatal origin for some cases of B-cell precursor ALL lacking specific clonotypic abnormalities.

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare but fatal disease in infancy. There are no previous reports on the clonality of T cells in FHL patients. We analyzed here the clonality of alphabeta-T cells in 5 FHL patients using an inverse reverse transcriptase-polymerase chain reaction (RT-PCR) of the T-cell receptor variable region gene (TCR V), a joining region gene of the beta chain (Jbeta)-PCR, a single-strand conformation polymorphism (SSCP), and sequence analysis. A high frequency (15%) of Vbeta and Valpha families was observed in 3 of 5 and 4 of 4 patients examined, respectively. In 19 Vbeta repertoires, including all highly frequent Vbeta, the Jbeta-PCR analysis showed restricted usage of the Jbeta family, indicating a marked bias to Jbeta1 subsets (the mean rate of Jbeta1:Jbeta2 was 87:13 in 65% of the alphabeta-T cells) in widespread alphabeta-T cells (in all patients but 1). In all patients, the clonality of specific Vbeta-Jbeta fragment expanded was confirmed by SSCP and sequence analysis. These results suggest that the existence of clonal expansion and restricted Jbeta1 usage of T cells in FHL is genetically associated with the pathogenesis and the immunodysfunction of the disease. These results help to explain some of the abnormal functional behaviors of T cells in FHL and raise new questions regarding the mechanisms responsible for the restricted clonal diversity.

The familial form of hemophagocytic lymphohistiocytosis (HLH) is a lethal disorder. Although the prognosis for Epstein-Barr virus-associated HLH (EBV-HLH) remains uncertain, numerous reports indicate that it can also be fatal in a substantial proportion of cases. We therefore assessed the potential of immunochemotherapy with a core combination of steroids and etoposide to control EBV-HLH in 17 infants and children who met stringent diagnostic criteria for this reactive disorder of the mononuclear phagocyte system. Treatment of life-threatening emergencies was left to the discretion of participating investigators and typically included either intravenous Ig or cyclosporin A (CSA). Five patients (29%) entered complete remission during the induction phase (1 to 2 months), whereas 10 others (57%) required additional treatment to achieve this status. In 2 cases, immunochemotherapy was ineffective, prompting allogeneic bone marrow transplantation. Severe but reversible myelosuppression was a common finding; adverse late sequelae were limited to epileptic activity in one child and chronic EBV infection in 2 others. Fourteen of the 17 patients treated with immunochemotherapy have maintained their complete responses for 4+ to 39+ months (median, 15+ months), suggesting a low probability of disease recurrence. These results provide a new perspective on EBV-HLH, showing effective control (and perhaps cure) of the majority of EBV-HLH cases without bone marrow transplantation, using steroids and etoposide, with or without immunomodulatory agents.

Activated ras genes are often associated with human myeloid leukemias with a monocytic differentiated phenotype. To investigate the role of the activated ras gene in leukemogenesis, a myeloid nontumorigenic cell line (FDC-P1) was infected with a selectable retroviral vector carrying the v-Ha-ras gene (H1neo). Infected FDC-P1 cells were not only tumorigenic, but also showed increased monocytic differentiation in vitro. Monocytic differentiation and tumorigenicity in vivo were correlated with several-fold increased levels of activated ras gene expression. Tumorigenic cells were arrested with respect to monocytic marker expression at a much later stage of macrophage differentiation than the parental noninfected FDC-P1 cells. These data thus suggest a model of how activated ras genes could be involved in the preferential induction of hematopoietic malignancies with a myelomonocytic phenotype.