Emergence of genetic instability in children treated for leukemia.

T cells from patients who had received chemotherapy for B-lineage acute lymphocytic leukemia were studied to determine whether genetic instability, a principal characteristic of cancer cells, can also occur in nonmalignant cells. Consistent with expectations for a genetic instability phenotype, multiple mutations were detected in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) reporter gene in independently isolated mutant T cells expressing identical rearranged T cell receptor beta (TCRbeta) gene hypervariable regions. These results indicate that cancer treatment can lead to genetic instability in nonmalignant cells in some individuals. They also suggest a mechanistic paradigm for the induction of second malignancies and drug resistance.

Effect of L-asparaginase administration on coagulation and platelet function in children with leukemia.

The use of L-asparaginase during remission induction in patients with leukemia is associated with coagulation abnormalities, which may present either as thrombosis or hemorrhage. However, because of the multiple pharmacologic and hematologic variables present in these patients, the exact contribution of L-asparaginase to these coagulation abnormalities is unclear. We studied platelet function and plasma coagulation parameters in 12 pediatric patients with acute lymphoblastic leukemia (ALL) receiving daily L-asparaginase as a single agent when in complete remission. Changes in the prothrombin time (PT), partial thromboplastin time (PTT), and fibrinogen, while statistically significant, remained within or close to the normal range during the study. Platelet function also remained normal during the study. In contrast, levels of protein C antigen decreased to a mean of 42%, a significant change from pretreatment values. Levels of antithrombin III (AT III) were likewise depressed to 15 mg/dL (34% of pretreatment value). Despite these changes in the levels of physiologic inhibitors of coagulation, this schedule of L-asparaginase administration was associated with only rare clinical thrombosis, and this study suggests that the development of this complication may be dependent on the presence of additional factors.

Accumulation of somatic mutations in proliferating T cell clones from children treated for leukemia.

There is continued controversy as to the sequential steps and mechanism(s) responsible for the in vivo acquisition of multiple mutations during neoplastic transformation. We investigated the in vivo clonality and mutational spectra of hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations in T cells from children with acute lymphocytic leukemia (ALL) to gain insight into the mutagenic mechanisms associated with leukemogenesis. We observed several instances of multiple, independent HPRT mutations accumulating in vivo in T cell receptor (TCR) gene defined clones that had undergone extensive pre- and/or post-thymic expansion following chemotherapy. In addition, we also detected the accumulation of multiple unique single mutations within distinct expanding post-thymic T cell clones. This pattern of clonally restricted hypermutability is compatible with extensive cell proliferation and selection alone without postulating genomic instability. These observations provide a paradigm for a continuum of cellular events that eventually results in the clonal accumulation of mutations in selected populations of cells in vivo and may provide insight into the primary genetic events associated with leukemogenesis, as well as the development of second malignancies and drug resistance following chemotherapy.

A method for the detection of multiple surface antigens on small heterogeneous cell populations.

A technic using fluorescent immunospheres was developed to identify simultaneously two surface antigens on individual lymphocytes while preserving Wright's stained cell morphology. Small numbers (10,000-20,000) of cells were studied from peripheral blood or bone marrow samples from normal control subjects and from patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). Samples were studied for surface antigens using OKT-11, OK-Ia-1, B1, and J5. Comparison was made with results obtained from the same patients by indirect immunofluorescence. Correlation between results obtained with immunospheres and indirect immunofluorescence was excellent (r = 0.97). In addition, 35 cerebrospinal fluid samples from children with ALL were tested using the immunosphere method alone. Results obtained with spinal fluid lymphocytes agreed with previously reported results using similar methodology. It is concluded that the use of fluorescent microspheres provides a method for the combined evaluation of cell morphology and surface antigens on small, heterogeneous cell populations.

Atypical background somatic mutant frequencies at the HPRT locus in children and adults with Down syndrome.

People with Down syndrome are 10-30 fold more likely to develop leukemia than the normal population. To date, little is known regarding the molecular mechanisms underlying this phenomenon. We have previously demonstrated that the spontaneous somatic mutant frequency (Mf) at a reporter gene, hypoxanthine-guanine phosphoribosyl transferase (HPRT), from a normal population showed a strict age dependency with an exponential increase in Mf from birth to late adolescents with a subsequent linear 2-5% increase per year in adults. In this study, we compared HPRT Mf in children and adults with Down syndrome using the HPRT T-cell cloning assay. We determined the Mf at the HPRT locus in 27 subjects with Down syndrome from ages 6 months to 53.4 years. Results demonstrated that background somatic Mf at the HPRT locus in children and adults with Down syndrome are not dependent on age as seen in a normal control population. Results also show that adults with Down syndrome have a significantly lower Mf than normal adults, and that children with Down syndrome have a significantly higher Mf than normal children, although the latter appears to be due to a decreased cloning efficiency (CE). These observations demonstrate that the frequency of spontaneous somatic mutations in children and adults with Down syndrome are atypical compared to normal controls, and suggest that the genetic mechanisms associated with background somatic mutational events in children and adults with Down syndrome may be different.

A solid phase urease-linked cellular immunosorbent assay for circulating polymorphonuclear binding immunoglobulin.

A cellular enzyme linked immunosorbent assay (CELISA) is reported for the detection of circulating polymorphonuclear granulocyte binding immunoglobulin (PBG) in patients' sera. The assay features a solid phase microtiter method in which the enzyme urease is fixed to the antihuman globulin conjugate reagent and uses 0.25 percent glutaraldehyde fixed normal human polymorphonuclear neutrophils (PMNs) as target cells. The assay gave positive results in four of 13 (31 percent) cases of idiopathic neutropenia in which an autoimmune etiology was suspected and one typical case of isoimmune neonatal neutropenia. In a group of 15 patients receiving multiple blood transfusions for chronic anemia, five (33 percent) showed significantly higher levels (p less than 0.001) of PBG than non-transfused normal donors. The PBG-CELISA appears potentially useful for the detection of autoimmune and isoimmune PMN antibodies and PMN binding IgG immune complexes.

An unusual central nervous system infection in a young immunocompromised host.

A 13-year-old girl with a ten-year history of lymphoblastic leukemia and several central nervous system (CNS) relapses developed a bone marrow relapse and accelerated CNS leukemia. Following treatment with CNS radiation and intravenous chemotherapy, she developed fever, pancytopenia, headache, and vomiting. Her neurological function deteriorated and she died on the 20th hospital day. Multiple CSF examinations failed to disclose either leukemic cells or organisms. Blood cultures obtained from a Broviac catheter yielded Micrococcus species. Postmortem examination showed meningoependymitis with intracellular coccal organisms. The pathology of this infection resembles intracranial Whipple's disease. Intracranial intracellular bacterial infection should be excluded in the infectious complications in the immunocompromised host.

Transient abnormal myelopoiesis of infancy associated with trisomy 21.

PURPOSE: A unique myelodysplastic syndrome referred to as transient abnormal myelopoiesis (TAM) has been reported to occur primarily in infants with Down's syndrome (DS) or other abnormalities of chromosome 21. This disorder raises basic questions regarding the pathogenesis of leukemia, yet its natural history is poorly documented and derives from small series and isolated case reports. PATIENTS AND METHODS: To better characterize TAM, we accumulated data on 35 cases identified through a questionnaire mailed to pediatric oncologists in the United States. These cases, pooled with two that we recently encountered, and 58 comparable cases reported in the literature comprise a series of 95 cases of TAM in DS. RESULTS: The patients in this series were notable for the high morbidity and mortality of this reportedly benign condition. Eleven percent of the patients died during the initial event, and the overall mortality for the entire series was 27%. Twenty-eight of the 85 patients (33%) who survived the initial event developed a subsequent hematologic disorder, most often acute nonlymphocytic leukemia, at a median age of 16 months. CONCLUSIONS: No initial clinical or hematologic features predicted the development of a subsequent hematologic disorder. However, those patients initially mosaic for the presence of trisomy 21 did not develop subsequent abnormalities. This series reviews questions regarding leukemogenesis in DS and underscores the importance of conducting future prospective studies of this unique hematologic disorder.

Defective megakaryocytopoiesis in the syndrome of thrombocytopenia with absent radii.

The syndrome of thrombocytopenia with absent radii (TAR) is a hereditary condition whose pathogenesis is poorly understood. In this investigation we evaluated a female infant with TAR and her parents using in vitro haematopoietic colony forming assays and an antiserum against platelet membrane glycoproteins (PGP) to label smears of her bone marrow. Megakaryocyte colony growth in vitro was virtually absent in optimally stimulated cultures of the patient's bone marrow progenitors. In contrast, erythroid and myeloid colony growth from the TAR infant's marrow cells was preserved. Staining of the patient's bone marrow smears with PGP antiserum detected no immature, small megakaryocyte precursors. A high level of megakaryocyte colony stimulating activity was detected in serum from the TAR infant, activity comparable to that present in sera from adults with aplastic anaemia. The elevated serum activity decreased by 6 months of age at which time partial platelet recovery had occurred. Evaluation of both peripheral blood haematopoietic progenitor cells and sera from the TAR infant's parents demonstrated no significant abnormalities. We conclude that the principle haematopoietic defect in this patient with TAR syndrome is the absence or arrested development of the committed megakaryocyte progenitor cell. Humoral regulation of megakaryocytopoiesis appears intact and is responsive to the degree of megakaryocytic hypoplasia.

Use of monoclonal antibodies to identify cerebrospinal fluid lymphoblasts in children with acute lymphoblastic leukemia.

The identification of small numbers of leukemic cells in the cerebrospinal fluid (CSF) presents a diagnostic problem in the treatment of children with acute lymphoblastic leukemia (ALL). We adapted a latex sphere rosetting technique to allow us to identify simultaneously cell surface markers and cell morphology in 199 CSF samples from 34 patients and 14 control subjects. In patients without leukemic meningitis, the majority of CSF lymphocytes (69%) were found to be mature T cells positive for OKT11. A much smaller number of cells (8%) were found to be B cells positive for la. In these children, only 3% of CSF lymphoid cells expressed the common acute lymphoblastic leukemia antigen (CALLA). Similar results were found in the control subjects. By contrast, 28 CSF samples from nine children with varying numbers of CSF lymphoblasts had much greater proportions of CALLA- and la-positive CSF cells (24% to 96%). Leukemic meningitis was present in one of these patients and later developed in four others. However, three patients with small numbers of lymphoblasts present but with low proportions of CALLA-positive CSF cells (less than 5%) subsequently had normal CSF examinations. We found the use of this rosetting technique valuable in providing information complementary to that obtained from cell morphology alone about the possible malignant nature of small numbers of lymphoblast-like CSF cells seen on cytocentrifuge preparations in children with ALL.

Adult-type CML in childhood: case report and review.

The clinical, hematologic, and cytogenetic features of ACML in children appear to be identical to Ph1-positive CML seen in adults. From our review of the literature, one could anticipate that a child with this condition would have a response to therapy and an anticipated survival similar to that seen in adults. This situation is quite different when one compares adults with ALL to children with the same disease. It has been suggested that Ph1-positive CML is an acquired, postzygotic abnormality induced by environmental agents. It is difficult to reconcile this hypothesis with the fact that this condition can be seen in infants as young as 5 months of age and the general belief that environmental carcinogens take many years to produce malignant changes in cells. Ph1-positive CML has been associated with atomic bomb exposure and it is of interest to note that two patients in the present series had received radiation. For both children and adults, bone marrow transplantation during the chronic phase is the most successful therapy if a suitable donor is available. Recently, successful marrow transplantation during the accelerated phase has also been reported. For patients without a suitable donor, control of the disease with either busulfan or hydroxyurea and attempts to induce a remission with chemotherapy during the accelerated or blast phase is the best current alternative. For patients whose blasts have lymphoid characteristics such as TdT activity, vincristine and prednisone may be successful. For those patients with a myeloid or mixed lymphoid-myeloid transformation, no chemotherapy regimen has been successful. An aggressive approach such as that described by Weinstein et al. for the treatment of acute nonlymphocytic leukemia might prove beneficial.

Aplastic presentation of acute lymphoblastic leukemia: evidence for cellular inhibition of normal hematopoietic progenitors.

Childhood acute lymphoblastic leukemia (ALL) may rarely present with blood and bone marrow findings suggestive of aplastic anemia. Although numerous examples of ALL presenting with this phenomenon have been reported, there is no accepted explanation for the pathogenesis of this preleukemic hypoplasia. We report a case of a child with ALL whose initial presentation was characterized by pancytopenia and bone marrow hypoplasia and who had repeated episodes of pancytopenia at times of systemic relapse. In vitro coculture experiments demonstrated that the leukemic cells from this patient were inhibitory for the growth of myeloid, erythroid, and megakaryocytic progenitor cells from normal peripheral blood. This inhibitory effect exhibited a dose-dependent relationship with the number of added lymphoblasts and persisted when the lymphoblasts were irradiated to prevent leukemic cell growth. Inhibitory activity was not present in media conditioned by the growth of the patient's lymphoblasts, nor was it present in lymphoblasts from three other children with ALL with similar immunophenotype but without marrow aplasia. These data suggest that the aplastic presentation of ALL may be attributable to inhibitory properties intrinsic to the leukemic cells rather than to other host factors.

Immunophenotypic characteristics of cerebrospinal fluid cells in children with acute lymphoblastic leukemia at diagnosis.

The presence of meningeal involvement in children with acute lymphoblastic leukemia (ALL) may have important prognostic and therapeutic implications. Conventional methods of diagnosing central nervous system (CNS) leukemia rely on the interpretation of cerebrospinal fluid (CSF) cell morphology, which may produce ambiguous results in the presence of minimal leukemic involvement. A methodology has been developed for immunophenotyping small numbers of CSF cells while preserving cell morphology. CSF samples from 33 children with CD10 (common ALL antigen [CALLA]) positive ALL were examined at initial presentation using both conventional morphology and this combined immunohistopathologic technique. Six (18%) of the samples contained lymphoblasts or cells considered morphologically suspicious for leukemic involvement. Nine additional samples (27% of the total) had normal CSF morphology, but contained increased numbers of CALLA positive cells. Twelve of the 33 samples were also examined for the simultaneous presence of nuclear terminal deoxynucleotidyl transferase (TdT) and demonstrated increased numbers of cells positive for both TdT and CD10. These data suggest that a large proportion of children with ALL may have abnormalities of CSF cells at initial diagnosis consistent with the presence of occult leukemic involvement.

Ifosfamide/etoposide combination in the treatment of recurrent malignant solid tumors of childhood. A Pediatric Oncology Group Phase II study.

BACKGROUND: The prognosis for children with recurrent or resistant malignant solid tumors remains dismal. More effective rescue therapy is needed for these children. METHODS: Between August 1987 and November 1990, 311 children with recurrent or resistant malignant solid tumors were treated by investigators in the Pediatric Oncology Group with intravenous infusions of 2.0 g/m2 of ifosfamide and 100 mg/m2 of etoposide (VP-16) plus mesna as uroprotection three times daily, with courses being repeated every 14-21 days for as long as the patients responded to therapy. RESULTS: Seventy-four percent of the 294 assessable patients entered in the study had metastatic disease and previously had been treated heavily. The complete response/partial response rate was 30%, and the overall response rate was 39.5%. Toxic effects included nephrotoxicity, mild liver dysfunction, neurotoxicity, and myelosuppression. Sixty-eight percent had an absolute neutrophil count (ANC) of less than 500/microliters. In 1606 courses of therapy administered, only 3.6% of patients developed a bacterial infection. Only two patients died of gram-negative sepsis. Four percent of the patients had gross hematuria (> 50 erythrocytes/high-power field), and 18.5% had microscopic hematuria (< 20 erythrocytes/high-power field). Fanconi syndrome developed in eight children. CONCLUSIONS: Ifosfamide/VP-16 is an active combination in children with recurrent malignant solid tumors. Although it was myelosuppressive, the incidence of infection was quite low (3.6%). Mesna was very effective in preventing the development of hematuria.