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.

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.

Incidence and prognostic significance of MDM2 oncoprotein overexpression in relapsed childhood acute lymphoblastic leukemia.

MDM2 overexpression by pediatric ALL cells at initial diagnosis has been linked to poor response to therapy. In the present study, we evaluated the incidence of MDM2 overexpression by ALL cells from pediatric patients at first relapse and compared MDM2 protein levels with in vitro response to adriamycin and with duration of initial complete remission (CR1). Since an important role of MDM2 in enhancing cell proliferation and survival appears to be inhibition of p53 activity, we also evaluated the status of p53 in these patients' leukemic cells. MDM2 protein levels were determined by Western blot analysis of leukemic bone marrow cells obtained from 42 patients with B cell precursor (BCP) ALL who relapsed during or following therapy on standard POG ALL protocols. Twelve of 42 (29%) cases have MDM2 levels >/=10-fold higher than those detected in normal bone marrow mononuclear (NMMC) cells, which express relatively low levels of protein. Thirty cases (71%) expressed MDM2 at levels <10-fold those in NMMC, including 24 MDM2-negative cases (57%). P53 mutations were detected by single-strand conformation polymorphism analysis in two cases. Overexpression of MDM2 (>/=10-fold) was significantly correlated with adriamycin resistance and decreased duration of CR1. Eight of 12 (75%) overexpressers showed high levels of in vitro resistance to adriamycin, compared to four of 30 (13%) non-overexpressers (P < 0.005). The median CR1 for MDM2 overexpressers was 20.5 months (range: 3-75 months) compared to 41 months (range: 8-98 months) for non-overexpressers (P < 0.01). Four of 42 patients failed to achieve CR following re-induction: leukemic cells from three of these patients either overexpressed MDM2 or contained a mutant p53. These results indicate that overexpression of MDM2 plays a significant role in refractory pediatric ALL and is associated with early relapse, adriamycin resistance, and failure to respond to re-induction therapy. Leukemia (2000) 14, 61-67.

Phase I/II trial of PIXY321 (granulocyte-macrophage colony stimulating factor/interleukin-3 fusion protein) for treatment of inherited and acquired marrow failure syndromes.

Fourteen paediatric patients with advanced amegakaryocytic thrombocytopenia (AMT) or other bone marrow (BM) failure syndromes were enrolled on one of two phase I/II dose escalation studies of PIXY321. PIXY321 was administered subcutaneously in doses ranging from 250 to 750 mg/m2/d. No dose-limiting toxicity was observed. Peak absolute neutrophil count (ANC) was higher than baseline in all patients. Most transfusion-independent patients demonstrated elevation in haematocrit and/or platelet count. Trilineage haemopoietic responsiveness was evident in the three transfusion-independent patients. In these paediatric populations PIXY321 is well tolerated and merits consideration as a potential therapy.

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.

Thrombocytopenia in the neonate.

Pediatricians caring for newborns will eventually be confronted with the problem of thrombocytopenia in the neonatal period. Familiarity with the differential diagnosis of neonatal thrombocytopenia and understanding the pathogenesis of the more common entities allows physicians to design a selective diagnostic and therapeutic plan to benefit these thrombocytopenic infants.

A pilot study of isotretinoin in the treatment of juvenile chronic myelogenous leukemia.

BACKGROUND: Juvenile chronic myelogenous leukemia (CML) is a rare myeloproliferative disease of infants and young children for which there is no effective therapy other than allogeneic bone marrow transplantation. In vitro, isotretinoin (13-cis-retinoic acid) attenuates both the spontaneous proliferation of leukemic peripheral-blood progenitor cells (granulocyte-macrophage colony-forming units) and their selective hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). We conducted a pilot study to evaluate the clinical efficacy of isotretinoin in juvenile CML. METHODS: To be eligible the patients had to have newly diagnosed untreated disease, leukocytosis with monocytosis, marrow with less than 25 percent blasts, hepatosplenomegaly, no chromosomal abnormalities, and negative viral cultures and antibody titers. Isotretinoin was administered orally in single daily doses of 100 mg per square meter of body-surface area. When possible, patients subsequently underwent bone marrow transplantation. RESULTS: Ten children (median age, 10 months) were enrolled in the study. In all 10 there was spontaneous colony formation of leukemic progenitor cells in vitro. In the eight patients tested there was hypersensitivity to GM-CSF. The only toxic effect of isotretinoin therapy was cheilitis in two patients. Four children had disease progression. Two children had complete responses to isotretinoin (normalization of the white-cell count and disappearance of organomegaly), three had partial responses (more than a 50 percent reduction in the white-cell count and degree of organomegaly), and one had a minimal response (more than a 50 percent reduction in the white-cell count, but a 26 to 50 percent reduction in the degree of organomegaly). The median duration of response was 37 months (range, 6 to 83). Three of the four children who had a complete or partial response and who did not undergo bone marrow transplantation were alive 36 to 83 months after the diagnosis of juvenile CML. The spontaneous colony formation in vitro was reduced in samples from the five patients in whom this factor was reassessed during treatment. There was also a reduction in the hypersensitivity of leukemic progenitor cells to GM-CSF in the two patients retested. CONCLUSIONS: Isotretinoin can induce durable clinical and laboratory responses in patients with juvenile CML.

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.

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.

Undifferentiated small cell hepatoblastoma with a unique chromosomal translocation: a case report.

Cytogenetic studies of pediatric tumors have revealed a number of reproducible karyotypic abnormalities, including del(p13) found in aniridia-Wilms' tumor association, t(8;14) in Burkitt's lymphoma, and t(11;22) in Ewing's sarcoma. To date, no consistent cytogenetic abnormality has been reported in association with hepatoblastoma. We report the case of a 7-month-old male infant with the undifferentiated small cell variant of hepatoblastoma. Immunohistochemistry revealed reactivity with antibodies to cytokeratin and vimentin throughout the tumor. Alpha-fetoprotein, neuron-specific enolase, and S100 stains were negative. Chromosomal analysis of metaphase cells from a culture of tumor tissue revealed a translocation of most of the long arm of chromosome 22 to the distal long arm of chromosome 10.

Philadelphia chromosome and monosomy 7 in childhood acute lymphoblastic leukemia: a Pediatric Oncology Group study.

During an 8-year period, 3,638 children from institutions of the Pediatric Oncology Group (POG) were diagnosed with acute lymphoblastic leukemia (ALL). Fifty-seven patients had Philadelphia chromosome-positive (Ph1) ALL. Blast cells obtained at diagnosis from 13 of these 57 cases (23%) were also found to have partial or complete monosomy 7 (-7). This subgroup of children with Ph1/-7 ALL was comprised primarily of older males with early B-lineage ALL. Bone marrow specimens from six Ph1/-7 patients were studied further using the polymerase chain reaction and primers that flank the ALL, and chronic myelogenous leukemia breakpoints to determine the molecular characteristic of the 9;22 translocation. Rearrangements were detected in RNA from bone marrow and/or peripheral blood cells of six patients, although four were in hematologic remission at the time of the analysis. Five cases showed the ALL breakpoint, while one child with Ph1/-7 showed the chronic myelogenous leukemia breakpoint. The induction failure rate was much higher in this subgroup (31%) as compared with Ph1-negative cases, and the projected duration of event-free survival reflected the aggressive nature of this subgroup because no children are projected to remain in remission at 2 years. ALL with both the 9;22 translocation and -7 appears to represent a unique and previously undescribed subgroup of childhood ALL associated with a particularly adverse outcome. Leukemic transformation in such patients may involve the interaction of a dominant oncogene (Ph1) and a tumor suppressor gene (-7).

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.

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.

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.

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.

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.

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.

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.

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.