Energy metabolism in human erythrocytes. I. Effects of sodium fluoride.

Exposure of red cells to fluoride produces a variety of metabolic alterations, most of which are based upon the secondary effects of enolase inhibition, which reduces pyruvate synthesis and interferes with the regeneration of diphosphopyridine nucleotide (NAD). Adenosine triphosphate (ATP) is consumed in the hexokinase and phosphofructokinase reactions but is not regenerated since the deficiency of NAD limits glyceraldehyde phosphate dehydrogenase. ATP depletion in the presence of fluoride and calcium induces a massive loss of cations and water. Of the other known sites of ATP utilization, membrane-bound ATPase is inhibited by fluoride, but the incorporation of fatty acids into membrane phospholipids is unaffected until ATP is depleted. The addition of methylene blue to fluoride-treated red cells regenerates NAD, permitting triose oxidation and the generation of 3-phosphoglycerate and 2,3-diphosphoglycerate. Enolase inhibition is then partially overcome by mass action, and sufficient glycolysis proceeds to maintain the concentration of ATP. This in turn prevents the massive cation and water loss, and permits membrane phospholipid renewal to proceed. Membrane ATPase activity is not restored by the oxidant so that normal cation leakage remains unopposed by cation pumping in red cells exposed to the combination of fluoride and methylene blue.

Energy metabolism in human erythrocytes. II. Effects of glucose depletion.

Normal red cells were incubated in the absence of glucose to develop a system in which total adenosine triphosphate (ATP) turnover could be assessed. After 1 hr, the triose pool had been completely consumed. Thereafter, the metabolism of 2,3-diphosphoglycerate (DPG) to pyruvate and lactate was the sole significant source of ATP synthesis.10(-3)M CuCl(2), which did not enter the cells, diminished ATP utilization by more than 50%. This could be only partially attributed to the inhibition by copper of residual acylation and cation pumping, which were already reduced by glucose depletion. Other membrane enzymes, which presumably function in the maintenance of membrane integrity, must, therefore, use a significant portion of erythrocyte ATP. The behavior of glucose-depleted red cells with respect to cation transport was complex. The addition of ouabain did not decrease ATP utilization in these red cells. Ouabain inhibitable potassium influx was nearly normal after triose depletion, but total potassium influx was decreased. In contrast, the ouabain inhibitable sodium efflux was markedly reduced after triose depletion, although the concentration of ATP was 70% of normal. The dissociation of monovalent cation pumping suggests that the energy for active sodium transport is derived from a specific source (such as the ATP produced by the phosphoglycerate kinase reaction) distinct from that for potassium transport.

Postremission therapy for children with acute myeloid leukemia: the children's cancer group experience in the transplant era.

We reviewed consolidation therapy results and analyzed postremission outcomes for 1464 children less than 21 years old at diagnosis in five consecutive Children's Cancer Group acute myeloid leukemia trials between 1979 and 1996. Children in remission were allocated to allogeneic bone marrow transplantation (BMT) (N=373) in first remission, if a matched family donor was available. Remaining children were assigned consolidation chemotherapy (N=688) or autologous purged BMT (N=217), or withdrew from study before assignment, or with unknown data (N=186). Overall and disease-free survival were superior for children assigned allogeneic transplants. High (>50,000/microl) diagnostic white blood cell (WBC) count was prognostic for inferior outcome, but French-American-British (FAB) subtypes were not. Inv(16) is a favorable karyotypic feature for children in first remission and t(8;21) is not. Allogeneic transplantation benefit was evident in most children, including those with high or low diagnostic WBC count, each FAB subtype, and t(8;21), but was not seen in children with inv(16). Therefore, these data suggest reserving matched related donor allogeneic transplantation for children with inv(16) for second remission, but not those with t(8;21).

Feasibility, toxicity, and biologic response of interleukin-2 after consolidation chemotherapy for acute myelogenous leukemia: a report from the Children's Cancer Group.

PURPOSE: Although remission can be achieved in 80% of children with acute myelogenous leukemia (AML), many patients experience relapse. Because interleukin-2 (IL-2) can induce remission in patients with overt evidence of AML, we hypothesized that IL-2 given to patients in first remission after intensive consolidation chemotherapy might prevent relapse. This study sought to determine whether such an approach was feasible. PATIENTS AND METHODS: Twenty-one patients in complete remission received IL-2 after completion of treatment on Children's Cancer Group (CCG) protocol 2941. Recombinant IL-2 9 x 10(6) IU/m2 daily by continuous intravenous infusion (c.i.v.) was given for 4 days. After 4 days rest, IL-2 1.6 x 10(6) IU/m2 daily c.i.v. was resumed for 10 days. We monitored patients for toxicity and measured absolute lymphocyte count, the absolute count of cells that express CD56 and CD3 antigen, and soluble IL-2 receptor alpha-chain (sIL-2R alpha) levels before the start of IL-2 and after completion of each of the two courses of IL-2. RESULTS: Observed toxicities included fever (57%), vascular leak (48%), hypotension (38%), tachycardia (14%), rash (29%), septicemia (5%), thrombocytopenia (29%), elevated transaminase (14%), electrolyte disturbance (29%), and hyperglycemia (10%). No patient required cardiac pressors or transfer to an intensive care unit. All patients studied developed an increase in lymphocyte count, CD56 count, CD3 count, and sIL-2R alpha levels after treatment with IL-2. CONCLUSION: This schedule of IL-2 was reasonably well tolerated by children with AML in first remission. After treatment, increased levels of sIL-2R alpha were observed. CCG is conducting a randomized prospective trial to assess the efficacy of IL-2 to prevent the relapse of AML (CCG-2961).

Chemotherapy for induction of remission of childhood acute myeloid leukemia followed by marrow transplantation or multiagent chemotherapy: a report from the Childrens Cancer Group.

PURPOSE: In an effort to evaluate the usefulness of bone marrow transplantation, the Childrens Cancer Group (CCG) initiated a multiinstitutional study comparing bone marrow transplantation versus chemotherapy after successful induction of remission for previously untreated children and young adults with acute myeloid leukemia. PATIENTS AND METHODS: From 1979 to 1983, 508 patients were entered onto this study and 490 were treated. After induction, patients with an HLA mixed leukocyte culture (MLC)-compatible sibling underwent bone marrow transplantation. Patients not eligible for bone marrow transplantation were eligible for randomization to two chemotherapy maintenance regimens. All patients undergoing bone marrow transplantation were conditioned with cyclophosphamide and total-body irradiation (TBI). Methotrexate was used to prevent or modify graft-versus-host disease (GVHD). RESULTS: Three hundred eighty-one patients achieved bone marrow remission (78%). Eighty-nine patients had an HLA/MLC-compatible sibling donor and were eligible for bone marrow transplantation, and 252 had no match. Comparison of survival estimates for patients eligible for transplantation versus not eligible at 3 years (52% v 41%), 5 years (50% v 36%), and 8 years (47% v 34%) showed a significant difference in favor of bone marrow transplantation (P < .05). Disease-free survival (DFS) demonstrated similar results. Application of a cure model to the results showed a better outcome for those eligible for transplantation (P = .04). Patients randomized between the two chemotherapy regimens did not show any significant difference between those treated with a continuous maintenance versus a cyclic regimen (P = .16). CONCLUSION: Children and young adults who successfully achieved a remission with multiple-agent chemotherapy who had an HLA/MLC-compatible donor and were thus eligible for an allogeneic bone marrow transplant had better survival than those not eligible for transplantation.

Prognostic variables in newly diagnosed children and adolescents with acute myeloid leukemia: Children's Cancer Group Study 213.

The objective of this study was to identify biologic parameters that were associated with either exceptionally good or poor outcome in childhood acute myeloid leukemia (AML). Among the children with AML who entered Children's Cancer Group trial 213, 498 patients without Down syndrome or acute promyelocytic leukemia (APL) comprise the basis for this report. Univariate comparisons of the proportion of patients attaining complete remission after induction (CR) indicate that, at diagnosis, male gender, low platelet count (< or =20 000/microl), hepatomegaly, myelodysplastic syndrome (MDS), French-American- British (FAB) category M5, high (>15%) bone marrow (BM) blasts on day 14 of the first course of induction, and +8 are associated with lower CR rates, while abnormal 16 is associated with a higher CR rate. Multivariate analysis suggests high platelet count at diagnosis (>20 000/microl), absence of hepatomegaly, < or =15% day 14 BM blast percentage, and abnormal 16 are independent prognostic factors associated with better CR. Univariate analysis demonstrated a significant favorable relationship between platelet count at diagnosis (>20 000/microl), absence of hepatomegaly, low percentage of BM blasts (< or =15%), and abnormal 16 with overall survival. Absence of hepatomegaly, < or =15% day 14 BM blast percentage, and abnormal 16 were determined to be independent prognostic factors associated with better survival.

Pneumococcal vaccination of recipients of bone marrow transplants.

A 14-valent pneumococcal vaccine was administered to 39 recipients of allogeneic bone marrow transplants, and their type-specific antibody responses were compared with normal control subjects. Preimmunization antibody levels in patients were twofold to 12-fold lower than those levels in control subjects for all serotypes. Mean postimmunization antibody levels for each serotype were also considerably lower in patients (range, 56 to 859 ng of antibody nitrogen per milliliter) than in control subjects (range, 727 to 5,626 ng/mL). Poor antibody responses were primarily associated with early vaccination after transplantation, corticosteroid therapy for graft-v-host disease and other illnesses, and the male sex. Antibody responses of patients not given corticosteroids and vaccinated more than seven months after transplantation improved with time after transplantation. Postvaccination infection occurred in five patients who were vaccinated early after transplantation. Pneumococcal vaccination has limited potential for providing protection in marrow transplant recipients except in the cases of those patients who are not receiving corticosteroids and are vaccinated more than seven months after transplantation.

Case problems in bone marrow transplantation. I. Graft failure in aplastic anemia: its biology and treatment.

An unusual case of graft failure following bone marrow transplantation for aplastic anemia is reported in which the patient had delayed and incomplete recovery of hematopoiesis despite documentation of sustained engraftment. The pathophysiology of graft failure following bone marrow transplantation is discussed and recent advances in pretransplant immunosuppressive therapy to prevent graft rejection are reviewed.

Autotransplantation after in vitro immunotherapy of lymphoblastic leukemia.

We have used in vitro immunotherapy before autologous bone marrow transplantation for three patients with acute lymphoblastic leukemia (ALL). Bone marrow was removed during remission, and mononuclear cells were separated by density-step centrifugation on Ficoll-Hypaque. The cells from each patient were treated with a heteroantiserum and complement to eliminate leukemic cells and were cryopreserved. Following chemotherapy and total body irradiation, the treated marrows were thawed and infused. All the patients showed positive evidence of returning marrow function before death. One patient who survived 4 months showed no evidence of leukemia at post mortem, and marrow sections demonstrated active hematopoiesis of all cell lines.

Radiation risk from screening mammography of women aged 40-49 years.

Although direct evidence of carcinogenic risk from mammography is lacking, there is a hypothetical risk from screening because excess breast cancers have been demonstrated in women receiving doses of 0.25-20 Gy. These high-level exposures to the breast occurred from the 1930s to the 1950s due to atomic bomb radiation, multiple chest fluoroscopies, and radiation therapy treatments for benign disease. Using a risk estimate provided by the Biological Effects of Ionizing Radiation (BEIR) V Report of the National Academy of Sciences and a mean breast glandular dose of 4 mGy from a two-view per breast bilateral mammogram, one can estimate that annual mammography of 100,000 women for 10 consecutive years beginning at age 40 will result in at most eight breast cancer deaths during their lifetime. On the other hand, researchers have shown a 24% mortality reduction from biennial screening of women in this age group; this will result in a benefit-to-risk ratio of 48.5 lives saved per life lost and 121.3 years of life saved per year of life lost. An assumed mortality reduction of 36% from annual screening would result in 36.5 lives saved per life lost and 91.3 years of life saved per year of life lost. Thus, the theoretical radiation risk from screening mammography is extremely small compared with the established benefit from this life-saving procedure and should not unduly distract women under age 50 who are considering screening.

Metabolic correlates of immune dysfunction in malnourished children.

Mononuclear cells of malnourished children contain diminished activity of phosphoglycerate kinase and pyruvate kinase (PK). The PK activity of these cells correlates well with the percentage of circulating thymus-derived lymphocytes (T-cells). Phytohemagglutinin causes an immediate increase in PK activity of mononuclear cells of malnourished patients. The correlation of PK activity with T-cells and the response of PK activity to phytohemagglutinin are in distinct contrast to observed perturbations of neonatal mononuclear cell metabolism. The relationship of the metabolic alterations to the pathophysiology of the immune system in malnutrition has not yet been defined.

Bone marrow transplantation using unrelated donors for patients with advanced leukemia or bone marrow failure.

Thirty-four patients received bone marrow transplants from unrelated donors. Donors and recipients were phenotypically matched for 6 of 6 HLA-A, B, and DR antigens in 27 cases and at 5 of 6 antigens in 7 cases. Twenty-three patients had leukemia, six had myelodysplasia, and five had aplastic anemia. Twenty-four patients had durable engraftment. Five died of sepsis prior to engraftment. Five patients failed to engraft; 2 of these patients had autologous bone marrow recovery. Seventeen patients developed grade greater than or equal to II acute graft-versus-host disease for an actuarial probability of 67 +/- 20%. The severity of acute graft-versus-host disease and its mortality appeared increased for recipients matched for 5 of 6 HLA-A, B, and DR antigens. Of the 34 patients, 13 (38%) are alive; actuarial survival beyond 6 months is 44 +/- 17%. None of the 25 leukemia and myelodysplasia patients achieving engraftment have relapsed. For leukemia and myelodysplasia recipients of 6 of 6 HLA-matched grafts, actuarial survival at 6 months was 55 +/- 21% compared with 14 +/- 26% for recipients matched for 5 of 6 HLA loci (P = 0.19). Infection and acute graft-versus-host disease were the primary causes of death in the engrafted patients. Survival for aplastic anemia patients was 20%. Late deaths due to pneumonia and bronchiolitis obliterans occurred after one year in 2 patients. Closely matched unrelated donor bone marrow transplants are associated with a higher incidence of graft failure and graft-versus-host disease than typically reported for transplants from HLA-identical siblings, but these preliminary data suggest a lower rate of relapse.

Normal neurodevelopment in four young children treated with bone marrow transplantation for acute leukemia or aplastic anemia.

Longitudinal neurodevelopmental studies of four consecutive young children treated by bone marrow transplantation for acute leukemia or aplastic anemia are presented. The children, the only four children less than 2 years of age who have received bone marrow transplants for these diseases at UCLA Medical Center, ranged in age from 36 weeks to 24 months at the time of transplantation. Conditioning involved high-dose cyclophosphamide treatment; three also had total body irradiation prior to bone marrow transplantation. Their respective outcomes after follow-up times of 28 months to 71 months posttransplantation are remarkable for normal somatic growth and normal development of intelligence, language, perception, and motor coordination. These findings indicate that future therapeutic studies of infants and young children with acute leukemia or aplastic anemia using total body irradiation, cyclophosphamide, and bone marrow transplantation are not contraindicated by risks of debilitating neurodevelopmental sequelae.

Occult recurrence of nasopharyngeal rhabdomyosarcoma manifesting as isolated increased intracranial pressure.

In this paper we report a child who had extensive neoplastic infiltration of the meninges which defied clinical detection. We believe that any patient with a past or present history of tumor who presents with increased intracranial pressure should be presumed to have intracranial neoplastic involvement, even in the face of negative diagnostic studies. As in this case, other causes of increased intracranial pressure should be systematically excluded.

Role and evaluation of mammography and other imaging methods for breast cancer detection, diagnosis, and staging.

Mammographic screening of women age 40 and older can reduce breast cancer deaths by at least 30% to 40%. However, not all cancers are detected by mammography. Although a new supplementary modality for screening could, in theory, fill in this detection gap, such utilization must be based on rigorous demonstration of its ability to consistently and frequently find early cancers missed by mammography, such as those occurring in dense breasts or rapidly growing interval cancers that surface clinically between mammographic screens. After an abnormality is found at mammographic screening, supplementary mammographic views and/or ultrasound are now used to match the finding with an ACR BIRADS final diagnostic assessment category to indicate the relative likelihood of a normal, benign, or malignant diagnosis so that routine screening, short interval follow-up, or biopsy can then be advised. Appropriate categorization will maximize early cancer detection and minimize false-positive biopsies. Application of a new imaging method to this type of diagnostic evaluation requires well-designed studies to determine its effectiveness for this purpose.

Non-calcified breast particles: a possible new marker of breast cancer.

Malignant and benign human breast tissue containing radiographic evidence of calcifications were studied by means of scanning electron microscopy and x-ray microanalysis. In addition to the well-known calcium-phosphorus deposits, discrete particles containing calcium with little or no phosphorus and calcium in combination with other elements were found. Moreover, particles containing elements other than calcium were observed. These include Al, Fe, Mg, Si, Cu, Zn, Cr, Ti, Ni, Pb, Au, Ag, Mo, Cl, I. Some of these "noncalcifications" contained a single element, others a combination of elements. These findings differ from the view that all breast particles are calcifications. Further study could lead to the development of new diagnostic techniques for the detection of breast cancer and/or chemotherapeutic agents.

Intracardiac right-to-left shunting and the risk of stroke during bone marrow infusion.

We describe a patient who developed a paradoxical embolus to the brain during infusion of bone marrow. She had a patent foramen ovale through which right-to-left shunting led to multiple cerebral emboli. This complication can be prevented by positioning the tip of the infusion catheter in the main pulmonary artery and reducing the volume of marrow product infused.

Marrow transplantation for thalassemia.

Ten patients with homozygous beta thalassemia, aged from 1 year 7 months to 13 years, underwent bone marrow transplantation from siblings or parents. The first case received 12 mg/kg busulfan, 120 mg/kg cyclophosphamide, and 300 cGy total body irradiation before transplantation; he survives, with a graft, more than 680 days after transplantation. The other nine patients received 16 mg/kg busulfan and 200 mg/kg cyclophosphamide. Two died of transplantation-related complications on days 30 and 55. Seven survive 170 to 580 days after transplantation. Three of the seven surviving patients have durable engraftment (greater than 230 to greater than 550 days) while four patients have autologous hematopoietic recovery. Four of five patients who had less than 50 prior transfusions achieved engraftment. Only one of five patients who had more than 50 prior transfusions achieved engraftment (P less than 0.05). The six-month actuarial survival was 80%; six-month actuarial disease-free survival was 40%. These data demonstrate that bone marrow transplantation may cure thalassemia, but engraftment may be jeopardized among patients who have been heavily transfused or have received marrow from a donor who is not HLA-identical.

B cell acute lymphoblastic leukemia (ALL) in donor cells following bone marrow transplantation for T cell ALL.

Leukemia recurred in a child with acute lymphoblastic leukemia after a bone marrow transplant. The pre-transplant leukemia was of T cell origin whereas the post-transplant relapse leukemia was of B cell origin. The recurrence was shown to be in donor cells by analysis of chromosomes and restriction fragment length polymorphisms. Although the mechanism of leukemia induction is uncertain, the possibilities of donor relapse due to EBV infection or chromosomal exchange were disproven. An alternative mechanism must account for leukemia induction in this patient.

Immune thrombocytopenia after umbilical cord progenitor cell transplant: response to vincristine.

An 8-month-old male with X-linked lymphoproliferative disease underwent an unrelated, partially matched (with major mismatch at DR locus), cord blood stem cell transplant. Four months following the transplant, he developed immune thrombocytopenia with hemolytic anemia (Evans syndrome). He received multiple courses of intravenous immunoglobulin, anti-Rh D immunoglobulin, a pulse of high-dose corticosteroids and cyclosporine with some improvement of hemolytic anemia, but no improvement of the thrombocytopenia. Addition of vincristine, resulted in long-term resolution of thrombocytopenia and anemia. No major toxicity was observed during treatment. Vincristine should be considered as a treatment for refractory immune thrombocytopenia after hematopoietic stem cell transplantation.