Ultrastructural, immunochemical, and cytochemical study of myeloperoxidase in myeloid leukemia HL-60 cells following treatment with succinylacetone, an inhibitor of heme biosynthesis.

Myeloperoxidase (MPO) is a heme-containing glycoprotein found in the primary granules (or azurophilic granules) of human polymorphonuclear leukocytes. In the present study, cultured myeloid leukemia HL-60 cells were exposed for 0-72 h to 250 microM 4,6-dioxoheptanoic acid (succinylacetone, SA), a specific inhibitor of heme biosynthesis, and the effects were evaluated using ultrastructural, immunochemical, and cytochemical methods. En bloc peroxidase staining of glutaraldehyde-fixed cells was accomplished with a 30-min exposure to 3,3'-diaminobenzidine (DAB) tetrahydrochloride. Ultrastructural examination revealed that peroxidase reactivity in the endoplasmic reticulum (ER) was relatively unchanged for 8 h and decreased between 12 and 24 h; however, ER lacked DAB-reactive peroxidase at 48-72 h. Peroxidase reactivity in the ER reappeared within 4 h after removal of SA. Seventy-two hours after exposure to SA the number of condensed cytoplasmic granules stained with DAB was significantly decreased, and many of the granules had a "target" appearance with a central DAB-reactive dense core. Staining of mitochondria was observed with overnight exposure to DAB and persisted in HL-60 cells treated 72 h with SA. Mitochondrial and nuclear morphology appeared unaltered. Immunostaining of MPO in thin sections of paraformaldehyde/glutaraldehyde-fixed unosmicated HL-60 cells, embedded in Lowicryl K4M, was accomplished with sequential exposure to an affinity-purified monospecific rabbit antibody to HL-60-MPO and protein A conjugated to 5- or 10-nm colloidal gold. Compared to untreated control HL-60 cells, cells exposed to SA for 48 h exhibited comparable to increased immunoreactive MPO in the ER, despite the absence of heme-dependent peroxidase reactivity. The data indicate that SA inhibits formation of enzymatically active MPO and that in the presence of SA, the ER contains a form(s) of MPO that lacks enzymatic reactivity.

Severe refractory anemia with ringed sideroblasts and bone marrow aplasia in a child.

Sideroblastic anemia is an extremely rare disorder in children. This report describes a 9-year 4-month-old girl with severe refractory anemia with ringed sideroblasts (RARS) that progressed to severe bone marrow aplasia. Ultrastructural studies revealed the presence of abundant intramitochondrial deposits of iron in erythroblasts similar to that observed in adults with this disorder. Although acid ferrocyanide staining confirmed the ferric valence of the iron deposits, they lacked morphologic and cytochemical characteristics associated with ferritin and hemosiderin. Bone marrow culture showed decreased or absent CFU-GEMM, CFU-GM, CFU-E, and BFU-E. Erythrocyte uroporphyrinogen I synthase, aminolevulinic acid dehydratase, uroporphyrinogen decarboxylase, urinary porphyrins, porphobilinogen, and aminolevulinic acid were normal. Free red cell protoporphyrin was increased. Therapy with corticosteroid and androgens was totally ineffective. The aplastic bone marrow in this child appeared to represent the end stage of RARS and differed from adults with RARS, who more frequently demonstrate a chronic course, often with the onset of leukemia as a terminal sequela. Although this case documents the occurrence of RARS in a child, additional reports of children with this disorder will be required to determine the prognosis and natural history of RARS in children.

Childhood undifferentiated leukemia with early erythroid markers and c-myb duplication.

Ultrastructural, flow cytometric, and molecular studies were performed on leukemia cells from bone marrow and pleural effusion of a 6-year-old boy diagnosed with undifferentiated (MO) leukemia, using routine histology and immunostains at diagnosis and relapse. Ultrastructurally, surface and/or intracellular ferritin particles were present on or in some blasts and the majority of blasts contained identifiable acid ferrocyanide reactive inorganic iron comparable to that seen in normal early erythroblasts. The cells lacked other evidence of differentiation, including diaminobenzidine-reactive or immunoreactive hemoglobin. Flow cytometric analysis of malignant cells showed a lack of lymphoid or myeloid markers. Anti-transferrin receptor antibody was positive on 93% of cells and antibody to glycophorin A reacted with 23% of cells. RNA blot analysis of leukemia cells with myeloperoxidase (MPO) showed an absence of appreciable levels of MPO mRNA. Chromosome analysis showed 51,XY, t(1;16)(p31;q24), +6, +10, +15, +19, +21. The oncogene c-myb, which is specifically expressed and regulated in hematopoietic cells and produces a DNA-binding protein responsible for myeloid differentiation, was found to be duplicated in the patient's tumor cells. Expression of c-jun, N-ras, c-myc, and p53 was normal. The data indicate that the malignant cells in this patient are of early erythroid lineage at diagnosis and relapse and that classification of cell lineage can be enhanced by ultrastructural Prussian blue staining. The failure of this otherwise undifferentiated leukemia to express or evolve into a myeloid phenotype is biologically and clinically distinct from previously described cases of erythroid and myeloid leukemia and may represent a previously unidentified phenotype which should be included in the spectrum of 'undifferentiated' childhood leukemia.

Cytogenetic and molecular evaluation of clinically aggressive esthesioneuroblastoma.

We report a 16-year-old boy with esthesioneuroblastoma that presented with a unilateral tumor extending to the maxillary sinus and periorbital region. Despite initial therapy with gross resection, 5,682 cGy to the tumor bed and chemotherapy, the patient subsequently had a rapid local recurrence with distant metastases. Immunocytochemical, ultrastructural, cytogenetic, and molecular techniques were performed to determine if this tumor was biologically similar to childhood neuroblastoma. Urinary excretion of vanillylmandelic acid (VMA) and homovanillic acid (HVA) were markedly elevated. Chromogranin and neuron specific enolase immunostaining of tumor cells was positive, as seen in neuroblastoma. Electron microscopic studies showed cells that were closely packed and connected by occasional cell junctions. The cell cytoplasm contained moderate amounts of filaments and microtubules. Numerous electron dense granules were observed; however, these granules lacked distinct nucleoids and generally reacted strongly for acid phosphatase, indicating a lysosomal rather than a secretory function. Tumor cells contained near-pseudotetraploid chromosomes, with all chromosomes represented at least three times, and chromosome 5 was present in multiples of eight. Clonal structural abnormalities included 2q+ and 5q+ and multiple double minutes. Northern blot analysis revealed both c-myc and N-myc expression; however, N-myc amplification was not demonstrated, and c-myc expression appeared increased, unlike cases of rapidly progressive neuroblastoma. These results suggest that despite biologic similarities to neuroblastoma in catecholamine excretion and some ultrastructural features, molecular genetic abnormalities differ in this comparatively aggressive case of estesioneuroblastoma.

Radiotherapy of pseudotumors of bone in hemophiliacs with circulating inhibitors to factor VIII.

Pseudotumors of bone in two hemophiliacs with severe factor VIII deficiency and a high level of circulating inhibitors are reported. Both had favorable response to radiation therapy after unsuccessful treatment with factor concentrates and remain free of recurrence at 18-62 months. A review of the literature of cases in which radiotherapy has been used is presented. Radiotherapy in the treatment of pseudotumors of bone in hemophiliacs should be strongly considered, particularly if coagulation factor inhibitors are present.

Postoperative chemotherapy for primary intracranial germ cell tumor.

Two children with primary intracranial mixed germ cell tumors are described who were successfully treated by partial resection of the tumor followed by sequential combination chemotherapy without radiation therapy. The chemotherapy consisting of VP-16 and cisplatin alternating with vincristine, methotrexate, and bleomycin resulted in apparent complete response after 6 to 7 months of treatment. Disease-free remission has continued 30-34 months off therapy. A small residual mass in one patient continues to decrease in size on magnetic resonance imaging and is presumed to represent postsurgical change rather than malignant tumor. This report demonstrates that chemotherapy may be effective in primary germ cell tumors of the suprasellar and pineal regions and could be considered for primary treatment instead of radiotherapy.