Morphology of human neutrophils: a comparison of cryofixation, routine gluteraldehyde fixation, and the effects of dimethyl sulfoxide.

Cell shape and density are critical to the evaluation of neutrophil function and/or activation. Dimethyl sulfoxide-cryofixation-freeze-substitution processing (DCF) instantly preserves cell processes and ultrastructural elements with fewer artifacts than routine chemical fixation with glutaraldehyde and postfixation osmium tetroxide (GO). This study morphometrically examined density-separated neutrophils to assess differences in DCF and GO processing procedures and studied the effect of dimethyl sulfoxide followed by GO fixation (DGO) on morphology. Fifteen consecutive neutrophils were analyzed using computerized planimetry for differences in DCF v. GO treatments (n = 4) and DGO v. GO treatments (n = 4). Cryofixed and DGO-fixed cells were significantly rounder than GO cells which had a more irregular surface with membrane projections. The cell volume of GO cells was 27-30% smaller than in DCF or DGO processing, while the surface area was similar. The increased volume in DCF and DGO cells did not appear to be due to abnormal cell swelling, since membranes, nuclear envelope, and mitochondrial cristae were more intact than in GO cells. Preservation of mitochondria as well as endocytic caveolae with a subplasmalemmal coating was best in DCF samples, moderate in DGO, and poorest in GO. Morphometric data showed that the nuclear compartment was 22% smaller, while the cytoplasm (and its associated compartments) was 29% smaller in GO compared to DCF-processed neutrophils. This was consistent with the more dense cytoplasm in GO cells. Pretreatment of neutrophils with dimethyl sulfoxide (DMSO) resulted in volume preservation and improved the morphology of GO fixation. In summary, DCF appears to be an excellent method for preserving neutrophil membranes and cytoplasmic organelles (particularly mitochondria), and prevents a number of artifacts caused by routine GO fixation. Morphology can also be improved by using DMSO in conjunction with GO.

Benign and malignant smooth muscle tumors containing Epstein-Barr virus in children with AIDS.

Smooth muscle tumors (leiomyosarcomas) are the second most prevalent malignancy of children with the acquired immunodeficiency syndrome (AIDS). We have investigated the tumors, plasma, and peripheral white blood cells of eight children with AIDS with smooth muscle tumors for evidence of tumor association with human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV). Very low levels of HIV were found in the tumors of the AIDS patients, probably resulting from blood-borne carriage of virus. These smooth muscle tumors had very high quantities of EBV in all the tumor cells by in situ hybridization, with an average of 4.5 EBV genomes per cell by quantitative polymerase chain reaction amplification. Increased amounts of EBV were found in the peripheral blood cells of two AIDS patients before the time of tumor diagnosis. EBV clonality studies demonstrated different monoclonal EBV infection of two separate colonic tumors from one patient, and dual or mixed monoclonal EBV infection in another patient. The muscle cells of leiomyomas and leiomyosarcomas of patients with AIDS demonstrated prominent staining with antibodies to the EBV receptor. The uniform distribution and striking amount of EBV in the tumor cells demonstrates that EBV is capable of infecting smooth muscle cells and that these cells support EBV replication. Clonal EBV proliferation suggests that EBV infection occurs at an early stage of tumor development. These findings indicate that EBV has a causal role in the oncogenesis of leiomyosarcomas of patients with AIDS.

Heterogeneity of peroxidase positive granules in normal and pathologic human neutrophils.

Studies have demonstrated significant heterogeneity in neutrophil granule morphology and physical density. Using cytochemical methods to localize peroxidase and vicinal glycol containing complex carbohydrates we examined the heterogeneity of neutrophil granules from intact human neutrophil granules in 13 isolated granule density fractions, calcium ionophore A23187 treated neutrophils and neutrophils from patients with Chediak-Higashi Syndrome and Specific Granule Deficiency. At least four distinct populations of peroxidase positive granules (PPG) were identified based on peroxidase staining, vicinal glycol staining, morphology, beta-glucuronidase and defensin content, and physical density characteristics. The smallest (0.15 micron diameter) PPG was the least dense granule, had a unique peroxidase/beta-glucuronidase ratio, reacted intensely for vicinal glycols, resisted ionophore degranulation and was not consumed in giant granule formation in Chediak-Higashi Syndrome. The largest (0.3 micron average diameter) and most physically dense PPG was rich in defensins, stained weakly for vicinal glycols, and was absent in specific granule deficiency. These studies demonstrate and correlate morphologic, biochemical, functional, and pathologic differences in PPG populations.

Evolution of AIDS and AIDS related malignancies in pediatric patients in the United States.

The pediatric AIDS epidemic began in the U.S.A. between 1983 and 1985. Hemophilia patients were among the first victims of this disease with the majority of these patients infected prior to 1984. At the South Texas Hemophilia Center 69 of 108 patients less than 21 years of age demonstrated serologic evidence of infection. Of these patients, 6 subsequently developed malignancies between 1987 and 1994. Between 1992 and 1996 data was subsequently accumulated on the development of malignancy in HIV positive patients through the Pediatric Oncology Group, which to date has enrolled 24 HIV positive children with malignancy. In these studies the majority of patients had B cell, non-Hodgkin's lymphomas, however approximately 20% of the patients were identified with leiomyosarcomas. Histologic studies of tumors of 6 children with AIDS and leiomyosarcomas or leiomyoma identified the EBV receptor or CD 21 in the tumor using immunoperoxidase techniques, whereas similar staining was not seen in smooth muscle tumors from HIV negative children. In situ hybridization techniques identified EBV-EBER probe in the tumors from HIV positive patients. In 2 patients with adequate tumor tissue EBV genome was present in high concentration using PCR techniques and Southern blot studies showed a monoclonal and biclonal proliferation. Other laboratories have reported similar EBV findings in lymphomas from AIDS patients. Thus EBV appears to be an important cofactor in development of malignancy in pediatric AIDS patients.

Neuroblastoma presenting as acute monoblastic leukemia.

PURPOSE: We report a 5-year-old boy with stage 4 neuroblastoma initially diagnosed as having acute monoblastic leukemia (FAB M5A, AMoL), based on bone marrow morphology, histochemistry, immunocytochemistry, immunophenotyping and cytogenetics, all consistent with AMoL. The patient also had circulating blasts at diagnosis. After failing initial therapy for AMoL and because of concerns about residual blasts with a clumped appearance in the bone marrow, urinary homovanillic acid (HVA) and vanillylmandelic acid (VMA) and N-myc amplification in tumor cells were evaluated and found to be positive, resulting in the diagnosis of neuroblastoma. Abdominal computerized tomography showed a left adrenal mass. A review of 10 reported cases of neuroblastoma with leukemic features showed that seven of them were misdiagnosed as having leukemia, and in six of the seven, the diagnosis of neuroblastoma was made postmortem. CONCLUSION: Neuroblastoma may be confused with acute leukemia, even with the use of modern techniques.

Association of Epstein-Barr virus with leiomyosarcomas in young people with AIDS.

BACKGROUND: Children with the acquired immunodeficiency syndrome (AIDS) have an unusually high incidence of smooth-muscle tumors (leiomyomas and leiomyosarcomas) in addition to malignant lymphomas. We tested the hypothesis that the smooth-muscle tumors in these children are associated with the Epstein-Barr virus (EBV). METHODS: Tissue specimens of five leiomyosarcomas and two leiomyomas from six children with AIDS were studied for evidence of the human immunodeficiency virus (HIV) and EBV by in situ hybridization and quantitative polymerase chain reaction (PCR). Comparison specimens included samples of leiomyosarcoma and leiomyoma from HIV-negative children. EBV clonality of leiomyosarcomas was determined by Southern blot analysis with oligonucleotide probes for EBV terminal-repeat fragments. Tumor specimens were tested by immunoperoxidase staining for infiltration by B lymphocytes and expression of the EBV receptor. Serologic testing for EBV was performed. RESULTS: In situ hybridization showed EBV genomes in all muscle cells of the five leiomyosarcomas and the two leiomyomas from the six HIV-infected children. Quantitative PCR demonstrated strikingly high levels of EBV in tumor tissue, with as many as 4.3 genome copies per cell. Two colonic leiomyosarcomas obtained from different sites at different times from one patient contained different episomal EBV clones, signifying the presence of distinct monoclonal EBV-related tumors. We found biclonal EBV infection in the leiomyosarcoma of another patient. No EBV was detected in normal muscle or tumor specimens from HIV-negative patients. Immunostaining for the EBV receptor was strongly positive in six of the seven leiomyomas and leiomyosarcomas from the patients with AIDS. CONCLUSIONS: EBV can infect smooth-muscle cells, at least in patients with AIDS, and it may contribute to the pathogenesis of leiomyomas and leiomyosarcomas in children with AIDS. EBV seems to play no part in smooth-muscle tumors in HIV-negative children.

Hereditary hemochromatosis: a prevalent disorder of iron metabolism with an elusive etiology.

Hereditary hemochromatosis is a prevalent inherited disorder with an estimated frequency of homozygosity of 0.2 to 0.45% in Caucasians. The disease is characterized by progressive iron overload until a massive accumulation of body iron occurs. Undetected, the disorder eventually can produce either cirrhosis, diabetes mellitus, cardiac disease, arthritis, or hepatocellular carcinoma or a combination of these manifestations. Early diagnosis and treatment prevents organ damage and normalizes life expectancy. Screening studies to detect hemochromatosis are most effectively accomplished by measurement of the serum iron and total iron binding capacity. Treatment is most effectively performed by frequent phlebotomy until body stores are empty and then 3 to 4 times yearly for life. The basic defect of hemochromatosis appears to increase iron absorption, decrease iron excretion, and produce preferential deposit of iron in hepatic parenchymal cells rather than Kupffer cells. The genetic abnormality of hemochromatosis is located on chromosome 6 in close association with the gene for HLA antigens. Recent speculation postulates that tumor necrosis factor may be involved in the etiology of this disease because of its location on chromosome 6 and its effect upon iron transport.

Phase I trial of continuous infusion carboplatin and etoposide in children with refractory acute leukemia: a Pediatric Oncology Group study.

PURPOSE: The purpose of this phase I study was to determine the toxicities and response to continuous infusion carboplatin in combination with a fixed dose of etoposide (VP-16) in children with refractory acute leukemia. PATIENTS AND METHODS: From January 1989 to February 1992, 20 patients received 28 courses of treatment. Each course of treatment consisted of a 1-hour intravenous (IV) infusion of VP-16 100 mg/m2/d for 5 days, followed by a 23-hour IV infusion of carboplatin each day. The initial, total 5-day dose of carboplatin (1,000 mg/m2) was escalated by 250- to 375-mg increments to a final, total dose of 1,875 mg/m2 over 5 days. RESULTS: Significant marrow suppression was observed in all patients, with prolonged marrow aplasia at the 1,875-mg/m2 dose level. Grade III diarrhea occurred in three patients; 10 patients experienced life-threatening infection and three had severe thrombocytopenic bleeding. Major marrow responses (two complete remissions and two partial remissions) occurred in four patients (20%). CONCLUSION: In view of the apparent antileukemic efficacy and minimal extramedullary toxicity, carboplatin deserves further study in a phase II trial.

Giant actin inclusions in hematopoietic cells associated with transfusion-dependent anemia and grey skin discoloration.

We evaluated a 13-month-old boy with cytoplasmic inclusions in hematopoietic cells, transfusion-dependent anemia, splenomegaly, and striking grey skin discoloration. Bright blue inclusions, 1 to 5 microns in diameter, were observed, primarily in the cytoplasm, of 30% to 40% of myeloid cells and in occasional monocytes, megakaryocytes, and lymphocytes on Wright Giemsa-stained bone marrow and blood smears. They occasionally involved the nucleus. The inclusions lacked lysosomes, polysaccharides, or lipids. Ultrastructurally, they lacked limiting membranes and consisted of tightly packed microfilaments averaging 7 nm in diameter, consistent with the size of actin monofilaments. On light microscopy, the inclusions stained with a monoclonal antibody to muscle-specific actin. Inclusion-positive cells contained increased F-actin content and were defective in chemotactic factor-activated actin polymerization; inclusion-negative cells polymerized actin normally. Neutrophil and platelet numbers and functional studies were mildly abnormal. Anemia and skin discoloration resolved spontaneously after 18 months, but the giant inclusions have persisted to the present. We conclude that this child has a previously unreported constellation of clinical and laboratory findings comprising severe anemia, intermittent neutropenia and thrombocytopenia, abnormal neutrophil migration and platelet aggregation, giant inclusions of actin in hematopoietic cells, and grey skin discoloration.

Ultrastructural morphology, cytochemistry, and morphometry of eosinophil granules in Chédiak-Higashi syndrome.

Lysosomal enlargement in Chédiak-Higashi Syndrome (CHS) occurs to varying degrees in different cell types and has provided insight into the pathophysiology of lysosomal granules. This study was undertaken to determine the extent of involvement of eosinophil crystalloid granules (CGs) and smaller non-crystalloid granules (NCGs) in giant granule formation. Eosinophils from two CHS patients were evaluated after glutaraldehyde fixation and staining for morphologic examination, peroxidase, and complex carbohydrate using uranyl acetate-lead citrate, diaminobenzidine-lead citrate, and periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) methods, respectively. Although many CGs appeared normal in shape and size, several CGs appeared enlarged and a few measured over 5 microns in diameter, consistent with giant granule formation in CHS. These giant granules either occasionally contained a single large crystalloid or, more frequently, contained numerous normal-size crystalloids. Enlargement of granules was also observed in some precursor CGs of bone marrow early eosinophils, indicating that giant granule formation was initiated during granule genesis. Almost all NCGs in late eosinophils were small granules and stained strongly with PA-TCH-SP in contrast to CGs. Most, but not all small granules were peroxidase-positive in eosinophil precursors, whereas the percentage of peroxidase-negative small granules increased in late eosinophils. This indicated the presence of at least two small granule populations. Morphometric studies indicated CHS selectively involved CGs and demonstrated that neither the average size nor numbers of NCGs were significantly different from normal eosinophils. Thus, these studies indicate that CHS selectively involves CGs, and demonstrate preservation of normal granule size and heterogeneity for NCGs in late eosinophils. These observations suggest that the underlying CHS pathophysiology does not involve all lysosomal subpopulations.

Heterogeneity of peroxidase-positive granules in normal human an Chédiak-Higashi neutrophils.

Studies have demonstrated significant heterogeneity in neutrophil granule morphology and physical density. This study evaluated the heterogeneity morphometrically, morphologically, cytochemically, and biochemically. Intact human peripheral blood neutrophils collected from normal volunteers and a patient with Chédiak-Higashi syndrome (CHS) and isolated normal neutrophil granules were processed for ultrastructural morphology and peroxidase staining. Intact cells, nuclei, and granule profiles were analyzed by computer-assisted planimetry. Peroxidase-positive granules (PPG) represented about 40% of normal neutrophil granules and covered the entire spectrum of granule size. PPG in the least-dense fractions of isolated granules were significantly smaller than in higher-density fractions. PPG in low- and intermediate-density fractions differed from high-density fraction by moderate to strong vicinal glycol staining with Thiéry's periodate-thiocarbohydrazide-silver proteinate method. Differing ratios of % beta-glucuronidase/% myeloperoxidase (MPO) across granule fractions indicated PPG heterogeneity. Morphometric analysis of neutrophils treated with 1 microM calcium ionophore A23187 did not show significant differences in PPG size or number. Biochemically analyzed MPO in these cells was preserved, although the number of peroxidase-negative granules (PNG) and levels of vitamin B12-binding protein were markedly decreased. In CHS, about 20% of granules were PPG. Analysis of CHS neutrophils revealed the persistence of microgranules similar to normals. PNG number and volume fractions of PPG and TG were not different from normals. Complex heterogeneity of normal PPG was quantitated using morphometry and appeared preserved in ionophore-treated cells but was uniquely modified in CHS.

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.

Comprehensive care for patients with hemophilia: an expanded role in reducing risk for human immunodeficiency virus.

Hemophilia is an inherited coagulation disease that affects approximately 1 in 5,000 to 10,000 males worldwide. Chronic joint disease and other long-term complications of recurrent bleeding persist in patients with hemophilia despite improved and more available clotting protein concentrates. The best care can be provided to patients who are followed regularly in specialized treatment centers. Services of every "comprehensive" hemophilia treatment center (HTC) have expanded since previous treatment with clotting factor concentrates infected many hemophilics with the human immunodeficiency virus (HIV). Each HTC offers therapeutic, educational, and counseling expertise in care for the complications of HIV. A nationwide network of specialists now provides care for patients with hemophilia and related congenital abnormalities. In Region VI (Texas, Oklahoma, and Arkansas), the treatment centers and their affiliates provide medical, psychosocial, orthopedic/physical therapy, dental, and case management services. Extramural funded research programs provide care and laboratory testing at no cost to individual subjects.

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.

Giant mitochondria distinct from enlarged mitochondria in secretory and ciliated cells of gerbil trachea and bronchioles.

Numerous mitochondria ranging from slightly larger than normal to several micrometers in diameter (giant) were found in about one-half the serous secretory cells in the surface epithelium of the normal gerbil trachea and proximal bronchi. Tracheal serous cells of mice also were found to contain numerous giant mitochondria. Clara cells of gerbil bronchioles contained abundant giant mitochondria in addition to normal tubular mitochondria and the second population of enlarged spherical mitochondria that have been described in Clara cells of several genera. In contrast, mouse Clara cells revealed the normal tubular and the enlarged spherical mitochondria but no giant mitochondria. A survey of a number of cell types in gerbils failed to disclose hypertrophied mitochondria outside tracheobronchial surface epithelium and bronchioles. The mitochondrial enlargement resulted from an increase of matrix but not cristae. The expansion of matrix displaced the relatively sparse cristae into small collections compressed against the outer membrane. The prevalence of giant mitochondria and of granular endoplasmic reticulum is similar among cells, and these two organelles are codistributed within cells. The megamitochondria and granular reticulum occupy a central stratum, whereas normal mitochondria occur in the apical and basal regions. The giant mitochondria are considered related to a normal biologic activity that is characteristic of respiratory tract epithelium of mice and gerbils selectively and is more prominent in secretory cells than in ciliated cells.

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.

Disseminated primitive neuroectodermal tumor: diagnosis using immunocytochemistry, electron microscopy, and molecular probes.

A patient with a disseminated small cell tumor presented with hyperuricemia, gingival hypertrophy, lymphadenopathy, and bone marrow replacement with tumor cells. Initial histologic examination and clinical presentation were consistent with presumed marker silent lymphoma/leukemia. Despite initial treatment with and response to lymphoma/leukemia therapy the patient relapsed in the testis, bone marrow, pancreas, and skin whereupon subsequent and retrospective immunocytochemical, ultrastructural, cytogenetic, and molecular analysis led to the diagnosis of primitive neuroectodermal tumor (PNET). Despite extensive investigation and autopsy no primary site of tumor could be found demonstrating that PNET should be considered in the differential diagnosis of disseminated small cell tumors without an apparent primary.