Ifosfamide plus doxorubicin in metastatic adult sarcomas: a multi-institutional phase II trial.

Between April 1986 and March 1987, 42 patients with advanced sarcoma were entered in this multi-institutional trial evaluating ifosfamide plus doxorubicin. The majority of patients had leiomyosarcoma and malignant fibrous histiocytoma although two patients with sarcomas of osseous origin were included. Doxorubicin was administered at a dosage of 60 mg/m2 by continuous push and ifosfamide 5.0 g/m2 by continuous infusion over 24 hours with mesna (7.5 g2 over 36 hours) with courses repeated every 3 weeks until progression, toxicity cumulative doxorubicin dosage of 450 mg/m2. Overall, 15 (36%) patients demonstrated objective remissions including three complete and 12 partial remissions (95% confidence limits, 21.5% to 52.0%). The median duration of remission was 7.0 months and the median survival time for all eligible patients was 8.0 months. Toxicity was predominantly hematologic with the median leukocyte nadir being 1,300 per microliter of blood and documented sepsis in six patients. These data support activity for ifosfamide plus doxorubicin in patients with advanced sarcoma, but the actual contribution of ifosfamide needs to be evaluated through prospective randomized trials which are currently underway.

Immunologically-mediated renal disease in Waldenström's macroglobulinemia.

A patient with Walderström macroglobulinemia associated with nephrotic syndrome is described. Serum cryoglobulin and rheumatoid factor were absent. Intramembranous electron-dense deposits were demonstrated in kidney biopsy material by electron microscopy. Deposits of immunoglobulin G (IgG), M (IgM) and the third component of complement (C3) were identified in kidney biopsy tissue by immunofluorescent staining methods. The serum immunoglobulins were characterized by chromatographic and immunochemical methods and showed a monocional IgM-K, IgG-K and gamma-chain piece of undefined structure. Free K- and gamma-chains were found in the urine. The IgM was not complexed to the IgG or vice versa, but the IgG was in an affregated form. Although it is not known which immunoglobulin initiated the tissue injury, IgG, IgM and complement deposits probably contributed to the renal dysfunction. The nephrotic syndrome diminished after treatemnt with chlorambucin and corticosteroids.

Follicular center-cell lymphoma with plasmacytic differentiation, monoclonal paraprotein, and peripheral blood involvement. Recapitulation of normal B-cell development.

A patient with a nodular and diffuse small-cleaved follicular center-cell lymphoma that exhibited definite plasmacytic differentiation, a related monoclonal gammopathy, and circulating population of small lymphocytes is presented. Aside from showing that the presence of numerous plasma cells is not a reliable criterion for the diagnosis of a reactive follicular proliferation, the case is an example of a lymphoma without a block in maturation. The "neoplastic" B-cells show development to follicular center cells and beyond, to functioning plasma cells, and probably also to recirculating "memory" cells. It also suggests that plasmacytoid lymphocytic lymphomas (Lukes-Collins classification) might represent a heterogeneous group of lymphoid neoplasms with some closely related to follicular center-cell lymphomas and others more closely related to small lymphocytic lymphoma/B-cell chronic lymphocytic leukemia.

Immunohistochemical classification of acute leukemias using peripheral blood smears.

Immunophenotypic classification of the acute leukemias (AcL) is of well documented value in those of lymphoid or uncertain origin and of increasing importance in those of nonlymphoid origin. Most of these studies have been performed on viable cell suspensions. To study the efficacy of a simpler immunohistochemical approach to the classification of the acute leukemias requiring only peripheral blood smears, 15 AcL (including three CGL-BC) were studied using an immunoalkaline phosphatase method and a panel of anti-lymphoid and anti-myeloid monoclonal antibodies. Routine cytochemistries were also performed (Sudan black, PAS). Using immunohistochemistry, five cases marked as common ALL (four were undifferentiated by cytochemistry, one ALL), eight cases as ANLL (all ANLL by cytochemistry) and two cases marked only with anti-HLA-DR (AUL by cytochemistry). These results show that immunophenotypic analysis of AUL, ALL and ANLL can be successfully performed even when only air dried peripheral blood smears are available.

Incorporation of inositol hexaphosphate into red blood cells mediated by dimethyl sulfoxide.

Inositol hexaphosphate (IHP) binds to deoxyhemoglobin and markedly decreases the affinity of hemoglobin for oxygen. We introduce here a method for incorporating this polyphosphate into erythrocytes, thus preparing very low affinity cells for use in respiration research. The method uses dimethyl sulfoxide (DMSO) to facilitate entry of IHP. The cells are exposed to a high concentration of DMSO which is rapidly diluted with IHP solution. During this dilution the cells become leaky and IHP enters. The influence of several variables at each step of the process has been investigated and the data support a transient osmotic gradient mechanism for IHP incorporation.

Amino acid requirements of a rat sarcoma as determined by a stem cell assay.

Knowledge of the amino acid requirements of a neoplasm is valuable in determining optimal nutritional support and antineoplastic therapy for the tumor-bearing host. The standard human tumor stem cell assay (HTSCA) was modified by reducing an individual amino acid below the normal plasma concentration of the Fischer 344 rat. All other amino acids were maintained at levels sufficient for normal HTSCA tumor colony growth. Twenty-two amino acids were tested at a mean concentration of 12% (range 3% to 35%) of their normal plasma level. Results indicated that all amino acids except L-glutamine and L-asparagine were present in sufficient quantity for normal tumor growth. Dose-response curves have shown more than 70% inhibition of tumor growth with a glutamine concentration of 50% and an asparagine concentration of 25%. Glutamine and asparagine levels of 4% and 1%, respectively, resulted in 100% inhibition. The data indicate that rat sarcoma stem cells are sensitive to decreased glutamine and asparagine concentrations.

Late intensification therapy in adult acute lymphoid leukemia. The Southeastern Cancer Study Group Experience.

One hundred ninety-two evaluable patients were treated on a multicenter protocol for adult acute lymphoid leukemia to determine in a prospective randomized fashion if late intensification chemotherapy beginning after about six months of treatment would improve remission duration and survival. The complete remission rate was 60%. The median remission duration was 13.5 versus 25.9 months (P = 0.31) for standard maintenance therapy and late intensification, respectively, and the median survival was 17.5 versus 34.7 months (P = 0.19) respectively. Although there was a suggestion that the late intensification strategy was helpful, relapse proved to be common during the early phases of treatment; thus, insufficient numbers of patients were available at the randomization point to conclusively address the possible value of late intensification. Intensive therapy earlier in remission should be evaluated.

Increased tyrosine protein kinase activity in hairy cell and monocytic leukemias.

Tyrosine protein kinases (TPK) help regulate cellular growth and differentiation. Several proto-oncogenes encode for protein products with associated tyrosine kinase activity. An assay for TPK activity was performed in cell extracts using a synthetic peptide substrate and [32P] adenosine triphosphate (ATP). TPK activity was elevated in K-562 cells, which possess an amplified c-abl oncogene, compared to normal blood mononuclear cells (K-562 = 9.37 +/- 1.72 [mean +/- standard deviation] pmol ATP/10(6) cells/min; normal = 1.14 +/- 0.46, p less than 0.01). TPK activity was measured in peripheral blood mononuclear cells from patients with hairy cell leukemia (HCL), myelomonocytic leukemia (MOL), acute myeloblastic leukemia (AML), and chronic lymphocytic leukemia (CLL). In patients with clinically active disease, elevated TPK activity was measured in mononuclear cells from five HCL patients (range 3.76-24.15) and from seven MOL patients. These elevated levels appeared to parallel disease activity, as low levels of TPK activity were measured in patients with inactive (treated) disease. Low levels of TPK were measured in mononuclear cells from active AML and CLL patients. Elevated TPK levels in patients with HCL and MOL may reflect the overexpression of a proto-oncogene or increased growth factor activity in immature or rapidly dividing leukemic cells. Serial TPK levels in HCL and MOL patients correlated with change in disease activity.

Phosphorylation of rat liver ribonucleic acid polymerase I by nuclear protein kinases.

Phosphorylation of rat liver RNA polymerase I occurred when intact rat liver nuclei were incubated with [gamma32P]ATP and N6,O2' dibutyryl cyclic 3':5'-AMP. In addition, partially purified RNA polymerase I could be phosphorylated in vitro by an endogenous protein kinase. Phosphorylation by either method was followed by extensive purification of the enzyme. This revealed that 32P remained bound to the enzyme throughout purification. Analysis of the homogeneous labeled protein by polyacrylamide gel electrophoresis under nondenaturing conditions followed by autoradiography revealed that only one of the two forms of RNA polymerase I in rat liver nuclei was phosphorylated. RNA polymerase II was not phosphorylated in intact nuclei. Polyacrylamide gel electrophoresis of the phosphorylated RNA polymerase I in the presence of 0.1% sodium dodecyl sulfate followed by autoradiography demonstrated that the 32P was located primarily on enzyme subunits SA1, SA3, and SA5-SA6. High voltage paper electrophoresis of a partial acid hydrolysate of phosphorylated RNA polymerase I revealed that both serine and threonine residues were phosphroylated. N6,O2'-Dibutyryl cyclic 3':5'-AMP stimulated endogenous RNA polymerase I activity and endogenous nuclear protein phosphorylation in intact nuclei. These results suggest that phosphorylation of RNA polymerase I by nuclear protein kinases may play a role in the control of transcription in mammalian cells.

Purification and properties of a nuclear protein kinase associated with ribonucleic acid polymerase I.

A cyclic AMP-dependent nuclear protein kinase was found to be closely associated with rat liver nucleolar RNA polymerase I throughout most of its purification. This protein kinase was purified to near homogeneity. It exhibits a number of unusual catalytic properties, including the inability to utilize Mn2+ when RNA polymerase is the substrate and the ability to phosphorylate both acidic and basic substrates. Phosphorylation of RNA polymerase I by this protein kinase results in the formation of phosphoester bonds characteristic of phosphoserine and phosphothreonine. Radioautography of polyacrylamide-gel electrophoretograms of the phosphorylated RNA polymerase I revealed that the 32P was located primarily on enzyme subunits SA1, SA3, SA5, and SA6 [nomenclature of Kedinger, Gissinger & Chambon (1974) Eur. J. Biochem, 44, 421-436].

The effect of INH-inhibited heme synthesis on globin synthesis.

In the rabbit reticulocyte cell-free system, optimal globin synthesis is dependent upon an adequate level of heme. The effect of heme is mediated by an inhibitor of globin synthesis initiation termed HCR. In addition to marked inhibition of total globin synthesis, HCR results in a decreased alpha/beta globulin synthesis ratio. We describe here the use of INH as a relatively nontoxic inhibitor of heme synthesis in intact rabbit reticulocytes with a resultant inhibition of globin synthesis. In parallel with the inhibition of globin synthesis in reticulocytes, an inhibitor of globin synthesis in the hemin-supplemented cell-free system is generated. No INH-induced alterations in alpha/beta synthesis ratio could be found in "stress" reticulocytes from phenylhydrazine-treated rabbits, but "normal" reticulocytes from untreated rabbits showed a decreased alpha/beta ratio. Inhibition of heme synthesis and the resulting decrease in globin synthesis and intracellular hemoglobin concentration may have application as a potential treatment of homozygous sickle cell disease.

Primary sideroblastic anemia masked by bleeding.

A patient with characteristic features of iron deficiency was unexpectedly found to have circulating siderocytes. Bone marrow iron stain at this time showed absence of both hemosiderin and ringed sideroblasts; electron microscopy revealed absence of mitochondrial iron loading but presence of cytoplasmic ferritin in normoblasts. Replenishment of iron stores led to development of typical sideroblastic anemia. These observations suggest that increased percentage of siderocytes in otherwise typical iron deficiency anemia may signify the presence of a sideroblastic process masked by iron deficiency due to bleeding.

Activation of renal erythropoietic factor by phosphorylation.

Renal erythropoietic factor (REF) serves as a substrate for a cyclic nucleotide-dependent protein kinase. The phosphorylation of REF is associated with increased erythropoietic activity. Conversely, REF isolated from hypoxic rats is a poor phosphate acceptor. These findings and the presence of a positive correlation between erythropoietin levels and urinary excretion of adenosine 3',5' monophosphate (cyclic-AMP) in anemic individuals suggest that the cyclic-AMP-protein kinase system plays an important role in the activation of REF.

Cobalamin (vitamin B12) deficiency detection by urinary methylmalonic acid quantitation.

A study was made to assess the value of cobalamin deficiency detection through quantitation of urinary methylmalonic acid (MMA). Urinary MMA was measured in 1118 patients suffering from megaloblastic anemia, other anemias, elevated red cell mean corpuscular volume, or unexplained neurologic disorders. Patients without proven cobalamin deficiency had urinary MMA levels less than 20 micrograms/ml. All patients (n = 27) confirmed to have cobalamin deficiency showed MMA levels greater than 20 micrograms/ml. Data are presented showing the Schilling test results, the comparison of serum cobalamin to urinary MMA levels, and other basic hematologic data. MMA levels are a good indication of cobalamin distribution and function since they are directly related to a cobalamin-dependent metabolic pathway. With rapid, reliable quantitation by mass spectrometry, urinary MMA can now be a useful clinical test.

Activation and partial characterization of a human reticulocyte heme-dependent eIF-Z alpha kinase.

An inhibitor of globin translation initiation that is activated under heme-deficient conditions and whose activity is associated with phosphorylation of the 38,000 dalton (alpha) subunit of the initiation factor eIF-2 has been previously described in rabbit reticulocytes. We describe here an analogous enzyme in human reticulocytes. The human enzyme can be activated in intact reticulocytes under conditions of heme synthesis inhibition or by incubation of lysates in the absence of hemin. Addition of hemin to lysates prevents activation of the eIF-2 alpha kinase. The partially purified kinase is associated with inhibition of globin synthesis in a rabbit cell-free system and phosphorylates the small subunit of highly purified rabbit eIF-2. We conclude that human reticulocytes contain a protein kinase that is analogous to the rabbit HCR and that may play a role in clinical heme deficiency states. l