ALBERT

Description: Cases of acute leukemia showing both terminal deoxynucleotidyl transferase (TdT) and myeloperoxidase (MPO) activities are usually classified as acute myelogenous leukemia (AML). Although some reports have implied overlap of TdT and MPO based on population percentages, direct evidence for simultaneous expression of TdT and MPO by a leukemic blast is lacking. By use of a simple new technique developed in our laboratory for identifying TdT and MPO in individual cells by light microscopy, we examined three cases of acute leukemia with both TdT and MPO positivity and found that the incidence of cells positive for both TdT and MPO was 0%, 1%, and 23%. Cytogenetic analysis showed a single leukemic clone in all patients, providing additional evidence that these leukemias arose from a single cell capable of expressing both MPO and TdT. These findings have implications for understanding the relation between MPO and TdT expression in leukemia.

Description: Biosynthesis and molecular structure of major histocompatibility complex (MHC) class II antigens of DR2/DR7 hairy cells were analyzed by two-dimensional polyacrylamide-gel electrophoresis (2D-PAGE). Two anti- human Ia monoclonal antibodies (mAb) were used to immunoprecipitate DR and DR-linked DC/DS molecules. Monoclonal antibody VI 15 C recognizes DR (I-E-like) molecules and CA 2.06 precipitates DR and DR-linked DC/DS (I-A-like) molecules in DR7 allotypes. Studies were performed on a pure population of hairy cells before and after culture with phorbol ester: 12-O-tetradecanoyl phorbol-13-acetate (TPA), 5 azacytidine (5 Aza), sodium butyrate (NA-BU), and phytohemagglutinin (PHA-P). Before any treatment, hairy cells expressed and synthesized DR antigens: DR alpha and beta subunits appeared both qualitatively and quantitatively normal by 2D-PAGE profile. In contrast, the hairy cells failed to express and synthesized any DC/DS molecule. The lack of DC/DS molecular expression was restored after culture in presence of TPA, sodium butyrate, and 5 azacytidine, but not after PHA-P treatment. Differential molecular expression of MHC class II antigens in leukemic cells provides a model to define further discrete stages of hemopoietic differentiation and study the role of these molecules in the cellular interactions occurring during differentiation.

Description: The activity of delta-aminolevulinic acid (ALA) dehydratase, an enzyme involved in heme biosynthesis, has been shown to increase in Friend virus-transformed murine erythroleukemia (MEL) cells during erythroid differentiation. In this study, the nature of the increase in ALA dehydratase activity in MEL cells was examined using a monospecific antibody directed to the enzyme. A sevenfold increase in ALA dehydratase activity was observed after cells had been treated with 1.5% Me2SO for 5 days. Ouchterlony double immunodiffusion analysis showed that lysates from untreated and Me2SO-treated MEL cells formed a single precipitin line with rabbit IgG directed to the normal mouse liver ALA dehydratase. A single arc of identity was also observed with the lysates from normal mouse erythrocytes, spleen, liver, and lysates from both uninduced and induced MEL cells. Rocket immunoelectrophoresis demonstrated that lysates from both uninduced and induced cells formed rockets with the IgG and that the peak height of the rocket was proportional to the ALA dehydratase activity applied. The slope of linear plots of rocket peak heights v ALA dehydratase activity was identical for lysates from uninduced and Me2SO-induced cells. Succinylacetone, a potent inhibitor of ALA dehydratase, was shown to markedly inhibit the activity of the enzyme, but did not interfere with the synthesis of ALA dehydratase induced by Me2SO treatment. Me2SO- induced increases in ALA dehydratase activity and the enzyme protein were both blocked by the simultaneous treatment of cells with 5-bromo- 2′-deoxyuridine (BrdU). BrdU-mediated repression of ALA dehydratase was partially overcome by treating the cells with thymidine. These data demonstrate that increased ALA dehydratase activity in MEL cells undergoing erythroid differentiation after Me2SO treatment is due to de novo synthesis of the same enzyme protein present in uninduced MEL cells as well as in normal erythrocytes. This represents the first direct demonstration of an increase in a heme biosynthetic pathway enzyme protein in erythroid cells undergoing differentiation.

Description: Hebbel and colleagues have proposed that increased adherence of sickle red cells to vascular endothelium may initiate vasoocclusive events in sickle cell disease. We have developed a micropipette technique to obtain direct, quantitative measure of the adherence of individual red cells to vascular endothelial cells. Using this technique, we found that the vast majority of sickle cells suspended in autologous plasma were strongly adherent to endothelial cells, whereas only a small fraction of normal cells were weakly adherent. Influence of plasma factors on adherence was determined by measuring adherence of sickle cells suspended in normal plasma and normal cells suspended in sickle plasma. Although over 90% of sickle cells adhered to endothelial cells in autologous plasma, the percentage of adherent cells decreased dramatically to less than 20% when the same sickle cells were suspended in normal plasma. In contrast, adhesion of normal red cells suspended in sickle plasma was only modestly increased compared to adhesion in autologous normal plasma. Our results provide direct evidence for markedly enhanced adherence of sickle cells to endothelial cells. In addition, they suggest that both cell membrane changes and plasma factors contribute to this interaction. The requirement for sickle plasma further implies that temporal changes in plasma factors may play an important role in determining the onset of vasoocclusive crisis.

Description: The effect of splenectomy on the response to random donor platelet transfusion in 15 multitransfused thrombocytopenic patients is presented. Eight patients responded poorly, with low corrected platelet count increments at 1 and 24 hours posttransfusion. These eight patients were clinically alloimmunized and had lymphocytotoxic antibody ( LCTAb ) in their sera. They responded well to closely HLA-matched transfusions. In contrast, seven splenectomized patients responded well to random donor platelets. Five of these patients had no LCTAb and no other evidence of immunization. Two patients who responded well to random donor platelets had “weak” LCTAb , and one responded to platelets presplenectomy in the presence of this antibody. Splenectomy does not improve the response to random donor platelets in alloimmunized recipients.

Description: Prenatal diagnosis of hematologic diseases can now be performed with fetal blood, fetal amniotic fluid cell DNA, and fetal chorionic villi DNA. Some hemoglobinopathies can be detected by all three methods, and the choice will depend on the available obstetric and laboratory techniques, as well as the time of presentation of the pregnancy. Hopefully, further development of molecular probes and techniques will soon expand these options to all of the globin disorders. Detection of coagulation disorders in utero currently requires samples of pure fetal blood. Gene cloning is accomplished for some (factor IX and antithrombin III) and is underway for others (factor VIII), and further investigation is necessary to determine whether deficiencies in these gene products are due to gene deletion or to mutant genes linked to polymorphic restriction enzyme sites of diagnostic use. Thus, molecular biology may be applied to prenatal diagnosis of the clotting problems, but this has not yet been accomplished. Disorders affecting the number and/or function of erythrocytes, leukocytes, and platelets can be diagnosed by analysis of fetal blood. Blood samples will continue to be required until more is known about the molecular biology of hematopoiesis. Syndromes that can be diagnosed by chromosome studies should be revealed in cultures of amniotic fluid cells, fetal blood lymphocytes, and chorionic villi cells. Cultured cells can be examined for karyotypes, Y-chromatin, spontaneous or induced chromosome breakage, DNA repair, SCEs, and translocations. The techniques for culturing amniotic cells and fetal blood white cells are established, and those for growing cells from chorionic villi are improving rapidly. Direct preparations of cells from villi only may suffice for some of the above analyses. The study of hematologic disease in utero has thus come full circle, from the use of amniotic cells to determine the sex in X-linked disorders, to fetal blood sampling for the analysis of gene products, then back to amniocentesis for DNA, and now earlier in gestation to chorionic villi. All of this has occurred in less than ten years, and it is anticipated that developments in the next ten years will be equally dramatic. The future should bring all prenatal testing into the first trimester, use molecular probes, and provide for both early diagnosis and early treatment of genetic hematologic disease.

Description: Methotrexate has been used as the mainstay therapy to prevent or ameliorate graft-versus-host disease (GVHD) in allogeneic bone marrow transplantation. We began a nonrandomized study in which methotrexate was not given routinely. Fifty-five patients underwent transplant for acute leukemia (44 patients), aplastic anemia (6 patients), and other malignancies (5 patients). Methotrexate was given to 34 patients (MTX +) and was withheld in 21 patients (MTX -). Median (range) age of patients was 12 (0.8–43) years in the MTX + group, and 16 (3–45) years in the MTX- group. Mean days (+/- SEM) to engraftment (neutrophils greater than 500/microL, and platelets greater than 20,000/microL untransfused) occurred earlier in the MTX- patients (19.6 +/- 1.4 v 24.9 +/- 1.8 days for granulocytes, and 19.3 +/- 1.5 v 27.4 +/- 2.8 days for platelets, P less than .05). There were no statistically significant differences between the patient groups for the incidence or severity of GVHD (10/34 in the MTX + group had grade O-l GVHD compared to 9/21 in the MTX- group). The interstitial pneumonitis occurred at a significantly increased rate in patients who received methotrexate (15/34) compared to those patients who did not (3/21) (P = .02). However, there was also a significant relationship between the interstitial pneumonitis and the preparative regimen: if the preparative regimen contained 1,000 rad single fraction total body irradiation, 8/14 patients were affected compared to 5/22 patients affected when 1,200 rad fractionated total body irradiation was used (P = .03). Because methotrexate significantly retards hematopoietic reconstitution, randomized trials for GVHD prevention are recommended.

Description: Acute graft-versus-host disease (GVHD), a major complication of allogeneic bone marrow transplantation (BMT), is probably mediated by T lymphocytes present in the marrow graft. In this study, the repopulation of the peripheral blood with T4+ and T8+ T cells was investigated during the period preceding the occurrence of acute GVHD. Twenty-four allogeneic and 11 autologous BMT recipients were monitored from day 4 post-BMT onward by the use of monoclonal antibodies, indirect immunofluorescence, and flow cytometry. The recipients of allogeneic transplants received methotrexate as GVHD prophylaxis. Similar recovery patterns for T4+ and T8+ T cells were found following autologous and allogeneic BMT. However, lymphoid repopulation occurred at a clearly faster rate after autologous BMT. T4+ T cells were the first to reappear in the peripheral blood, followed by T8+ T cells 4–7 days later. The T8+ T cell reconstitution occurred at an even faster rate in patients who were to develop grade II-IV GVHD, as compared with those with grade O-I GVHD, thus leading to an earlier decrease in the T4/T8 ratio. Of 10 patients with a T4/T8 ratio less than 2.5 at day 19, 9 developed grade II-IV GVHD and 1 showed no GVHD. Of 14 patients with a ratio greater than 2.5 at that time, only 2 developed grade II-IV and 12 grade O-I GVHD (p less than 0.001). In the 11 patients developing grade II-IV GVHD, the T4/T8 ratio decreased to values less than 2.5 before the first clinical symptoms of GVHD in 9; it coincided in one and occurred later in another patient. Thus, early monitoring of the T4/T8 ratio can distinguish patients at risk of developing grade II-IV GVHD.

Description: We have studied the role of factor VIII-von Willebrand factor (FVIII- vWF) in both platelet adherence to subendothelium and ristocetin- induced platelet aggregation using monoclonal antibodies to human FVIII- vWF. Twenty-five monoclonal antibodies were obtained, two of which were directed to the factor VIII moiety of FVIII-vWF; one of these two completely inhibited the procoagulant activity (FVIII:C). The remaining 23 monoclonal antibodies were directed to the von Willebrand factor moiety of FVIII-vWF. The ability of the latter monoclonal antibodies to inhibit platelet adherence to arterial subendothelium was investigated with a perfusion model. According to the number of platelets adhering to the subendothelium, three groups of monoclonal antibodies could be discerned: (A) antibodies not affecting platelet adherence; (B) antibodies that inhibited platelet adherence to the level as observed when von Willebrand's disease plasma was tested; and (C) antibodies that completely inhibited both platelet adherence to subendothelium and ristocetin-induced platelet aggregation. The two antibodies present in group C competed for the same or closely related epitope(s) present on FVIII-vWF. These results demonstrate that a domain is present on the FVIII-vWF molecule that is associated both with ristocetin-induced aggregation and with the ability of FVIII-vWF to support platelet adherence to the subendothelium. Based on these observations, it is concluded that ristocetin-induced binding of FVIII-vWF to platelets reflects, at least in part, a physiologic mechanism regulating the function of FVIII-vWF in primary hemostasis.

Description: We have analyzed a cloned beta O-thalassemia (beta O-thal) gene from a patient doubly heterozygous for hemoglobin Lepore and beta O- thalassemia. Studies of 3H-uridine incorporation into beta-globin mRNA in this patient's erythroblasts suggested an intranuclear defect in both beta and Lepore (delta beta) mRNA synthesis, as did S1 nuclease analysis of nuclear RNA. However, the nucleotide sequence of the beta O- thal gene revealed only a single base change in codon 39 (CAG----UAG), which created a premature translation termination codon. The 5′ flanking sequence, including transcription promotor boxes and the mRNA initiation (CAP) site, were normal. The unexpected effect of this mutation on intranuclear beta-mRNA synthesis in vivo was studied by insertion of the cloned gene into a plasmid expression vector and transfection into tissue culture (COS-1) cells. beta-Globin mRNA produced by the transfected cells was assessed by S1 nuclease analysis. The beta O-39 thalassemia gene generated five- to tenfold less beta- mRNA than a normal beta-gene in both nuclear and cytoplasmic RNA, simulating the results observed in vivo. Moreover, the small amount of beta O-39 mRNA produced was as stable as normal beta-mRNA during an actinomycin D chase, ruling out rapid cytoplasmic turnover as a cause of the reduced accumulation. Cotransfection of the beta O-39 thalassemia gene with a mutant tyrosine suppressor tRNA gene resulted in restoration of the beta O-39 mRNA accumulation to near-normal levels. On the basis of these results, we suggest that the low levels of beta-mRNA known to exist in the common form of beta O-thalassemia, beta O-39 thalassemia, result from a lesion in transcription, or early posttranscriptional processes; the defect appears to be corrected by restoration of proper translational potential to the mutant mRNA, at least in a gene transfer-expression system in tissue-culture cells.

Description: Two new cell surface antigens specific for the erythroid lineage were defined with cytotoxic IgM monoclonal antibodies (McAb) (EP-1; EP-2) that were produced using BFU-E-derived colonies as immunogens. These two antigens are expressed on in vivo and in vitro derived adult and fetal erythroblasts, but not on erythrocytes. They are not detectable on resting lymphocytes, concanavalin-A (Con-A) activated lymphoblasts, granulocytes, and monocytes or granulocytic cells or macrophages present in peripheral blood or harvested from CFU-GM cultures. Cell line and tissue distributions distinguish McAb EP-1 and EP-2 from all previously described monoclonal antibodies. McAb EP-1 (for erythropoietic antigen-1) inhibits the formation of BFU-E and CFU-E, but not CFU-GM, colonies in complement-dependent cytotoxicity assays. By cell sorting analysis, about 90% of erythroid progenitors (CFU-E, BFU-E) were recovered in the antigen-positive fraction. Seven percent of the cells in this fraction were progenitors (versus 0.1% in the negative fraction). The expression of EP-1 antigen is greatly enhanced in K562 cells, using inducers of hemoglobin synthesis. McAb EP-2 fails to inhibit BFU-E and CFU-E colony formation in complement-dependent cytotoxicity assays. EP-2 antigen is predominantly expressed on in vitro derived immature erythroblasts, and it is weakly expressed on mature erythroblasts. The findings with McAb EP-1 provide evidence that erythroid progenitors (BFU-E and CFU-E) express determinants that fail to be expressed on other progenitor cells and hence appear to be unique to the erythroid lineage. McAb EP-1 and EP-2 are potentially useful for studies of erythroid differentiation and progenitor cell isolation.

Description: This study was undertaken to examine the interaction of platelet size and age in determining in vitro platelet function. Baboon megakaryocytes were labeled in vivo by the injection of 75Se- methionine. Blood was collected when the label was predominantly associated with younger platelets (day 2) and with older platelets (day 9). Size-dependent platelet subpopulations were prepared on both days by counterflow centrifugation. The reactivity of each platelet subpopulation was determined on both days by measuring thrombin-induced aggregation. Platelets were fixed after partial aggregation had occurred by the addition of EDTA/formalin. After removal of the aggregated platelets by differential centrifugation, the supernatant medium was assayed for remaining platelets and 75Se radioactivity. Comparing day 2 and day 9, no significant difference was seen in the rate of aggregation of a given subpopulation. However, aggregation was more rapid in the larger platelet fractions than in the smaller ones on both days. A greater percentage of the 75Se radioactivity appeared in the platelet aggregates on day 2 than on day 9. This effect was independent of platelet size, as it occurred to a similar extent in the unfractionated platelets and in each of the size-dependent platelet subpopulations. The data indicate that young platelets are more active than older platelets. This study demonstrates that size and age are both determinants of platelet function, but by independent mechanisms.

Description: We have measured the fully carboxylated (native) prothrombin antigen and the undercarboxylated (abnormal) prothrombin antigen in patients treated with sodium warfarin using specific immunoassays to evaluate a new approach for monitoring oral anticoagulant therapy. Plasma and serum samples (391) were assayed for the prothrombin time, native prothrombin antigen, and abnormal prothrombin antigen. The results were correlated with the presence of bleeding or thromboembolic complications at the time of phlebotomy. The native prothrombin antigen correlated with the occurrence of complications in 95% of samples. Of 13 samples from patients with bleeding complications, 13/13 (100%) had a native prothrombin of 12 micrograms/mL or lower. Of seven samples from patients with thromboembolic complications, 6/7 (86%) had a native prothrombin of 24 micrograms/mL or greater. By comparison, a prothrombin time index of 1.5 to 2.5, 1.5 to 2.2, 1.5 to 2.0, or 1.3 to 1.8 identified 6/20 (30%), 9/20 (45%), 11/20 (55%), or 12/20 (60%) patients at risk, respectively. Although the prothrombin time index did correlate with the presence of bleeding complications, the native prothrombin antigen correlated closely with the presence of bleeding and thromboembolic complications. According to these results, the native prothrombin antigen, maintained in a range of 12 to 24 micrograms/mL by regular adjustment of the warfarin dosage, may be associated with a reduced risk of complications due to excessive or insufficient warfarin therapy. On the basis of these preliminary data, we recommend that the native prothrombin antigen be considered to monitor warfarin therapy.

Description: High proliferative potential macrophage progenitor cells (HPP-CFC) in 5- fluorouracil (FU) treated and normal mouse bone marrow (BM) have been shown to be less sensitive to inhibition of proliferation by prostaglandins of the E series (PGE) than low proliferative potential macrophage progenitor cells (LPP-CFC) in normal BM in agar cultures. The growth of large colonies (diameter greater than 0.5 mm) derived from HPP-CFC in FU BM, which require a combination of macrophage colony- stimulating factor (CSF-1) plus a new growth factor called synergistic activity (SA), are inhibited by 50% in the presence of 5.5 X 10(-6) M PGE1. On the other hand, LPP-CFC in normal BM, which form smaller colonies (diameter less than or equal to 0.5 mm) in the presence of CSF- 1 alone, require only 5 X 10(-8) M PGE1 for the same level of inhibition. Addition of appropriate concentrations of PGE1 to the agar culture assay should improve detection of HPP-CFC by inhibiting the proliferation of LPP-CFC. These observations suggest that the apparent negative feedback control of macrophage production by PGE operates largely on the LPP-CFC, which respond to CSF-1 alone, and is probably not involved in the regulation of the more primitive HPP-CFC.

Description: We used both radiolabeled and fluorescein-labeled antiglobulins in assays to detect antibodies against platelets in multiply transfused patients to determine the value of these tests in predicting the outcome of platelet transfusion in such patients. In 15 allosensitized patients, we studied 68 single-donor platelet transfusions, 43 (63%) of which had a poor outcome, defined as a corrected count increment (CCI), less than 10,000. The results obtained with either test were significantly correlated with the CCI following transfusion (p less than 0.001), but the assay using the radiolabeled antiglobulin had slightly better sensitivity, specificity, and predictive value. When the assays were used in combination, there was again significant correlation with the CCI of the transfusion, p less than 0.001. When both assays predicted failure of the transfusions, 31/31 (100%) such transfusions resulted in a CCI of less than 10,000, and when both assays predicted success of the transfusions, 14/15 (93%) such transfusions resulted in a CCI of greater than 10,000. Both assays are useful in predicting the outcome of the platelet transfusions; when the assay results were concordant, almost total predictive accuracy was obtained.

Description: A 42-yr-old woman with systemic lupus erythematosus without bleeding diathesis developed a prolonged activated partial thromboplastin time that was not corrected by normal plasma. An inhibitor that acted rapidly and inactivated 0.5 U/ml plasma thromboplastin antecedent (PTA, factor XI) at a 1:200 plasma dilution was demonstrated. In addition to a low titer of PTA (less than 0.01 U/ml), plasma assayed at 20-fold dilution also showed low titers of Hageman (factor XII, 0.02 U/ml), Fletcher (plasma prekallikrein, 0.02 U/ml), and Fitzgerald (high molecular weight kininogen, less than 0.01 U/ml) factors. The titer of these factors, except PTA, returned to normal upon further plasma dilution or upon removal of the inhibitor by protein A adsorption. Thus, the inhibitor appeared to interfere with these clotting factor assays, possibly by inactivating PTA in the substrate plasmas in the test system. Its specificity was further confirmed. The inhibitor did not interfere with surface-induced proteolytic cleavage of Hageman factor. Surface-induced generation of plasma kallikrein activity (amidolysis of H-D-pro-phe-arg-pNa and cold-promoted factor VII activity enhancement) requires only Hageman, Fletcher, and Fitzgerald factors and was normal. Reactions requiring all 4 contact phase factors, including PTA, such as surface-induced generation of plasmin activity (amidolysis of H-D-val-leu-lys-pNa) and activated Christmas factor (factor IXa) activity, were defective. Furthermore, the inhibitor bound to agarose-protein A inactivated and removed PTA selectively from normal plasma. The inhibitor was an IgG-lambda autoantibody that precipitated PTA. The inactivated activated PTA (factor XIa) without the requirement for an additional cofactor. Furthermore, it inhibited surface-induced activation of PTA by interfering with its proteolytic cleavage upon glass surface exposure and with its binding onto the reactive surfaces.

Description: Because clinical disorders of spontaneous iron overload have no experimental counterpart, we studied iron distribution (atomic absorption analysis) and intestinal absorption (59Fe) in mice with hereditary alpha-thalassemia. Mice heterozygous for a radiation-induced alpha-Hb gene deletion exhibit a mild hemolytic anemia, like the human condition, with microcytosis, reticulocytosis, splenomegaly, and chemical evidence of defective alpha-chain synthesis. Quantitative iron determination showed that total iron content in spleen, liver, and kidney, but not heart or lung, of adult alpha-thalassemic mice was greater (P less than .05) than that in unaffected littermates. Iron concentration was also increased in liver (P less than .001), spleen (P = .025), kidney (P = .058), and heart (P = .010); in general, the greater the iron concentration in liver, the greater that in spleen (r = .39, P = .009), kidney (r = .70, P less than .001), and heart (r = .46, P less than .001). In mice examined 8 months postoperatively, splenectomy, as compared to sham operation, significantly raised iron content in extrasplenic tissues, but did not affect total body iron. At 10–11 weeks of age, but no longer at 12–14 weeks, thalassemic mice showed higher rates of iron absorption than age-matched controls. Thus, alpha-thalassemic mice display an early occurring iron absorption defect, leading to a modest, sustained, nonprogressive iron overload, and thereby represent a valuable model for exploring disorders of iron homeostasis.

Description: The role of divalent cations in platelet adherence to deendothelialized human arteries in flowing blood was investigated in an annular perfusion chamber. Spreading of platelets on the subendothelium was impaired below 30 microM of free Ca2+ ions (Ca2+). When Ca2+ was replaced by Mg2+, adherence was unchanged in perfusates without exogenous factor VIII-von Willebrand factor (FVIII-vWF), but the ability of FVIII-vWF to support platelet adherence was lost. Binding of FVIII-vWF to the vessel wall was independent of divalent cations, but bound FVIII-vWF was only able to mediate adherence after exposure to Ca2+. Pretreatment of FVIII-vWF with the calcium chelator EGTA (10 mM) resulted in loss of the ability to facilitate platelet adherence, while the ristocetin cofactor activity remained intact. Full restoration of the ability to mediate platelet adherence could only be obtained by prolonged dialysis against Ca2+ in the millimolar range. These data indicate that divalent cations have at least two separate roles to play in supporting platelet adherence: (1) platelet spreading on the subendothelium requires Ca2+ or Mg2+; (2) FVIII-vWF should be exposed to Ca2+ to obtain its optimal biologic activity in supporting platelet adherence.

Description: Peripheral blood granulocytes from patients with chronic myelogenous leukemia (CML) were studied for accessibility of membrane sialic acid and galactose residues to sodium borohydride-3H radiolabeling after oxidation with sodium metaperiodate (PI/B3H4) or with galactose oxidase (GO/B3H4). Granulocytes from untreated patients with chronic myelogenous leukemia showed increased radiolabeling with PI/B3H4, and decreased labeling with GO/B3H4 when compared to normal granulocytes. Granulocytes from leukemic patients receiving chemotherapy showed normal labeling patterns. Gel electrophoresis of membrane extracts showed that the changes in PI/B3H4 and GO/B3H4 reactivity of CML cells were distributed over all membrane protein bands. Our data suggest that CML granulocyte membrane proteins are aberrantly sialylated, with decreased accessibility of galactose residues, and that these changes may be reversed by clinical drug treatment.

Description: We present a colony assay system that allows in situ identification of human basophil/mast cell (basophil) colonies. In methylcellulose culture, in the presence of phytohemagglutinin-leukocyte conditioned media (PHA-LCM), human peripheral blood and bone marrow cells form colonies that can be distinguished by their unique morphological characteristics. Pure basophil colonies are diffuse, small colonies containing small, round, highly refractile cells. These characteristics of the constituent cells led us to the observation that a significant number of basophils are found in combination with eosinophils. The mixed eosinophil/basophil colonies have the distinctive elements of pure eosinophil and pure basophil colonies. Usually, these are diffuse colonies with compact clusters of slightly larger, darker-appearing cells. We also found colonies that contained basophils and neutrophils/monocytes, but this type could not be consistently identified by in situ morphology. Cytochemical analysis confirmed the metachromatic nature of the granules in the basophils. The presence of IgE receptors on the cells was documented by indirect immunofluorescent staining after passive sensitization with purified human IgE. Peripheral blood cells from six healthy volunteers formed 5.7 +/- 1.0 (mean +/- SEM) pure colonies in 2 X 10(5) cells. Cultures of bone marrow cells from patients with various types of anemia had 9.0 +/- 1.5 colonies in 10(5) cells. This is the first description of a colony assay system for in situ identification of a pure population of basophilic granulocytes.

Description: Morphological, immunologic, and functional properties of peripheral blood cells from two patients with chronic proliferations of granular lymphocytes are described. Cells from both patients showed a heterogeneous pattern from both a morphological and immunologic standpoint, indicating a polyclonal, rather than a monoclonal, expansion of these cells. In fact, both large and small-to-medium-sized granular lymphocytes were observed, and different percentages of positivity were found in the analysis with a large panel of monoclonal antibodies. Serologic and histologic features support the hypothesis that this lymphocytosis could be secondary to bacterial or viral infections rather than a primary event, suggesting that these patients may have chronic reactive immunoregulatory disorders.

Description: This report describes the experience of the Southeastern Cancer Study Group (SECSG) with a transport medium used for immunologic phenotyping of non-Hodgkin’s lymphomas. In a 2-mo pilot study, portions of 53 specimens of non-Hodgkin's lymphoma from four member institutions of the SECSG and affiliated community hospitals were sent by regular mail to a central laboratory. Immunologic phenotyping was carried out using a frozen section immunoperoxidase technique. In 48 of the cases, a clear-cut immunologic phenotype was obtained. Thirty-four tumors were of B cell origin and 7 had T cell markers. Six of the remaining lymphomas had neither B nor T cell markers, and the seventh had both. In 12 cases, phenotyping was also carried out at the originating institution using conventional cell suspension techniques; agreement between the two methods was excellent. The immunologic results were correlated with histopathologic diagnosis standardized using the Working Formulation for non-Hodgkin’s lymphomas. It was found that the low grade tumors were all B cell, but that the intermediate grade tumors were very heterogeneous immunologically. About one-fourth of the diffuse, intermediate grade or miscellaneous tumors had T cell markers. Our results indicate that immunologic phenotyping may be performed satisfactorily on transported material, making multiinstitution studies on the prognostic significance of immunologic phenotype in non- Hodgkin’s lymphomas feasible.

Description: In the baboon (Papio species), the two nonallelic gamma-genes produce gamma-chains that differ at a minimum at residue 75, where isoleucine (I gamma-chain) or valine (V gamma) may be present. This situation obtains in baboons that are sometimes designated as Papio anubis, Papio hamadryas, and Papio papio. However, in Papio cynocephalus, although the I gamma-chains are identical with those in the above mentioned types, the V gamma-chains have the substitutions ala----gly at residue 9 and ala----val at residue 23. The V gamma-chains of P. cynocephalus are called V gamma C to distinguish them from the V gamma A-chains of P. anubis, etc. A single cynocephalus animal has been found to have only normal I gamma-chains and I gamma C-chains (that is, glycine in residue 9, valine in 23, and isoleucine in 75). When HbF is produced in response to stress with 5-azacytidine, P. anubis baboons respond with greater production than do P. cynocephalus, and hybrids fall between. Minimal data on P. hamadryas and P. papio suggest an even lower response than P. cynocephalus. As HbF increases under stress, the ratio of I gamma to V gamma-chains changes from the value in the adult or juvenile baboon toward the ratio in the newborn baboon. However, it does not attain the newborn value. The V gamma A and V gamma C-genes respond differently to stress. In hybrids, the production of V gamma A- chains exceeds that of V gamma C-chains. A controlling factor in cis apparently is present and may be responsible for the species-related extent of total HbF production. It may be concluded that the more primitive the cell in the erythroid maturation series that has been subjected to 5-azacytidine, the more active is the I gamma-gene.

Description: In the baboon (Papio species), the two nonallelic gamma-genes produce gamma-chains that differ at a minimum at residue 75, where isoleucine (I gamma-chain) or valine (V gamma) may be present. This situation obtains in baboons that are sometimes designated as Papio anubis, Papio hamadryas, and Papio papio. However, in Papio cynocephalus, although the I gamma-chains are identical with those in the above mentioned types, the V gamma-chains have the substitutions ala----gly at residue 9 and ala----val at residue 23. The V gamma-chains of P. cynocephalus are called V gamma C to distinguish them from the V gamma A-chains of P. anubis, etc. A single cynocephalus animal has been found to have only normal I gamma-chains and I gamma C-chains (that is, glycine in residue 9, valine in 23, and isoleucine in 75). When HbF is produced in response to stress with 5-azacytidine, P. anubis baboons respond with greater production than do P. cynocephalus, and hybrids fall between. Minimal data on P. hamadryas and P. papio suggest an even lower response than P. cynocephalus. As HbF increases under stress, the ratio of I gamma to V gamma-chains changes from the value in the adult or juvenile baboon toward the ratio in the newborn baboon. However, it does not attain the newborn value. The V gamma A and V gamma C-genes respond differently to stress. In hybrids, the production of V gamma A- chains exceeds that of V gamma C-chains. A controlling factor in cis apparently is present and may be responsible for the species-related extent of total HbF production. It may be concluded that the more primitive the cell in the erythroid maturation series that has been subjected to 5-azacytidine, the more active is the I gamma-gene.

Description: Fibrin prepared from 15 pathologic thrombi was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the extent and pattern of fibrinolysis that occurs in vivo. Two groups of patients could be distinguished on the basis of the polypeptide chain composition of fibrin in their thrombi. Those patients who presented with acute vascular obstruction, either arterial or venous, showed a minimal degree of fibrin degradation, with a dominance of intact, undegraded crosslinked gamma-gamma dimers. On the other hand, patients with long-standing symptoms associated with chronic aortic aneurysms had thrombi containing extensively degraded fibrin. Thrombi in large aortic aneurysms were dissected into concentric layers that showed different degrees of fibrinolysis. The luminal surface consisted of fresh, red thrombus and contained undegraded crosslinked fibrin similar to that found in patients with acute occlusive disease. Deeper layers of the thrombus showed gamma-gamma chain degradation throughout, indicating that this portion was undergoing active thrombolysis. The findings demonstrate that the variability in the pathophysiologic balance between coagulation and fibrinolysis is reflected in vivo by the polypeptide chain composition of crosslinked fibrin in thrombi. The results support the hypothesis of a dynamic equilibrium between clotting and lysis, but indicate that the balance between these two processes may be distinctly different in separate areas of a single clot.

Description: The fucose-binding lectin from Lotus tetragonolobus ( FBL -L) has been previously shown to bind specifically to normal cells of the myeloid and monocytic lineages. The purpose of this study was to explore the utility of fluoresceinated FBL -L as a leukemia differentiation marker in conjunction with a panel of other frequently used surface markers (Fc receptor, HLA-DR, OKM1, and antimonocyte antibody). FBL -L reacted with leukemic cells in 8/9 cases of clinically recognized acute myeloid leukemia, including myeloid blast crisis of chronic granulocytic leukemia, 3/3 cases of chronic phase chronic myelogenous leukemia, and in 2/7 cases of clinically undifferentiated acute leukemia. Correlations were noted between reactivity with FBL -L, and DR and Fc receptor expression. Among continuous cell lines, FBL -L bound with high intensity to a majority of HL-60 and U937 cells. The less well differentiated myeloblast cell lines, KG-1, KG1a , and HL-60 blast II, exhibited less FBL -L binding than HL-60 and U937. A moderate proportion of K562 cells exhibited low level binding of FBL -L. Several lymphoblastic cell lines exhibited a pattern of low intensity binding that was distinguishable from the high intensity binding pattern of the myeloblastic lines. FBL -L reactivity of U937 was enhanced by induction of differentiation with leukocyte conditioned medium, but not dimethylsulfoxide. Such treatments induced contrasting patterns of change of HL-60 and U937 when labeled with OKM1, alpha-Mono, and HLA- DR. These studies demonstrate the application of FBL -L to analysis and quantitation of myelomonocytic leukemic differentiation.

Description: To investigate the cellular events that accompany erythroid hyperplasia, we studied several effects of erythropoietin (Epo) on marrow CFU-E in sickle cell anemia (SCA). We measured CFU-E number, CFU- E growth as a function both of Epo exposure time and of Epo concentration, and suppression of Epo-induced CFU-E formation by anti- Epo antiserum. With 0.5 U Epo/ml, the number of CFU-E was elevated in SCA (1,087 +/- 520) compared to normal (430 +/- 130). CFU-E were formed even when Epo was immediately neutralized by a 1/150 dilution of anti- Epo. After 40 hr of Epo exposure, only 2% of total CFU-E were expressed in normal marrow, whereas 12%-40% of CFU-E were expressed in SCA. Inhibition of CFU-E growth required at least 1/50 dilution of anti-Epo in SCA and a 1/300 dilution in normal marrow. In contrast to normal, a small number (5%-20%) of CFU-E were expressed in the absence of added Epo in SCA, and this pool required a 1/150 dilution of anti-Epo for inhibition. The Epo dose-response curve in SCA revealed a peak in colony formation around 0.1 U Epo/ml and 0.5 U Epo/ml, whereas only one peak at 0.5 U Epo/ml was seen in normals. These data strongly suggest that, in response to the demands of chronic erythroid hyperplasia in SCA, a pool of CFU-E is present characterized by increased in vitro sensitivity to Epo.

Description: Thirteen patients with acute leukemias that were difficult to classify by the use of cytochemical staining and terminal deoxyribonucleotidyl transferase (TdT) activity are reported. The phenotype of the leukemic cells was characterized by the presence of mature or early monocyte lineage antigens and intense Ia antigen expression detected by monoclonal antibodies, terminal deoxytransferase activity, and cytochemical features, including both Sudan black B and periodic acid- Schiff activity. The mean age of this group of patients was 60 years. Five patients had leukemia occurring after chemotherapy or radiotherapy of a prior malignant disease, and two patients had a refractory anemia prior to development of acute leukemia. These patients had a low response rate to chemotherapy. This series of leukemia appears to form a distinct nosologic entity, representing a leukemic transformation among early cells of the monocyte lineage, resulting in a predominant neoplastic cell that is less mature than either the French-American- British M4 acute myelomonocytic leukemia or M5 acute monoblastic leukemia. The presence of terminal deoxytransferase activity was interpreted as indicating the primitive state of the cells in the differentiation sequence, rather than as implying any significance with respect to lineage.

Description: Levels of platelet-associated IgG (PA-IgG) were studied in 72 patients with Hodgkin–s (HD) and non-Hodgkin–s lymphoma (NHL). Thirty-nine percent of patients with HD and 20% of patients with NHL had elevated PA-IgG levels. There was a positive correlation between disease activity and the presence of PA-IgG in HD and NHL. In patients with HD, PA-IgG strongly correlated with extent of disease and may serve as a marker of disease activity. PA-IgG may have facilitated platelet destruction in 5 of 11 thrombocytopenic patients with HD and increased PA-IgG and in 2 patients with HD and increased PA-IgG who developed severe thrombocytopenia when treated with chemotherapy.

Description: Sedimentation analysis of factor VIII complex was performed in the analytical ultracentrifuge using partition cells. This method allowed for the calculation of three different sedimentation coefficients from each run: one based on ristocetin agglutination activity for von Willebrand protein, SWF; one based on coagulant activity for factor VIIIC, SVIIIC; and one based on the schlieren or adsorption data for protein concentration, Sconc. In most cases, there was no agreement between the three values calculated from the same run, indicating a heterogeneous system. The calculated functional sedimentation coefficients give values that require the molecules to be highly asymmetric to be consistent with a glycoprotein of high molecular weight, which is in agreement with results observed in electron microscope studies. The dissociation of VIIIC into a smaller form can be demonstrated by this method. Determination of the three sedimentation coefficients in a series of fractions from gel filtration indicates a uniform size for the VIIIC activity but not for the WF activity. These observations are in agreement with the concept of a copolymer between WF and VIIIC and also with the concept of separate polymers for the two activities.

Description: The malignant monoclonal population in hairy cell leukemia (HCL) has been variously ascribed to be of myeloid, B, or even T cell origin. Recent data have been interpreted as suggesting that hairy cells (HC) may concomitantly or serially express both B and T surface determinants, a phenomenon which, if verified, would be unique among the lymphoproliferative malignancies. Data described here, however, demonstrate that (1) at least the majority of HCL are phenotypically of B cell derivation, and (2) the initial B cell phenotype is retained and solely expressed on cultured as well as phytohemagglutinin (PHA) activated monoclonal malignant HC.

Description: To study the influence of a biologic environment on cultured human leukemia cells, KG-1, KG-1a, and HL-60 cells were inoculated subcutaneously into newborn nude mice. The cells developed myelosarcomas at the site of inoculation and in lungs and kidneys. KG-1 and HL-60 myelosarcomas were successfully passaged through adult nude mice, whereas KG-1a tumors proliferated only after transplantation into newborn hosts. The human nature of the cells forming myelosarcomas in mice was assessed by chromosomal analyses and detection of cross- reactivity with an antibody to the human leukemia cell line K562. We undertook electron microscopic and cytochemical examinations of the cells proliferating in vitro and in the mice. The granules of KG-1 cells in vivo did not react for acid phosphatase, as observed in vitro, and the HL-60 cells proliferating in mice lost the perinuclear myeloperoxidase (MPO) demonstrated in cultured cells. Although the influence of an in vivo selection of cell subpopulations cannot be ruled out, the enzymatic changes are compatible with induced cell differentiation. Conclusive evidence of differentiation in vivo was observed in the KG-1a cell subline. The undifferentiated KG-1a blasts developed cytoplasmic granules and synthesized MPO during proliferation in vivo. These observations indicate that human leukemia cells from established cell lines proliferate in nude mice and may acquire new differentiated properties in response to the in vivo environment.

Description: The Southeastern Cancer Study Group conducted a post-remission induction randomized trial in adult acute myelogenous leukemia to assess the efficacy of alternate drug therapy during consolidation and of immunotherapy during maintenance. Of 508 evaluable patients entered into the study, 335 (66%) achieved a complete remission treated with a 7-day infusion of cytosine arabinoside at a dose of 100 mg/sq m/day and 3 days of daunorubicin at a dose of 45 mg/sq m/day. Those in remission were randomized to receive 3 courses of 1 of 3 consolidation regimens: (A) a continuous infusion of 5-azacytidine, 150 mg/sq m/day for 5 days; (B) 5-azacytidine plus beta-deoxythioguanosine, 300 mg/sq m/day for 5 days; or (C) cytosine arabinoside, 100 mg/sq m/day intravenously, and thioguanine, 100 mg/sq m orally every 12 hr, plus daunorubicin, 10 mg/sq m every 24 hr daily for 5 days. There was no difference in relapse rate among the 3 arms. Those completing consolidation and remaining in remission were randomized to 1 of 3 maintenance regimens: (D) chemotherapy, 5-day infusion of cytosine arabinoside and 2 days of daunorubicin (same doses as induction) given every 13 wk for 1 yr; (E) BCG given twice weekly for 1 mo and then monthly for 1 yr; or (F) the combination of regimens D and E. The median duration of remission was significantly better on regimen D (17.4 versus 9.4 and 9.5 mo), and median survival was 29 mo compared to 21 mo for the other regimens. Those given different drugs during consolidation than used for induction (regimens A and B) and subsequent chemotherapy for maintenance (regimen D) had the longest remission durations and survival. Immunotherapy was not as good as intensive chemotherapy for maintenance.

Description: Samples of leukemic cell DNA from 14 children with acute nonlymphocytic leukemia (ANLL) and 4 human myeloid leukemia cell lines were analyzed for rearrangement in the heavy chain region of the immunoglobulin gene. The diagnosis of ANLL was confirmed in all patients by morphological, cytochemical, and immunologic studies. By restriction endonuclease digestion and hybridization with cloned heavy chain immunoglobulin gene probes for the constant (Cmu) and joining (JH) regions, the DNA of 2 patients and 1 cell line (ML-1) was found to contain rearrangements. The DNA from the remaining 12 patients and 3 cell lines was not rearranged (germline configuration). Both patients with apparent immunoglobulin gene rearrangement achieved complete remission on therapy for ANLL. Immunoglobulin gene rearrangement in phenotypically defined ANLL suggests (1) that such changes may not be limited to lymphoid leukemia of B cell lineage, or (2) that, in some patients, the leukemic transforming event may involve stem cells capable of both B cell and myeloid differentiation.

Description: Patients with multiple myeloma (MM) are at an increased risk for infections with bacteria that require opsonization with complement. Because Streptococcus pneumoniae is the most frequently encountered pathogen in these patients, we investigated the ability of serum from patients with MM to mediate the binding of C3b, the major opsonin of the complement system, to S. pneumoniae. S. pneumoniae types 3, 14, and 25 were chosen for study, since S. pneumoniae type 3 activates primarily the classical complement pathway (CCP), type 25 primarily the alternative complement pathway (ACP), and type 14 both pathways. S. pneumoniae were treated with normal serum or serum from 17 patients with MM, and the bound C3b was quantified with fluorescein-conjugated anti-C3 in a spectrophotofluorometric assay. Despite normal or elevated serum concentrations of C3, total hemolytic complement, and C-reactive protein in all of the MM sera, factor B in 16/17 such sera, and C4 in 14/17 MM sera studied, all 17 sera demonstrated a defect in C3b binding to type 3 (32.7% +/- 6% of normal). In addition, serum from 15/17 patients bound decreased amounts of C3b to types 14 (39.6% +/- 8%) and 25 (52.2% +/- 8%). Mixing normal serum with MM serum restored MM C3b binding activity to all three S. pneumoniae types, suggesting that the defect was related to a deficiency rather than an inhibitor of C3 activation. Although MM patients are unable to produce specific antibodies to bacterial antigens, the addition of anti-S. pneumoniae antibodies to MM serum did not enhance C3b binding to any of the S. pneumoniae types. However, when S. pneumoniae were opsonized in a mixture of MM serum and C3-depleted normal serum, C3b binding was restored to all three S. pneumoniae types, demonstrating that MM C3 functions normally in the presence of other normal serum factors. In the present studies, the MM C3b binding defect appeared to correlate with the incidence of S. pneumoniae infections. Serum from patients with a history of an S. pneumoniae infection bound significantly less C3 (20.5% +/- 4%) than those study patients without a history of an S. pneumoniae infection (55.8% +/- 8%) (p less than 0.0025). Thus, MM serum has a defect in the activation of C3, and this may contribute to the increased susceptibility of MM patients to S. pneumoniae infections.

Description: The effects of hemin on the conversion of pyruvate kinase (PK) isozymes from the M2-type to the L-type in K562 cells were investigated. Immunofluorescence, ion exchange chromatography, and electrophoretic studies showed that the untreated K562 cells contained only the M2-type PK, while eight to 20 days after induction with hemin, concomitant with hemoglobin F synthesis, L-type PK levels increased while M2-type PK levels decreased. Electrophoretic study revealed three hybrid isozymes of the L-type and M2-type PK. We conclude that the conversion of PK isozymes from the M2-type to the L-type in erythroid precursor cells occurs in the early stage of maturation.

Description: In this study, the effects of 5-aza-2′-deoxycytidine on differentiation of human leukemic cells in primary suspension culture are reported for the first time. Morphological and functional differentiation was induced in cells from two acute monoblastic leukemias and two of three acute myeloid leukemias following repeated exposures to 1 mumol/L 5-aza- 2′-deoxycytidine. The observation that nontoxic concentrations of the drug are able to induce the in vitro differentiation of both monoblastic and myeloblastic leukemic cells into mature elements may encourage the exploitation of the differentiating properties of 5-aza- 2′-deoxycytidine in chemotherapy protocols for acute non-lymphoblastic leukemias.

Description: We describe four patients with impaired platelet aggregation and 14C- serotonin secretion during stimulation with adenosine diphosphate (ADP), epinephrine, collagen, and platelet-activating factor. The response to arachidonic acid was normal in all patients with regard to aggregation and in three of the four with regard to 14C-serotonin secretion. The total platelet adenosine triphosphate (ATP) and ADP content and the ATP to ADP ratio was normal in all patients, thereby excluding storage pool deficiency as the cause of the secretion defect. Studies with 3H-arachidonic acid-labeled platelets revealed that the thrombin-induced liberation of arachidonic acid from membrane-bound phospholipids was impaired in these patients. Further, platelet thromboxane B2 production, measured using a radioimmunoassay, was diminished during stimulation with ADP and thrombin, but was normal with arachidonic acid, indicating that the oxygenation of arachidonic acid was normal and that the diminished thromboxane production was due to a defect in the liberation of arachidonic acid. Release of arachidonic acid is mediated by phospholipases that are Ca++ dependent. To examine whether these patients may have a defect in making intracellular Ca++ available, another Ca++-dependent process, myosin light chain phosphorylation, was studied during thrombin stimulation. Platelets from three of the patients were found to behave the same as normal ones, suggesting that the deficiency in phospholipase activity may not be due to impaired Ca++ mobilization. Our studies demonstrate a novel group of patients with platelet secretion defects associated with impaired liberation of arachidonic acid from phospholipids. These patients exemplify a congenital defect, other than deficiencies of cyclooxygenase and thromboxane synthetase, by which thromboxane production may be impaired in platelets.

Description: Restriction endonuclease mapping defined a partial deletion of about 1.35 kb in the beta-globin gene of a black American patient with hemoglobin S-beta zero-thalassemia and in his uncle with a beta zero- thalassemia trait. The 5′ endpoint of the deletion is about 600 bases upstream from the cap site, and the 3′ endpoint lies within about 500 bases from the 5 splice junction of the second intervening sequence. The deletion is different from that of a previously reported Indian beta zero-thalassemia allele, where 0.6 kb is deleted at the 3′ end of the beta-globin gene.

Description: Hereditary persistence of fetal hemoglobin (HPFH) is a genetically heterogeneous and clinically benign condition characterized by persistent expression of fetal hemoglobin (Hb F) into adulthood. In the G gamma beta + type, no major deletions in the globin gene cluster occur; adult heterozygotes produce approximately 20% Hb F, which results from overproduction of G gamma chains, with no apparent increase in production from the adjacent A gamma gene. We have recently described a point mutation 202 base pairs 5′ to the cap site of the G gamma gene in an individual with G gamma beta + HPFH. This mutation abolishes a normal ApaI restriction endonuclease site, and thus can be detected by blotting of genomic DNA. We present here further data on the ApaI mutation: (1) It occurs in six of seven families with G gamma beta + HPFH. (2) In three families, detailed haplotype analysis using 11 polymorphic restriction sites in the beta globin cluster has been done. The two that carry the missing ApaI site are identical but the third, which has a normal ApaI pattern, differs from the other two in at least two sites, one of which is a new polymorphic Nco I site between the delta and beta globin genes. This suggests the possibility of a different HPFH mutation in the third family. (3) The haplotype of the G gamma beta + HPFH chromosome carrying the ApaI mutation is different from that of 108 beta A chromosomes of black individuals that have been tested. (4) The G gamma ApaI site is normal in 61 beta A and 109 beta S alleles from non-HPFH black individuals, including 22 who share the same haplotype for the intragenic G gamma, A gamma HindIII polymorphisms. These data add support to the possibility that the -202 mutation is actually causative of the G gamma beta + HPFH phenotype.

Description: Potential limitations of prenatal diagnosis of hemophilia B, as compared to hemophilia A, include (1) occurrence of far more frequent defects with abnormal circulating antigen, (2) lower levels of factor IX in fetal plasma at 16 to 20 weeks gestation, and (3) the presence of factor IX antigen in amniotic fluid. In addition, proteolysis could occur, especially with amniotic fluid contamination of fetal plasma. A sensitive polyclonal immunoradiometric assay for factor IX antigen was used to characterize the range of levels in amniotic fluids and fetal plasma samples. To assess for altered forms, factor IX species were compared to those of a homologous clotting factor, prothrombin. Fourteen postmortem abortus blood samples from fetuses of 14 to 23 weeks gestation had factor IX antigen levels that averaged 5.1 U/dL and ranged from 1.7 to 15 U/dL. Amniotic fluid factor IX antigen averaged 2.9 U/dL, with a range from 1.4 to 8.5 U/dL in 19 separate amniocentesis samples. Thus, in a male fetus at risk of hemophilia B and with a low circulating level of gene product, mixture of fetal plasma with amniotic fluid could severely limit prenatal diagnosis, assuming that the amniotic fluid factor IX is of maternal origin. Despite rapid processing of amniotic fluid samples, the prothrombin was extensively cleaved, suggesting that it had been activated in vivo. On gel electrophoresis of amniotic fluid samples, however, factor IX was only minimally cleaved. In the postmortem fetal blood specimens, prothrombin was partially cleaved. On crossed-immunoelectrophoresis, fetal plasma prothrombin showed decreased migration in calcium, compared to EDTA, indicative of mature gamma-glutamyl carboxylation. The latter presumably resulted from fetal hepatic synthesis.

Description: Colony-forming cells in ten cases of acute myeloid leukemia (AML) were studied with six cytotoxic monoclonal antibodies that react with antigens expressed at discrete stages of differentiation of normal and leukemic hematopoietic cells. The reactivity of the whole leukemic population was measured by indirect immunofluorescence, and the reactivity of the colony-forming cells was established by complement- mediated cytotoxicity and by fluorescence activated cell sorting. Comparison of the immunofluorescent reactivity with cytotoxicity and cell sorting showed that colony-forming cells were found within a fraction of the leukemic subpopulations that expresses these antigens. This finding implies that immunofluorescence reactivity of the total leukemic population does not necessarily predict the phenotype of the clonogenic cells. When the surface phenotype of the clonogenic leukemic cells was compared to that previously established for normal marrow hemopoietic clonogenic cells, several patterns were seen: (1) in four of ten cases, the clonogenic cells expressed a phenotype like that of relatively mature normal granulocyte-macrophage colony-forming cells (late CFU-GM) or, (2) in two cases, a phenotype similar to the less mature colony-forming cells (early CFU-GM or CFU-GEMM), and (3) in four cases, a composite phenotype of early and late CFU-GM. Thus, the level of impairment of differentiation in AML may vary from case to case. In those cases phenotypically similar to the late CFU-GM, it may be possible to separate leukemic clonogenic cells from less mature normal clonogenic cells using monoclonal antibodies selectively cytotoxic for the late CFU-GM.

Description: We evaluated 37 patients with moderate or severe hemophilia A and six patients with severe factor IX deficiency for clinical or laboratory evidence of immune abnormalities. Patients were assigned to one of four groups according to the type of clotting factor replacement. Twenty patients had received only cryoprecipitate during the two years preceding the evaluation (group I); 11 additional patients were treated predominantly with cryoprecipitate but had also received up to nine bottles of factor VIII concentrate (group II); six patients received factor VIII concentrate (group III); six patients received factor IX concentrate (group IV). There was no clinical or laboratory evidence of immunodeficiency among the 43 patients. The mean absolute number of Th cells was normal in all patient groups, but the mean absolute number of Ts cells was increased compared with controls, both in patients treated with cryoprecipitate and in patients treated with factor VIII or factor IX concentrate. There was no correlation between the Th/Ts ratio and patient age, alanine aminotransferase level, hepatitis serology, in vitro lymphocyte function, or amount of clotting factor administered. Our observations demonstrate that the volunteer or commercial origin of clotting factor replacement cannot fully explain the alterations in lymphocyte subset distribution previously described in patients with hemophilia A.

Description: Human monocytes generate the procoagulant tissue factor (MTF) following exposure to a variety of immune stimuli in vitro. The generation of MTF is modified by T cells, lymphokines, and immunoregulatory lipoproteins, and recent studies have shown that MTF can be activated in an immune- specific manner following exposure to antigen. We have examined the role of serum factors in the regulation of MTF generation. Low concentrations (less than 1%) of heat-inactivated normal human serum greatly enhanced MTF generation in cultures of normal peripheral blood mononuclear cells. The stimulatory effect was observed in cultures of both unstimulated cells and cells exposed to bacterial lipopolysaccharide. Stimulation was not observed at high serum concentrations (greater than 10%) and could not be explained by endotoxin contamination or activation of the assay system. Stimulatory activity was present in plasma and BaSO4-adsorbed plasma as well as autologous and allogeneic serum, was not abolished by removal of serum lipoproteins, and did not require the presence of T cells for its expression. Sera from 28 different normal volunteers were screened for stimulatory activity and demonstrated a wide variation in potency. These results suggest that a potent factor is present in sera that enhances the expression of MTF activity in vitro. This factor is distinct from previously described lipoprotein regulators and may play a role in the initiation of coagulation in both normal hemostasis and pathologic states.

Description: Non-Hodgkin's lymphoma (NHL) is a very rare complication of acute lymphoblastic leukemia (ALL). We present the pathologic, clinical, immunologic, and ultrastructural features of the third reported example of NHL following successfully treated ALL. This white girl developed ALL with predominantly L1 cells at 3.5 yr of age. The lymphoblasts were terminal deoxynucleotidyl transferase (TdT) positive and were non-B, non-T cells. She achieved a complete remission with standard induction therapy and has remained in continuous complete remission. Four and one- third years after the onset of ALL, she developed multifocal, pleomorphic large cell lymphoma of the small bowel, which resulted in episodes of intussusception and obstruction. These pleomorphic and frequently multinucleated lymphoma cells lacked TdT, common ALL antigen, and all tested markers of B cell, T cell, and histiocyte differentiation. Following three small bowel resections, systemic multiagent chemotherapy, and abdominal irradiation, she is currently free of disease.

Description: Leukemia in the newborn is an infrequent disease that has not been well defined using modern laboratory techniques. We describe two infants, one at birth and one at four weeks, with acute lymphoblastic leukemia. The blasts from each patient were studied in great detail, using a battery of cytochemical and immunologic procedures in addition to ultrastructural studies. Immunologic cell marker studies, not previously reported in congenital leukemia, showed the lymphoblasts from each infant to be of the pre-B cell phenotype. Each infant relapsed, one after a 17-week clinical remission and the other after a 44-week remission. The former has died while the latter is in a second remission. The subtype of pre-B cell acute lymphoblastic leukemia (ALL) which in childhood appears to confer an unfavorable prognosis, may have the same significance in neonatal ALL.

Description: The proliferation and differentiation of granulocyte and monocyte progenitor cells (CFU-C) in vitro is dependent on the presence of a group of closely related glycoproteins termed colony-stimulating factors (CSF). In order to investigate the interaction of these factors with CFU-C, we purified CFU-C from the peripheral blood of chronic myeloid leukemia patients with an immune rosette technique using specific monoclonal antibodies (mean 74-fold enrichment, 45% cloning efficiency). Colony formation by purified CFU-C demonstrated an absolute dependence on an exogenous source of CSF. Liquid culture of small aliquots of enriched CFU-C with CSF-containing medium resulted in a rapid, time- and concentration-dependent induction of DNA synthesis as measured by 3H-thymidine incorporation. This specific CSF induction of DNA synthesis by enriched CFU-C was used to develop a microassay system for CSF activity. CSF activity could be reproducibly quantitated in 24–48 hr. The proliferating cells in this assay system were shown to be myeloid progenitor cells by examining the morphology of their progeny and by determining the surface antigen phenotype of the responding cells (Ia+, T3-, B1-, Mo1-). This microassay provides a quantitative assessment of CSF activity that may be useful in the purification of human CSF and in the generation of monoclonal antibodies to CFU-C surface structures.

Description: Colony-forming cells in ten cases of acute myeloid leukemia (AML) were studied with six cytotoxic monoclonal antibodies that react with antigens expressed at discrete stages of differentiation of normal and leukemic hematopoietic cells. The reactivity of the whole leukemic population was measured by indirect immunofluorescence, and the reactivity of the colony-forming cells was established by complement- mediated cytotoxicity and by fluorescence activated cell sorting. Comparison of the immunofluorescent reactivity with cytotoxicity and cell sorting showed that colony-forming cells were found within a fraction of the leukemic subpopulations that expresses these antigens. This finding implies that immunofluorescence reactivity of the total leukemic population does not necessarily predict the phenotype of the clonogenic cells. When the surface phenotype of the clonogenic leukemic cells was compared to that previously established for normal marrow hemopoietic clonogenic cells, several patterns were seen: (1) in four of ten cases, the clonogenic cells expressed a phenotype like that of relatively mature normal granulocyte-macrophage colony-forming cells (late CFU-GM) or, (2) in two cases, a phenotype similar to the less mature colony-forming cells (early CFU-GM or CFU-GEMM), and (3) in four cases, a composite phenotype of early and late CFU-GM. Thus, the level of impairment of differentiation in AML may vary from case to case. In those cases phenotypically similar to the late CFU-GM, it may be possible to separate leukemic clonogenic cells from less mature normal clonogenic cells using monoclonal antibodies selectively cytotoxic for the late CFU-GM.

Description: A complement (C)-dependent antiglobulin assay was utilized to determine the reactivity of non-C-fixing monoclonal antibodies (MoAb) with human granulocyte-macrophage progenitor cells (CFU-GM). The variables of the assay were analyzed with non-C-fixing MoAb against Ia antigens, including CR11–462, which recognizes the same (or spatially close) determinant identified by the C-fixing anti-Ia MoAb Q5/13. The sensitivity of the antiglobulin assay was influenced by dilutions of anti-mouse Ig xenoantiserum and of rabbit C. Five non-C-fixing MoAb to Ia antigens, seven non-C-fixing MoAb to HLA-A,B antigens, and one non-C- fixing MoAb to beta 2-microglobulin induced marked inhibition of human CFU-GM in the antiglobulin assay. The activity of non-C-fixing MoAb in the antiglobulin assay was comparable to that of C-fixing anti-Ia and anti-HLA-A,B MoAb in the standard cytotoxicity assay. In addition, the cytotoxic effect of dilute C-fixing anti-Ia MoAb was enhanced when the antiglobulin technique was employed. The results of this study indicate that the antiglobulin assay is a rapid and simple technique for the characterization of antigens on human hematopoietic progenitors. Our data also indicate that Ia antigens are expressed on most CFU-GM and that the conflicting results in the literature (that is, those suggesting that Ia antigens are expressed on a smaller proportion of CFU-GM) may reflect differences in the cytolytic activity of the MoAb and rabbit C used.

Description: Serum cobalamin (vitamin B12) and unsaturated B12 binding capacity (UBBC) have been measured in 24 cases of hypereosinophilia: 16 were cases of hypereosinophilic syndrome (HES) and 8 of secondary eosinophilia. The two groups were similar with respect to absolute eosinophil counts. Serum cobalamin and UBBC were found to be markedly increased in most cases of HES and normal in secondary eosinophilia. This elevation of UBBC was mainly related to the increase of R binders (transcobalamins I and III). The elevated serum cobalamin and R binders in HES were due neither to a higher intracellular content of R binders nor to an increased release of these binders from eosinophils of HES. Pure fractions of eosinophils obtained from HES and secondary eosinophilia did not exhibit any difference in vitamin B12 binders. On the other hand, neutrophil-rich fractions from the same patients showed a higher content of intracellular B12 binding proteins than pure eosinophil fractions, irrespective of the cause of eosinophilia. These findings suggest that the increased serum vitamin B12 and UBBC could reflect an expanded pool of both eosinophils and neutrophils in HES and, thus, provide an additional argument for the inclusion of this syndrome in the group of myeloproliferative disorders.

Description: The present studies report erythropoietin (Ep) production in primary cultures of a human renal carcinoma from a patient with erythrocytosis that has been serially transplanted to BALB/c nude mice. The levels of erythropoietin in the culture media were estimated using the exhypoxic polycythemic mouse assay (EHPCMA), fetal mouse liver erythroid colony- forming technique (FMLC), and a radioimmunoassay (RIA). The spent culture media of the exponentially growing cells contained less than 10 mU/ml of Ep measured by RIA. However, after the cells became confluent, Ep levels (RIA) in the spent media showed a marked increase to approximately 300 mU/ml. Ep levels estimated using the FMLC and EHPCMA were approximately 2/3 and 1/10, respectively, of those measured by RIA. Rabbit antiserum to highly purified human urinary Ep (70,400 U/mg protein) was utilized for immunocytochemical (peroxidase-antiperoxidase method) localization of Ep in the cultured cells. Very few of the cells in exponential growth exhibited Ep-like immunoreactivity, whereas intense Ep-like immunoreactivity was observed in the cytoplasm of the cells maintained in culture for a prolonged period after reaching confluency. The most intense staining was observed in some of the cells forming domes. The domes developed after the cells reached confluency, and their numbers increased with increasing time in confluent culture, in parallel with the increase in Ep levels in the spent media. This primary cell culture system of a renal cell carcinoma maintained in nude mice, which produces immunologically and biologically active Ep, may provide a useful model for studies of the mechanism of Ep production.

Description: We assessed the number of Langerhans cells (LC) before and after bone marrow transplantation (BMT) in 27 patients in order to study the fate and behavior of these dendritic antigen-presenting cells following allogeneic BMT. LC were identified using monoclonal antibody OKT6 on skin biopsies performed on days - 10, 0, 11, 25, 39, 120, and 365. In a control group composed of 15 healthy adults aged 20–37 yr, the mean number of LC (+/- SEM) was 25.6 +/- 1.17/0.1 sq mm of epidermal surface. Our study shows that pretransplant, the number of LC in patients with aplastic anemia or leukemia was lower than that of controls. The finding of low numbers of LC in patients with untreated aplastic anemia is suggestive of a medullary origin of LC in man. Moreover, during the early posttransplant period, nearly all patients present a severe deficit in LC. This deficit may delay the maturation of their immune system. The number of LC reaches nearly normal levels 4– 12 mo after BMT. Finally, we have noted a significant impairment of LC reconstitution in patients with acute graft-versus-host disease (GVHD), providing evidence that this defect may be an important mechanism involved in acute GVHD-related immunodeficiency.

Description: The acute effects of a single intravenous dose of L-asparaginase on protein synthesis were studied in normal rabbits and in animals that had received turpentine to stimulate fibrinogen production. Male New Zealand rabbits received L-asparaginase (500 U/kg) 16 hr before the injection of the radiolabeled amino acid [75Se]selenomethionine (75SeM). Incorporation of 75SeM into fibrinogen and serum proteins in the L-asparaginase-treated rabbits was the same as for saline-treated controls, with fibrinogen representing approximately 5% of the labeled plasma proteins. In turpentine-treated rabbits, the maximal incorporation of 75SeM into serum proteins remained unchanged, whereas 75SeM-fibrinogen increased sixfold and accounted for 25% of the labeled proteins. Animals that received L-asparaginase at the same time as turpentine or 14 hr later showed significant decreases in synthesis of both serum proteins and fibrinogen. 75SeM-fibrinogen that was purified from L-asparaginase-treated rabbits underwent normal catabolism when injected into normal recipient rabbits. These data indicate that L- asparaginase can acutely cause partial inhibition of both serum protein and fibrinogen synthesis when administered to rabbits shortly before or during a period of increased fibrinogen production. Fibrinogen that is synthesized in the presence of L-asparaginase does not have an abnormal rate of catabolism.

Description: 1,(2-Chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and other chloroethylnitrosourea anticancer agents in clinical use produce severe and cumulative bone marrow toxicity. Chlorozotocin, a glucose analogue, has demonstrated reduced hematologic toxicity while retaining full antitumor activity. The biochemical-pharmacologic properties of chlorozotocin and CCNU were compared in human bone marrow. After a 2-hr incubation with a 0.1-mM drug concentration, total cellular uptake of chlorozotocin in whole marrow was 2.47 +/- 0.80 pmole/10(4) cells and was not significantly different compared to the uptake of 1.94 +/- 0.53 pmole/10(4) cells with CCNU. The quantitative alkylation of bone marrow DNA by chlorozotocin, 22.8 +/- 1.2 pmole/mg DNA, was equivalent to that produced by CCNU, 22.9 +/- 0.5 pmole/mg DNA. Bone marrow was separated into 14 fractions by centrifugal elutriation. CCNU uptake was found to be greater than that of chlorozotocin in 3 fractions that were primarily composed of lymphocytes, monocytes, and normoblasts. Chlorozotocin uptake was greater than CCNU in 6 fractions that contained primarily mature and immature myeloid cells as well as the highest CFU-GM activity. The two drugs produced a comparable degree of DNA strand breakage and DNA-protein cross-linking as measured by alkaline elution of pooled fractions of elutriated bone marrow. DNA interstrand crosslinking was not found with either drug. The most significant finding of this study is the differences in the site of drug alkylation by chlorozotocin and CCNU in bone marrow chromatin. Endonuclease digestions with MCN, DNase I, and DNase II showed nonrandom alkylation of specific regions of chromatin by the two drugs: CCNU demonstrated a preferential binding to the transcriptionally active regions of chromatin, whereas chlorozotocin predominantly alkylated the transcriptionally inactive regions. These data suggest that the lethal damage of nitrosourea alkylation in human bone marrow is principally expressed in transcriptionally active regions of chromatin.

Description: Tumor burden in adult patients with acute leukemia is assessed using the percentage of blast cells in the bone marrow or blood. It is clear, however, that not all blast cells are leukemic cells, especially during rapid marrow regeneration. Similarly, some leukemia cell lines have been shown to differentiate in vitro, and the same process also occurs in vivo. Therefore, the leukemic burden may be due to more differentiated cells as well as to blast cells. The purpose of this study was to investigate whether the human malignancy-associated nucleolar antigen (HMNA) could be used as a marker for leukemic cells and to examine its potential as a diagnostic tool. The proportion of HMNA-positive cells in the bone marrow of patients with acute leukemia was determined by indirect immunofluorescence with antibodies to HMNA and was compared with the differential counts routinely made in the clinic laboratory. The percentages of HMNA-positive cells among the nucleated cells in the marrow of 72 patients with clinical evidence of leukemia were significantly higher (range 9%-98%, median 83%) than those observed for nonleukemic individuals (range less than 0.05%-2.5%, median 1%) or for fractions of marrow cells from normal volunteers enriched for normal early progenitor cells (less than or equal to 2%). Patients with leukemia in remission had a lower percentage of HMNA- positive cells (range 0%-83%, median 3%). The percentage of HMNA- positive cells increased as patients approached relapse. Although the percentage of HMNA-positive cells was related to the percentage of blast cells in the bone marrow of the patients with leukemia, some partially differentiated cells were also HMNA-positive in some specimens, and some blastic cells were HMNA-negative in other specimens. These studies indicate the potential usefulness of HMNA as a marker for leukemic cells.

Description: Kidney allografting was performed in a group of ten beagles, and viable leukocytes infiltrating the transplanted organs were isolated during episodes of acute rejection 5 or 6 days postoperatively. These infiltrate populations, consisting predominantly of lymphocytes and monocytes/macrophages, were found to have significantly increased amounts of procoagulant activity relative to control leukocytes isolated from circulating blood and lymph. Using nonspecific esterase staining in an agar microclot assay, procoagulant activity in the infiltrate leukocytes was found to reside in monocytes/macrophages rather than other coisolated cell types. By contrast, control monocytes from blood had no activity in this microclot assay. Procoagulant activity in the infiltrate cells was characterized as tissue factor. Increased amounts of this activator of the extrinsic pathway, as found in infiltrate monocytes/macrophages, may initiate clotting reactions and fibrin deposition within allografts.

Description: A case of transcobalamin II deficiency with several unique features is described. The clinical presentation was typical, except for a slightly delayed age at presentation and the occurrence of apparent neurologic dysfunction from the beginning. The unusual biochemical feature was a low serum cobalamin level (97 pg/ml). Several cobalamin-binding protein abnormalities coexisted and antedated cobalamin therapy. Chief among these was the complexing of all serum R binder (transcobalamin I), leaving the patient with no detectable R binder. This defect appeared to be transient. Noteworthy, too, was a prominent binder of 70,000 mol wt that also carried the bulk of his serum cobalamin after therapy; it was prominent in his presumably heterozygous relatives too. The interrelationship between all these abnormalities is intriguing but unclear. The abnormality in transcobalamin II deficiency is clearly not limited solely to deficiency of transcobalamin II. It is also evident that this entity must now be considered in the differential diagnosis of low serum cobalamin levels in infancy.

Description: We have shown previously that the cause of anemia in healthy elderly subjects can usually not be identified. In this study, hematopoiesis was examined in 18 healthy elderly subjects with unexplained anemia and in 15 young and 15 healthy elderly individuals without anemia. No reduction in circulating testosterone was noted, making decreased androgen levels as a cause for the anemia unlikely. The 2,3 diphospho- glycerate (2,3DPG) levels in the anemic subjects were significantly higher than their corresponding controls, suggesting that the anemia was pathologic, as no increase would be expected if the low hemoglobin was a physiologic adjustment to age. The anemia was associated with a reduction in marrow normoblast and CFU-E number, but no decrease in BFU- E levels was seen. This suggests that the mechanism of the anemia is a decrease in stem cell proliferation. This could be caused by a reduction in circulating erythropoietin or a defect in end organ response. A second possibility is that a basic cellular abnormality exists. The presence of an overall reduction in hematopoiesis in anemic elderly (decreased peripheral blood counts, reduced marrow myeloid precursors, and CFU-C levels) makes this especially likely. The abnormality may be caused by a mechanism unrelated to the aging process. The fact that nonanemic elderly also have reductions in hematopoiesis suggests that age contributes to the defect.

Description: A simple technique using an aggregometer and fixed washed human platelets (FWP) and fibrillar collagen has been used to evaluate the contribution of the two components of the factor VIII (FVIII) complex to platelet-collagen interactions. FWP bound individually to collagen fibrils in suspension, and both the total number of FWP bound and the rate of adhesion increased with increasing collagen concentration. Von Willebrand's disease (vWD) type I or normal plasma immunoadsorbed with anti-factor VIII-related antigen (anti-FVIIIR:Ag) antiserum gave 20% and vWD type IIa gave 50% of the rate of adhesion obtained with normal, hemophilia A, or hemophilia A with inhibitor plasma, but the same percent adhesion was found with all plasmas. The rate of adhesion of both vWD type I and type IIa was corrected by the addition of purified FVIII complex. These results indicated that the FVIIIR:Ag and not the factor VIII coagulant activity (FVIII:C) in normal plasma or purified FVIII complex caused an accelerating effect on the rate at which FWP bound to collagen. Collagen fibrils not only bound FWP, but also adsorbed the FVIII complex with preferential adsorption of the forms of FVIIIR:Ag with the greatest ristocetin cofactor (FVIIIR:RCoF) activity. Saturation of collagen with FWP did not change the adsorption pattern of the FVIII complex. Also anti-FVIIIR:Ag blocked the accelerating effect of the FVIII complex but not the adhesion of FWP. Thus, FWP and FVIIIR:Ag appeared to bind to separate sites on collagen.

Description: The infusion of 1-deamino-(8-D-arginine)-vasopressin (DDAVP) causes not only an elevation in factor VIII-related antigen (FVIIIR:Ag), but also a marked elevation of plasma von Willebrand antigen II (vWAgII). vWAgII reaches a peak concentration at 60 min and is elevated 3–8-fold over basal levels in normal individuals and individuals with type I, IIA, and IIB von Willebrand's disease. As the mechanism of hemostatic alteration brought about by DDAVP might be due to release of endothelial cell proteins, endothelial cell cultures were performed. The cultures demonstrated synthesis and secretion of vWAgII, as evidenced by the incorporation of 35S-methionine into the vWAgII molecule. Thus, vWAgII, like FVIIIR:Ag, is an endothelial cell protein.

Description: The level of assimilation of dietary iron is believed to have an important influence on iron status. To examine the effect of enhancing the availability of dietary iron on iron balance, 17 adult volunteer subjects were given 2 g of ascorbic acid daily with meals for 16 weeks. Serum ferritin levels before and after the study averaged 46 and 43 micrograms/L, respectively, indicating a negligible effect on iron stores. When vitamin C supplementation was continued for an additional 20 months in five iron-replete and four iron-deficient subjects, serum ferritin determinations again failed to indicate any significant effect of the vitamin C on iron reserves. These findings were not explained by intestinal adaptation to the enhancing effect of the vitamin, because radioisotopic measurements of nonheme iron absorption showed no reduction in the enhancing effect of 1 g of ascorbic acid after four months of megadoses of vitamin C. It is concluded that altering the availability of nonheme dietary iron has little effect on iron status when the diet contains substantial amounts of meat.

Description: Potential limitations of prenatal diagnosis of hemophilia B, as compared to hemophilia A, include (1) occurrence of far more frequent defects with abnormal circulating antigen, (2) lower levels of factor IX in fetal plasma at 16 to 20 weeks gestation, and (3) the presence of factor IX antigen in amniotic fluid. In addition, proteolysis could occur, especially with amniotic fluid contamination of fetal plasma. A sensitive polyclonal immunoradiometric assay for factor IX antigen was used to characterize the range of levels in amniotic fluids and fetal plasma samples. To assess for altered forms, factor IX species were compared to those of a homologous clotting factor, prothrombin. Fourteen postmortem abortus blood samples from fetuses of 14 to 23 weeks gestation had factor IX antigen levels that averaged 5.1 U/dL and ranged from 1.7 to 15 U/dL. Amniotic fluid factor IX antigen averaged 2.9 U/dL, with a range from 1.4 to 8.5 U/dL in 19 separate amniocentesis samples. Thus, in a male fetus at risk of hemophilia B and with a low circulating level of gene product, mixture of fetal plasma with amniotic fluid could severely limit prenatal diagnosis, assuming that the amniotic fluid factor IX is of maternal origin. Despite rapid processing of amniotic fluid samples, the prothrombin was extensively cleaved, suggesting that it had been activated in vivo. On gel electrophoresis of amniotic fluid samples, however, factor IX was only minimally cleaved. In the postmortem fetal blood specimens, prothrombin was partially cleaved. On crossed-immunoelectrophoresis, fetal plasma prothrombin showed decreased migration in calcium, compared to EDTA, indicative of mature gamma-glutamyl carboxylation. The latter presumably resulted from fetal hepatic synthesis.

Description: This article describes three patients with megakaryoblastic leukemia, in whom the blast cells were identified as megakaryoblasts by the platelet peroxidase (PPO) reaction. More than 70% of the blasts in these patients were positive for the PPO reaction. Ultrastructurally, acid phosphatase activity in the megakaryoblasts was detected in the nuclear envelope, the endoplasmic reticulum, and in a few granules, but not in the Golgi cisternae. Some blast cells were identified by immunofluorescence or immunoalkaline phosphatase, using monoclonal antiplatelet glycoprotein IIb/IIIa antibody. In one patient, most of the blasts were positive for anti-HLA-DR monoclonal antibody. The possible order of the appearance of markers in the maturation of the megakaryocytic cell lineage is postulated, based on the data from the present cases and those previously reported. PPO activity appears in very immature cells, which retain Ia-like antigens. Platelet-specific glycoprotein IIb/IIIa is seen in immature cells that are only recognized by PPO activity.

Description: Prothrombin deficiency has been known to occur in association with lupus inhibitors for over 25 years. We studied 21 patients with lupus inhibitors and found that four of five with prothrombin deficiency and ten of 16 with quantitatively normal prothrombin had abnormal prothrombin crossed-immunoelectrophoresis (CIEP) characterized by material moving slower in the first dimension of electrophoresis than normal prothrombin. In two patients with prothrombin deficiency, all prothrombin measured by quantitative assay and all slow-moving material on CIEP were removed by treatment with Staphylococcal protein A (SPA). These patients had free antibody, which bound to normal plasma prothrombin, forming larger amounts of slow-moving material on CIEP. A third patient with prothrombin deficiency had only partial removal of prothrombin after SPA treatment. Two patients with quantitatively normal prothrombin had all slow-moving material on CIEP and about one fourth of the prothrombin by quantitative assay removed by SPA treatment. There was no correlation among the strength of the inhibitor, the presence of a “cofactor effect,” and the prothrombin abnormality. These data suggest that heterogeneous antiprothrombin antibodies, with or without prothrombin deficiency, are present in the majority of patients with lupus inhibitors.

Description: Two patients with chronic myeloid leukemia (CML) showed previously undescribed variants of a “masked” Ph1 abnormality. The first patient had the karyotype 46,XY, + 21, -9, -22, +mar9,mar18 at presentation in the chronic phase. The dicentric marker 9 was interpreted as representing the usual translocation of 22q11 to 9q34, followed by translocation of the Ph1 chromosome (the deleted 22) to 9p and probable translocation of 9p to the distal long arm of the marker. The patient developed clones containing 2 and 3 copies of the “Ph1-containing” marker 9 concomitant with the metamorphosis of his disease to a more aggressive phase. The second case presented with the karyotype 46,XY,- 9,-22,+two D-group markers. A complex rearrangement of chromosomes 9 and 22 is postulated, with interstitial insertion of either 9p or distal 9q into chromosome 22q11. This patient is still in the chronic phase of his disease 9 mo after presentation. The common denominator in these unusual “masked” cases is the 22q11 breakpoint. The paucity of published reports of duplication of 9q + without concurrent duplication of the Ph1 chromosome, supported by the findings in our first case, leads us to conclude that the amplification of genes on the Ph1 chromosome are more important for the evolution of the abnormal stem cell in CML than the chromosome 9 derivative.

Description: Thrombin Metz and normal thrombin, resulting from activation of the respective prothrombins by factor Xa in the presence of calcium, phospholipid, and factor Va, were purified by chromatography on sulfopropyl Sephadex. By physicochemical criteria, thrombin Metz is identical to normal thrombin. Its functional properties were investigated in some reactions in which thrombin is classically involved. Thrombin Metz exhibits less than 4% of fibrinogen clotting activity. Both Km and Kcat, determined on S2238, are abnormal. Titration with the high-affinity competitive inhibitor of thrombin, DAPA, shows that fluorescence enhancement of the probe is only 34% in binding to thrombin Metz when compared to that observed in binding to normal thrombin. High-performance liquid chromatography has been used to measure the simultaneous rate of release of fibrinopeptides A and B. A decreased release rate for both fibrinopeptides, more marked for fibrinopeptide B, results in a slow fibrin polymerization, as followed by absorbance at 450 nm. Thrombin Metz is less than 5% as effective as normal thrombin in inducing platelet aggregation. Interaction with antithrombin III is slower than normal when followed by SDS gel electrophoresis and inhibition of the amidolytic activity of thrombin on S2238. This abnormality is not observed in the presence of heparin. However, thrombin Metz binds less tightly to a heparin-Sepharose column, and the direct inhibition of heparin on its activity on S2238 is weaker. From these results, we can predict that the defect in thrombin Metz affects the catalytic site or its vicinity and, jointly or consequently, the region of interaction of thrombin with antithrombin III and heparin.

Description: Granulocyte-macrophage colony growth depends on the presence of colony- stimulating activity (CSA). Phorbol esters induce concentration- dependent colony formation in the absence of exogenous CSA. We questioned whether phorbol esters mimicked the action of CSA by directly stimulating colony growth, or whether phorbol esters acted indirectly by inducing marrow cells to release CSA. First, after incubating human bone marrow cells with phorbol 12,13-dibutyrate (PDB) for 3 days, we separated PDB from the protein peak of the conditioned medium by Sephadex G-10 gel filtration and tested this peak for the presence of CSA. When diluted 1:10 in the agar colony assay, this material induced 133 +/- 15 colonies/10(5) bone marrow cells. Second, to determine whether bone marrow cells required the continued presence of PDB in order to release CSA, PDB was removed from bone marrow cells by washing, and these cells were reincubated in fresh medium in the absence of PDB. CSA was found in the medium of these cultures; its release was maximal after preincubation of bone marrow cells with 5 X 10(-8) M PDB for 3 days, followed by incubation for 3 days in the absence of PDB. This CSA stimulated granulopoiesis out of proportion to monocytopoiesis, with 85% +/- 17% of the colonies being granulocytic (as indicated by histochemical staining for chloroacetate esterase), and 12% +/- 3% being monocytic (as indicated by nonspecific esterase). Inhibitors of monocyte colony formation, including PGE1, were not present in the medium that contained this CSA. These studies demonstrate that normal human bone marrow cells exposed to PDB release CSA and that this CSA selectively stimulates granulopoiesis in vitro.

Description: Two patients with chronic myeloid leukemia (CML) showed previously undescribed variants of a “masked” Ph1 abnormality. The first patient had the karyotype 46,XY, + 21, -9, -22, +mar9,mar18 at presentation in the chronic phase. The dicentric marker 9 was interpreted as representing the usual translocation of 22q11 to 9q34, followed by translocation of the Ph1 chromosome (the deleted 22) to 9p and probable translocation of 9p to the distal long arm of the marker. The patient developed clones containing 2 and 3 copies of the “Ph1-containing” marker 9 concomitant with the metamorphosis of his disease to a more aggressive phase. The second case presented with the karyotype 46,XY,- 9,-22,+two D-group markers. A complex rearrangement of chromosomes 9 and 22 is postulated, with interstitial insertion of either 9p or distal 9q into chromosome 22q11. This patient is still in the chronic phase of his disease 9 mo after presentation. The common denominator in these unusual “masked” cases is the 22q11 breakpoint. The paucity of published reports of duplication of 9q + without concurrent duplication of the Ph1 chromosome, supported by the findings in our first case, leads us to conclude that the amplification of genes on the Ph1 chromosome are more important for the evolution of the abnormal stem cell in CML than the chromosome 9 derivative.

Description: Recent evidence suggests that HLA-A,B antigens present on human platelets, are acquired from plasma. Using hypertonic acid chloroquine (200 mg/ml, pH 5.0, 1 hr, 4 degrees C), we demonstrate elution of most or all HLA-A2 and HLA-B7,40 immunoreactive material from platelets. Corresponding antigens on human lymphocytes were not affected by this treatment. These findings are consistent with the hypothesis that HLA- A,B antigens are adsorbed onto platelet surfaces.

Description: Washed and gel-filtered human platelets were dose-dependently aggregated by the addition of cationized ferritin (CF). Ca++ and plasma factors were not necessary to induce the aggregation. Immediately after the addition of CF, CF particles were attached to the surface of platelets that showed discoid form, as observed electron microscopically. Some platelets were connected to each other through the CF particles located on their membranes. After the addition of CF, the following was observed: at 15 sec after, platelets showed a round form and were aggregated to each other; at 3 min after, centralization of granules was clearly seen and the aggregates increased their size during the time course; at 3–5 min after, the CF-connected aggregates were found locally. Around the aggregates, other platelets were aggregated, though not through the membrane-located CF. Observing with a lumiaggregometer, the aggregation showed a biphasic curve associated with adenosine triphosphate (ATP) release. The second part of aggregation curve was inhibited by PGI2, PGE1, aspirin, N- ethylmaleimide, and apyrase. The first part of the aggregation curve was inhibited only by heparin. Neuraminidase treatment also inhibited the aggregation dose-dependently. These findings suggest that neutralization of the platelet surface negative charge by a positively charged macromolecule can trigger platelet aggregation, which is followed by the release reaction.

Description: The relation between platelet buoyant density and beta-thromboglobulin (beta-TG), a marker for platelet alpha-granule content, was assessed by three independent approaches. (1) Platelets were separated on iso- osmolar discontinuous Stractan density gradients into five fractions, ranging in density from 1.061 g/ml to 1.091 g/ml (20 degrees C). The beta-TG content (mean +/- SD, n = 17) increased with the platelet density from 27.8 +/- 8.6 micrograms beta-TG/10(9) cells (20% less- dense platelets) up to 65.6 +/- 15.5 micrograms beta-TG/10(9) cells (15% most-dense platelets). (2) Activation of platelets in platelet- rich plasma with thrombin, adenosine diphosphate, collagen, or epinephrine resulted in a decreased density of the platelets. This was only seen when there was simultaneous secretion of beta-TG. (3) The less-dense and the more-dense platelet fractions, after isolation by density gradient centrifugation, were separately treated with thrombin. After complete degranulation, the density distribution of the originally less-dense and more-dense platelets were identical and were much narrower than the density distribution of resting platelets.

Description: Previous reports have shown that serum beta-2-microglobulin (S beta2M) is a reliable marker of presenting tumor mass, response to chemotherapy, and prognosis of patients with multiple myeloma (MM). In order to more thoroughly evaluate the optimal use of S beta2M in plasma cell dyscrasias (PCD), S beta2M levels were serially measured in 160 patients with MM, in comparison with 37 normal controls (NC) and 28 patients with monoclonal gammopathy of undetermined significance (MGUS). In MGUS, S beta2M did not differ significantly from that of NC, but was significantly lower than that of MM (p less than 0.001), including low cell mass MM (p less than 0.02). In MM, S beta 2M was highly correlated with the total body burden of myeloma cells as derived from the staging of Durie and Salmon, both at diagnosis and in remission (residual tumor mass) (p less than 0.001). During the plateau phase, S beta 2M remained very stable and was always within the normal range for patients with greater than or equal to 75% tumor regression. The most striking finding was that S beta 2M gave an extremely reliable fit for survival prediction at (1) diagnosis, (2) remission, and (3) early relapse, with higher S beta 2M levels in each instance being in favor of poorer prognosis. We conclude that S beta 2M is an extremely useful marker in initial stratification and follow-up of patients with MM.

Description: Human neutrophil elastase is thought to play an important role in connective tissue destruction in diseases such as emphysema and arthritis. In this article, it is demonstrated that the elastase activity of mature human peripheral blood neutrophils can be rapidly increased in vitro by treatment of the cells with lipopolysaccharide from E. coli and that this increase is inhibited by corticosteroid pretreatment of the cells and by inhibitors of protein synthesis. Alterations in intracellular enzyme content of the mature polymorph in response to bacterial products or other stimuli may be important for amplification of the inflammatory response.

Description: Chronic idiopathic thrombocytopenic purpura (ITP) is caused by an antibody reactive with platelet-associated antigens. The present studies provide direct evidence that some patients with chronic ITP have autoantibodies against the platelet glycoprotein (GP) IIb/IIIa complex. Microtiter wells, coated with a monoclonal antibody (2G12) specific for GPIIb/GPIIIa were reacted with GPIIb/GPIIIa contained in a platelet extract. Control wells containing the same antibody were reacted with a cell extract containing no GPIIb/GPIIIa. After washing, the wells were reacted with patient or control plasma, and IgG binding was detected using 125I-Fab2-anti-human IgG. Assay values were expressed as binding ratios (cpm GPIIb/GPIIIa wells/cpm control wells). Plasma from 5 of 56 patients with chronic ITP had ratios (1.36–3.14) greater than 3 standard deviations above the mean (+/- SD) of control plasmas--0.93 +/- 0.12. Elevated values were also noted in two patients with anti-P1A1 antibody (ratios greater than 30) and in one patient with Hodgkin's disease and an ITP-like syndrome (ratio 1.53). Normal values were noted in 34 patients with a variety of immune and nonimmune diseases. Plasma from two of the positive ITP patients was reacted with 125I-surface-labeled platelets and, after solubilization, the IgG and bound antigen were precipitated with Staph-A. Autoradiographs from SDS- PAGE electrophoresis of the Staph-A-bound proteins shows two radioactive bands consistent in size with GPIIb and GPIIIa.

Description: Temporary or long-lasting decrease of one or more blood cell lineages following allogeneic hematopoietic stem cell transplant (allo-HSCT) can lead to increased morbidity and mortality. Post-transplant cytopenia is often secondary to drug toxicity, viral infections, graft versus host disease (GVHD), or relapse. Less is known about the significance of cytopenia of unknown cause. Here we retrospectively analyzed 91 consecutive adult patients (median age 46 years; range: 19-63) who had received allo-HSCT conditioned with myeloablative fludarabine/ I.V. busulfan at our institution. Diagnoses included: acute myeloid leukemia (AML) or myelodysplatic syndrome (MDS) (n=57), acute lymphoblastic leukemia (ALL) (n=15), chronic myeloid leukemia (CML) (n=9), non-Hodgkin lymphoma (NHL) (n=5), B cell chronic lymphocytic leukemia (B-CLL) (n=2), myelofibrosis (n=1), unclassified myeloproliferative neoplasms (MPN) (n=2). Stem cell source was granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood stem cells (PBSC) in 81, bone marrow in 8 and cord blood in 2 patients. Median follow up for the entire cohort was 20 months (range 1-162 months). Patients with cytopenia of unknown cause were identified if they had full donor chimerism, negative cytomegalovirus (CMV) by molecular analysis, no active infections, acute GVHD

Description: Deregulated gene expression due to genetic alterations, such as gene fusions affecting transcription and/or epigenetic factors is the hallmark of acute myeloid leukemia and the basis for the differentiation block of hematopoietic progenitors. Acute megakaryoblastic leukemia (AMKL) is a subtype of poor prognosis acute myeloid leukemia (AML) affecting primarily young children. Recently, the ETO2-GLIS2 fusion has been identified in 20-30% of de novo AMKL and associated with the worst prognosis in this subtype of AML. To characterize the transformation induced by ETO2-GLIS2, we first defined the consequences of ETO2-GLIS2 expression on hematopoietic progenitors and the contribution of ETO2 and GLIS2 on differentiation and self-renewal. Using methylcellulose replating assays and phenotype characterization, we show that the GLIS2 moiety drives the megakaryocytic phenotype whereas both the ETO2 and GLIS2 moieties are required for maintaining self-renewal. Global expression profiling and comparison to patients' signature consistently identify ETO2-GLIS2-mediated deregulation of major transcriptional regulators of hematopoiesis and leukemogenesis, including overexpression of the ERG oncogene. ChIP-seq analysis reveals that ETO2-GLIS2 is recruited at normal ETO2 complexes sites and also at GLIS2-specific targets through binding via GLIS2 DNA-binding domain. We demonstrate that ETO2-GLIS2 fusion localize at half of H3K27Ac-dense enhancers, so called super-enhancers, to control transcription of associated genes. We show that interaction of ETO2-GLIS2 with ETO2 complexes is an essential node for the transcriptional control by the fusion at enhancer elements. Indeed, ETO2-GLIS2 dimerizes and interacts with endogenous ETO2 via its NHR2 domains. An NHR2 peptide-interference strategy inhibits oligomerization, reverses the transcriptional activation at enhancers, promotes megakaryocytic differentiation and abrogates human AMKL cells maintenance in vivo. Finally, upregulation of ERG by ETO2-GLIS2 further strengthen enhancers formation as ERG is co-recruited generating a feed forward loop at these elements and its knockdown or genetic inactivation downregulates expression of ETO2-GLIS2 targets required for leukemic cells survival. We propose that the megakaryocytic differentiation arrest and self-renewal controlled by ETO2-GLIS2 results from an imbalance in the expression of master transcription factors imposed by aberrant chromatin structures at enhancers that may be disrupted by targeting the NHR2 interface. Disclosures No relevant conflicts of interest to declare.

Description: Multipotent mesenchymal stromal cells (MSCs) have immunomodulatory properties and have been successfully used for treatment of autoimmune diseases and acute or chronic graft-versus-host disease. Therapy with MSCs is not always effective. It has been shown that MSCs immunomodulatory properties can be improved by means of various agents, such as IFN-g, TNF-a, IL-17. After 4 hours of IFN-g exposure the expression level of immunomodulatory genes increased - IDO1 300, CSF1 - 7, and IL6 - 2.4 times. MSCs typically express low levels of MHC class I, and no MHC class II or co-stimulatory molecules (e.g., B7-1, B7-2, or CD40), making them partially immunoprivileged. However, treatment with IFN-g leads to increased expression of HLA-DR antigens on MSCs. After injection to the patient the characteristics of MSCs differ from those which have been studied in culture due to their interactions with other cells in the bloodstream and tissues. In this study the model of MSCs and MSCs treated with IFN-g (IFN-g-MSC) interactions with allogeneic lymphocytes in vitro was developed. The aim of the study was to identify the changes in MSCs and IFN-g-MSCs characteristics after co-cultivation with lymphocytes in vitro in dynamics. Materials and methods MSCs were isolated from 13 bone marrow (BM) samples used for allogeneic hematopoietic cells transplantation and cultured by a standard method in aMEM with 10% fetal bovine serum (FBS). MSCs on 2-3-d passages were seeded 105 cells per flask with 25 cm2 bottom area and a day later 500 units/mL of IFN-g were added for 4 hours to half of the cultures. Then the media was changed on RPMI-1640 with 10% FBS. Some cultures were seeded with 106 allogeneic lymphocytes, to half of these cultures 5 mg/ml phytohemagglutinin (PHA) was added for lymphocytes activation. All flasks were cultured up to 4 days at 37°C and 5% CO2. After 1, 2, 3 and 4 days lymphocytes were washed from MSCs. MSCs were removed from the flasks with trypsin and the number of viable cells was determined by dye exclusion method (trypan blue). For each of the MSCs cultures the mean fluorescent signal intensity level (MFI) of HLA-DR was determined by direct immunofluorescent staining with anti-HLA-DR APC (BD Pharmingen) antibodies and measured on flow cytometer BD FACS Canto II (BD Biosciences, USA). Data are presented as mean ± standard error. Statistical analysis was performed using Student's t-test (considered reliable p

Description: Mixed Lineage Leukemia gene rearrangements (MLL-r) account for nearly 10% of human acute leukemia cases and are generally associated with poor prognosis. Previous studies have revealed an essential role of the histone H3K79 methyltransferase Disruptor of Telomeric Silencing-1 Like (DOT1L) in MLL-r leukemogenesis. Our recent report (Chen et al. 2015 Nature Medicine) further identified a role for histone acetylation in DOT1L dependent gene expression driven by MLL-fusion proteins including MEIS1 and HOXA cluster genes. A first-in-human Phase I clinical trial demonstrated clinical activity of DOT1L inhibition in MLL-r leukemia patients, thus providing a potential opportunity for treating these malignant diseases. Nevertheless, the incomplete silencing of the leukemic program by only targeting DOT1L motivates the need for additional and perhaps combinational approaches to improve therapies against MLL-r leukemias. To enhance the efficacy of DOT1L inhibition, we sought to identify genes whose suppression would synergize with the DOT1L inhibitors to suppress the proliferation of mouse bone marrow progenitors transformed with MLL-AF9. We conducted a pooled RNAi screen using a customized library composed of 2,252 shRNA targeting 468 epigenetic regulators (i.e. writers, readers, and erasers of chromatin modifications; Fig 1). The integrated shRNA sequences were assessed using high-throughput sequencing. By comparing the change in frequency of each shRNA construct cultured in control vs. an IC50 DOT1L inhibitor EPZ4777, we identified several candidate modulators of DOT1L dependency, which had multiple shRNAs selectivity depleted only in the DOT1L suppressed condition. Notably, using a network correlation study, we found that one of the top candidate genes Plant Homeodomain Finger Protein 20 (PHF20) is highly associated with histone acetylation in the mammalian epigenome. Knockdown of PHF20 drastically increased the sensitivity of MLL-AF9 leukemic blasts to DOT1L inhibitors through enhanced myeloid differentiation and reduced cell proliferation, colony formation, and re-plating capacity. Similar phenotypes were also observed in PHF20-deficient MLL-AF9 cells generated by CRISPR/Cas9-mediated gene knockout. PHF20 is an epigenetic adaptor protein that has no predicted enzymatic activity. To investigate the role of PHF20, we conducted a CRISPR functional domain screen and identified the requirement of the chromatin reader domains in PHF20, including the Tudor domains and the PHD-finger, in supporting the survival of MLL-r leukemic cells upon DOT1L inhibition. We also performed RNA-seq and found that suppression of PHF20 facilitated the silencing of the MLL-AF9 leukemic program induced by DOT1L inhibitor treatment. Chromatin immunoprecipitation and sequencing (ChIP-seq) analyses validated that PHF20 contributes to the maintenance of histone acetylation including H3K9ac and H4K16ac at MLL-AF9 target loci. In line with the profound loss of histone acetylation at MLL-AF9 target loci in PHF20-depleted cells, we found that knockdown of a known PHF20 interacting partner KAT8 (a histone acetyltransferase; also known as MOF or MYST1) phenocopies the effects observed in PHF20-knockdown cells. Finally, we showed that pharmacological inhibition of DOT1L and KAT8 synergistically suppresses the proliferation and survival of MLL-AF9 leukemic cells. These data collectively highlight the involvement of a novel DOT1L-PHF20-KAT8 axis in mammalian gene regulation and MLL-r leukemogenesis. In summary, our studies show that MLL-rearrangements may drive leukemic transformation by coordinating an epigenetic network involving several histone modifications associated with gene transcription (e.g. H3K79 methylation and H3K9/H4K16 acetylation). Our results also suggest that simultaneous targeting of multiple components of this epigenetic feed-forward loop including DOT1L and PHF20/KAT8 may provide a novel and more effective approach against MLL-r leukemia. Disclosures Bradner: Novartis Institutes for BioMedical Research: Employment. Armstrong:Epizyme, Inc: Consultancy; Vitae Pharmaceuticals: Consultancy; Imago Biosciences: Consultancy; Janssen Pharmaceutical: Consultancy.

Description: Background In younger and fit multiple myeloma (MM) patients (pt), autologous stem cell transplantation (ASCT) remains the gold standard treatment. Mobilization chemotherapy is usually administered in an inpatient regimen and Cyclophosphamide (CY) at different doses is the most used chemoterapy for collecting peripheral blood stem cells (PBSC) in MM. Clinical trials have demonstrated that intermediate dose CY (3 and 4 g/m2, ID-CY) combined with G-CSF, is an efficient mobilizing regimen with less toxicity compared with high dose CY (7 g/m2, HD-CY) in term of neutrophil recovery, thrombocytopenia, need of transfusions and IV antibiotics. (Fitoussi et al, BMT 2001; Goldschmidt et al, BMT 1996). Objective To evaluate the safety of mobilization therapy administered in an outpatient regimen, with the prospect to lower costs and minimize patient inconvenience, maintaining an optimal yield. Methods 92 pt with newly diagnosed MM underwent outpatient stem cell mobilization between 2002 and 2016 with CY 3 g/m2 (82%) or 4 g/m2 (18%) + G-CSF after induction therapy with bortezomib-based (79%) or VAD-like (21%) regimens. No antibiotics prophylaxis was routinely used. Day 0 was defined as the CY infusion day. CY was administered in 2-4 consecutive 1h infusions (depending on total dose). Hyper-hydration (3.5/4 l), antiemetics and the uroprotectant Uromitexan were began IV 1 hour before CY infusion. Subsequently, Uromitexan was continued at home orally in the next 12h. Furthermore, the patient was advised to drink 2.5/3 l of water in the next 24h. G-CSF 10 mcg/Kg was started by day +5 and continued until completion of apheresis. Blood count was monitored at day + 4 and daily from day +7. CD34+ cells were counted on peripheral blood by day 7; apheresis was started at leukocyte rise and with a value of at least 20 CD34+/μl. Number of apheresis depended on the number of CD34+ cells collected to obtain al least 4x106 CD34+/Kg. Results Median age at diagnosis of was 56y (range 34-68). MM isotype was IgA, IgG and micromolecular respectively in 18%, 58% and 24%. Prior MGUS was present in 37 cases (43%). LDH was elevated in 7 pt (11%), whereas ISS was 1/2/3 in 47%/30%/23%. Bone disease was detectable in 74% of pt, with 56% having 3 or more osteolysis. Median bone marrow plasma cell at diagnosis was 60% (range 10-95%). Pt received induction with bortezomib-based regimens (79%) or chemoterapy, mostly VAD (21%). 8 pt (9%) required second line therapy before mobilization. Response prior of mobilization was CR/sCR in 15%, VGPR in 59%, PR in 24%, and SD in 2%. Stem cell collection was successful in 98% of pt, with a median CD34+ harvest of 9.8x106/Kg. Chemotherapy was very well tolerated. Most frequently observed adverse events (AEs) were nausea and vomiting of grade 1-2. 2 pt experienced cystitis (one grade 1, one grade 2), 2 pt infections, 2 pt hyperthermia regressed rapidly without therapy, 1 patient diarrhea. 3 pt had neurological symptoms: in 2 cases they were aspecific (headache, instability); the other case presented a sudden appearance of 7th cranial nerve deficit at the end of mobilization chemotherapy infusion with negative imaging and successively regressed in few hours, interpreted as transient ischemic attack not correlated with Cy. Only 2 patient required hospitalization for AEs: 1 patient for fever grade 3 without microbiological findings, rapidly regressed with IV antibiotics; the second one for 7th cranial nerve deficit. These were the only grade 3 AEs, no grade 4 AEs verified. There were no other significant AEs related to chemotherapy. All pt except 2 proceeded to stem cell harvest and reached CD34+ target, but 5 pt required administration of Plerixafor on demand. The 2 pt not reaching CD34+ target successfully mobilized afterwards, 1 with different chemoterapy and the other with G-CSF and Plerixafor. After mobilization, 88 pt proceeded to single (45%) or double (55%) ASCT. Conclusion In conclusion, outpatient mobilization with ID-CY appears to be an efficient and safe procedure, with minimal and manageable side effects and low rate of hospitalization. Outpatient mobilization could ameliorate the quality of life of pt and reduce costs, avoiding or minimizing the hospitalization rate, without compromising the safety profile and the success of PBSC collect. Disclosures No relevant conflicts of interest to declare.

Description: Background: Histone acetylation plays a key role in regulating gene expression and in control of cellular activities in multiple pathways involved in normal and cancer cell growth.Panobinostat (pano) is a pan histone de-acetylase inhibitor (HDAC-i) approved by the FDA on February 23, 2015 for use withbortezomib (btz) and dexamethasone (dex) for patients with multiple myeloma (MM) who have had at least 2 prior lines of therapy including bothbtz and an immunomodulatory agent (IMiD). The combination ofpano withIMiDs and proteasome inhibitors (PIs) has been found to demonstrate enhanced anti-myeloma activity in clinical trials (Berdeja JG et al, 2015,Haematologica;Mateos M et al, 2010, ASCO Abstract 8030, JCO 28:15s). The goal of this retrospective study is to evaluate the real world experience on efficacy and safety ofpano in combination with a variety of FDA approved agents including a PI, anIMiD or a monoclonal antibody-based regimen in patients with relapsed/refractory MM. Methods: Between February 23, 2015 and July 1, 2016, 34 consecutive patients with relapsed/refractory MM who were treated with commercialpano were identified from the JohnTheurer Cancer Center. Charts were analyzed for response and safety data. The study was approved by the institutional review board. Results: Median age was 63 (range 27-78), with 58% percent men. Thirty-one patients (91.2%) wereDurie-Salmon stage II or III. Ten (30%) had high-risk FISH as defined byt(14;16), t(4;14), del p53, and gain 1q21. Median number of prior lines was 5 (range 2-9). All patients were relapsed/refractory to their last line of therapy, and 18 (53%) werebtz-refractory, 25 (74%) werelenalidomide-refractory, 27 (79%) werepomalidomide-refractory, and 29 (85%) were carfilzomib-refractory. Twenty-five (74%) were refractory to the combination of carfilzomib with anIMiD. Five patients (14.7%) had priordaratumumab, and 4 (12%) had prior HDAC-i therapy. Median number of cycles withpano was 1 (range 1-5). The overall response rate (≥ partial response (PR)) was 23.5% and the clinical benefit rate (≥ minor response (MR)) was 67.6%. The median duration of response (≥ stable disease (SD)) was 3 months. The median progression-free survival (PFS) for all patients was 2.3 months (95% CI: [1.27 - 4.07]). See Figure 1. Median overall survival (OS) from initiation ofpano through 7/27/16 was 5.5 months (95% CI: [3.93, NA]). See Figure 2. Of the 4 patients who were refractory to a prior HDAC-i, 1 achieved PR (4 cycles), 1 achieved MR (5 cycles) and 2 had disease progression. Only 1 patient discontinuedpano due to toxicities. Grade 3 and 4 non-hematologic toxicities were diarrhea (N=1), and hypoxia/respiratory failure (N=1). Grade 3 and 4 hematologic toxicities occurred in 11 (32%) patients, with 5 (15%) anemia, 9 neutropenia (26%), and 8 (24%) thrombocytopenia. Serious adverse events included acute kidney injury, GI bleed, and febrile neutropenia in 3 patients, respectively. Conclusions: These observations demonstrate that real-world use ofpano outside of the FDA indication in combination with PI andIMiD-based regimens has activity and is well tolerated in heavily pretreated patients with relapsed/refractory MM, even those who have exhausted conventional treatments. Further assessment in a larger prospective study is warranted. Figure 1 PFS of all patients receivingpanobinostat-based regimens Figure 1. PFS of all patients receivingpanobinostat-based regimens Figure 2 OS of all patients receivingpanobinostat-based regimens from time of initiatingpanobinostat Figure 2. OS of all patients receivingpanobinostat-based regimens from time of initiatingpanobinostat Disclosures Biran: Takeda: Speakers Bureau; Celgene: Speakers Bureau; Novartis: Speakers Bureau; Amgen: Speakers Bureau. Vesole:Janssen: Speakers Bureau; Novartis: Speakers Bureau; Takeda: Speakers Bureau; Celgene: Speakers Bureau; Amgen: Speakers Bureau. Richter:Celgene: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Jannsen: Speakers Bureau. Siegel:Celgene: Honoraria, Speakers Bureau; Merck: Honoraria; Takeda: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau.

Description: Genomic stability and integrity in Hematopoietic Stem Cells (HSCs) is maintained via DNA damage checkpoints, DNA proofreading and DNA repair (Moehrle et al., 2015; Cell Rep). Despite these mechanisms, recurring and non-recurring mutations accumulate in HSCs upon aging, which correlate with an elevated incidence of myeloproliferative diseases (Rossi, Bryder, and Weissman, Exp. Gerontol. 2007) as well as changes in clonality (Akunuru and Geiger, 2016; Trends in Mol. Med.). The rate at which such mutations accumulate in individual HSCs and the selection advantage/disadvantage that they provide is unclear and is an active area of investigation. Evolutionary theory supports a strong influence of the aged niche on the selection of HSCs clones upon aging (Rozhok, Salstrom, and DeGregori, 2014; Aging). We hypothesized that variant profiling of single HSCs based on RNA transcripts will reveal mutational signatures adapted to the selection pressure of the aging microenvironment. We performed Single cell RNA-seq of daughter cell pairs from young and aged murine HSCs (LSK, CD34-, flk2-). The Genome Analysis Toolkit (GATK; Broad Institute) RNA-seq variant/mutation calling algorithm pipeline was applied with some modifications. Only variants that were observed in both daughter cells of a given pair were selected, which significantly decreased our false discovery rate (tested by a Monte Carlo simulation). First and most interestingly, we observed no significant difference in the overall number of variants/mutations between young and aged HSCs, further supporting our recently published observations on the frequency of DNA mutations in HSCs upon aging (Moehrle et al., 2015; Cell Rep). We then used an approach that takes into account the 3' and 5' bases flanking a variant to generate motifs whose frequencies can be mathematically analyzed to deduce characteristic mutational patterns, termed as mutational signatures (Nik-Zainal et al., 2012; Cell). We employed a non-negative matrix factorization (nmf) and principal component analysis (pca) algorithms to generate 10 mutational signatures that explained 〉 95% of the variance in the dataset. We then analyzed the signatures in a pairwise fashion and selected two signatures with the highest discrimination score between young and aged HSC. Based on this, cells fell into two major groups: group 1 predominantly contained aged single cells (~90% of the cells in this group) whereas, interestingly, group 2 contained a mix of young and aged HSCs. The segregation of young and aged single HSCs counts between groups 1 and 2 was tested using Fisher's exact test and was statistically significant (p-value 0.0029). These data indicate that while the overall mutational load is not elevated, majority of aged HSCs acquire a mutational signature distinct from young HSCs, while a proportion of aged HSCs present with a young-like HSC signature. Furthermore, our results show that even those cells that have acquired an aging signature aren't homogeneous and show sub-clustering tendencies, providing the first hint that they may potentially evolve further into more distinct clones. In conclusion, our results show that individual HSCs reflect a mixed mutational profile reminiscent of a non-uniform accumulation of variants. As such signatures are a reflection of underlying mechanisms by which the mutations accumulate (Nik-Zainal et al., 2012; Cell), the proportion of aged HSCs sharing similar mutational signatures but distinct from the young HSCs reveal an aging signature that indicates specific mutational factors and selection pressure of the aging microenvironment. Disclosures Mulaw: NuGEN: Honoraria.

Description: Autologous stem cell transplant (ASCT) remains the standard of care for Multiple Myeloma (MM) patients younger than 70 years old. The role of induction therapy is crucial within a program of high-dose therapy since deeper is the response before, higher is the outcome of transplant. In this study, we analyzed a real life setting of patients treated with three different induction approaches: VAD (Vincristine-Adriamycin-Dexamethasone), VD (Bortezomib - Dexamethasone), and VTD (Bortezomib-Thalidomide-Dexamethasone) in terms of depth of response, 2 years therapy-free rate and toxicity. One hundred and sixty-three MM patients (pts) were included in the analysis: 62 pts treated with VAD (38%), 44 with VD (27%) and 57 with VTD (35%). In VTD group 49 pts (86%) received Bortezomib subcutaneously. As shown in Table 1, patients of the three groups were similar for D&S stage (p 0.59), a higher rate of ISS stage 3 was observed in VAD group (p=0.019), patients in VTD group were significantly older (p=0.024), median follow-up was significantly lower in VTD pts (p

Description: Introduction: Neutrophil extracellular traps (NETs) are structures composed of DNA, histones, and bactericidal factors that are expelled by neutrophils in order to trap and neutralize bacteria. NETs play a role in host defense by trapping and killing infecting bacteria and inactivating bacterial virulence factors. Activation of the coagulation cascade by these components can lead to "immunothrombosis" and facilitate the containment and destruction of bacteria within a fibrin clot. Although extracellular nucleosomes (structures consisting of DNA wound around a histone protein core) within NETs can contribute to host defense, they can also play a role in disease pathology by leading to inflammation, endothelial damage, and pathological thrombosis. Disseminated intravascular coagulation (DIC) is a condition characterized by systemic activation of the coagulation and fibrinolytic systems that can occur in conjunction with several underlying conditions, including sepsis. Links between infection, host response, and systemic coagulation, extracellular nucleosomes may play a significant role in the pathophysiology of sepsis-associated DIC. The purpose of this study was to quantify extracellular nucleosomes in the plasma of patients with sepsis-associated DIC. Materials and Methods: Citrated, de-identified plasma samples were collected from patients with sepsis and suspected DIC at ICU admission and on ICU days 4 and 8 under an IRB approved protocol. DIC score was evaluated in each sample using the ISTH scoring algorithm incorporating platelet count, PT/INR, fibrinogen (Recombiplastin, Instrumentation Laboratory, Bedford, MA), and D-Dimer (HyphenBioMed,Neuville-Sur-Oise, France). Plasma from healthy individuals was purchased from a commercial laboratory (George King Biomedical, Overland,KS). Nucleosomes in plasma were measured using the Cell Death Detection ELISA (Roche Diagnostics, Indianapolis, IN). The correlation of variation for both intra-assay and inter-assay variation was 0.2, p

Description: Background: Erythropoiesis Stimulating Agents (ESAs) are FDA approved for chemotherapy induced anemia and anemia of chronic kidney disease. These indications are more frequent in patients with multiple myeloma. Use of ESAs has been associated with an increased risk of heart attack, stroke, venous thromboembolism (VTE), and all-cause mortality. In patients with cancer, ESA's have been associated with worse progression free survival (PFS) and overall survival (OS) Previous research regarding ESA use in patients with multiple myeloma has been limited and conflicting. In this study, we evaluate the effects of ESA use in patients with multiple myeloma. Additionally, we examined the frequency of ESA usage after the 2007 FDA safety update revising product labeling for ESAs. Methods: A retrospective chart review was conducted on patients diagnosed with active multiple myeloma between January 1st, 2000 and December 31st, 2015 at Gundersen Health System in La Crosse Wisconsin. Collected data include patient demographics, medications, lab tests, comorbidities, and the dates of any VTE, stroke, or myocardial infarction. Both a logistic regression and a matched case-control analysis were used to compare rates of complications in patients using ESAs to those that did not. ESA effect on median survival time was also calculated for each International Staging System (ISS) stage of disease. Results:There were 278 patients included for demographic analysis (Table 1), of which 268 were included in the logistic regression analysis and 124 (62 pairs) in the matched case-control analysis. A logistic regression model constructed via stepwise selection found that bone lesions at diagnosis (Odds Ratio (OR): 2.5 [1.2-5.1]), antiplatelet drug use (OR: 3.7 [1.2-11.3]) and ESA use (OR: 4.7 [2.2-9.9]) were associated with increased risk of VTE in our patient population. Use of an Angiotensin Converting Enzyme (ACE) inhibitor (OR: 0.4 [0.2-0.9]) was associated with a decreased risk of VTE. Increased odds of VTE with ESA usage (OR: 5.9 [1.9 - 18.8]) were also noted in the matched case-control analysis. There was no association found between rate of stroke and ESA usage in either logistic or matched case-control analysis. No significant association between ESA use and overall survival was noted in either logistic or matched case-control analysis. When comparing outcomes based upon pre and post FDA revised product labeling, we found a 50% reduction in ESA usage within our institution. In this same time period, the percentage of patients with multiple myeloma developing VTEs has been significantly reduced (18.6% vs 12.8% [p

Description: Introduction Multiple myeloma (MM) is a largely incurable plasma cell malignancy characterised by marked genomic heterogeneity, in which chromosome 1q21 amplification (amp1q21) associates with poor prognosis. Genomic analysis using next generation sequencing has identified recurrent mutations, but no universal acquired somatic mutation(s) have emerged in MM, suggesting that understanding pathways of survival will require analysis of individual tumours in distinct disease subsets. To compound complexity of the problem, intraclonal variation (ICV), known as a major driver mechanism in cancer plasticity, in which clonal competitor cells undergo selection during disease evolution and progression by Darwinian principles, will need to be fully mapped at the genome level. Identifying the true level of ICV in a tumour will thus require analysis at the level of whole exome sequencing (WES) in single cells (SCs). In this study, we sought to establish WES methodology able to identify ICV in SCs in an index case of amp1q21 MM. Methods Cell selection and sequencing CD138+ tumour cells and CD3+ T-cells were isolated from a presentation case of amp1q21 MM as bulk populations to high purity (〉97%). Single MM cells and normal T cells were individually isolated and used for single cell (SC) whole exome sequencing (WES). Whole genome amplification (WGA) was performed by multiple displacement amplification (Qiagen REPLI-g Mini kit), and exome capture was performed using Agilent SureSelect. Libraries were then 90 bp paired end sequenced on an Illumina HiSeq2000 (BGI, China). Data analysis Data was produced for bulk (1000 cells) MM and bulk germline T cells, twenty MM SCs and five T cell SCs. Raw data was aligned to hg19 reference sequence using NovoAlignMPI (v3.02.03). Variant calling was performed using SAMtools (v1.2.1) and VarScan (v2.3.6) and variants were annotated using ANNOVAR. High confidence variants were called in the bulk tumour WES by pairwise comparison with bulk germline WES. Variant lists were also cross-searched against various variant databases (CG46, 1000 genomes, dbSNP, esp650 and in-house database) in order to exclude variants that occur in the general population. Multiple quality control measures were employed to reduce the number of false positive calls. Results and Discussion Data and bioinformatics pipelines are of a high quality SC WES generated raw data reads that were similar to bulk WES of 1000 cells, with comparable mapping to Agilent SureSelect target exome (69-76% SC vs. 70% bulk) and mean fold coverage (56.8-59.1x vs. 59.7x bulk). On average, 82% of the exome was covered sufficiently for somatic variant (SV) calling (often considered as ≥ 5x), which was higher than seminal published SC WES studies (70-80%) (Hou et al., Cell, 2012; Xu et al., Cell, 2012). We identified 33 potentially deleterious SVs in the bulk tumour exome with high confidence bioinformatics, 21 of which were also identified in one or more SC exomes. The variants identified include suspected deleterious mutations in genes involved in MAPK pathway, plasma cell differentiation, and those with known roles in B cell malignancies. To confirm SV calls, randomly selected variants were validated by conventional Sanger sequencing, and of 15/15 variants in the bulk WES and of 55/55 variants in SCs, to obtain 100% concordance. Intraclonal variation in MM Significantly, ICV was apparent from the SC exome variant data. Total variant counts varied considerably among SCs and most variant positions had at least several cells where no evidence of the variant existed. Bulk WES lacks crucial information We identified an additional 23 variants that were present in 2+ SC exomes, but absent in the bulk MM tumour exomes. Of these, 30% (7 variants) were examined for validation, and were amplifiable in at least one cell to deliver 100% concordance with variant calls. These variants are of significant interest as they reveal a marked occurrence of subclonal mutations in the MM tumour population that are not identified by bulk exome sequencing. They indicate that the mutational status of the MM genome may be substantially underestimated by analysis at the bulk tumour population level. Conclusion In this work we establish the feasibility of SC WES as a method for defining intraclonal genetic variation in MM. Disclosures No relevant conflicts of interest to declare.

Description: Background: Glucocorticoids has been a backbone of various treatment regimens for multiple myeloma (MM). The repeated and chronic use of high doses of glucocorticoids is associated with development of secondary adrenal insufficiency (AI), and AI could be a major problem in critically-ill patients. However, there has been no specialized data about incidence and clinical significance of secondary AI in hospitalized patients with MM. Purpose: The objective of this retrospective study is to evaluate incidence, predictive factors, and clinical significance of secondary AI in hospitalized patients with MM. Methods: We retrospectively evaluated medical records of MM patients who were hospitalized in Chonnam National University Hwasun Hospital, South Korea from December 2014 to December 2015. The definite AI was diagnosed when the peak cortisol concentration was less than 〈 500 nmol/L (18 mcg/dL) after ACTH administration. Results: Between December 2014 and December 2015, 77 patients were hospitalized, and 58 underwent rapid ACTH stimulation test. The most frequent cause of hospitalization was infection (70.7%), followed by weakness (24.1%), and the others (5.3%). The definite AI was confirmed in 19 patients (32.7%). To evaluate the predictive factors of AI, all variables including clinical characteristics, laboratory results, cumulative steroid dose, and treatment duration at hospitalization were analyzed, but there were no significant predictors for AI. In addition, the patients with AI had a significantly poor survival outcomes compared to those without AI (the median overall survival of 42.3 months vs. 82.7 months; P = 0.037) (Figure 1). Conclusions: This study showed that the secondary AI is not a rare condition among hospitalized patients with MM, and there was no specific predictable symptoms or signs. In addition, development of AI in the treatment period is associated with a poor prognosis. This study suggests that evaluation of AI is routinely needed in hospitalized patients with MM. Disclosures No relevant conflicts of interest to declare.

Description: Background and objective: Multiple Myeloma (MM) is a plasma cell malignancy with a well documented immune dysfunction. Previous reports in murine models and our previous work in MM patients, showed that myeloid cells, both granulocyte-myeloid derived suppressor cells (G-MDSC) and neutrophils sedimenting on top of red cells after density gradients, defined as high-density neutrophils (HDN) are increased and exert an immunosuppressive activity. Moreover, we showed that myeloid precursors could be activate and acquire G-MDSC phenotype if cultures in presence of MM mesenchymal cells. Thus, we characterized functionally HDN to gauge their contribution in immunesuppression, angiogenesis and bone resorption in MM microenvironment. Methods: In HDN freshly-isolated from a series of 60 newly-diagnosed MM, 30 smoldering MM and 30 MGUS we investigated by RT-PCR, flow-cytometry, western-blot and immunofluorescence genes and proteins related to immunesuppression (Arg-1, IDO), angiogenesis (BV8, VEGF, MMP9) and bone resorption (IL1-b, MMP9, CHI3L). Results: First, MM-HDN exhibited an increased expression of immunesuppressive molecules Arg-1 and IDO compared to MGUS and healthy subjects (25.5 vs 6.2 vs 1 fold changes in gene expression, p=0.003), confirmed by functional assay of enzymatic activity, immunostaining and WB, positively correlated with advanced disease. Second, MM-HDN showed increased expression of BV8 and MMP9 (p=0.01, p=0.05) but not VEGF, as confirmed by immunofluorescence. In line with this observation, by using migration assay (CIM plate) MM-HDN induced neo-vasculogenesis in vitro by forming more and aberrant tubes than healthy or MGUS-HDN, thus to disclosure a novel proangiogenic mechanism driven by BV8. Third, MM-HDN showed increased expression of IL1-b, MMP9, CHI3L, associated to increased bone resorption activity, evaluated by dentin-disk assay. The same findings could be obtained treating bone matrix with MM-HDN-conditioned media, suggesting the crucial role of molecules released by MM-HDN but no from MGUS-HDN. Conclusion: taken together, our observations disclosure the contribution of aberrantly activated HDN in MM clinical features, contributing to immunosuppression, angiogenesis and bone resorption. Disclosures No relevant conflicts of interest to declare.

Description: Background:Novel insights into the biology of myeloma cells have led to the identification of relevant prognosis factors.Cytogenetic abnormalities (CA) has become one of the most important prognostic factors, and the presence of t(4;14), t(14;16) or del(17p) are associated with poor prognosis. Although there are some reports indicating that 1q gains may be considered as a poor-risk feature, the information is not uniform. Furthermore, there are important controversies about whether or not novel agents-based combinations are able to overcome the poor prognosis of CA. In the relapse setting, the combinations including proteasome inhibitors and immunomodulatory drugs have shown to improve, and some of them to overcome, the outcome of patients with high-risk CA. Here we report a preplanned analysis, in a series of elderly newly diagnosed myeloma patients included in the Spanish GEM2010 trial and receiving VMP and Rd, in a sequential or alternating approach, in order to evaluate the influence of CA by FISH on the response rate and outcome. Patients and methods: 242 pts were randomized to receive a sequential scheme consisting of 9 cycles of VMP followed by 9 cycles of Rd or the same regimens in an alternating approach (one cycle of VMP alternating with one Rd, up to 18 cycles. VMP included the IV administration of weekly bortezomib (except in the first cycle that was given twice weekly) at 1.3 mg/m2 in combination with oral melphalan 9 mg/m2 and prednisone 60 mg/m2once daily on days 1-4. Rd treatment consisted on lenalidomide 25 mg daily on days 1-21 plus dexamethasone 40 mg weekly. FISH analysis for t(4;14), t(14;16), del(17p) and 1q gains was performed at diagnosis according to standard procedures using purified plasma cells. Results: In 174 out of the 233 patients evaluable for efficacy and safety, FISH analysis at diagnosis were available and two groups were identified: high-risk group (n= 32 patients with t(4;14) and/or t(14;16) and/or del(17p)) and standard-risk group (n=142 patients without high-risk CA). The rates of CA was similar in both treatment arms. Response Rates (RR) were no different in the high-risk vs standard-risk groups, both in the sequential (74% vs 79% RR and 42% vs 39% CR) and alternating arms (69% vs 86% RR and 39% vs 38% CR). After a median follow-up of 51 months, high-risk patients showed shorter PFS as compared to standard risk in the alternating arm (24 versus 33 months, p=0.03) and this also translated into a significantly shorter OS (38.4m vs not reached, p=0.002). However, in the sequential arm, high-risk and standard-risk patients showed similar PFS (29.5 months vs 31.5 months, p=0.9) and OS (46m vs 63m, p=0.1). This beneficial effect observed in the sequential arm applied to both t(4;14) or del(17p). As far as 1q gains is concerned, 151 patients had 1q information and 76 of them had 1q gains (50.3%), defined as the presence of more than 3 copies in at least 10% of plasma cells. The rate of 1q gains was well balanced in both sequential and alternating arms. The ORR was similar in patients with or without 1q gains (83% vs 80%) as well as the CR rate (45% vs 31%), and no differences were observed between sequential and alternating arms. Patients with or without 1q gains had a similar PFS (36 months vs 29 months) and 4-years OS (63% vs 68%) in the whole series and no differences were observed between the sequential and alternating arms. This effect has been observed in patients with 1q gains as isolated CA and the outcome was slightly but not significantly worse when 1q gains were present plus either t(4;14) and/or del17p. Conclusions: The total therapy approach including VMP and Rd administered in a sequential approach is able to overcome the poor prognosis of the presence of high-risk CA in elderly patients with newly diagnosed MM. The presence of 1q gains has no impact in the PFS and OS of elderly patients treated with VMP and Rd. Disclosures Mateos: Janssen, Celgene, Amgen, Takeda, BMS: Honoraria. Martínez-López:Novartis: Honoraria, Speakers Bureau. Oriol:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Paiva:Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding.

Description: Introduction: Idelalisib (IDELA) is a selective, small molecule inhibitor of PI3Kd that has shown significant efficacy in treatment of patients (pts) with relapsed chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL). A common adverse event (AE) observed in IDELA studies is diarrhea/colitis (DC): grade ≥3 ~15%. Published preclinical data suggests that PI3Kd plays a critical role in regulating the function and development of regulatory T-cells (T-regs). This biomarker analysis aimed to evaluate possible immune mechanisms that may have contributed to DC in IDELA-treated pts. Methods: Longitudinal absolute peripheral blood T (CD4+ and CD8+), NK (CD16+/CD56+) cell subsets, cytokines, and chemokine levels from patients treated with IDELA were analyzed (Table 1). Since absolute numbers of T-reg cells were not available, we utilized epigenetic qPCR method (Kleen T. et. al. J Immunother Cancer 2015) to assess the status of T-regs by quantifying FOXP3 utilizing banked peripheral blood mononuclear cells (PBMCs). The following cytokines and chemokines were measured: IL-12p40, IL-17A, IFNγ, TNFα, G- CSF, MIP1α (CCL3), CCL5 (RANTES), IL-10, IL-1RA, IL-6, IL-7, IL-8, IL-15, CRP, and IP-10 (CXCL10). We evaluated the association of changes from baseline of these biomarker(s) with the occurrence and severity of DC events during IDELA treatment. Association of cytomegalovirus (CMV) with DC was not addressed in this study and is being presented separately. Results: There were no differences in absolute numbers of T (CD4+ or CD8+) and NK cells between pts treated with IDELA in both trials with grade ≥3 DC vs those with no DC. Consistently, results from epigenetic qPCR analysis also demonstrated no differences in temporal profiles for peripheral T-cell subsets (CD3+, CD8+, or FOXP3+) in CLL pts treated with IDELA with grade ≥3 DC vs no DC. Baseline and on-treatment changes in peripheral T-cell subsets were not predictive of DC. Analysis of T-cell subsets from the visit immediately prior (t-1) to the first occurrence of grade ≥3 DC was not predictive, and revealed no differences compared to pts with no DC. Lower levels of CD3+, CD8+, and FOXP3+ were noted longitudinally as well as at t-1 visits in grade 1/2 DC vs non-DC pts, but these changes were not predictive of grade 1/2 DC. Increased levels of circulating pro-inflammatory cytokines (IL-15, IFN-γ, and CLL5) were noted in both CLL and indolent non-Hodgkin lymphoma (iNHL) pts treated with IDELA. IL-17A level was significantly higher at the t-1 visit in CLL pts with grade ≥3 DC vs no DC. However, Receiver Operating Characteristic analysis deemed that neither individual cytokine/chemokine or in combination was not predictive for DC occurrence. CLL/iNHL pts with grade ≥3 DC vs no DC were noted to have higher on treatment IL-8. CLL pts presented lower baseline IL-6 and G-CSF levels in patients with grade ≥3 DC vs no DC (Table 2). There were no associations between baseline circulating plasma markers and DC in pts with iNHL. Conclusion: With currently available data, no single circulating immune biomarker is associated with or is predictive for the development of DC during treatment with IDELA. Lower levels of CD3+, CD8+, and FOXP3+ were noted longitudinally in grade 1/2 DC vs no DC pts. No differences were observed in temporal profiles for T-cell subsets in pts with grade ≥3 DC vs those with no DC. However, higher on-treatment IL-8 and lower baseline IL-6 and G-CSF were noted in the relapsed CLL pts with grade ≥3 DC when compared with no DC pts. While quantitative analysis of these T-cell subsets was not associated with grade ≥3 DC, the qualitative function of T-cells may play a role in mediating DC. Functional assays for T-cells were not explored in this study. In addition, our concurrent analysis of colonic biopsies and association with CMV in pts with IDELA associated DC will be presented separately. Disclosures Furman: Pharmacyclics: Consultancy, Speakers Bureau; Gilead Sciences: Consultancy; Janssen: Consultancy; Genentech: Consultancy; Abbvie: Consultancy, Honoraria. Hallek:Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau. Sharman:Gilead Sciences, Inc.: Honoraria, Research Funding. Hillmen:Pharmacyclics: Research Funding; Janssen: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Research Funding. Zelenetz:Gilead Sciences: Research Funding. Flinn:Janssen: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Gilead Sciences: Research Funding; ARIAD: Research Funding; RainTree Oncology Services: Equity Ownership. Jurczak:Gilead Sciences: Research Funding; Janssen: Research Funding; Celltrion, Inc: Research Funding; Acerta: Research Funding; Bayer: Research Funding. Munugalavadla:Gilead Sciences: Employment, Equity Ownership. Xiao:Gilead Sciences: Employment, Equity Ownership. Zheng:Gilead Sciences: Employment, Equity Ownership. Rao:Gilead Sciences: Employment, Equity Ownership. Dreiling:Gilead Sciences: Employment, Equity Ownership. Salles:Roche/Genentech: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Gilead: Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Mundipharma: Honoraria. O'Brien:Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria.