Effect of neuraminidase treatment on serum reactivity to autologous leukemic blast cells

Summary The sera of 35 patients with acute lymphoblastic leukemia (ALL) and acute non-lymphoblastic leukemia (ANLL) were tested for reactivity against cell surface antigens of autologous leukemic blast cells by protein A assay (PA), immune adherence assay (IA), and anti-C3 mixed hemadsorption assay (C3-MHA). Autologous serum reactivity was detectable by PA in four cases and by LA and C3-MHA in about half the patients. Autologous serum reactivity occurred more often in ALL than in ANLL. Absorption studies revealed that in one patient only the autologous reactivity was directed against a restricted antigen, which could be detected only on the individual T-ALL blast cells. All other autologous antibodies detected unspecific antigens. Neuraminidase treatment had two effects: first, it increased antibody attachment to antigens which are also present on untreated cells; secondly, after neuraminidase treatment an antigen was detectable on the cell surface which could also be demonstrated on neuraminidase-treated non-leukemic cells (e.g., erythrocytes). Neither of these two effects of neuraminidase treatment seems to be tumor-specific. Possible therapeutic effects of neuraminidase are probably caused by unspecific adjuvant effects of the enzyme.     

Neoplastic meningosis in immature cell leukemias and malignant lymphomas

162 patients with acute leukemias or malignant non-Hodgkin lymphomas were examined for meningeal and cerebral manifestations of their disease. A clinically manifest disease could be found in 13 patients, meningosis was additionally detected by autopsy in 32 patients. The highest frequency was found in acute lymphatic leukemia followed by lymphoblastic lymphomas and acute myeloic leukemias. Less frequently there was a meningeal involvement in low-grade malignant lymphomas which becomes clinically manifest only in some rare cases. In this respect, non-lymphoblastic, high grade-malignant lymphomas take an intermediate position. On principle, meningosis prophylaxis is imperative for acute lymphoblastic leukemias and advanced lymphoblastic lymphomas.     

Correlation between plasma fibronectin level and experimental rat heat stress mortality

Reticuloendothelial system (RES) clearance function correlates with the mortality rate associated with stresses that can induce shock. Likewise, experimental rat heat stress (ERHS) mortality rate is altered by modulation of RES function. Since plasma fibronectin (PF) in many instances appears to mediate in vivo phagocytosis by the RES, the relationship between mean plasma fibronectin level (MPFL) and ERHS mortality was examined. A comparison of MPFLs prior to ERHS revealed that rats which ultimately comprised the survival group had a MPFL of 269.0 +/- 11.2 micrograms/ml, whereas that of the nonsurvivors was 252.9 +/- 11.9 micrograms/ml. Both groups had elevated MPFLs up to 12 h following ERHS. However, after this time, MPFL began to decline. The decline was more severe for the nonsurvivors, with MPFLs at 15, 18, and 20.3 h significantly (P less than 0.01) lower than the values for the survival group. Even the lowest MPFL (256.0 +/- 30.7 micrograms/ml) noted for the survival group was still significantly (P less than 0.01) higher than the value (159.3 +/- 13.3 micrograms/ml) determined for agonal samples collected from nonsurvivors. Furthermore, grouping rats according to their preheat PF level demonstrated that rats with levels exceeding 300 micrograms/ml had significantly (P less than 0.05) reduced mortality rates (12.5 vs. 51.3%) compared with rats with levels below this value. It was concluded that elevated PF levels prior to ERHS correlated with thermotolerance.     

Cold synchronization for the study of peripheral blood and bone marrow chromosomes in leukemias and other hematologic disease states

Summary A method of cold-shock synchronization of immature granulocytic cells from peripheral blood or bone marrow is described. It is shown that this method provides an increased yield of early metaphases and offers advantages over others currently employed.The peaks of mitotic activity following the cold-shock treatment differ for patients with acute non-lymphoblastic leukemia (ANLL) and patients with chronic myeloid leukemia (CML) by an interval of 2 h.This method is considered to be suitable for routine cytogenetic studies on hematological patients.     

Characteristics of the silver-stained nucleoli in blast elements of various diameters from patients with acute leukemia

Using silver staining, a study was made of the activity of ribosomal cistrons (RC) in proliferating and non-proliferating subpopulations of bone marrow and peripheral blood blasts, taken from 33 patients with acute non-lymphoblastic leukemia and from 17 patients with acute lymphoblastic leukemia. All the blasts including non-proliferating ones were revealed to synthesize ribosomal RNA (rRNA). The RC activity of large blasts was higher than that of small ones. A significant difference (P less than 0.01) in RC activity between lymphoid and non-lymphoid blasts was found. The relation between nucleolar organizer activity of blasts and their proliferating potentials is discussed, in addition to a possibility for practical use of the above mentioned data.     

Second malignancies in Hodgkin's disease: a complication of certain forms of treatment.

A total of 764 patients with Hodgkin''s disease treated with radiotherapy (RT) or chemotherapy or both were reviewed 3-186 months (median 43 months) after initial treatment to assess the incidence of second malignancies. Incidence of solid tumours and acute non-lymphoblastic leukaemia (ANLL) were calculated by a life-table method and percentages of patients affected derived from life-table plots. Within 10 years after initial treatment the overall incidence of second solid tumours was 7.3%, and over a comparable period 2.4% of patients developed ANLL. Solid tumours occurred only in patients given RT with or without adjuvant chemotherapy, and ANLL occurred only after treatment with MOPP (mustine, vincristine, procarbazine, and prednisolone) or modified MOPP regimens. Neither solid tumours nor ANLL occurred in patients given ABVD (adriamycin, bleomycin, vinblastine, and dacarbazine). The highest incidence of leukaemia (5.4%) occurred after treatment with extensive RT plus (5.4%) occurred after treatment with extensive RT plus MOPP; hence the benefits of this approach in Hodgkin''s disease must be weighed against its carcinogenic potential.     

CFU-gm colony formation of peripheral blood and bone marrow in adult acute leukemia at presentation, during remission, and at relapse

Granulocyte/macrophage colony-forming unit (CFU-gm) formation was studied simultaneously in bone marrow and peripheral blood of 52 previously untreated adult patients with acute non-lymphocytic (ANLL) and 36 with acute lymphoblastic leukemia (ALL). They were followed during induction therapy at monthly intervals while in remission and in 19 ANLL and 22 ALL cases, until relapse. Patients showing a decreased colony number in the marrow but normal or increased colony numbers in the peripheral blood had a high probability of entering remission. Non-responding patients displayed an opposite pattern. The higher the degree of marrow repopulation with granulocytic progenitor cells after induction treatment, the longer remission duration and survival for ANLL patients and the longer survival for ALL patients. CFU-gm formation returned to normal in the early stages of complete remission, but then declined progressively. At ANLL and ALL relapse, colony growth was reduced markedly while cluster formation remained normal. The number of marrow colonies and clusters in ANLL were significantly higher at first and second relapse compared to the growth pattern at first presentation. A similar trend had been observed in ALL, suggesting a selection advantage.     

OAP combination in the treatment of elderly leukaemic patients with preexisting severe internal disease

Sixteen elderly patients affected by acute non lymphoblastic leukaemia (ANLL) with a preexisting severe internal disease were treated with a low systemic toxicity drugs combination: OAP (Vincristine, Cytarabine and Prednisone). Complete remission was achieved in 5 patients (31%) after 2 OAP courses. The mean duration of remission was 18 weeks. Six patients were resistant to the therapy. Six patients died during the treatment: 5 in induction phase and 1 in consolidation phase. Even though the duration of remission was short we retain that OAP combination may be still considered a good therapeutical approach in elderly ANLL patients with associated severe internal disease.     

Influences of clotting factors (thrombin, factor XIII) and of fibronectin on the growth of tumor cells and leukemic cells in vitro

Thrombin, factor XIII and fibronectin were incubated with cultures of mouse sarcoma cells, human cervix carcinoma cells (HeLa cells) and cells of an acute lymphoblastic leukemia. Thrombin induced a significant increase of 3H-thymidine uptake into cells with a 1,5- to 2-fold increase of cell count. The cells of an acute lymphoblastic leukemia showed a similar response to the influence of thrombin. Factor XIII in tumor cells merely induced an increase of 3H-thymidine uptake, the cell count remained constant. The cells of an acute lymphoblastic leukemia showed under the influence of factor XIII a significant increase of cell count and thymidine uptake. HeLa cell growth was optimal at low fibronectin concentrations. Fibronectin concentrations of 1 mg/ml to 3 mg/ml inhibited HeLa and mouse sarcoma cell growth.     

Effect of antihypertensive treatment on kidney function in diabetic nephropathy.

The effect of long term, aggressive antihypertensive treatment on kidney function in diabetic nephropathy was studied prospectively in 11 insulin dependent diabetics (mean age 30). During the mean pretreatment period of 32 (range 23-66) months the glomerular filtration rate decreased significantly and albuminuria and the arterial blood pressure increased significantly. During the 72 (range 32-91) month period of antihypertensive treatment the average arterial blood pressure fell from 143/96 mm Hg to 129/84 mm Hg and albuminuria decreased from 1038 micrograms/min to 504 micrograms/min. The rate of decline in the glomerular filtration rate decreased from 0.89 (range 0.44-1.46) ml/min/month before treatment to 0.22 (range 0.01-0.40) ml/min/month during treatment. The rate of decline in the glomerular filtration rate was significantly smaller during the second three years compared with the first three years in patients who received long term antihypertensive treatment (greater than or equal to 6 years). One patient died from acute myocardial infarction (glomerular filtration rate 46 ml/min/1.74 m2). Effective antihypertensive treatment postpones renal insufficiency in diabetic nephropathy.     

Fractionated total body irradiation and high dose cyclophosphamide: a preparative regimen for bone marrow transplantation for patients with hematologic malignancies in first complete remission

We treated 73 patients with hematologic malignancies in first complete remission (acute lymphoblastic leukemia = 23 patients; acute non-lymphoblastic leukemia = 25 patients; chronic myelogenous leukemia in first chronic phase = 20 patients, and high grade lymphoma = five patients) with a uniform preparative regimen consisting of fractionated total body irradiation (1,320 cGy) and high dose cyclophosphamide (100 mg/kg), followed by allogeneic bone marrow transplantation. By radiation dosimetry we demonstrated that the calculated doses were delivered accurately and reproducibly. Actuarial survival rates (+/- SEM) in complete remission were as follows: Acute lymphoblastic leukemia = 74 +/- 9%; acute nonlymphoblastic leukemia = 50 +/- 11%; and chronic myelogenous leukemia = 55 +/- 11%. Actuarial relapse rates for these three diagnoses were 19 +/- 9%, 17 +/- 11%, and 0% respectively. Three of the five lymphoma patients are alive in complete remission at 22+, 28+, and 54+ months. Overall probability of survival for the 73 patients was 59 +/- 7%. Interstitial pneumonia, usually associated with cytomegalovirus infection and graft-versus-host disease, and relapse of the underlying malignancy were the major causes of death.     

Clinical significance of fibrinopeptide A in acute lymphocytic and non-lymphocytic leukaemia

Fibrinopeptide A (FPA) was systematically investigated in 74 patients with acute leukaemia at different stages of the disease (50 with non-lymphocytic leukaemia, ANLL; 24 with lymphocytic leukaemia, ALL). At diagnosis, 75% of the cases had high FPA levels (86% in ANLL and 54% in ALL) with significantly higher levels in ANLL than in ALL (13.4 vs 4.4 ng/ml; p less than 0.001). Patients with DIC (20 cases in ANLL and 1 case in ALL) had significantly higher levels (p less than 0.001). FPA levels were neither correlated with fibrinogen or FDP levels nor with blast cell count. During chemotherapy, median FPA did not show significant changes whereas, at the end of therapy, a return toward normality was generally observed both in ALL and ANLL apart from the group of patients with acute promyelocytic leukaemia. Among the 24 patients who entered post-remission follow-up (13 ANLL and 11 ALL), 10 cases out of the 11 relapsing (6/6 with ANLL and 4/5 with ALL) had increased FPA 1 to 2 months before the ascertainment of the relapse. However, 16% and 9% of the samples obtained on different occasions, respectively from ANLL and ALL cases in maintained first remission, showed FPA above the normal limit. This study demonstrates that subclinical activation of blood coagulation, as indicated by high FPA level, is common both in lymphocytic and non-lymphocytic leukemia and suggests that this phenomenon is related to disease activity.     

In vitro exposure of leukemic cells to low concentration Arabinosyl Cytosine: no evidence of differentiation inducing activity

Low dose Arabinosyl Cytosine (LD ARA-C) is widely used for treatment of acute non-lymphocytic leukemia (ANLL) and myelodysplastic syndrome (MDS) in the elderly, based on a favorable response rate and on the hypothesis that LD ARA-C can induce differentiation or maturation of leukemic cells. We investigated the effect of low concentration of ARA-C on the growth of marrow cells that were obtained from 6 cases of MDS and from 11 cases of ANLL by using the in vitro culture system for normal granulo-monocyte precursors (CFU-GM). At ARA-C concentrations equal to or higher than 1 ng/ml cell growth was inhibited in a dose-dependent manner. At ARA-C concentration of 0.1 ng/ml cell growth was slightly affected, but colony number and colony cell composition were identical to control cultures. This experiment did not support the hypothesis that ARA-C can induce leukemic cells to recover any normal growth patterns but confirmed that even very low ARA-C concentrations can inhibit or slow down leukemic cell proliferation.     

Detection of factor XIII deficiency in acute leukemia with resonance thrombography

In 16 patients affected with acute leukemia (7 patients with acute lymphatic leukemia and 9 patients with acute myeloid leukemia) the resonance thrombogramme was recorded during cytostatic induction therapy, coagulation factor XIII (subunit XIII-A, XIII-S) and further hemostasiological parameters were determined. Subunit XIII-A was lowered to 36%, subunit XIII-S to 65% and the fibrin formation time of the resonance thrombogramme was extended to 9 minutes. There exists a negative correlation between component XIII-A and fibrin formation time r = -0.48 (p less than 0.01). The influence exerted by diminishing factor XIII and fibrin(ogen) splitting products on the fibrin formation time was investigated in in-vitro tests. A diminution of factor XIII below 10% will extend the fibrin formation time to about 10 minutes, an increase of fibrin(ogen) splitting products to 100 micrograms/ml to about 3 minutes.     

Activated terminal complement in cerebrospinal fluid in Guillain-Barré syndrome and multiple sclerosis

A quantitative enzyme-linked immunosorbent assay was used to measure the concentration of fluid-phase complement C5b-9 complexes (SC5b-9) in the cerebrospinal fluid (CSF) of 14 patients with acute monophasic Guillain-Barré Syndrome (GBS), 21 patients with multiple sclerosis (MS), and 11 patients with noninflammatory central nervous system (CNS) diseases. SC5b-9 complexes were detected in the CSF of 13 of 14 patients with acute GBS (mean, 3.08 micrograms/ml; range, 0 to 7.1 micrograms/ml) and 16 of 21 patients with MS (mean, 1.83 micrograms/ml; range, 0 to 7.5 micrograms/ml). In the control group of patients with noninflammatory CNS diseases, SC5b-9 was not detected in eight of 11 and was present in low concentrations in the remaining three patients (mean, 0.28 micrograms/ml; range, 0 to 1.7 micrograms/ml). The finding of SC5b-9 complexes in the CSF of patients with GBS and MS suggests that terminal complement components may participate in the tissue-damaging processes in these diseases.     

Levels of selenium, zinc, copper, and antioxidant enzyme activity in patients with leukemia

Essential elements, mainly selenium and zinc, were involved in protection against oxidative stress in cells. Oxidation could lead to the formation of free radicals that have been implicated in the pathogenesis of many diseases, including leukemia. Leukemia is a neoplastic disease that is susceptible to antioxidant enzyme and essential elements alterations. This study was undertaken to examine the levels of essential elements, antioxidant enzymes activities, and their relationships with different types of leukemia. Serum selenium, zinc, and copper concentrations, red blood cell glutathione peroxidase (GPx) activities, plasma Cu−Zn superoxide dismutase (Cu−Zn SOD) activities and lipid peroxidation (LPO) levels were determined in 49 patients with different types of leukemia before initial treatment. Serum selenium and zinc concentrations were lower in leukemia patients than those of controls (p<0.01). Serum copper concentration was higher in leukemia patients than that of controls (p<0.01). The activities GPx and Cu−Zn SOD were significantly increased in leukemia patients, especially with acute leukemia (AL), acute lymphoid leukemia (ALL), and acute nonlymphoid leukemia (ANLL) (p<0.05), whereas no difference was found between those of chronic myelogeneous leukemia and the controls. The levels of LPO were normal as controls. Serum selenium concentration was not correlated with GPx, and serum levels of zinc and copper were not related to Cu−Zn SOD. Serum zinc levels had a negative correlation with the absolute peripheral blast cells, whereas serum copper had a positive correlation with the absolute peripheral blast cells. Increased GPx and Cu−Zn SOD activities and normal levels of LPO, which were a protective responses, were an indicator of mild oxidative stress; it mights indicate that the essentials elements alterations in leukemia patients were mostly dependent on tumor activity. Changes of their levels demonstrated that there are low selenium, zinc, and high copper status in leukemia patients. The decrease of plasma zinc and increase of the Cu/Zn ratio could be the index that showed an unfavorable prognosis of acute leukemia.     

A radioimmunoassay for the measurement of aminopeptidase (microsomal) in human serum

A radioimmunoassay for the measurement of aminopeptidase (microsomal) (AP) in human serum was developed by using antiserum to human kidney AP. AP purified from kidney and AP present in normal serum and in serum from a patient with obstructive jaundice gave parallel logit-log transformation lines, suggesting immunological identity. The mean concentration of AP in normal serum (n = 104) was 1.33 +/- 0.30 (mean +/- SD) micrograms/ml. Men had significantly higher serum AP levels (1.41 +/- 0.30 micrograms/ml) (p less than 0.005) than women (1.24 +/- 0.28 micrograms/ml). Serum AP levels of patients with hepatoma (2.26 +/- 0.87 micrograms/ml) and cancer of the pancreas or the biliary tract (2.90 +/- 0.67 micrograms/ml) were significantly higher (p less than 0.005) than those of normal subjects. Patients with acute and chronic hepatitis (2.06 +/- 0.66 micrograms/ml) also had significantly higher serum AP levels (p less than 0.005) than normal subjects. In pregnant women, however, the increase in AP activity without the increase in AP concentration showed that the increased AP activity was due to an enzyme other than AP. The enzyme levels and activities in normal serum as well as in patients' sera were significantly correlated (normal, r = 0.77; patients, r = 0.95). Based on the specific activity of AP purified from human plasma, the enzyme activity splitting L-alanyl-beta-naphthylamide is due almost completely to AP in normal subjects and in patients with hepatobiliary diseases.     

Morphometric assessment of bone marrow fiber content in acute nonlymphatic leukemia at presentation

The number of intersections of reticulin fibers per sq mm of fat cell-free marrow parenchyma with the lines of a grid ocular (i/sq mm) represents an objective measure of the bone marrow reticulin fiber content. This method was used to assess the reticulin fiber content of bone marrow biopsies from 50 cases of acute nonlymphatic leukemia (ANLL) at presentation and 20 controls. Seventeen (34%) of the 50 patients with ANLL showed fibrosis, i.e., had a reticulin fiber score above the upper 99% confidence limit of the mean of 20 normal control biopsies. The frequencies of marrow fibrosis, as defined above, were 47% (16 of 34) in the combined subtypes of undifferentiated (M0), myeloid (M1), myelomonocytic (M4) and monocytic (M5) acute leukemia and 7% (1 of 15) in the combined subtypes of acute myeloid leukemia with partial maturation (M2) and acute promyelocytic leukemia (M3) (P less than .01). The fibrosis scores of M0/M1/M4/M5 patients were significantly higher than those of M2/M3 patients (P less than .05) and of controls (P less than .005). Finally, the survival of patients with and without fibrosis was not different.     

The role of cholesterol in the glucocorticoid-mediated inhibition of cell cycle progression in human acute lymphoblastic leukemia cells

Two glucocorticoid receptor-containing clones of human acute lymphoblastic leukemia, one (CEM-C7) sensitive and one (CEM-C1) resistant to dexamethasone (dex) were studied in an effort to identify the time course of the biochemical changes responsible for dex-induced growth inhibition of CEM-C7 cells. Cells were synchronized by treatment with 0.25 mM (C7) or 0.50 mM (C1) thymidine for 12 h followed by 0.025 micrograms/ml (C7) or 0.050 micrograms/ml (C1) colcemid for 12 h, then released either in the presence or absence of 1 microM dex. The inhibition of cellular proliferation which occurs at 48 h after release in the dex-treated CEM-C7 cells was preceded by an inhibition of acetate incorporation into cholesterol, first evident at 24 h, inhibition of protein synthesis at 30 h, and the development of a cell cycle block in G1 at 36 h. No inhibition of any of these parameters was seen in the resistant CEM-C1 cells. Thus the inhibition of cholesterol synthesis in the sensitive cells may be one of the earliest parameters affected by glucocorticoids.     

Antibodies against 9-O-acetylated sialic acids in childhood acute lymphoblastic leukemia: a two-year study with 186 samples following protocol MCP 943

Initial studies have revealed an enhanced surface expression of 9-O-acetylated sialoglycoconjugates (9-OAcSGs) on lymphoblasts concomitant with high titers of antibodies (anti-9-OAcSGs) in childhood acute lymphoblastic leukemia (ALL). This study was undertaken in 186 coded samples from 69 ALL patients to evaluate if antibodies against these sialoglycans could monitor response to the treatment. An ELISA was developed using bovine submaxillary mucin (BSM) containing high % of 9-O-acetylated sialic acids (9-OAcSA) as the capture antigen, to investigate serum levels of anti 9-OAcSGs in a single-center series of pediatric, clinically-diagnosed and immunophenotypically confirmed ALL patients, as compared to 130 healthy controls. At presentation, a 3.8-fold increase in anti-9-OAcSGs levels was detected in 63/69 ALL patients (mean +/- SEM was 102.8 +/- 6.3 microg/ml) as compared to normal controls (27.17 +/- 0.76 microg/ml), assay sensitivity being 91.3%. On an individual basis (n = 25) in patients who were longitudinally monitored for two years, a significant decline in their mean +/- SEM of OD405 was observed from 0.85 +/- 0.06 to 0.28 +/- 0.03. Additionally, a dot-blot was developed to evaluate the proportion of immune-complexed 9-OAcSGs in these patients employing achatinin-H, a 9-OAcSA-binding lectin. Our data indicate that these economically viable ELISA-based approaches allow for reliable, sensitive and rapid diagnosis of ALL. We contend that these disease-specific antibodies could be considered as potential markers both for the initial diagnosis of ALL and possibly for longitudinal monitoring of the disease.