Immunoreactive calcitonin in leukaemia.

A radioimmunoassay was used to measure concentrations of immunoreactive human calcitonin (HCT) in plasma and leucocytes from patients with various leukaemic and myeloproliferative disorders. Plasma immunoreactive HCT concentrations were increased in 32 out of 33 patients with chronic granulocytic leukaemia (CGL) and in all eight patients with acute myeloid leukamia (AML) at presentation or in relapse. Out of 11 patients with other myeloproliferative disorders, eight had increased plasma immunoreactive HCT concentrations. Buffy-coat-cell extracts and culture media from peripheral leucocytes of patients with CGL also contained increased immunoreactive HCT concentrations. In contrast, plasma from patients with chronic lymphocytic leukaemia, acute lymphoblastic leukaemia, and AML in remission had low or undetectable immunoreactive HCT concentrations. Increased plasma and cellular concentrations of immunoreactive HCT may be a consequence of abnormal proliferation of myeloid cells and might prove to be valuable in predicting relapse in patients with myeloid leukaemias.     

Acute leukemia in adults: assessment of remission induction with combination chemotherapy by clinical and cell-culture criteria.

Remission induction was assessed by clinical and cell-culture criteria for 65 patients with acute myelogenous leukemia (AML), 11 patients with chronic myelogenous leukemia (CML) in blast crisis and 19 patients with acute lymphoblastic leukemia (ALL). Cyclophosphamide, cytosine arabinoside and vincristine (CAV) therapy resulted in complete remission in 23 of 50 previously untreated patients with AML and in 3 of the 11 patients with CML. Fourteen patients with ALL responded to vincristine-prednisone induction therapy and two to induction therapy with CAV. The median duration of survival of the responding patients was 2.2 years, compared with 4 months for the patients who did not respond to treatment. Granulopoietic colony formation, assessed by assay of colony-forming units dependent on colony-stimulating activity in culture (CFU-C), was abnormal in 37 of 42 bone marrow aspirates from patients with AML before treatement. CFU-C concentration increased when leukocyte-conditioned medium (LCM) was added to the cultures; 13 cultures had normal or elevated CFU-C concentration with LCM. Marrow cells of patients with ALL or CML in blast crisis demonstrated a similar pattern. Serial studies of marrow CFU-C concentration of 31 patients with AML demonstrated a change to a normal pattern with successful remission induction. Results of this study suggest that administration of purified LCM to leukemic patients might increase granulocyte production from potential but unstimulated granulopoietic precursors. This therapy would lessen the probability of death from infection during remission induction.     

An abnormal CD34+ myeloid/CD34+ lymphoid ratio at the end of chemotherapy predicts relapse in patients with acute myeloid leukemia.

During conventional follow-up of patients with acute myeloid leukemia (AML), the emergence of cytopenias is considered to be a sign of impending relapse, and it represents an example of how leukemic hematopoiesis affects normal hemopoietic differentiation. In the present study, we have explored the possible value of the analysis of the distribution of CD34+ myeloid and CD34+ lymphoid progenitor cells in follow-up complete remission bone marrow samples from de novo AML patients as a prognostic parameter for predicting relapse. A total of 213 bone marrow samples from 36 AML patients in morphological complete remission, obtained at the end of induction, consolidation, and intensification therapy and every six months thereafter were analyzed. The normal CD34+ myeloid/CD34+ lymphoid ratio ranged between 2.4 and 8.9. In contrast, in most AML cases an abnormally high ratio (> or =10) was observed at the end of induction and consolidation therapy: 96% and 75% of cases, respectively. On the other hand, at the end of intensification, 70% of the patients displayed a normal CD34+ ratio. Patients with a myeloid/lymphoid CD34+ ratio higher than 10 at the end of intensification showed a significantly lower overall survival (median survival of 19 months versus median not reached, P = 0.05), as well as a lower disease-free survival (median of 7 months versus 30 months, P = 0.0001). Regarding sequential studies, 67% of the relapses were preceded by the re-appearance of an abnormal CD34 ratio, whereas relapse was not predicted in four patient with leukemia classified as M3 undergoing maintenance therapy. From the remaining 18 patients who are still in continuous complete remission, all except 3 cases (17%) displayed a normal CD34 myeloid/lymphoid ratio. In summary, the present study shows that the persistence at the end of chemotherapy of an abnormally high (> or =10) ratio between CD34+ myeloid and CD34+ lymphoid progenitors in the bone marrow of AML patients is associated with high risk of relapse and a shorter overall survival.     

Acquired neutrophil myeloperoxidase deficiency: an indicator of subclinical activation of blood coagulation?

Using an automated cytochemical analyzer used for routine differential counts, we have been able to demonstrate acquired myeloperoxidase deficiency in 102 patients at our institution. Clinical and laboratory data on these patients showed a high incidence of diabetes mellitus (25.5%) and thrombotic diseases (24.5%), as well as a strikingly constant hyperfibrinogenemia (mean = 635 mg/100 ml; range = 360-1015 mg/100 ml). In 4 additional acute leukemia patients in complete remission, a close time correlation was noted between acquired MPO deficiency, diffuse intravascular coagulation and relapse. These findings indicate the importance of the relationships between neutrophil granulocytes and blood coagulation, and suggest that similar changes in neutrophil MPO activity may represent an early morphological indicator of subclinical activation of blood coagulation.     

Cytofluorometric study of myeloperoxidase (MPO) expression in neutrophilic granulocytes of subjects with primary or secondary MPO deficiency: analysis of the histographic antigen distribution using the Kolmogorov-Smirnov mathematical model]

Neutrophil granulocytes from 12 subjects with primitive myeloperoxidase (MPO) deficiency (6 totally deficient) and 16 patients with secondary partial MPO deficiency were tested using two different anti-MPO monoclonal antibodies (MoAbs), in combination with a flow cytometer. Results demonstrated three different patterns of immunoreactivity with the MPO protein:i) a bright MPO antigenic expression, typical of patients with secondary MPO deficiency (comparable with that observed in the control group); ii) a medium MPO antigenic expression, typical of subjects with hereditary partial MPO deficiency; and iii) a dim MPO antigenic expression, characteristic of individuals with hereditary total MPO deficiency. No significant differences in granulocyte MPO reactivity were demonstrated for the two MoAbs. Furthermore, in two individuals with complete primitive deficiency, the single histogram analysis of MPO fluorescence seemed to show that only 38% (case 1) and 44% (case 2) of neutrophil were reactive with the MoAbs anti-MPO: the use of multiple histogram analysis in combination with Kolmogorov-Smirnov statistics allowed us to demonstrate that all the cells express a low density of MPO antigen. These data suggest that patients with primary MPO deficiency have different amount of MPO antigens in the neutrophils, and the levels of MPO fluorescence seem to decline concurrently with enzyme activity, thereby suggesting the presence of a diminished MPO production. On the contrary, in most cases of acquired MPO deficit, the reduced cytochemical activity contrasts with normal antigenic reactivity: this might be the result of the presence of an inactive enzyme.     

Identification of neutrophil alkaline phosphatase-inducing factor in cystic fluid of a human squamous cell carcinoma as granulocyte colony-stimulating factor

We have previously shown that a factor termed neutrophil alkaline phosphatase-inducing factor (NAP-IF) has the capacity to induce neutrophil alkaline phosphatase (NAP) in postmitotic granulocytes (PMGs). This factor has characteristics similar to those of granulocyte colony-stimulating factor (G-CSF), suggesting that the two factors assayed by different methods may be attributable to an identical macromolecule. In a preliminary experiment, we showed that purified natural G-CSF (nG-CSF) could induce NAP in vitro in the presence of 10% (v/v) fetal calf serum (FCS). In this study, purified human nG-CSF and recombinant G-CSF (rG-CSF) induced NAP in granulocytes from both normal individuals and patients with chronic myelogenous leukemia in a dose-dependent fashion in serum-free and serum-containing culture conditions. The induction of NAP by G-CSF was detectable at 0.4 ng/ml and became maximal between 10 and 20 ng/ml. Anti-G-CSF serum incubated with either NAP-IF or rG-CSF inhibited induction of NAP. Morphological examinations revealed that granulocytes cultured with G-CSF were more mature than those cultured without G-CSF, indicating that G-CSF promoted maturation of granulocytes in parallel with NAP induction. These results indicate that NAP-IF in the cystic fluid of a human squamous cell carcinoma is identical to G-CSF and that induction of NAP by G-CSF is really a reflection of cell maturation promoted by G-CSF.     

Flow cytometric determination of atypical antigen expression in acute leukemia for the study of minimal residual disease.

The aim of this study was to investigate to which extent acute leukemias could be monitored for residual disease by using atypical antigen combinations as leukemia-related markers. Atypical antigenic features were determined by double color flow cytometry and included coexpression of lymphoid and myeloid related antigens, unphysiological coexpression of immature and mature antigens, and lack of an antigen that is normally expressed during maturation. Atypical immunophenotypes were detected in 35 of 68 patients with AML (51.5%) and 15 of 24 patients with ALL (62.5%). When 12 patients with leukemia-associated markers were again analyzed at relapse, the relevant antigen combinations were retained in 11 of them. The sensitivity of this two color flow cytometric assay as determined in dilution experiments was 1 in 10(3) to 10(4) cells. Follow-up studies of bone marrow samples revealed that, after induction chemotherapy cells with leukemia-associated markers were detectable in several patients at a frequency of 0.5 to 4%, but only patients in whom the cells with atypical antigens never disappeared suffered from relapse. In contrast, patients who became negative for the atypical cells remained in complete remission (median remission duration after the first negative bone marrow assessment by flow cytometry 52 weeks, range 20-102). We conclude that atypical antigen combinations, which are present in a meaningful number of acute leukemias, are a valuable means of monitoring acute leukemia patients during follow-up. This flow cytometric approach can complement other strategies to get a more accurate definition of remission in acute leukemia.     

Immunocytochemical identification of abnormal polymorphonuclear neutrophils in patients with leukemia.

High titer, monospecific antibodies to human granulocyte myeloperoxidase, cathepsin G, elastase, lysozyme, and lactoferrin were conjugated with fluorescein and rhodamine and used for immunofluorescent staining of mature neutrophils obtained from 25 patients with acute and chronic leukemia. In 11 (44%) of the patients, two populations of mature neutrophils were detected. The abnormal cells were identified by complete deficiency of one or more markers and constituted 10%-100% of the total number of neutrophils. This immunocytochemical approach may permit recognition of mature cells derived from leukemic clones, and serial determinations of the ratio of normal to abnormal cells may be useful in the management of patients with leukemia.     

Pulmonary complications in patients receiving granulocyte transfusions and amphotericin B.

To evaluate the possibility that in febrile granulocytopenic patients amphotericin B given along with granulocyte transfusions could increase the incidence of pulmonary complications, we studied 43 severely granulocytopenic patients during 46 episodes of fever. Granulocytes were administered as part of the clinical protocol to all 19 patients who had clinically or microbiologically documented infection; the other 24 patients were randomly allocated to treatment with granulocytes (13 patients) or without granulocytes (11 patients). In all, 32 patients received granulocyte transfusions during 35 episodes of fever. Pulmonary complications developed in six patients in each of the two randomized groups. The incidence of pulmonary complications was not influenced by the number of granulocyte transfusions or by the number of granulocytes per transfusion. Pulmonary complications were significantly more likely to occur in patients with fungal infections. Amphotericin B was given according to clinical indications; 21 patients in all received it. Survival was significantly poorer in patients with pulmonary complications, but the administration of amphotericin B was not related either to survival or to the incidence of pulmonary complications. We conclude that pulmonary complications and poor prognosis are related to underlying pulmonary fungal infection and not to any interaction between amphotericin B and granulocyte transfusions.     

The immunoregulatory abilities of polymorphonuclear neutrophils in the course of multiple sclerosis.

The polymorphonuclear neutrophils (PMN) possess sufficient potential to affect both immune response and inflammation, however it has not been yet described in the course of multiple sclerosis (MS). We have studied binding of fluorescein isothiocyanate (FITC)- stained TNF-alpha by PMN, the expression of CD11a, CD11b, and CD18 molecules of beta2-integrines and the expression of CD10 (neutral endopeptidase-NEP) and of CD13 (aminopeptidase N; APN) antigens on PMN in three different groups of MS patients. The control group included neurological patients (OND) with noninflammatory diseases. The obtained results have proved that during MS exacerbation and in the course of chronic progressive MS, PMN reveal several forms of preactivation, including significantly higher stained-TNF-alpha binding, higher expression of CD11b and CD18, as well as CD10 and CD13 antigens, in comparison with MS remission or OND. We suggest that the increased expression of these molecules on PMN of MS patients in exacerbation of the disease and to a lower degree in the course of CP-MS is a result of PMN priming, and directly prove the PMN involvement in the disease pathogenesis.     

Two functional domains in the phagocyte membrane glycoprotein Mo1 identified with monoclonal antibodies

Granulocytes from patients genetically deficient in the leukocyte glycoprotein family, Mo1, LFA-1, and Leu-M5 (P150,94), have defective complement receptor type III (CR3) activity as well as abnormal adhesion-dependent functions such as spreading, chemotaxis, and phagocytosis. To determine the contribution of the Mo1 heterodimer deficiency to the various functional aberrations observed in deficient granulocytes, we mapped the functional domains of Mo1 using several monoclonal antibodies to this molecule. In addition to iC3b binding, two granulocyte adhesion functions were examined: Cell spreading on plastic coverslips and chemotaxis. One monoclonal antibody to Mo1, 44, inhibits all three functions. Other monoclonal antibodies (903, Leu-15, and OKM10) inhibit iC3b binding to granulocytes but have no effect on cell spreading and/or chemotaxis. Another antibody, 904, has no significant inhibition of iC3b binding but inhibits spreading on plastic and chemotaxis. These studies suggest the presence of two functional domains in Mo1: one involved in iC3b binding and the other in enhancing certain granulocyte adhesion-dependent functions.     

Preferential loss of mismatch repair function in refractory and relapsed acute myeloid leukemia： potential contribution to AML proqression

Acute myeloid leukemia （AML） is an aggressive hematological cancer. Despite therapeutic regimens that lead to complete remission, the vast majority of patients undergo relapse. The molecular mechanisms underlying AML development and relapse remain incompletely defined. To explore whether loss of DNA mismatch repair （MMR） function is involved in AML, we screened two key MMR genes, MSH2 and MLH1, for mutations and promoter hypermethylation in leukemia specimens from 53 AML patients and blood from 17 non-cancer controls. We show here that whereas no amino acid alteration or promoter hypermethylation was detected in all control samples, 18 AML patients exhibited either mutations in MMR genes or hypermethylation in the MLH1 promoter. In vitro functional MMR analysis revealed that almost all the mutations analyzed resulted in loss of MMR function. MMR defects were significantly more frequent in patients with refractory or relapsed AML compared with newly diagnosed patients. These observations suggest for the first time that the loss of MMR function is associated with refractory and relapsed AML and may contribute to disease Datho8enesis.     

Seven day cytosine arabinoside administered subcutaneously plus daunorubicin for remission induction in AML

51 patients (mean age 40, range 17-71) with previously untreated AML (25% of M4-6 FAB type) were given Ara-C subcutaneously 100 mg/m2 every 12 h for 7 days and DNR 45 mg/m2 on days 1-3 i.v. for inducing remission. After 52 days 49% of patients on an average reached CR. The complete responders were given a 5 day treatment pulses monthly until relapse, which consisted of Ara-C s.c. plus one of the following drugs added in cyclic rotation: 6-TG, cyclophosphamide, CCNU or DNR. After 2 years the intervals between cycles were prolonged step by step. The mean/median of CR duration equals 26/19 months and of the survival of complete responders 32.9/27 months. The relapse rate and the CNS relapse rate equaled 1.9% and 0.36% per patient/month of observation. T and B cell depletion was observed during maintenance. The results indicate that the subcutaneous route of administering Ara-C is effective during the induction of remission.     

The light microscopic demonstration of hydroperoxidase-positive Phi bodies and rods in leukocytes in acute myeloid leukemia.

Unique fusiform or spindle-shaped particles (Phi bodies) and rods with hydroperoxidase (catalase and/or peroxidase) activity are present in human granulocyte precursors only in acute myelogenous leukemia (AML). These newly recognized particles are much more numerous and prominent than Auer rods. They may be rapidly and readily identified using the microscope in marrow or peripheral blood films when the procedures recommended in this paper for fixation, incubation for hydroperoxidase demonstration in 3,3'-diaminobenzidine (DAB)/H2O2 medium, copper salt treatment and counterstaining (optional) with the Papanicolaou method are employed. Films prepared in the same manner but treated with benzidine/H2O2 medium for myeloperoxidase did not reveal these particles. We believe that Phi bodies are pathognomonic of AML since they are almost invariably present in AML patients with active disease. Their presence serves to distinguish AML from acute lymphocytic leukemia and from chronic granulocytic leukemia in blast crisis. Since the particles disappear in disease remission and reappear upon relapse, the recommended procedure is not only useful in diagnosis but in guiding therapy. When a very rapid diagnosis is needed, it is not necessary to counterstain the preparations, but the nuclei, cytoplasm and plasmalemma can readily be observed in the granulocyte precursors when they are counterstained by the Papanicolaou method. This treatment does not diminish the clarity of the Phi bodies and rods which stain by virtue of their peroxidatic activity. This cytochemical diagnostic procedure should be considered for adoption by hematology laboratories.     

Spontaneous injury in isolated sheep lungs: role of resident polymorphonuclear leukocytes.

Perfusion of isolated sheep lungs with homologous blood caused pulmonary hypertension and edema that was not altered by depletion of perfusate polymorphonuclear (PMN) leukocytes (D. B. Pearse et al., J. Appl. Physiol. 66: 1287-1296, 1989). The purpose of this study was to evaluate the role of resident PMN leukocytes in this injury. First, we quantified the content and activation of lung PMN leukocytes before and during perfusion of eight isolated sheep lungs with a constant flow (100 ml.kg-1.min-1) of homologous blood. From measurements of myeloperoxidase (MPO) activity, we estimated that the lungs contained 1.2 x 10(10) PMN leukocytes, which explained why the lung PMN leukocyte content, measured by MPO activity and histological techniques, did not increase significantly with perfusion, despite complete sequestration of 2.0 x 10(9) PMN leukocytes from the perfusate. MPO activities in perfusate and lymph supernatants did not increase during perfusion, suggesting that lung PMN leukocytes were not activated. Second, we perfused lungs from 6 mechlorethamine-treated and 6 hydroxyurea-treated sheep with homologous leukopenic blood and compared them with 11 normal lungs perfused similarly. Despite marked reductions in lung PMN leukocyte concentration, there were no differences in pulmonary arterial pressure, lymph flow, or reservoir weight between groups. Extravascular lung water was greater in both groups of leukopenic lungs. These results suggest that resident PMN leukocytes did not contribute to lung injury in this model.     

Marrow culture studies in adult acute myeloid leukemia at diagnosis and during complete remission

We used the human placental conditioned medium stimulated single layer agar culture technique to study the in vitro growth of marrow cells from 62 adult patients with acute myeloid leukemia (AML). Bone marrow cells were cultured from 50 patients at the time of initial diagnosis, 19 patients in early remission and 20 patients during their full complete remission. Marrow cultures from untreated patients exhibited heterogeneous growth patterns ranging from complete growth failure to excessive microcluster formation. We classified the growth patterns into 4 groups: (1) Gr I: normal growth, (2) Gr II: no growth, (3) Gr III: decreased growth, (4) Gr IV: excessive growth of microclusters. At presentation, none had Gr I growth; Gr II growth was observed in 23; Gr III in 14 and Gr IV in 13. A predominance of no growth were seen in M1 and M3 subtypes, while Gr IV growth was more commonly observed in M2 or M4 subtype. We were unable to correlate the culture findings with age or white cell count. The present results not only indicated that AML at diagnosis was characterized by abnormal granulopoiesis but also demonstrated that leukemic progenitor cells were heterogeneous with different capacities to express their proliferating potential in vitro. Except few with decreased growth, the growth characteristics generally returned to normal with successful remission induction. Both Gr II and Gr IV growth patterns were not observed either in early remission or during full complete remission.     

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.     

Cell separation in the buffy coat

One of the most rapid methods to determine cell counts in whole blood is by way of layer thickness measurements of the buffy coat. The purpose of this study was to determine the separation and purity of blood cells in the different layers of the buffy coat. Blood samples were centrifuged at 10,000 g in microhematocrit tubes with an inserted float to expand the buffy coat region. Whole blood from normal laboratory individuals separates by density into four regions: platelets, a layer of lymphocyte and monocytes, granulocytes and erythrocytes. A thin band of highly swollen red cells was discovered between the buffy coat layers of many normal volunteers and patients. Stereological analysis of electron micrographs showed that mixing of formed elements within the layers is less than 2% with the exception of some erythrocytes, which can make up a higher volume fraction in the lymphocyte/monocyte and granulocyte layers. The red cell column contains about 95.7% erythrocytes and is depleted of platelets and leukocytes. In approximately 5% of hospital blood samples, the granulocyte-erythrocyte interface was feathered and undetectable, and a significantly higher volume fraction of red cells was found among the granulocytes. Cell mass density determinations indicate that the erythrocytes in these abnormal granulocyte layers have a lowered mass density, overlapping with that of the granulocytes.     

Quantitative evaluation of leukemic mitochondria with a computer-controlled image analyzer

Mitochondria from 25 patients with acute lymphoblastic leukemia (ALL) and 25 patients with acute myelogenous leukemia (AML) were compared in terms of their number, area, and shape index using a computer-controlled image analyzer. The number of mitochondria was greater in the AML than in the ALL patients. However, their size, as measured in electron micrographic profiles was similar in the two groups, in disagreement with conventional reports that mitochondria are small in granulocytes but large in lymphocytes. Two ALL patients had giant mitochondria. The mitochondria of the ALL cells were more irregular than those of the AML cells, and furthermore, within the ALL group, the degree of the irregularity was greater in those with a poor prognosis than in those in longstanding remission. The number of mitochondria was significantly greater in B-cell ALL than in null cell and T-cell ALL.     

Quantitative behavior of eosinophil granulocytes in the bone marrow of children with acute lymphoblastic leukemia]

Of 31 children affected with acute lymphoblastic leukaemia the quantitative behaviour of eosinophilie granulocytes was examined in the course of the disease. Nearly all patients were treated according to a chemotherapy scheme (Memphis IV). During this therapy the eosinophils greatly diminished initially increased significantly to subnormal values and to the values of healthy persons with persisting full remission. Another significant decrease occurred during the relapse and in the pre-final stage. During each following relapse a greater diminution of bone-marrow eosinophils could be observed. Simultaneously the decrease of eosinophils led to a shift in the degree of maturation. In this connection the similar behaviour of neutrophilic and eosinophilic granulocytes of the bone-marrow must be stressed. Eventually, the lbast excrescence in the bone-marrow and its therapy cannot solely be decisive for the findings made. Relations to the lymphocytic system can be referred to.