A simple one-tube assay for immunophenotypical quantification of leukemic stem cells in acute myeloid leukemia.

Relapses after initial successful treatment in acute myeloid leukemia are thought to originate from the outgrowth of leukemic stem cells. Their flow cytometrically assessed frequency is of importance for relapse prediction and is therefore assumed to be implemented in future risk group profiling. Since current detection methods are complex, time- and bone marrow consuming (multiple-tubes approach), it would be advantageous to have a broadly applicable approach that enables to quantify leukemia stem cells both at diagnosis and follow-up. We compared 15 markers in 131 patients concerning their prevalence, usefulness and stability in CD34(+)CD38(-) leukemic stem cell detection in healthy controls, acute myeloid leukemia diagnosis and follow-up samples. Ultimately, we designed a single 8-color detection tube including common markers CD45, CD34 and CD38, and specific markers CD45RA, CD123, CD33, CD44 and a marker cocktail (CLL-1/TIM-3/CD7/CD11b/CD22/CD56) in one fluorescence channel. Validation analyses in 31 patients showed that the single tube approach was as good as the multiple-tube approach. Our approach requires the least possible amounts of bone marrow, and is suitable for multi-institutional studies. Moreover, it enables detection of leukemic stem cells both at time of diagnosis and follow-up, thereby including initially low-frequency populations emerging under therapy pressure.

Peripheral blood minimal residual disease may replace bone marrow minimal residual disease as an immunophenotypic biomarker for impending relapse in acute myeloid leukemia.

As relapses are common in acute myeloid leukemia (AML), early relapse prediction is of high importance. Although conventional minimal residual disease (MRD) measurement is carried out in bone marrow (BM), peripheral blood (PB) would be an advantageous alternative source. This study aims to investigate the specificity of leukemia-associated immunophenotypes used for MRD detection in blood samples. Consistency of PB MRD as compared with BM MRD was determined in flow cytometric data of 205 paired BM and PB samples of 114 AML patients. A significant correlation was found between PB and BM MRD (r=0.67, P<0.001), while median PB MRD percentage was factor 4-5 lower compared with BM MRD. Primitive blast (CD34+/CD117+/CD133+) frequency was significantly lower in PB (median factor 23.7), indicating that PB MRD detection is more specific than BM. Cumulative incidence of relapse 1 year after induction therapy was 29% for PB MRD-negative and 89% for PB MRD-positive patients (P<0.001). Three-year OS was 52% for MRD-negative and 15% for MRD-positive patients (P=0.034). Similar differences were found after consolidation therapy. As PB MRD appeared to be an independent predictor for response duration, the highly specific PB MRD assay may have a prominent role in future MRD assessment in AML.

Measurement of primary in vivo IgM- and IgG-antibody response to KLH in humans: implications of pre-immune IgM binding in antigen-specific ELISA.

The antigen Keyhole Limpet Hemocyanin (KLH) is often used to test the primary in vivo antibody response capacity in humans. However, measurement of IgM anti-KLH antibodies in ELISA is complicated by the presence of natural antibodies in human serum. This problem occurs particularly at low antibody levels, i.e. after immunization with low doses of antigen and, under these conditions, it was found to be impossible to assess a dose-response curve by immunizing a series of individuals with different suboptimal doses of KLH. This problem was circumvented by choosing conditions for minimal binding of pre-immune IgM and to correct for such binding. Although signal-to-background ratios were markedly improved by modifying the ELISA conditions, pre-immune IgM still showed binding to KLH due to interaction with polysaccharide determinants. This non-specific binding was correlated with the total IgM content of the samples. When anti-KLH activities before and after immunization were expressed relative to total serum IgM, a significant correction was achieved, resulting in a diminished inter-individual variability with respect to both pre-immune and post-immunization values. As with IgG-class antibodies to KLH, virtually no binding was observed in pre-immune sera. After expression of the anti-KLH response as a ratio between the post-immunization and pre-immunization titres, a dose of 50 micrograms was found to be sufficient to evoke a detectable IgG-antibody response in the 10 subjects tested. To elicit a positive IgM response, a minimal dose of 250 micrograms was required.

Evaluation of a method of production and purification of monoclonal antibodies for clinical applications.

A procedure is described for the purification of monoclonal antibodies (Mab) from ascitic fluids, which meets the quality control required for in vivo applications of immunoglobulins (Ig) in man. Additional assays were performed to calculate viral and DNA content of the purified Mab. These studies are important to prevent the possible side effects, oncogenic events and virus-related diseases which could follow immunotherapy with Mab.

Immunoperoxidase procedures to detect monoclonal antibodies against cell surface antigens. Quantitation of binding and staining of individual cells.

An immunoperoxidase method has been developed which allows accurate and sensitive quantitation of the binding of monoclonal antibodies to cell surface antigens. Monolayers of fixed cells were prepared in wells of Terasaki micro-test plates and monoclonal antibodies bound to cell surface antigens were identified by the unlabeled antibody-enzyme method of Sternberger (1974). The cell-bound peroxidase could either be quantified per well or visualized on individual cells by the use of appropriate substrates for peroxidase. Experimental procedures are described in detail and results obtained with several monoclonal antibodies with specificity for different target cells are shown. Limitations and applications of the technique are discussed.

Density separation of spleen cells increases fusion frequency and yield of Ig-producing hybridomas.

The efficiency of hybridoma formation and growth after cell fusion can be much improved by fractionation of the mouse splenocytes. A simple procedure is described in which splenocytes with a specific gravity of more than 1.065 g/cm3 are selected by centrifugation on a Percoll gradient. The resulting cell suspension is largely depleted of macrophages and fibroblasts while the cell viability is improved. In fusion experiments performed with these cells, overgrowth of hybridomas by macrophages, fibroblasts and P-cells is avoided. The fusion efficiency and the frequency of immunoglobulin-secreting hybridomas is increased compared with fusions carried out with unfractionated spleen cells.

The role of interleukin-2 in proliferative responses in vitro of T cells from patients after bone marrow transplantation. Evidence that minor defects can lead to in vitro unresponsiveness.

We have studied lectin-induced interleukin-2 (IL-2) production and proliferation of peripheral blood mononuclear cells from patients who had undergone a successful allogeneic bone marrow transplantation. Shortly after transplantation, the T cells show a decreased proliferative response and a decreased IL-2 production. However, addition to the culture of exogenous IL-2 does not result in restoration of the proliferative response, which indicates that the low proliferative response is not due to decreased IL-2 production alone. Longitudinal studies show a substantial variation between patients in the time in which the capacity to produce IL-2 is restored; however, in all patients there is a period in which IL-2 production is still diminished, but the proliferative capacity, as measured upon addition of exogenous IL-2 to the culture, is almost within the normal range. Also during this period, the proliferative response of the T cells can be restored by the addition of irradiated "feeder cells" obtained from the bone-marrow donors, as these cells secrete IL-2 without consuming it. Because peripheral blood samples from patients after bone marrow transplantation show great imbalances in the distribution of T4/T8 subpopulations, we have studied the influence of an artificially produced "reverse T4/T8" ratio on the proliferative response to mitogen and (allos-)antigen stimulation of healthy donor T lymphocytes. Even at very low proportions of T4 cells, normal responses were obtained in the proliferation assays with polyclonal mitogens. Only the response to soluble antigens, such as tetanus toxoid, was impaired. However, a low proportion of T4 cells resulted in a low IL-2 production so that, when IL-2 is a limiting factor due to intrinsic defects of patient cells, an inverse T4/T8 ratio can cause a nonresponsiveness in in-vitro assays.

Stepwise amplified immunoperoxidase (PAP) staining. I. Cellular morphology in relation to membrane markers.

A novel procedure for the assay of monoclonal antibodies is described. The technique is based on a combination of three principles. Unlabeled (sheep) antiserum to mouse immunoglobulin (Ig) and complexes of peroxidase with mouse monoclonal antiperoxidase (monoclonal PAP complexes) are used as reagents in a variant of the unlabeled antibody enzyme (PAP) method, described by Sternberger. The amount of peroxidase eventually bound to a monoclonal antibody can be varied over a wide range by repetition of incubation cycles with anti-mouse Ig and monoclonal PAP complexes. During the assay, incubations and wash steps are performed by immersion of whole slides. The influence of repetitive incubation cycles with anti-mouse Ig and monoclonal PAP complexes on background staining and detection of monoclonal antibodies at low concentrations was quantitated in a model system. At a given primary antibody concentration, a linear relationship was found between peroxidase activity and the number of incubation cycles. Application of the technique to the detection of monoclonal antibodies bound to cell-surface antigens is described. Peripheral blood cells were labeled in suspension with monoclonal antibodies. Cytocentrifuge preparations of labeled cells were prepared, and such preparations were fixed before stepwise-amplified PAP staining. Cells showed intense specific staining. Morphological detail of stained and unstained cells is preserved, allowing morphological analysis of labeled cells and rapid analysis of monoclonal antibody specificity. Because the reagents used in the assay can be produced in large quantities with uniform quality, the technique can be readily automated. This, together with the possibility to increase the sensitivity of antibody detection in a controlled, stepwise fashion to levels that cannot be reached with "single-step" techniques, may further expand the applications of monoclonal antibodies.

Colony-forming cells in chronic granulocytic leukemia--I. Proliferative responses to growth factors.

Peripheral blood cells from 2 patients with chronic granulocytic leukemia were separated by density centrifugation. Mononuclear cells of low density (d less than 1.062g cm-3) with blast-cell morphology were cryopreserved before culture in vitro. Upon culture in conventional colony assays, up to 20% of the cells formed hemopoietic colonies. Although the spectrum of colony types resembled that of normal bone-marrow cells, there were large differences between the patients with respect to the number, type and size of colonies that were observed. Colony formation required the addition of hemopoietic growth factors, such as colony-stimulating activity and erythropoietin to the culture medium. The cells were used to assay hemopoietic regulatory molecules. Both erythropoietin and colony-stimulating activity induced a strong proliferative response as measured by thymidine incorporation. Maximal stimulation was observed when erythropoietin and the supernatant of mixed lymphocyte cultures were added simultaneously. The difference between the cells from the 2 patients in clonal assays was reflected by the different response to individual hemopoietic regulators. The time course of maximal stimulation followed distinct patterns dependent on the source of stimulator. The stimulation was linearly dependent on the input cell number. Taken together, cryopreserved blast cells from patients with chronic granulocytic leukemia appear to be very useful for the characterization of factors regulating hemopoiesis, as well as for studies of hemopoiesis in general.

Inhibition, by vinca alkaloids and colchicine, of antigenic modulation induced by anti-CD19 monoclonal antibodies.

Several clinical trials have been reported in which monoclonal antibodies (McAb) were used for therapy of lymphoid malignancies. Such trials have shown that infusion of McAb recognizing lymphoid antigens, is well-tolerated, and leads to the coating of tumor cells and tumor regression in some patients. However, the tumoricidal capacity of a McAb is hampered by the presence of circulating free antigen, antigenic modulation, development of human anti-mouse antibodies, emergence of antigen-negative variants of tumor cells and the inadequacy of host-effector cell mechanisms. We have studied the antigenic modulation induced by immunoglobulin (Ig) heavy chain switch variants of anti-CD19 McAb. Modulation of CD19 molecules was not related to the IgG subclass of the McAb. Immunofluorescence studies on the Burkitt tumor cell line Daudi showed that CD19 molecules are internalized after incubation by anti-CD19 McAb. Next, the effect of cytoskeleton inhibitors on antigenic modulation was studied. We found that antigenic modulation on Daudi cells and on an Epstein-Barr virus-transformed B-cell line was completely inhibited by vinca alkaloids (VA) or by colchicine. Interestingly, antigenic modulation of tumor cells from a VA-resistant patient, was not inhibited by VA or colchicine. These findings provide information for the rational design of more effective clinical trials with McAb.

Colony-forming cells in chronic granulocytic leukemia--II. Analysis of membrane markers.

Membrane markers and functional properties in vitro of blast cells from the peripheral blood of 2 patients with chronic granulocytic leukemia were studied. Buffy-coat cells were enriched for colony-forming cells by density centrifugation (d less than or equal to 1.062 g cm-3). Upon culture, a large proportion of the (cryopreserved) low-density cells from both patients formed hemopoietic colonies that were heterogeneous with respect to size and cellular composition. Expression of membrane markers on the cells, which had the morphology of undifferentiated blasts, was studied using flow cytometry with a panel of monoclonal antibodies. A striking heterogeneity was observed in that variable numbers of cells were found to express myelomonocytic, megakaryocytic and erythroid membrane markers. Antigenic properties of colony-forming cells were studied by sorting of cells with a fluorescence activated cell sorter. Low numbers of cells (10, 4 and 1, respectively) were sorted directly into the wells of Terasaki microtest plates. With this system, it was shown that myeloid colony-forming cells from patient 1 were exclusively present in HLA-DR-positive cell fractions. Colony formation from the level of a single sorted cell was documented. Sorting of cells labeled with anti-blood-group-H antibody showed that small erythroid colony-forming cells from patient 2 were blood-group-H antigen-positive. These cells did not express HLA-DR. The other colony-forming cells from this patient and essentially all colony-forming cells from patient 1 were HLA-DR-positive and blood-group-H-negative. Although only 2 patients were tested, our studies clearly demonstrate that low-density cell fractions from the blood of patients with CGL provide distinct advantages for the study of membrane properties of hemopoietic cells and of hemopoietic differentiation in general.

CD4 to CD8 ratio and in vitro lymphoproliferative responses during experimental gingivitis in pregnancy and post-partum.

The absolute numbers and percentages of peripheral T, B, and NK cells were assessed in 7 women, both during the second trimester of pregnancy and 6 months post-partum. Furthermore, the in vitro responses of peripheral blood lymphocytes (PBL) to several mitogens and a preparation of Prevotella intermedia were compared in a period of experimentally-induced gingivitis during pregnancy and post-partum. Clinically, the periodontal pocket bleeding index (PPBI) was found to be higher during pregnancy than post-partum. The absolute numbers of CD3, CD4, and CD19 positive cells appeared to be decreased during pregnancy as compared to post-partum. However, the results did not indicate any evidence for a reduced in vitro PBL response to several mitogens and a preparation of P. intermedia during pregnancy.

Stimulation of lymphocytes in vitro by Bacteroides intermedius and Bacteroides (Porphyromonas) gingivalis sonicates.

The present study was designed to assess whether the in vitro stimulation of lymphocytes by sonicates of Bacteroides intermedius and Bacteroides (Porphyromonas) gingivalis is antigen specific or non-specific. In addition, the role of T and B lymphocytes in these responses was assessed. Peripheral blood lymphocytes obtained from healthy volunteers were cultured in the presence of these bacterial preparations and the proliferative response was measured. In similar experiments the response of umbilical cord blood lymphocytes did not exceed background values. In limiting dilution experiments only 1:4000, 1:6800, and 1:8200 of the lymphocytes initially reacted to B. intermedius, which strongly argues for the antigen-specificity of the response. Purified T cells, in the presence of monocytes, proliferated when stimulated with B. intermedius and B. gingivalis. As for B cell stimulation, the bacterial extracts were capable of inducing IgM production, which appeared to be T cell dependent. These findings support the notion that B. intermedius and B. gingivalis induce specific T cell activation; secondarily, a T cell dependent, polyclonal B cell activation may occur.

Tumor heterogeneity makes AML a "moving target" for detection of residual disease.

Detection of minimal residual disease is recognized as an important post-therapy risk factor in acute myeloid leukemia patients. Two most commonly used methods for residual disease monitoring are real-time quantitative polymerase chain reaction and multiparameter flow cytometry. The results so far are very promising, whereby it is likely that minimal residual disease results will enable to guide future post-remission treatment strategies. However, the leukemic clone may change between diagnosis and relapse due to the instability of the tumor cells. This instability may already be evident at diagnosis if different subpopulations of tumor cells coexist. Such tumor heterogeneity, which may be reflected by immunophenotypic, molecular, and/or cytogenetic changes, can have important consequences for minimal residual disease detection, since false-negative results can be expected to be the result of losses of aberrancies used as minimal residual disease markers. In this review the role of such changes in minimal residual disease monitoring is explored. Furthermore, possible causes of tumor instability are discussed, whereby the concept of clonal selection and expansion of a chemotherapy-resistant subpopulation is highlighted. Accordingly, detailed knowledge of the process of clonal evolution is required to improve both minimal residual disease risk stratification and patient outcome.

Tumor heterogeneity makes AML a "moving target" for detection of residual disease.

Detection of minimal residual disease is recognized as an important post-therapy risk factor in acute myeloid leukemia patients. Two most commonly used methods for residual disease monitoring are real time quantitative polymerase chain reaction and multiparameter flow cytometry. Results so far are very promising, whereby it is likely that minimal residual disease results will enable to guide future post-remission treatment strategies. However, the leukemic clone may change between diagnosis and relapse due to instability of the tumor cells. This instability may already be evident at diagnosis if different subpopulations of tumor cells coexist. Such tumor heterogeneity, which may be reflected by immunophenotypic, molecular and/or cytogenetic changes, can have important consequences for minimal residual disease detection, since false-negative results can be expected to be the result of losses of aberrancies used as minimal residual disease markers. In this review the role of such changes in minimal residual disease monitoring is explored. Furthermore, possible causes of tumor instability are discussed, whereby the concept of clonal selection and expansion of a chemotherapy resistant subpopulation is highlighted. Accordingly, detailed knowledge of the process of clonal evolution is required to improve both minimal residual disease risk stratification and patient outcome. © 2013 Clinical Cytometry Society.