C. Gattringer

Detection of activity of P-glycoprotein in human tumour samples using rhodamine 123

Summary Based on the fluorescent properties of the dye rhodamine 123 (Rh123), which is transported by the membrane efflux pump P‐glycoprotein (P‐gp), we developed a functional flow cytometric assay for the detection of multidrug‐resistant (MDR) cells. Using drug sensitive cell lines (KB‐3–1) and MDR mutants (KB‐8–5, KB‐C1) experimental conditions were established that enabled demonstration of significant differences in Rhl23 efflux and accumulation. Subsequently we investigated the applicability of this functional assay for the prediction of MDR in human peripheral blood and bone marrow samples. Using two‐colour flow cytometry, the leukaemic blast cells of six patients suffering from acute myeloid leukaemia (AML) were analysed. In three cases the blast cells showed a rapid and marked Rh123 efflux. In the presence of MDR inhibitors these cells retained Rh123. To determine whether the efflux of Rhl23 was associated with P‐gp expression, the leukaemic cells were stained with the monoclonal antibody MRK‐16. In addition extracted RNA was analysed by polymerase chain reaction to evaluate the expression of mdr 1 mRNA. In all three Rh123+ cases mdr 1 mRNA was detectable whereas only one AML case expressed P‐gp. In comparing Rh123 with daunorubicin, which also allows the detection of MDR cells, accumulation studies proved Rh123 to be the more sensitive drug for flow cytometric MDR screening. Additionally, two‐colour flow cytometry was much easier to perform with Rh123 than with daunorubicin.

Recombinant human granulocyte-macrophage colony-stimulating factor applied locally in low doses enhances healing and prevents recurrence of chronic venous ulcers

Background Chronic venous leg ulcers have a major medical and economic impact on the elderly worldwide. Healing of the large ulcers (>10 cm2 ) occurs only in two‐thirds of the patients and reulceration of healed ulcers recurs in one‐third within 1 year. Because both healing and relapse rate influence greatly a patient’s quality of life and the overall cost of treatment, every effort should be made to improve these two parameters.

Expression of the neural cell adhesion molecule (CD56) by human myeloma cells.

Recent studies in multiple myeloma indicate that molecules associated with different haematopoietic lineages may be expressed aberrantly by myeloma cells. In order to investigate this phenomenon further, we studied the immunophenotype of bone marrow cells from 21 patients with multiple myeloma using a panel of monoclonal antibodies against T.B. myelomonocytic, and natural killer (NK)‐cell antigens. Leu‐19/NK.H1 (CD56), a molecule identical to N‐CAM, which is normally expressed by neuroectodermal and NK cells, was found in 13 patients (62%). Dual‐parameter flow cytometry was used to correlate N‐CAM positivity with DNA aneuploidy or cytoplasmic immunoglobuiin expression as markers of myeloma cells. When N‐CAM was found positive, other haematopoietic antigens were expressed only in three out of 13 cases (23%). In contrast, myeloma cells not expressing N‐CAM frequently exhibited pre‐B cell markers, myeloid antigen, and HLA‐DR, respectively (seven out of eight cases, 88%). Six out of eight N‐CAM‐negative myelomas were of the IgG lamba isotype, otherwise no clearcut association with basic clinical and laboratory parameters was noted. We conclude that N‐CAM expression is a common finding in multiple myeloma. Whether its expression and the observed antigenic heterogeneity is just a manifestation of malignancy or N‐CAM may play a role in the biology of multiple myeloma regarding tumour cell spread, remains to be explained.

Long‐term follow‐up of patients with hairy cell leukaemia treated with pentostatin: lymphocyte subpopulations and residual bone marrow infiltration

Summary. Peripheral blood lymphocyte (PBL) subsets and bone marrow biopsies were analysed in six patients with hairy cell leukaemia (HCL) treated with 2′‐deoxycoformycin (pentostatin, DCF) according to a phase II trial of the EORTC Leukemia Cooperative Group. All patients responded to DCF with four complete and two partial remissions according to conventional criteria.

Diagnostic and prognostic value of immunohistological bone marrow examination: Results in 212 patients with lymphoproliferative disorders

SummaryCryostat sections of 246 consecutive bone marrow biopsies from 212 patients with lymphoproliferative disease were investigated using a panel of monoclonal antibodies (MOAbs) and an immunoperoxidase technique. Bone marrow involvement was demonstrated by immunohistological examination in 121/160 patients (76%) with non-Hodgkin lymphomas (NHL) and 16/23 patients (70%) with plasma cell malignancies; the definite immunological diagnosis could be performed in 77% and 88%, respectively. Reactivity with the MoAb Ki-67 correlated with clinical parameters: in all cases exhibiting more than 5% positive cells an unfavourable course was seen, independent of the histological subtype. Another MoAb of potential prognostic relevance is KiM4b, which reacts with follicular dendritic cells (FDC). Besides the presence of FDC in germinal center tumors (CB/CC and CC-NHL) we found FDC in a minority of cases with B-CLL (5/44) and IC lymphoma (4/18). In the latter group 3/4 patients showed a favourable clinical course (vs 2/14 without FDC). The MoAb Tü1 could discriminate between the lymphoplasmocytoid (11/12 positive) and the lymphoplasmocytic (0/6 positive) subtype of IC lymphoma and has proven of diagnostic importance. Expression of IL-2 receptors, detected by MoAb anti-Tac (CD25), was demonstrated on leukemic cells from patients with hairy cell leukemia (100%), B-CLL (82%), IC (61%), CC (50%) and CB/CC lymphoma (50%). A considerable number of reactive T-lymphocytes (5%–60% of tissue cells) were identified among the neoplastic B cells with a predominance of CD4+ cells in most cases with NHL, whereas the CD4+/CD8+ ratio was significantly lower in myelomas and non-infiltrated bone marrows. The potential meaning of these findings is discussed. The immunohistological bone marrow analysis represents an important additional method in the diagnostic procedures of lymphoproliferative diseases involving the bone.

Flow cytometric detection of cytoplasmic antigens in acute leukemias: Implications for lineage assignment

This study aimed at optimizing the conditions for flow cytometric detection of myeloperoxidase (MPO), cytoplasmic CD3 (cCD3), and cytoplasmic CD22 (cCD22), which seem to be more reliable lineage-associated markers in acute leukemia than surface antigens. Fixation methods employing saponin as detergent resulted in accurate detection of MPO and cCD3, whereas cCD22 was detectable only after buffered-formaldehyde-acetone fixation. MPO was detected in 16/17 AML, but only in 1/6 ALL, the MPO positive ALL being also CD13 positive. MPO was detectable in 3/4 AML with T-lymphoid features; a case of myeloid antigen-positive T-ALL, however, was MPO-negative. cCD3 was expressed only in T-ALL, and five cases of lymphoid antigen-positive AML were cCD3-negative. We suggest that these flow cytometric assays are useful for the lineage assignment of poorly differentiated leukemias and contribute to the identification of truly biphenotypic acute leukemias.

Growth fraction of tumour cells and infiltrationdensity with natural killer-like (HNK1+) cells in non-Hodgkin lymphomas

Summary. Two markers for cells in the growth fraction, the T9 antigen (i.e. the transferrin receptor) and the T10 antigen were investigated in frozen tissue sections of 105 non‐Hodgkin lymphomas (NHL). The results were correlated with the histological subtype and the pattern of tumour infiltration by reactive cells. Special attention was directed to the density of natural killer (NK)‐like cells using the anti‐HNK1 (Leu7) antibody since the transferrin receptor (tfr) or other growth‐associated membrane structures may serve as target for NK cells. Our study confirms a relationship between number of tumour cells with the T9 marker and tissue infiltration by HNK1 cells in NHL of low (chroniclymphocytic leukaemia, hairy‐cell leukaemia, immunocytic lymphoma, centroblastic‐centrocytic lymphoma) and intermediate (centrocytic and centroblastic lymphoma) but not in NHL of high malignant grade (immunoblastic and lymphoblastic lymphoma). Comparable results were obtained with the T10 antigen although the correlation was less close. The percentage of cells in the growth fraction, defined by the expression of the T9 and T10 marker, corresponded with prognostically unfavourable subgroups with the remarkable exception of follicular NHL of centroblastic‐centrocytic type. This lymphoma showed high numbers of cells with the T9 and the T10 marker in a microenvironment resembling normal germinal centres in many aspects.

Spontaneous and conA-induced suppressor lymphocytes: a comparative study.

Suppressor monocytes, Concanavalin A(ConA)-induced suppressor T cells, and short-lived suppressor lymphocytes have been describe in humans. The present study was performed to evaluate spontaneous suppression in a test system similar to that employed for the demonstration of ConA-induced suppressor cells: Lymphocytes were either stimulated by ConA (= induced suppressor cells) or immediately mitomycin-treated (= spontaneous suppressor cells). Both preparations were tested for their capacity to suppress mitogen-induced proliferation of autologous cells. Depletion of monocytes or B lymphocytes did not affect spontaneous suppression. The active cells were short-lived in vitro. Therefore the net increase in suppressor activity generated by preculture with ConA is in part related to a loss of spontaneous inhibitory activity in the control cultures kept without mitogen. Spontaneous suppressor cell activity was comparable to that of ConA-induced suppressor cells.

A Micro-Culture System for Cloning Human T Lymphocytes in Agar

A simple and reproducible single-layer micro-agar culture system for cloning of human T lymphocytes has been described. The system consists of an agar layer, in which mononuclear cells from peripheral blood were suspended, and a liquid overlayer containing the mitogenic substance. The advantages of the described method are a low incubation volume (0.5 ml) and the liquid overlayer. The addition of different test substances to the liquid overlayer is simple and easily controllable. Depending on the agar concentration a different number of formed colonies can be found floating in the liquid phase. The morphological, cytochemical and immunological characteristics of the cells from those aggregates could be easily studied. The T-cell characteristics of formed clusters and colonies was confirmed by immunofluorescence and E rosette formation. The effects of agar, serum and cell concentrations, as well as the mitogenic activation caused by three lectins on the development and number of colonies were studied on day 7, 10 and 14 of incubation.

Abnormal expansions of granular lymphocytes: Reactive lymphocytosis or chronic leukemia? case report and literature review

SummaryA case of chronic lymphoproliferative disorder is presented, wherein a morphologically homogeneous population of lymphoid cells displayed properties similar to those described for large granular lymphocytes (LGL). Besides their LGL-like phenotype (VEP 13+, OKM 1+, OKT 10+ Fc-IgG-receptor+, OKT 3−), the proliferating cells were cytotoxic to NK targets as well as to antibody-coated target cells. Clinically, our patient presented low-grade lymphocytosis, splenomegaly, neutropenia, hyperimmunoglobulinemia and recurrent infections. Based upon this and 32 similar cases reported in the literature, we conclude that lymphoproliferative disorders involving GL encompass a variety of clinical entities, ranging from reactive GL lymphocytoses to overt lymphocytic malignancies.