Clinical value of bone marrow cultures in childhood pure red cell aplasia.

PURPOSE: We assessed the value of marrow cultures for defining the pathophysiology, diagnosis, and therapeutic response to immunosuppressive therapy in childhood pure red cell aplasia (PRCA). PATIENTS AND METHODS: Patients were evaluated either at diagnosis (n = 23) or at the time of treatment failure (n = 2). Twelve patients had transient erythroblastopenia of childhood (TEC), 4 had Diamont-Blackfan anemia (DBA), and 9 had acquired sustained PRCA (A-Su-PRCA). Bone marrow mononuclear cells were cultured with combination of human recombinant (rhu) erythropoietin (EPO), granulocyte monocyte colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), Interleukin 3 (IL-3), either with or without stem cell factor (SCF), and burst forming unit of erythroid (BFU-E) growth was assessed. RESULTS: The combination of growth factors without SCF failed to induce any erythropoiesis (BFU-E < 10/10(5) mononuclear cells) in 10 patients (2 with TEC, 2 with DBA, and 6 with A-Su-PRCA), although the growth of erythroid colonies was substantially lower in the remaining patients than in controls (45.5 +/- 15.4 versus 91.7 +/- 12.7, p < 0.05). Addition of SCF restored erythropoiesis in all but 6 patients (5 with A-Su-PRCA and 1 with DBA). Five of 6 nonresponders did not respond to any immunomodulating therapy; of the 5, 3 had or developed some evidence of myelodysplasia. CONCLUSION: Our data indicate that in vitro colony studies might prove to be a useful diagnostic tool, because erythropoiesis' poor response to growth factors, including SCF, may suggest the diagnosis of myelodysplasia. Moreover, it may have predictive value; in cases of PRCA, regardless of etiology, poor growth of erythropoietic colonies may predict refractoriness to immunomodulating therapy.

Differential diagnosis based on immunological-phenotyping in suspected malignant bone marrow involvement in childhood.

The diagnostic value of immunophenotyping (IP) as a first-line diagnostic method in diseases that infiltrate the childhood bone marrow (BM) or mimic infiltrated BM was examined. Two hundred and fifty unselected BM samples from 250 children suspected to have a malignancy infiltrating their BM were evaluated by means of IP and conventional morophological-cytochemical (MC) studies. We applied the alkaline phosphatase anti-alkaline phosphatase method for IP using a panel of monoclonal antibodies (Mabs) against leukocyte-associated antigens, neuroectodermal antigens, and intermediate filament antigens. Four cases of neuroblastoma, two cases of Ewing sarcoma, and one case of rhabdomyosarcoma were diagnosed by IP but not by MC studies. In nine cases of acute leukemia bone marrow blasts could not be ascribed to a specific lineage on the basis of blast morphology or histochemistry. Eight samples without morphological evidence of malignant infiltration revealed an increased percentage of immature B cell precursors (CD10+, TdT+) suggesting acute lymphoblastic leukemia. None of these children has developed malignant lymphoproliferative disease. Our data suggest that the immunological evaluation of BM in childhood is highly capable of discriminating between different malignant populations but it does not recognize malignancy and therefore supplements but cannot replace conventional methods for diagnosis.

2',3'-Dideoxycytidine preferentially inhibits in vitro growth of granulocyte-macrophage colony-forming cells from patients with chronic myeloid leukemia.

The effect of three nucleoside analogs, including 2-methyl-2'-deoxyadenosine (MDA), 5-amino-2'-deoxyuridine (ADU) and 2',3'-dideoxycytidine (DDC) on colony formation from unfractionated human bone marrow obtained from volunteers expressing parameters typical for normal cells (NBM) and from patients with the diagnosis of chronic myeloid leukemia (CMLBM) was observed. For the clonal growth of granulocyte-macrophage colony-forming cells (GM-CFC), a semisolid fibrin clot culture medium supplemented with 20% fetal bovine serum and 10% human placental conditioned medium was used. DDC has been shown to be at least a 10-fold more potent inhibitor for the growth of GM-CFC from CMLBM than from NBM. On the other hand, the effect of MDA and ADU on CMLBM did not differ markedly from the effect on NBM. These results suggest that DDC inhibits preferentially progenitor cells from CMLBM.

Expression of markers shared between human haematopoietic cells and neuroblastoma cells.

Neuroblastoma (NB) is a solid tumor of childhood with a relatively bad prognosis, with the exception of young infants (less than 1 year), in whom spontaneous regression of tumor burden occurs. The reasons for this are still unknown but immune mechanisms may be involved. In this study, we have examined the ability of several monoclonal antibodies (MoAbs), which recognize markers predominantly expressed on human haematopoietic cells, to react with four human neuroblastoma cell lines (UKF-NB 1-4) and SK-N-SH as control cell line. In order to define the phenotype of NB cells, we used a large panel of MoAbs consisting of 2 major groups: a) well characterized MoAbs raised against antigens of neuroectodermal origin from the Kemshead-serie (e.g. UJ 13A, UJ 127.II, UJ 167.11, UJ 181.4, UJ 223.8, A2B5), b) monoclonal antibodies which have been considered to react with haematopoietic cells (HLA-DR and anti-CD-molecules CD1, CD7, CD9, CD10, CD13, CD16, CD19, CD20, CD24, CD57). The phenotypic analyses were performed at various times of culture by an immunoenzymatic procedure (APAAP-technique). Most of the MoAbs used against neuroblastoma cells showed a strong reactivity pattern with the NB cell lines. None of the antibodies against T-lymphocytes bound to any of the NB cells assayed in our study, with the exception of anti-CD 1. On the contrary, B-cell markers BA-2 (CD9) and BA-1 (CD24) cross-reacted with the NB cells just as well as the marker for NK-cells (CD57), but they did not express reactivity with Leu-11b (CD16), anti-CALLA (CD10) and anti-HLA-DR.

Immunological detection and definition of minimal residual neuroblastoma disease in bone marrow samples obtained during or after therapy.

Immunological staining by the alkaline phosphatase/anti-alkaline phosphatase (APAAP) technique has been used to recognize low levels of neuroblastoma cells in bone marrow mononuclear cells. Immunological phenotyping with 11 well characterized monoclonal antibodies was performed on 16 children with neuroblastoma and BM involvement during or after therapy. Neuroblasts were detected in 11 of 16 patients (0.1-5%), whereas BM biopsies on six of these patients were classified as normal. Aspirates, stained conventionally, were positive for pathological cells in three patients only. The comparison of the phenotype of the neuroblastoma cells at the time of diagnosis to the phenotype of the residual cells within one patient revealed differences. The phenotype of residual disease in different patients on the other hand showed a unique pattern. The above mentioned results lead to the conclusion that the immunological procedure is particularly suitable for the analysis of minimal residual neuroblastoma since the technique allows very minor cell populations to be identified in BM samples.

[Immunologic identification of undetectable neuroblastoma cells by current cytohistological studies of bone marrow samples]. / Immunologische Identifizierung von durch herkömmliche zytohistologische Untersuchungen an Knochenmarksproben nicht erkennbare Neuroblastomzellen.

Immuno-alkaline phosphatase staining (APAAP technique) has been used to identify neuroblastoma cells on bone marrow samples from 12 children at various stages of the disease. On 72 occasions immunological analyses were performed using a panel of monoclonal antibodies which selectively bind to cells of neuroectodermal origin. In 57 of these procedures, tumor cells were detected, whereas histological and cytological analyses revealed pathological cells in 45 and 37 cases respectively. Reactivity of minimal residual tumor cells--mainly with three MAbs (UJ13A, UJ167.11, A2B5)--points to the fact that these cells belong to a resistant neuroblastoma clone, which remains in bone marrow despite intense therapy. Our study demonstrates that immunological staining may identify and define a small number of neuroblastoma cells which are not yet detectable by traditional histological and cytological criteria.