Differential adhesiveness between blood and marrow leukemic cells having similar pattern of VLA adhesion molecule expression.

Functional adhesion of blood and marrow leukemic cells from 14 acute myeloid leukemia patients presenting with hyperleukocytosis was evaluated by performing cytoadhesion assays on purified (extracellular matrix proteins) and non-purified supports (MRC5 fibroblastic cell line). Results, in 30-min chromium release assay, show a mean +/- S.D. adhesion to fibronectin, collagen, and laminin respectively of 30 +/- 17%, 20 +/- 13%, 25 +/- 17% for blood leukemic cells and 18 +/- 11%, 11 +/- 10%, 11 +/- 8% for marrow leukemic cells. These differences between blood and marrow cells were statistically significant (respectively P = 0.005, P = 0.01 and P = 0.002), while no difference was noted regarding adhesion to non-purified supports. The higher adhesion of blood blast cells to purified supports was observed regardless of CD34 expression. No significant difference was observed in the expression of cell surface VLA-molecules (CD29, CD49b, CD49d, CD49e, CD49f) between blood and marrow blast cells. The addition of GM-CSF or G-CSF induced increased adhesion of marrow blasts and decreased adhesion of blood blasts leading to a loss of the difference between blood and marrow cells. In a 60-min chromium release assay, marrow blasts adhered even more than blood leukemic cells to fibronectin. In contrast, marrow blasts from 'aleukemic' acute myeloid leukemia patients did not show any modification regarding their adhesion to extracellular matrix proteins when co-cultured with growth factors.

Influence of malignant cell clonogenic capacities and position along the maturation pathway on their susceptibility to lymphokine-activated killer cell cytotoxicity.

In order to investigate the sensitivity of malignant target cells to lysis by LAK cells according to their clonogenic capacities and their position along the maturation pathway, we compared clonogenic and chromium release cytotoxicity assays performed on human hematopoietic cell lines using Effector: Target ratios of 1:1, 3:1, 6:1, 12:1, 24:1, 48:1 and 96:1, and studied the sensitivity of HL-60 and U937 human cell lines after exposure to different factors including GM-CSF, SCF, IFN, Retinoic acid (RA), DMSO, and TPA which are able to recruit cells into the cell cycle or to induce cell differentiation. There was a good correlation between the lysis of the target cells using 51Cr release and the growth inhibition in semisolid medium. The degree of inhibition was significantly higher using the colony growth assay (p = 0.006). Regarding the effects of culturing cell lines with proliferating and differentiating agents on the sensitivity of these cell lines to LAK cytolysis, a correlation was noted between the proliferative response of the U937 cell line and susceptibility to LAK cell lysis (p = 0.01), while results appeared close to significance with HL-60. The most significant effects were a decreased sensitivity of HL-60 to LAK lysis with RA (p < 0.001) and TPA (p < 0.001), and an increased susceptibility of U937 to LAK lysis with GM-CSF (p < 0.0001). In studies planned to investigate whether susceptibility of treated cells to LAK activity was a consequence of a downregulation of adhesion molecules expressed on target cell surface, the proportion of cells expressing adhesion molecules was not significantly changed, except for CD54 expression on HL-60 cells which showed a higher expression, after cells were treated with RA or DMSO. We conclude that clonogenic cells are more sensitive to LAK cell lysis and that cell line sensitivity to LAK cytolysis can be modulated by a variety of agents of potential therapeutic use. The poor correlation between adhesion molecules expression and sensitivity to LAK lysis suggests that molecules other than CD54, CD56, CD58, and CD106 may possibly be more central to the processes involved.

Immunocytochemical detection of neuroblastoma cells infiltrating clinical bone marrow samples.

To evaluate the feasibility and clinical usefulness of immunocytochemical detection of bone marrow metastases in neuroblastoma, we studied bone marrow samples from patients undergoing intensive therapy, followed in the majority of cases by autologous bone marrow rescue. Two monoclonal antibodies were used in an indirect immunoenzymatic assay to test 384 samples collected from multiple bone marrow sites during 79 staging procedures in 48 patients. Of 578 immunocytochemical tests, 59 (10%) yielded non-evaluable results. Analysis by individual bone marrow sites showed an agreement between cytological and immunocytochemical examinations in 276 of 309 (89%) evaluable tests with 5 A7 and in 179 of 210 (85%) with UJ 13 A. Infiltration by neuroblastoma cells was reported in 9% of samples by cytology, in 6% by immunochemistry with 5 A7 and in 16% with 13 A. Analysis of results by staging demonstrated agreement between cytological examination and immunocytochemical detection with both monoclonal antibodies in 60 of 75 (80%) evaluable stagings. Bone marrow metastasis was detected by cytology in 22% of stagings, by immunochemistry with 5 A7 in 23%, with UJ 13 A in 25%. Detailed analysis of discordant results revealed that they were related partly to bone marrow sampling variability associated with focal and minimal metastasis of neuroblastoma cells. These data suggest the clinical usefulness of immunocytochemical detection as a complementary test to cytological examination for accurate evaluation of bone marrow infiltration in patients with disseminated neuroblastoma.

[The significance of immunological analysis for the detection of residual neuroblasts in bone marrow]. / Intérêt de l'analyse immunologique pour la détection de neuroblastes résiduels dans la moelle osseuse.

Immunological analysis is complementary to morphological investigation to detect bone marrow (BM) metastases in neuroblastoma patients. It is essential at time of BM harvest for an autograft, since it is the only examination which allows a precise evaluation of the graft contamination by malignant cells. Simple indirect immunofluorescence staining with anti-neuroblastoma monoclonal antibodies (UJ13A and HSAN 1-2) allows a final detection of about 1% malignant cells in the BM, and 1% when cells are gathered in clumps. The use of an immunocytochemical method (alkaline phosphatase) together with double immunofluorescence staining permits to detect as few as one residual neuroblastoma cell in 10(4) normal ones. These two methods used together have allowed to assess the neuroblastoma nature of rare isolated cells with pseudo-lymphocytic aspect.

[Detection of residual cells contaminating the bone marrow in neuroblastoma. Apropos of 80 bone marrow evaluations]. / Détection des cellules résiduelles contaminant la moelle osseuse des neuroblastomes. A propos de 80 bilans médullaires.

Eighty bone marrow studies (each including 4 aspirates and 4 trephine biopsies) were performed in 37 children with stage IV neuroblastoma to assess the most accurate means for detection of invasion by neuroblastoma cells. Among 38 abnormal results, only trephine biopsy(ies) were found positive in 24 cases (63%), only aspirate(s) in 5 cases (13%), and both in 9 cases (24%). In 37% of abnormal results, only 1 of the 8 tests performed was found positive. No benefit was obtained from either associated touch imprints of iliac biopsies (67 investigations), or exploration of extra-iliac sites (66 sternums, 53 tibias). Two monoclonal antibodies, claimed to be specific for detection of neuroblastoma cells in bone marrow, were used in 56 investigations; they could detect minimal residual disease in some cases, but they were unreliable when no staining was obtained if initial phenotype of neuroblastoma had not been assessed, or when few isolated cells were observed. Prospective studies using immunocytology and immunohistology are thus warranted.

Experimental evaluation of an immunomagnetic bone marrow purging procedure using the Burkitt lymphoma model.

In order to test the numerous variable biological parameters which may contribute to the efficiency of the immunomagnetic depletion method, experiments have been carried out with different Burkitt lymphoma cell lines. The Hoechst dye method, a sensitive assay able to detect 3 log elimination, and a liquid culture assay suitable for identifying an elimination greater than 4 logs, were used to measure malignant cell elimination. These assays showed that the separation of bone marrow mononuclear cells on Ficoll-hypaque, the use of beads in excess, and a double treatment of the samples are needed to obtain optimal depletion of malignant cells. Under these conditions, the immunomagnetic procedure is capable of depleting 1% tumour cell infiltration at the lower limit of detection of a very sensitive culture assay (i.e. greater than 4 log elimination).

EBV-negative and -positive Burkitt cell lines variably express receptors for B-cell activation and differentiation.

The expression of receptors for proliferation and differentiation factors was analyzed by indirect immunofluorescence on 29 Burkitt lymphoma (BL) cell lines previously classified into 3 groups on the basis of their reactivity with 8 monoclonal antibodies (MAbs), including anti-CALLA, BL13 and TU1. BL13 and HB5 antibodies recognize different epitopes of the EBV/CR2 receptors. The determinant recognized by BL13 has been previously shown to be expressed only on cell lines of the first two groups, supposed to derive from the germinal center and to be negative on a third group of lines of putative BM origin and established from sporadic cases of BL. In contrast, and as expected from its reactivity on normal B cells in the BM or in the lymph nodes, HB5 antibody reacts with all BL lines except one. The receptor for transferrin is expressed on the 29 lines. Two new MAbs, Bac-1 and B1H5, could recognize respectively receptors for BCGF1 and BCGF2. Bac-1 reacts with 15 of 17 BL lines belonging to the first two groups and 7 of 12 BL lines of the third group; 14 of 15 EBV + lines express Bac-1. No BL line expresses B1H5. The IL2 receptor is weakly expressed on 5 EBV + cell lines and one EBV (-) line. All delta are BCGF1-positive. The almost constant expression of BCGF1 receptor on EBV + cell lines is the only strict relation between the expression of receptors for growth factors and their characteristics (i.e. EBV association, translocation, ethnic origin and clinical presentation). The maturation stage or the origin of BL cell lines in relation to the expression of growth factor receptors and the functional significance of these receptors will be discussed.

Histological, cytological and immunological analyses are complementary for the detection of neuroblastoma cells in bone marrow.

On 80 occasions 4 iliac biopsy trephines and 4 iliac aspirations were performed in 37 children with neuroblastoma at various stages of the disease. In 38 of these procedures, tumour cells were detected. In 24% of cases, both trephines and aspirates were positive, whereas in 63% neuroblastoma cells were detected only on the trephines and in 13% only on the aspirates. In addition, in 37% of the stagings, only one out of the 8 investigations was abnormal. Only in one of 33 pathological cases, was BM involvement diagnosed on trephine imprint. No involvement was ever observed on tibial and sternal aspirates without iliac involvement. Immunological studies with two monoclonal antibodies HSAN 1-2 and UJ13A were performed on 56 occasions. Cytohistological and immunological studies were concordant in 39. In 3 studies, the antigens recognized by the two monoclonal antibodies were not expressed by the initial tumour and in 3 additional studies immunological results were falsely negative; but in 11 cases monoclonal antibodies identified residual malignancy despite normal cytohistology. From this study, biopsies appear more helpful to detect malignant cells than aspirates. Immunological staining clearly leads to a better definition of tumour cells in aspirates.