Correlation between expression of CD56/NCAM and severe leukostasis in hyperleukocytic acute myelomonocytic leukaemia.

OBJECTIVE: The possible contribution of surface molecules to the development of leukostasis syndrome in hyperleukocytic acute myeloid leukaemia (AML) was assessed by routine immunophenotyping and grading of the probability of clinical leukostasis. METHODS: Fifty-three patients (23 women, 30 men, median age 59 yr) with hyperleukocytic AML [white blood count (WBC) above 50 x 10(9)/L] were graded for the probability of clinical leukostasis according to the severity of neurologic, pulmonary and other symptoms possibly caused by leukostasis using a recently published scoring system. Age, WBC, absolute blast count, haemoglobin, cytogenetic risk group, infection, relative CD56 expression and absolute count of CD56 positive blasts were analyzed in multivariate stepwise backward logistic regression analysis. RESULTS: In patients with acute monocytic leukaemia (AML M4/M5) the absolute count of leukaemic blasts expressing CD56/NCAM was highly associated with the development of symptoms graded as highly probable leukostasis and all three patients succumbing to early death were CD56 positive. Only the absolute count of CD56 positive blasts was a significant predictor of risk of severe leukostasis (P = 0.020). This was not found in AML without monocytic involvement (AML M1, M2, M3v). CONCLUSIONS: The expression of CD56/NCAM, a surface marker used in routine immunophenotyping of AML, may help to predict severe and potentially fatal leukostasis in hyperleukocytic acute myelomonocytic leukaemia. These results emphasize the usefulness of this four-stage clinical grading scale for analysing the factors, which lead to severe leukostasis in hyperleukocytic patients. We extend previous findings that the mechanisms of leukostasis are different depending on the involvement of the monocytic lineage.

Grading of symptoms in hyperleukocytic leukaemia: a clinical model for the role of different blast types and promyelocytes in the development of leukostasis syndrome.

OBJECTIVE: Patients with hyperleukocytic leukaemia were graded according to the severity of symptoms possibly caused by leukostasis to evaluate the effectiveness of therapy and to test the relative contribution of blast type and count of blasts and promyelocytes in the development of leukostasis syndrome. METHODS: Ninety-five patients (59 male, 36 female, median age 52 yr) with hyperleukocytic leukaemia [leukocytes above 50 x 10(9)/L, 48 acute myeloid leukaemia (AML), 31 chronic myeloid leukaemia (CML), 13 acute lymphoblastic leukaemia (ALL), three chronic myelomonocytic leukaemia (CMML)] were grouped according to the presence or absence and severity of neurologic, pulmonary and other symptoms into four categories (no, possible, probable and highly probable leukostasis syndrome). Age, white blood count (WBC), haemoglobin, blast count and total of blasts plus promyelocytes of these groups were compared by Mann-Whitney U-test. RESULTS: Patients with myeloid leukaemia (AML M1/M2, CML) which scored as highly probable leukostasis showed significantly higher WBC (P = 0.011), lower haemoglobin (P = 0.004), higher peripheral blast counts (P = 0.004) and higher total of peripheral blasts plus promyelocytes (P < 0.001) compared with the lower probability groups. In leukaemia involving the monocytic lineage (AML M4/M5, CMML) no significant differences were found in any of these factors between patients with highly probable leukostasis and the other patients. CONCLUSIONS: Our results show that a four-stage clinical grading scale is a valuable tool for analysing hyperleukocytic patient populations and evaluate the effectiveness of therapy more precisely. We further demonstrate that the mechanisms of leukostasis are different in myeloid leukaemia as compared with leukaemia with involvement of the monocytic lineage.

Failed or inadequate bone marrow aspiration: a fast, simple and cost-effective method to produce a cell suspension from a core biopsy specimen.

Failure to aspirate bone marrow (BM) diminishes diagnostic accuracy and efficiency because BM cell suspensions are crucial for modern haematological diagnostic methods such as cytomorphology, flow cytometric immunophenotyping (FCI), cytogenetics or fluorescent in situ hybridization (FISH). We mechanically disaggregated unfixed BM core biopsies with the Dako Medimachine in 65 cases of macroscopically suspected dry taps. Cytospins, three-colour FCI and in some cases karyotyping and FISH were performed successfully. Most cytospins (34 of 50; 68.0%) were of good quality, while a further 18.0% showed moderate but still informative quality. FCI showed good quality in 36 of 60 (60.0%) cases; in 13.3% quality was moderate, but diagnostically useful results were obtained. Surprisingly, all four cases of formerly undiagnosed BM-carcinosis could be clearly detected on cytospins. Finally, five of seven (71.4%) attempts yielded analysable metaphases mostly in cases where no metaphases could be obtained from BM or peripheral blood. The described method of mechanical disaggregation of unfixed BM core biopsies compares favourably with other published approaches, allowing the application of all techniques where BM cell suspensions are needed. Thus, it can help to establish the underlying diagnosis in patients with abnormalities in peripheral blood and unsuccessful marrow aspirations.

Disease- or therapy-related bone marrow damage cannot be overcome by changes in stem cell source or dose in allogeneic transplantation.

OBJECTIVE: To test whether the functional impairment of the host bone marrow (BM) microenvironment pre-existing at the time of transplantation could be overcome by the increased content of immature cells in allogeneic peripheral blood stem cell transplantation (PBSCT) when compared with bone marrow transplantation (BMT). METHODS: Cobble stone area forming cells (CAFC) were assayed in normal BM and BM after allogeneic BMT and PBSCT after stable engraftment. Groups were compared by two-tailed t-test. RESULTS: While BM from 11 normal controls contained an average of 778.8 CAFC-d35 per 10(6) low density bone marrow cells (LDBMC, range 453-1231 per 10(6) LDBMC), BM from patients after BMT contained an average of 123.7 CAFC-d35 per 10(6) LDBMC (range 38-257) per 10(6) LDBMC. BM from patients transplanted with PBSC after myeloablative conditioning contained 128.3 (range 46-305) CAFC-d35 per 10(6) LDBMC (P = 0.89 compared with BMT). Similar results were obtained when patients after PBSCT with non-myeloablative conditioning were included (P = 0.62 compared with BMT). CAFC numbers in patients transplanted in early stages of myeloid leukaemia (acute myeloid leukaemia first remission, chronic myeloid leukaemia first chronic phase) were significantly higher than CAFC numbers in patients transplanted in more advanced stages (P = 0.008) or myelodysplastic syndrome (P = 0.023). The lowest CAFC numbers were found in two cases of retransplantation. CONCLUSION: Our findings indicate that the functional state of the BM microenvironment rather than stem cell dose or source is limiting for the homing and engraftment of immature haemopoietic cells in clinical transplantation.

Characterization of two novel pre-B-cell lines (LK63 and LiLa-1): potential models of pre-B-cell differentiation.

In this report we describe two newly isolated pre-B acute lymphoblastic leukaemia cell lines. Both cell lines lack EBV as detected by the EBNA-1 gene probed Southern-blots. Neither cell line expressed the B-cell-specific CD20 antigen on the cell membrane. However surface expression of CD20 was induced by phorbol ester (TPA) on both LiLa-1 and LK63 cell lines. Other pre-B and B-cell lines, such as Reh, Nalm-1, and BALL-1 did not exhibit these changes in phenotype. Previous immunoprecipitation studies have noted that a broad 50-55 kD band co-precipitates with the characteristic 33-37 kD CD20 protein. We demonstrate that, while the 33-37 kD CD20 species was undetectable on resting LiLa-1 and LK63 cells, in each case a 50-55 kD protein was immunoprecipitated by the CD20 antibody. However, the failure to detect any cell surface CD20-associated antigen on the control cells by immunophenotyping indicated that the CD20 epitope of the 50-55 kD molecule was not expressed on the cell surface. Following exposure to TPA the 50-55 kD species was reduced over 48-72 h while the level of the p33-37 CD20 protein was increased. Northern-blot analysis showed that the 50-55 kD protein was not a cryptic form of CD20 as the uninduced cells contained no detectable CD20 mRNA. The decrease of the 50-55 kD protein and the acquisition of the mature CD20 molecule were paralleled by a decline in proliferative activity in both cell lines. As expression of CD20 by normal pre-B cells also coincides with the cessation of cell division and maturation towards a mature B-cell phenotype, these cell lines appear to represent models for a discrete stage of B-cell differentiation which may be valuable in defining the signals regulating pre-B-cell proliferation.

The effect of cytokines on the expression of MHC antigens and ICAM-1 by normal and transformed synoviocytes.

We report the expression on synovial cells of cell surface molecules known to be involved in T cell activation by antigen presenting cells. Normal human synovial fibroblasts and a human synovial cell line transformed with the SV40 large T antigen were used for in vitro stimulation studies with recombinant cytokines. We demonstrate an increase in MHC-A, B, C expression in normal synovial cells in response to recombinant interferon gamma (r gamma IFN), tumour necrosis factor alpha and beta (rTNF alpha and beta) and interleukin-1 (rIL-1 alpha). Intercellular adhesion molecular-1 (ICAM-1) expression was increased in parallel with MHC Class I. The combination of r gamma IFN and rTNF alpha was additive in its effect on ICAM-1 expression. Northern blot analysis suggests that ICAM-1 expression in synovial cells is controlled at the level of transcription. In contrast, MHC Class II (HLA-DR) was only significantly induced by r gamma IFN. Other stimuli including interleukin-4 (IL-4), interleukin 6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF) and prostaglandin E2 (PGE2) did not affect the expression of ICAM-1 or MHC Class I and II. Leucocyte function antigen 3 (LFA-3) expression was not affected by any of the stimuli tested. Immunoperoxidase staining of rheumatoid synovial tissue confirmed enhanced in vivo expression of ICAM-1 in rheumatoid arthritis. These changes are discussed in the context of T cell activation in inflammatory arthritis.

Cloned stromal cell lines derived from human Whitlock/Witte-type long-term bone marrow cultures.

We report a human bone marrow culture technique that initially parallels the murine Whitlock/Witte culture system. As in the murine system, B cells predominate over other cell types, and all differentiation stages from pre-B to plasma cell are observed. Although these human long-term cultures pass through stages resembling phases I to III of murine Whitlock/Witte cultures, no outgrowth of nonadherent cells was seen after cultures had reached the "crisis" phase unless Epstein-Barr virus (EBV)-transformants appeared. The stromal cells persisted well beyond crisis, but they could not be maintained and passaged as cell lines, limiting their use in molecular analysis. Transfection of these stromal cells with plasmid DNA containing the simian virus 40 (SV40) early region yielded 124 cloned cell lines. Analysis of these lines showed that all expressed SV40 large T antigen, but they retained most phenotypic markers found on non-transformed stromal cells. When adherent and T-cell-depleted bone marrow cells were cultured on either nontransformed stromal layers or transformed cell lines they proliferated actively and soon yielded predominantly lymphoid nonadherent populations. Moreover, prolonged survival of acute lymphoblastic leukemia cells of pre-B phenotype was regularly achieved on both normal and transformed adherent cell layers. Although the liquid culture system favored lymphocytes, transformed stroma supported colony formation by both human and murine hemopoietic progenitors when marrow was added in agar medium. This was not explained by colony-stimulating factor (CSF) production, because striking heterogeneity in the levels of granulocyte CSF (G-CSF) and granulocyte-macrophage CSF (GM-CSF) secretion by the lines was noted. Some lines that did not produce detectable CSF demonstrated good support of fresh bone marrow growth and acute lymphoblastic leukemia (ALL) cell survival. The heterogeneity of these cell lines and their capacity to support hemopoiesis suggest that they will be useful in studying the molecular basis of in vitro lymphohemopoiesis in man.