Cytogenetic evolution patterns in CML post-SCT.

The cytogenetic evolution patterns in chronic myeloid leukemia (CML) after allogeneic (allo) stem cell transplantation (SCT) are different from the ones observed in non-transplanted patients, a phenomenon suggested to be caused by the conditioning regime. We reviewed 131 CMLs displaying karyotypic evolution after SCT (122 allo, nine autologous (auto)), treated at Lund University Hospital or reported in the literature. Major route abnormalities (i.e., +8, +Ph, i(17q), +19, +21, +17 and -7) were seen in 14%, balanced aberrations in 61%, hyperdiploidy in 19%, pseudodiploidy in 79%, divergent clones in 14%, and Ph-negative clones in 21%. The breakpoints involved in secondary structural rearrangements clustered at 1q21, 1q32, 7q22, 9q34, 11q13, 11q23, 12q24, 13q14, 17q10 and 22q11. Cytogenetic abnormalities common in AML after genotoxic exposure, that is, der(1;7)(q10;p10), del(3p), -5, del(5q), -7, -17, der(17p), -18, and -21, were only rarely seen post-SCT. Comparing the cytogenetic features in relation to type of SCT revealed that balanced aberrations were significantly more common after allo than after auto SCT (64 and 22%, respectively, P=0.03). In addition, there was a trend as regards hyperdiploidy being more common after auto (P=0.07) and pseudodiploidy being more frequent after allo SCT (P=0.09). Possible reasons for these differences are discussed.

Modulation of the production of leukemia associated inhibitor (LAI) and its interaction with granulocyte-macrophage colony-forming cells.

Leukemia associated inhibitor, LAI, reversibly inhibits DNA synthesis in normal human granulocyte-macrophage colony-forming units (CFU-GM). LAI is produced by myeloid leukemia cells, a subpopulation of normal nonadherent low-density mononuclear cells in peripheral blood and bone marrow, as well as by the human promyelocytic cell line HL-60. Normal low-density marrow cell absorbed LAI at 37 degrees C from HL-60 cell-conditioned medium. When normal marrow cells were treated with trypsin or chymotrypsin they lost their capacity to absorb LAI and also became insensitive to the inhibitory effect of LAI. These observations were taken as circumstantial evidence for the existence of a trypsin-sensitive LAI receptor on normal marrow cells, including CFU-GM. Glucocorticoid steroids (hydrocortisone, prednisolone, and dexamethasone) inhibited LAI production by acute myeloid leukemia (AML) cells, normal LAI-producing cells, and HL-60 cells. The fact that prostaglandin E1 (PGE) totally inhibited LAI production by normal cells and that indomethacin abrogated the inhibitory effect of adherent cells on LAI production suggested a role for adherent monocytic cells and PGE in the regulation of LAI production.

IL-10 as an autocrine regulator of CSF secretion by monocytes: disparate effects on GM-CSF and G-CSF secretion.

In previous studies of endogenous granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) production, we found several differences in the secretion pattern within and between different cell systems; for example, CSF secretion by endothelial cells is not affected by any major downregulatory factors, whereas monocyte CSF secretion is modulated by several mechanisms. In this study, we characterized the factors that inhibit CSF secretion by monocytes. Three cytokines have inhibitory effects: interleukin (IL)-4, IL-10, and IL-13. Among these, IL-4 and IL-10 have higher potency than IL-13. IL-4 and IL-13 affect GM-CSF and G-CSF secretion to the same extent. In contrast, exogenously added IL-10 has a stronger inhibitory effect on GM-CSF secretion than on G-CSF secretion. We also found that monocytes produce IL-10 with an autocrine downregulatory effect, and that this autocrine IL-10 reaches concentrations at which in most cases only GM-CSF (not G-CSF) secretion is significantly affected. We postulate that the disparate effect of IL-10 on monocyte secretion of the two CSFs reflects their physiological functions, with GM-CSF being mainly a proinflammatory cytokine working in the local compartment and G-CSF functioning mainly as a cell recruiting factor.

T lymphocytes downregulate granulocyte-macrophage colony-stimulating factor secretion from stimulated monocytes by increasing the secretion of monocyte-derived interleukin-10.

In previous studies we characterized the cytokine regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) secretion by endothelial cells and monocytes and found differences in secretion pattern within and between these cell systems. In this study, the regulatory effect of T lymphocytes on CSF secretion was examined. T lymphocytes had no effect on CSF secretion by endothelial cells. In contrast, the addition of T lymphocytes significantly and dose dependently downregulated GM-CSF, but not G-CSF, secretion by monocytes. In one of our previous studies it was shown that interleukin-4 (IL-4) and interleukin-10 (IL-10) were the most potent inhibitory cytokines of CSF secretion by monocytes. Both these cytokines are produced by T lymphocytes. However, the downregulating effect on monocyte GM-CSF secretion was not due to increased secretion of T-lymphocyte-derived IL-4 or IL-10. Instead, the presence of T lymphocytes increased the secretion of monocyte-derived IL-10. It was shown earlier than IL-10 regulates CSF secretion by monocytes in an autocrine manner. Our data indicate that T lymphocytes might interfere with this autocrine regulation and thereby influence monocyte function in immune response and cell proliferation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in serum in bone marrow transplanted patients.

Colony-stimulating factors (CSF) are being increasingly used to accelerate hematopoietic recovery after bone marrow transplantation. To study the endogenous serum levels of CSF in bone marrow transplanted patients we have used immunoassays measuring granulocyte-macrophage colony-stimulating factor (GM-CSF) with a sensitivity of 0.10 ng/ml and granulocyte colony-stimulating factor (G-CSF) with a sensitivity of 0.05 ng/ml. Serum samples, taken from the conditioning treatment until engraftment, were analysed in 13 patients receiving allogeneic transplants and in eight patients receiving autologous transplants. Ten patients had acute myeloid leukemia, seven acute lymphoblastic leukemia, one acute undifferentiated leukemia, two non-Hodgkin's lymphoma and one multiple myeloma. Samples were taken 1-2 times before transplantation and 1-2 times per week after transplantation (median of 46 days in allotransplant recipients and 32 days in autotransplant recipients); 17% of the allogeneic transplanted patients and 35% of the autologous transplanted patients had detectable levels of G-CSF. In both types of transplantation the G-CSF concentrations were low: median 0.06 (range 0.05-0.14) and 0.08 (range 0.05-0.40) ng/ml respectively. GM-CSF was detected only in one analysed sample in all patients. There was no evidence of increased CSF levels related to engraftment or documented infections.

Fludarabine-based preparative protocol for unrelated donor cord blood transplantation in children: successful engraftment with minimal toxicity.

Unrelated umbilical cord blood (UCB) is a new source of hematopoietic stem cell (HSC) for allogeneic transplantation. The optimal conditioning for UCB transplant has not been defined. Intensive immunosuppression is frequently employed because of the higher risk of graft failure. Fludarabine monophosphate is shown to be an effective agent for facilitating allogeneic HSC engraftment. We have recently encountered three children who were not ideal candidates for 'conventional' conditioning protocols. TBI followed by fludarabine and melphalan were used for transplant preparation. The UCB units were one HLA-antigen (n = 1) and two HLA-antigen mismatched with the recipients. All three patients engrafted successfully. There was mild extramedullary toxicity. Two patients are alive with complete chimerism 8 and 20 months after transplant. A fludarabine-based protocol may be considered for selected cases of UCB transplants.

No prevention of cytomegalovirus infection by anti-cytomegalovirus hyperimmune globulin in seronegative bone marrow transplant recipients. The Nordic BMT Group.

A randomized multicentre study was conducted to evaluate the effect of anti-CMV hyperimmune globulin in the prophylaxis of CMV infections in CMV seronegative allogeneic BMT patients who received a transplant from a seropositive donor or who had received blood products unscreened for CMV during the treatment before BMT. Twenty-eight patients were included in the study. Thirteen were randomized to receive and 15 not to receive intravenous CMV hyperimmune globulin. A dose of 0.4 g/kg of immunoglobulin was given on day -8 and 0.2 g/kg on days -1, +7, +14, +21, +28, +35, +42, +56 and +70 in relation to the day of transplantation. Among the 15 patients not given immunoglobulin CMV was isolated in three, and two of them developed clinical CMV disease. In addition, one more patient developed CMV antibodies without virus isolation. In five of the 13 patients given immunoglobulin the virus could be isolated, and four of them developed CMV disease. One additional patient showed seroconversion but no other findings of CMV infection. The incidence of acute and chronic GVHD was similar in the two arms. There was no significant difference in survival. In conclusion, the present results do not indicate a beneficial effect of CMV hyperimmune globulin infusions in the prophylaxis of CMV infection or disease in seronegative allogeneic bone marrow transplant recipients from a seropositive donor.

More efficient mobilisation of peripheral blood stem cells with HiDAC+AMSA+G-CSF than with mini-ICE+G-CSF in patients with AML.

We have compared the efficacy of two PBSC mobilisation regimens, mini-ICE+filgrastim (second consolidation) and HiDAC+AMSA+filgrastim (third consolidation), in two consecutive cohorts of patients with AML CR1 receiving treatment according to a joint protocol. Group A: 18 patients, aged 41 (21-65) years, were mobilised with mini-ICE (idarubicin 8 mg/m(2)+cytarabine 800 mg/m(2)+etoposide 150 mg/m(2) days 1-3) followed by filgrastim 300-480 microg once daily s.c. from day 11 after start of chemotherapy. Only four patients reached >5 CD34+ cells/microl blood (B-CD34+) and were able to undergo leukaphereses. Two out of 18 (11%) reached the defined target of >/=2.0 x 10(6) CD34+ cells/kg after 1-3 leukaphereses. Group B: 20 patients, aged 50 (29-67) years, received HiDAC+AMSA (cytarabine 3 g/m(2) b.i.d. days 1, 3, 5+amsacrine 150 mg/m(2) q.d. days 2, 4) followed by filgrastim at a similar dose starting on day 7. A total of 18 patients reached B-CD34+ >5/microl and underwent PBSC harvesting, starting on day 23 (14-29) and yielding 4.0 (0.9-21) x 10(6) CD34+ cells/kg. Of 20 patients, 17 (85%) reached the defined target of >/=2.0 x 10(6) CD34+ cells/kg after 1-3 leukaphereses. We conclude that HiDAC+AMSA+G-CSF - in contrast to mini-ICE+G-CSF - is an efficient regimen for mobilising PBSC in patients with AML CR1.

Trisomy 13 as a primary chromosome aberration in acute leukemia.

Four patients with acute leukemia displayed trisomy 13 as the primary chromosome abnormality. The two patients with acute nonlymphocytic leukemia FAB-type M1 (ANLL-M1) had the karyotypes 47,XY,+13/48,XY,+13,+13 and 47,XX,+13, a patient with the hypogranular form of ANLL M3 had 47,XX,+13, and the fourth patient, who had acute undifferentiated leukemia (AUL), had the karyotype 47,XY,+13/48,XY,+8,+13. Including these four cases, a total of 24 hematologic neoplasms with an extra chromosome 13 as the sole aberration have now been reported. Except for the AUL, all have been of myeloid origin--20 ANLL, one myelodysplastic syndrome, and two chronic myeloproliferative disorders. Trisomy 13 as the sole acquired karyotypic abnormality therefore seems to be strongly associated with myeloid differentiation of the neoplastic cells and with a differentiation block leading to acute leukemia.

Endogenous production and peripheral blood levels of granulocyte-macrophage (GM-) and granulocyte (G-) colony-stimulating factors.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) are two important granulopoietic growth factors. This review will focus on the endogenous production of human GM-CSF and human G-CSF and its possible reflection in circulating levels in peripheral blood. When adequately stimulated a variety of cell-types such as monocytes/macrophages. T-lymphocytes, endothelial cells and fibroblasts can produce CSFs in vitro. G-CSF can increase to detectable levels in peripheral blood when there is a demand for granulocyte production such as acute neutropenic in conjunction with hematological disorders, chronic neutropenic conditions and acute infectious diseases in patients with or without underlying hematological disorders. G-CSF in peripheral blood is detected more often and in higher concentrations than GM-CSF. An independent regulation of GM-CSF and G-CSF secretion, quantitative differences in production and/or differences in elimination or distribution might be of importance.

Outcome for patients with leukemia, multiple myeloma and lymphoma who relapse after high dose therapy and autologous stem cell support.

Relapses after autologous transplantation are a serious clinical problem in patients with haematological diseases. The decision making for handling of such patients is difficult and the aim of this retrospective analysis of posttransplant relapses was 1) to obtain information of practical importance for the management of future relapses and 2) to evaluate the basis for clinical phase I-II trials of salvage therapy combined with biological modifiers. Included in the study were 283 patients with acute leukemia, multiple myeloma and malignant lymphoma who relapsed after autologous transplantations during a five year period from 1989 to 1994. Chemo- and radiotherapy was given to 229 patients after relapse or due to progressive disease and the response evaluated after 90 days. Fifty four patients (24%) obtained a complete remission and 44 patients (19%) partial responses. The overall median survival from relapse was 5 months. In the group given salvage treatment the median survival was 7 months and in the 54 patients who obtained remission the median survival was 15 months. So far 6 of 14 patients in continuous complete remission have a remission time after relapse longer than the time in remission after transplantation. Survival after relapse depended upon the time from transplantation to relapse, primary disease and if salvage therapy was given. In conclusion posttransplant relapses can be treated but the strategy has to be evaluated in future clinical trials.

[Umbilical cord blood from unrelated donor. An alternative to bone marrow transplantation]. / Navelsträngsblod från icke besläktad givare. Alternativ till benmärgstransplantation.

Unrelated umbilical cord blood (UCB) as a source of haematopoietic stem/progenitor cells has been used for the first time in Sweden for allografting two boys (6 and 11 1/2 years old) with acute leukaemia in complete remission. Double class-II HLA-mismatch UCB units, from the Milan and New York cord blood banks, respectively, were used. Pre-transplant preparation consisted in fractionated total body irradiation in both cases, followed by cytoreductive high-dose cyclophosphamide for the 6-year-old with acute lymphocytic leukaemia (ALL) and fludarabine-melphalan for the 11 1/2-year-old with acute non-lymphocytic leukaemia (ANLL). Despite delayed haematopoietic recovery (probably due to HLA disparity) or reactivated cytomegalovirus infection in one boy, the immediate post-graft course was uneventful and extramedullary toxicity mild in both cases. Only transient acute graft-versus-host disease occurred, and complete donor chimerism was confirmed. Unfortunately, the out-come was unfavourable in both cases, the ANLL patient succumbing on day 96 due to pneumonia followed by multiorgan failure, and the ALL patient on day 140 due to combined medullar and central nervous system relapse. Although UCB transplantation needs further evaluation, it may contribute substantially to successful salvage procedures. Thus the introduction of cord blood banking in Sweden merits consideration.

Cell-mediated inhibition of granulopoiesis in vitro in patients with acute myeloid leukemia in remission.

We investigated the in vitro granulopoiesis in 11 patients with acute myeloid leukemia (AML) in complete remission 3-80 months after diagnosis (median 8.5 months). 3 of the patients had subnormal levels of bone marrow-derived CFU-GM. 6 of 10 patients tested had defective recloning capacity of d-7 CFU-GM, suggesting a stem cell defect. Most patients (7/11) showed an increased colony growth of bone marrow-derived CFU-GM after T-cell depletion by E-rosetting, while readdition of isolated autologous T cells to T-cell depleted marrow caused a dose-dependent inhibition of colony formation; bone marrow T cells were more effective in this inhibition than peripheral blood T cells. Experiments using cells depleted of either CD4- or CD8-positive cells and CD4/CD8-enriched cell populations showed that both CD4- and CD8-positive cells had the capacity to inhibit colony growth. Long-term culture of bone marrow cells in suspension showed that the production of CFU-GM declined at about the same rate as in normal controls. Our findings suggest that there are persisting stem cell defects in patients with AML in remission and that the cell growth regulatory systems may be altered. These abnormalities could possibly be an effect of residual damage to the hematopoietic system caused by intensive chemotherapy.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in serum during induction treatment of acute leukaemia.

Granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) are increasingly used to stimulate granulopoiesis in neutropenic patients but in most cases without any knowledge of the endogenous CSF-levels. With the purpose to define serum levels of GM-CSF and G-CSF during induction chemotherapy and haematological reconstitution in patients with acute leukaemia we have used enzyme-linked immunosorbent assay (ELISA) techniques to measure these growth factors in 18 patients with acute myeloid leukaemia (AML) and eight patients with acute lymphoblastic or undifferentiated leukaemia (ALL/AUL). G-CSF above 0.05 ng/ml was detected in 54% of the analysed AML samples, median 0.29 (range 0.05-2.80) ng/ml; and in 40% of analysed ALL/AUL samples, median 0.09 (range 0.05-3.00) ng/ml. In patients with AML there was a clear correlation between an elevated serum concentration of G-CSF and documented infections. On the other hand, 15/18 of the patients with acute myeloid leukaemia and 8/8 patients with ALL/AUL had non-detectable levels of GM-CSF (less than 0.10 ng/ml). Two patients had measurable levels of GM-CSF in all samples, median 0.71 (range 0.26-1.18) ng/ml and in these patients the levels successively decreased during and after chemotherapy and did not increase in response to infections. In normals detectable levels of GM-CSF were found in 2/35 individuals and G-CSF in 0/10 individuals.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) secretion by adherent monocytes measured by quantitative immunoassays.

The kinetics of GM-CSF and G-CSF secretion by purified adherent human monocytes were studied by quantitative immunoassays. Interleukin-1 (IL-1); 4-40 ng/ml and E. coli lipopolysaccharide (LPS); 0.1-1.00 ng/ml, were the most effective stimuli and induced dose-dependent secretion of both GM-CSF and G-CSF. Secretion of newly synthesized CSF was detectable 3-6 hours after stimulation and continued for approximately 24 h. Twenty minutes pulse exposure to LPS was sufficient to induce half maximum secretion of GM-CSF, and after 24-36 h the adherent monocytes could not be restimulated. Neither GM-CSF nor TNF could down-regulate the secretion of GM-CSF. IL-3 induced a minor secretion of GM-CSF whereas TNF, G-CSF, M-CSF and IFN-gamma were unable to induce GM-CSF secretion. In addition to LPS and IL-1, GM-CSF and to a minor degree TNF induced G-CSF secretion. Enriched T lymphocytes secreted GM-CSF, but not G-CSF, after stimulation with PHA or staphylococcal enterotoxin A (SEA), whereas LPS and IL-1 were without stimulatory effects. We also noted that enriched T lymphocytes added to LPS-stimulated adherent monocytes at ratios of 1:10 or more inhibited, in a dose-dependent fashion, GM-CSF secretion by 13-55%. These findings add new quantitative data on CSF secretion by human monocytes.

Venous volume in offspring of hypertensive patients.

Twenty normotensive healthy male offspring of hypertensive patients were compared to 20 adequate controls regarding venous volume, the effect of ouabain on venous volume and signs of left ventricular hypertrophy. Among offspring there were slightly increased amplitudes in the orthogonal ECG as compared to controls but the differences were not statistically significant. Using a plethysmographic technique we found a significantly lower venous volume of the forearm in the offspring than in controls. There was, however, no effect of ouabain on the venous volume. The decreased venous distensibility among normotensive offspring might be an early manifestation of essential hypertension.