Kinetics of hemopoietic recovery after peripheral blood stem cell transplantation: impact of stem cell purification and G-CSF

We investigated the role of stem cell purification and G-CSF (early vs. delayed vs. no G-CSF) administration on hemopoietic recovery and supportive care requirements after stem cell transplantation. Thirty-two patients submitted to autologous CD34(+) peripheral blood stem cell transplantation (PBSCT) were studied, and data were compared to patients undergoing unfractionated peripheral blood stem cell transplantation (uPBSCT) matched for age, disease, and conditioning regimen. Except for PMN, hemopoietic recovery was significantly slower and supportive care requirements were significantly higher after CD34(+) PBSCT. Median time to PMN >0.5 x 10(9)/l was 13 days (range 9-27) and 13 d (range 9-23); reticulocytes (Ret) >1% was 14.5 d (range 12-34) and 12 d (range 10-27); high-fluorescence reticulocytes (HFR) >5% was 12 d (range 9-26) and 9 d (range 7-11); platelets >50 x 10(9)/l and >100 x 10(9)/l was 20 d (range 10-240), 12 d (range 9-60) and 33 d (range 15-720), 15 d (range 11-210). When the analysis was performed on subgroups of patients (early/delayed/no G-CSF), early G-CSF significantly promoted PMN recovery (>0.5 x 10(9)/l and >1.0 x 10(9)/l) compared to no G-CSF, without affecting RBCs or platelet recovery. Delayed G-CSF did not improve PMN recovery compared to patients not receiving G-CSF, did not result in a significant reduction of drug requirements, and had a negative impact on erythroid and platelet recovery. In conclusion, these preliminary data suggest that G-CSF is useful if given early after CD34(+) PBSCT. CD34(+) PBSCT may overall require a significant increase of resource utilization that should be outweighed by proven clinical benefit.     

Outcome of allogeneic peripheral blood stem cell transplantation using matched sibling donors in patients with high-risk hematological diseases

Although the use of peripheral blood stem cells instead of bone marrow is still a matter of debate in transplantation from HLA-identical sibling donors, allogeneic peripheral blood stem cell transplantation (PBSCT), with a stronger graft-versus-leukemia (GVL) effect, may be preferable as a source of stem cells, especially in the case of advanced hematologic diseases. As such, the current paper reports on the outcomes of 27 consecutive patients with high-risk hematologic diseases treated with allogeneic PBSCT. The median dose of CD34+, CD3+ cells, and MNC infused was 8.18 x 10(6)/kg (range: 2.78-14.93), 1.50 x 10(8)/kg (range: 0.06-4.25), and 7.17 x 10(8)/kg (range: 0.95-15.85), respectively. The median time taken for the ANC and platelets to reach 500 and 20,000 x 10(6)/microL was 15 (range: 9-25) and 16 d (range: 10-56), respectively. Three patients (11.1%) experienced transplant-related mortality within 90 d of transplantation, and 15 (62.5%) of 24 evaluated patients developed chronic graft-versus-host disease (GVHD; six limited, nine extensive). There was a significant difference in overall survival (OS) between the group with chronic GVHD and the group without chronic GVHD (P = 0.0253). The causes of death included relapse (six cases) and non-relapse mortality (infection: four cases, chronic GVHD-related death: three cases). The 4-yr OS rate and disease-free survival rate was 43.3 +/- 10.9% and 35.8 +/- 10.2%, respectively. Accordingly, chronic GVHD was found to have a positive role in patients with high-risk hematologic diseases that received allogeneic PBSCT.     

Treatment with campath-1H for relapsed chronic lymphocytic leukemia after allogeneic peripheral blood stem cell transplantation does not abrogate the development of chronic GVHD

The graft vs. leukemia (GVL) effect is one of the most important factors of anti-tumor activity after allogeneic hematopoetic stem cell transplants (alloSCT). Its effectiveness depends mainly on the tumor biology as well as the tumor burden. Patients with a high tumor burden may not respond to GVL-effect despite otherwise sensitive biology. Campath-1H is known as an effective treatment of chronic lymphocytic leukemia (CLL). Due to its ability to induce profound immunosuppression, it has also been used as part of conditioning regimens before alloSCT. We report a patient, who received campath-1H in combination with docetaxel for treatment of chemotherapy and donor lymphocyte infusion resistant CLL after alloSCT, who developed shortly after discontinuation of treatment with campath-1H severe eosinophilia of the peripheral blood and typical clinical as well as histological signs of cutaneous chronic graft vs. host disease followed by complete clearance of CLL. The clinical course demonstrates the impact of the tumor burden on the GVL-effect, as well as the effectiveness of campath-1H in the presence chemo-resistance in a patient with CLL. Furthermore, the GVL effect was not abrogated by the use of campath-1H.     

Analyses of minimal residual disease based on Flt3 mutations in allogeneic peripheral blood stem cell transplantation

Purpose Activating Flt3 mutations are observed in about 30% of patients with acute myeloid leukaemia (AML) and individual Flt3 mutations are applicable for minimal residual disease (MRD) analyses.Methods We investigated the MRD status in four AML patients carrying different Flt3 mutations (three patients with Flt3 length mutations of the juxtamembrane domain, one patient carrying a mutation of the Flt3 tyrosine kinase domain, i.e. Flt3-TKD mutation) who underwent allogeneic peripheral blood stem cell transplantation (PBSCT). Residual leukaemia cells were retrospectively determined by real-time PCR at different time points.Results We can demonstrate a good correlation between the course of MRD status and clinical events in all four investigated patients.Conclusion These examples demonstrate the potential impact of Flt3 based MRD status not only after but also prior to allogeneic PBSCT.     

Fluctuations in plasma macrophage colony‐stimulating factor levels during autologous peripheral blood stem cell transplantation for haematologic diseases

Plasma macrophage colony‐stimulating factor (M‐CSF) levels were measured in 13 haematologic patients treated with autologous peripheral blood stem cell transplantation (PBSCT). Six of the patients showed an increase in M‐CSF peak levels (> 3000 pg/ml) during the conditioning and stem cell infusion period. The peak levels of M‐CSF in this phase correlated with thrombomodulin levels, indicating the endothelial origin of plasma M‐CSF. However, the M‐CSF levels were not influenced by TNFα. More patients with high M‐CSF levels (> 5000 pg/ml) suffered from organ failure than those with lower M‐CSF levels. These results suggest that high M‐CSF levels may correlate with cellular or organ damage in patients treated with PBSCT.     

Loss of cooperative function of transforming growth factor-beta signaling proteins, smad3 with embryonic liver fodrin, a beta-spectrin, in primary biliary cirrhosis.

Modulation of fibrogenesis, epithelial, and mesenchymal cell fates are prominent effects of transforming growth factor-beta (TGF-beta) signaling by Smad proteins. We have previously shown that Smad2 and Smad3 insufficiency leads to a loss of bile ducts. In addition, Smad3/4 activity is mediated by embryonic liver fodrin (ELF), a beta-Spectrin. In mouse elf(-/-) mutants and in liver explant cultures, loss of ELF function results in T lymphocytic proliferation and absent intrahepatic bile ducts. A similar phenotype is seen in a number of cholestatic diseases with progressive loss of intrahepatic bile ducts and fibrosis. However, the expression patterns of Smads or role of ELF in cholestatic and fibrotic liver diseases are not yet known. METHODS/RESULTS: We investigated the role of ELF in primary biliary cirrhosis (PBC), autoimmune hepatitis C, chronic viral hepatitis and in livers from mice deficient in Smad2/Smad3. We generated elf(+/-) mutant mice and analyzed for chronic liver disease and hepatocellular cancer (HCC) from 6 to 12 months. Perturbations in ELF expression were consistently seen only in PBC tissues. ELF expression was similarly aberrant in tissues from Smad2(+/-)/Smad3(+/-) mutant mice. Further studies indicated that ELF mislocalization is correlated with aberrant localization of Smad3 in some PBC tissues. Thirteen of 17 elf(+/-) mutant mice developed steatosis, fibrosis, hepatic dysplasia, with HCC in two mice. CONCLUSIONS: These results suggest that a compromised cytoarchitecture and polarized trafficking of TGF-beta signaling molecules, ELF and Smad3 are involved in the pathogenesis of PBC as well as HCC.     

Halofuginone inhibits multiple myeloma growth in vitro and in vivo and enhances cytotoxicity of conventional and novel agents

Multiple Myeloma (MM), a malignancy of plasma cells, remains incurable despite the use of conventional and novel therapies. Halofuginone (HF), a synthetic derivative of quinazolinone alkaloid, has recently been shown to have anti-cancer activity in various preclinical settings. This study demonstrated the anti-tumour activity of HF against a panel of human MM cell lines and primary patient-derived MM cells, regardless of their sensitivity to conventional therapy or novel agents. HF showed anti-MM activity in vivo using a myeloma xenograft mouse model. HF suppressed proliferation of myeloma cells alone and when co-cultured with bone marrow stromal cells. Similarly, HF induced apoptosis in MM cells even in the presence of insulin-like growth factor 1 or interleukin 6. Importantly, HF, even at high doses, did not induce cytotoxicity against CD40 activated peripheral blood mononuclear cells from normal donors. HF treatment induced accumulation of cells in the G(0) /G(1) cell cycle and induction of apoptotic cell death associated with depletion of mitochondrial membrane potential; cleavage of poly (ADP-ribose) polymerase and caspases-3, 8 and 9 as well as down-regulation of anti-apoptotic proteins including Mcl-1 and X-IAP. Multiplex analysis of phosphorylation of diverse components of signalling cascades revealed that HF induced changes in P38MAPK activation; increased phosphorylation of c-jun, c-jun NH(2)-terminal kinase (JNK), p53 and Hsp-27. Importantly, HF triggered synergistic cytotoxicity in combination with lenalidomide, melphalan, dexamethasone, and doxorubicin. Taken together, these preclinical studies provide the preclinical framework for future clinical studies of HF in MM.     

Serum levels of soluble CD30 in autologous peripheral blood stem cell transplantation

High-dose chemotherapy with peripheral blood stem cell transplantation (PBSCT) has become an important method of treatment for hematological and solid tumors. We examined levels of interleukin(IL)-4, interferon (IFN)γ, soluble (s) CD30, and sIL-2 receptor (R) before and after autologous PBSCT, and compared findings for PBSCT with those for bone marrow transplantation (BMT). We found significantly higher IL-4 levels 1 and 3 weeks after PBSCT than before PBSCT, while IFNγ levels remained almost unchanged after PBSCT. IFNγ levels were increased 3 weeks after BMT, although no increase in IL-4 level was observed. The serum sCD30 level was significantly higher 3 weeks after PBSCT, but not following BMT. For 34 samples on days 0 and 21 from 17 patients undergoing PBSCT, a strong correlation was observed between sCD30 and sIL-2R levels (r = 0.43, P < 0.01). However, no significant correlation between sCD30 and sIL-2R levels was found for BMT patients. These findings suggest that sCD30 is a useful marker for evaluating immunological activity following autologous PBSCT, and that the immunological conditions after autologous PBSCT may be associated with helper-T-cell-2-type immune responses. Received: 31 May 1999 / Accepted: 20 September 1999     

Peripheral monocytes and CD4+ cells are potential sources for increased circulating levels of TGF-beta and substance P in autoimmune myelofibrosis

Myelofibrosis is an uncommon phenomenon associated with a variety of neoplastic and inflammatory processes. Although there is evidence that cytokines elaborated by clonal malignant hematopoietic cells are implicated in myelofibrosis in primary hematologic disorders, there has been little data to date on the pathophysiology of myelofibrosis in autoimmune disorders. Here we report a case of autoimmune myelofibrosis with pancytopenia. Peripheral blood monocytes and CD4-positive lymphocytes produced significantly elevated levels of transforming growth factor beta (TGF-beta) compared to similar cells from healthy volunteer controls. TGF-beta has been implicated in the pathogenesis of myelofibrosis associated with primary hematological malignancies. Furthermore, substance P, previously linked to myelofibrosis, was also detected in elevated levels in the patient's serum and correlated negatively with the levels of the patient's blood counts. These findings suggest a role for both TGF-beta and substance P in the pathophysiology of autoimmune myelofibrosis. This is the first report of deregulated production of TGF-beta by monocytes in the pathobiology of autoimmune myelofibrosis.     

CD34 cell selection of peripheral blood progenitor cells using the CliniMACS device for allogeneic transplantation: clinical results in 102 patients

The present study investigated the effects of CD34(+) cell selection in 102 patients using the CliniMACS device. Patients were at high risk for the development of graft versus host disease (GvHD) because of age, or the use of a haploidentical, mismatched or unrelated donor (UD). The median age of the patients was 44 years. The CliniMACS procedure yielded 8.0 x 10(6) CD34(+) cells/kg and the number of residual T cells was 1.3 x 10(4)/kg (median). The median follow up was 20.6 months. The probability of graft failure was 7%. The rate of acute GvHD was low (compatible family donors 10%, UDs 17%, and haploidentical donors 26%) with no patient enduring more than grade II disease. The cumulative incidence of chronic GvHD at the median follow up after transplant was 15% for the compatible family donor group, 40% for the UD group and 78% in the group transplanted from a haploidentical donor Treatment failure was mainly because of transplant-related mortality, especially aspergillus infection, and not due to relapse. The probability of disease-free survival, stratified for the risk of treatment failure, was 27% for the high risk, 46% for the intermediate risk and 83% for the low risk group.     

Th1/Th2 cytokine profiles and their relationship to clinical features in patients following nonmyeloablative allogeneic stem cell transplantation

An imbalance in helper T-cell type 1 (Th1) and type 2 (Th2) cytokines is suggested to play an important role in the pathogenesis of acute graft-versus-host disease (aGVHD). The aim of this study was to investigate the cytokine bias acquired by T cells after transplantation and its possible influence on relapse of original malignancy. Cytokine levels by peripheral CD4 and CD8 T cells were tested at various pre- and post-transplant time points with fluorescein isothiocyanate-based intracellular cytokine assay after short-term in vitro mitogenic stimulation (phorbol myristate acetate + ionomycin). In both CD4+ and CD8+ cells, interferon (IFN)-gamma-producing cell populations increased, indicating a shift to a Th1 cytokine profile with aGVHD. IFN-gamma-producing T cells was significantly lower in patients who experienced relapse of original disease compared to those who showed no signs of relapse and compared to normal controls. Our studies demonstrate that aGVHD correlates with a Th1 bias and that Th1 response may potentiate an effective immune surveillance.     

Favourable effect of the combination of acute and chronic graft-versus-host disease on the outcome of allogeneic peripheral blood stem cell transplantation for advanced haematological malignancies

To assess the influence of graft-versus-host disease (GVHD) on the outcome of patients with advanced haematological malignancies (AHM) who received a primary, unmodified allogeneic peripheral blood progenitor cells transplant (allo-PBT) from a human leucocyte antigen (HLA) identical sibling donor, we analysed 136 patients with myeloid neoplasms (n = 70) or lymphoproliferative disorders (n = 66), transplanted at 19 Spanish institutions. Median age was 35 years (range 1-61). The cumulative incidence of relapse for all patients was 34% (95% CI, 26-42%), 41% (95% CI, 33-49) for patients without GVHD and 14% (95% CI, 3-25) (P = 0.001) for patients with acute and chronic GVHD. After a median follow-up of 11 months (range 2-49), 60 (44%) patients remained alive with an actuarial probability of overall survival and disease-free survival (DFS) at 30 months of 31% (95% CI, 21-41%) and 28% (95% CI, 17-39%) respectively. In patients surviving > 100 d, the low incidence of relapse in those with acute and chronic GVHD led to a DFS of 57% (95% CI, 38-76%) compared with a DFS of 34% (95% CI, 17-51%) in the remaining patients (P = 0.03). Our results indicate a reduced incidence of relapse for patients with AHM receiving an unmodified allo-PBT and developing acute and chronic GVHD, which results in an improved DFS.     

Production of B-cell growth factor interleukin 2 and gamma interferon by peripheral blood lymphocytes from patients with chronic lymphocytic leukemia of B-cell type

Chronic lymphocytic leukemia of B-cell type (B-CLL) is a malignant disease characterized by monoclonal proliferation of small lymphocytes of B-cell origin, usually associated with suppression of polyclonal B-cell activation (i.e., proliferation and differentiation). Normal human B-cell proliferation is controlled by different T-cell-derived lymphokines, including interleukin 2 (IL2) and gamma interferon (gamma-IFN), that account for the majority of the B-cell growth factor (BCGF) activity produced by mitogen-activated peripheral blood mononuclear cells (PBMCs). We have previously shown an increased and dysregulated secretion of IL2 in peripheral blood from patients with B-CLL. BCGF, IL2, and gamma-IFN productions by phytohemagglutinin (PHA)-stimulated PBMCs were investigated in 13 patients with active untreated B-CLL and 11 healthy donors. B-CLL PBMCs produced a significant amount of BCGF (6 U/ml) despite the low percentage of T cells (10%) associated with this disease as compared with that found in healthy donors (61%). BCGF production in normal controls and B-CLL patients was tripled after irradiation of PBMCs or addition of indomethacin. gamma-IFN secretion in B-CLL patients was decreased when compared with normal controls. Therefore, when gamma-IFN was calculated per fixed number of T cells, production was significantly higher in B-CLL patients than in normal controls, showing a dilution of the productive cells. This study suggests that T cells from B-CLL patients are functional in terms of BCGF production despite their decreased percentage and abnormalities in surface markers.     

Multiple myeloma: relationship between light chain isotype suppression, labelling index of plasma cells, and CD38 expression on peripheral blood lymphocytes

In this study we have investigated the relationship between the labelling index of plasma cells, the expression of CD38 positive lymphocytes in the peripheral blood, and light chain isotype suppression. This study confirms the relationship between plateau-phase disease and light chain isotype suppression (LCIS) and documents an inverse relationship between LCIS and CD38 positive lymphocytes (.001 less than P less than .01), which is similar to the relationship we have described with the expression of CD10 positive lymphocytes. PCA-1 is rarely expressed in the peripheral blood of patients with myeloma and does not fulfill a role as a marker of active vs. stable disease. There is no relationship between the labelling index of plasma cells and LCIS, because many patients can enter a stage of progressive disease and yet have a labelling index of less than 1% at that time, although a labelling index less than 1% is present in the majority of patients with LCIS. beta-2-microglobulin (beta 2M) also fails to differentiate these two phases of disease in myeloma and does not have a relationship with LCIS, CD38 expression, or CD10 expression. These data suggest that myeloma, like chronic granulocytic leukemia (CGL), can be considered as having two phases of disease: a stable or chronic phase disease, as identified by the presence of LCIS, the absence of CD10 and CD38 positive lymphocytes in the peripheral blood, and a low labelling index, and progressive disease, which is associated with the loss of LCIS and of, CD10 and CD38 positive lymphocytes in the peripheral blood and a high labelling index, although in many cases of progressive disease, the labelling index may also be low. beta 2M does not differentiate between these states.     

Transplantation with higher dose of natural killer cells associated with better outcomes in terms of non-relapse mortality and infectious events after allogeneic peripheral blood stem cell transplantation from HLA-matched sibling donors

BACKGROUND: Little is known about the role of the CD56+ natural killer (NK) cell dose on the outcome of allogeneic peripheral blood stem cell transplantation (PBSCT). Recently, higher dose of NK cells has been associated with a lower incidence of severe graft-versus-host disease (GVHD). The current study attempted to evaluate the effect of the NK cell dose on transplant outcomes in allogeneic PBSCT setting. METHODS AND MATERIALS: Sixty-one cytokine mobilized PBSC recipients were analyzed according to the infused dose of CD34+ cells and NK cells in relation to overall survival (OS), non-relapse mortality (NRM), GVHD, and infectious events. RESULTS: The group received a higher dose of NK cells (> or =5 x 10(7)/kg) showed a lower incidence of NRM (P = 0.0186) and infectious events (P = 0.0107). In a multivariate analysis, a higher dose of NK cells was correlated to better transplant outcomes for NRM (P = 0.042) with CD34+ cell dose (P = 0.018), and for infectious events (P = 0.013) with CD34+ cell dose (P = 0.016). Higher NK cell infusion group also showed a faster immune recovery in serial measurements at days +90, +180, and +365. CONCLUSIONS: High dose of NK cells may play an important role in improving transplant outcomes, in terms of reducing NRM and infectious events together with CD34+ cells.     

Peripheral blood CD5-positive B lymphocytes (B-1a cells) after allogeneic stem cell transplantation for acute myeloid leukaemia in humans

Background

Only few data are available in literature regarding the reconstitution of B-1a cells after allogeneic bone marrow transplantation performed for haematological malignancies.

Methods

In this study we used flow cytometry to assess the reconstitution of the peripheral blood B-1a cell compartment after allogeneic peripheral blood stem cell transplantation. Cytometric analyses were performed over time on 11 consecutive patients undergoing allogeneic peripheral blood stem cell transplantation for acute myeloid leukaemia in our Haematology Unit and the results were compared with available data regarding B-1a cell reconstitution after allogeneic bone marrow stem cell transplantation.

Results

In spite of an earlier recovery of B-1a cells in the peripheral blood after allogeneic bone marrow transplantation, the reconstitution of this B-cell subset was similar, regardless of the source of stem cells employed.

Conclusions

Further studies are necessary in order to clarify the origin of B-1a cells in humans in health and illness.     

Polymorphisms in the transforming growth factor-beta gene (TGF-beta) and the risk of advanced alcoholic liver disease.

BACKGROUND/AIMS: There are wide interindividual differences in the risk of developing alcoholic cirrhosis. Transforming growth factor beta(1) (TGF-beta(1)) is the main cytokine involved in liver fibrogenesis. The TGF-beta(1) gene is polymorphic at several sites and these polymorphisms are probably related to differences in the rate of TGF-beta(1) synthesis. Our aim has been to analyse the influence of the TGF-beta(1) gene polymorphisms in the predisposition to advanced alcoholic liver disease (ALD) in ethanol abusers. METHODS: TGF-beta(1) single nucleotide polymorphisms at positions -509 (C or T), +869 (C or T, codon 10), and +915 (C or G, codon 25) were examined in 165 alcoholics with advanced ALD and in 185 healthy controls. RESULTS: Among the 94 male patients with oesophageal varices, those carrying the GG genotype at position +915 were diagnosed at an older age than the remaining patients (age 52.1 years, standard deviation (SD) 9.9 vs. 45 SD 13.4, P=0.012). No other statistically significant differences were found in the distribution of the three TGF-beta(1) polymorphisms analysed individually or as combined haplotypes. CONCLUSIONS: The polymorphisms at the TGF-beta(1) gene analysed in this study are probably not related to the risk of advanced ALD.