Steve Marley

Long-term clinical results of autologous infusion of mobilized adult bone marrow derived CD34+ cells in patients with chronic liver disease

Abstract.  Evidence is growing in support of the role of stem cells as an attractive alternative in treatment of liver diseases. Recently, we have demonstrated the feasibility and safety of infusing CD34+ adult stem cells; this was performed on five patients with chronic liver disease. Here, we present the results of long‐term follow‐up of these patients. Between 1 × 106 and 2 × 108 CD34+ cells were isolated and injected into the portal vein or hepatic artery. The patients were monitored for side effects, toxicity and changes in clinical, haematological and biochemical parameters; they were followed up for 12–18 months. All patients tolerated the treatment protocol well without any complications or side effects related to the procedure, also there were no side effects noted on long‐term follow‐up. Four patients showed an initial improvement in serum bilirubin level, which was maintained for up to 6 months. There was marginal increase in serum bilirubin in three of the patients at 12 months, while the fourth patients serum bilirubin increased only at 18 months post‐infusion. Computed tomography scan and serum α‐foetoprotein monitoring showed absence of focal lesions. The study indicated that the stem cell product used was safe in the short and over long term, by absence of tumour formation. The investigation also illustrated that the beneficial effect seemed to last for around 12 months. This trial shows that stem cell therapy may have potential as a possible future therapeutic protocol in liver regeneration.

Optimal timing for processing and cryopreservation of umbilical cord haematopoietic stem cells for clinical transplantation

Some of the factors that may influence the number and quality of cord blood haematopoietic progenitor cells available for transplantation have been investigated including site of collection, delayed processing after collection and cryopreservation protocol. We used the granulocyte–macrophage progenitor (CFU-GM) and erythroid burst-forming unit (BFU-E) assays to quantify progenitors. The capacity of CFU-GM to produce secondary colonies was used as a measure of progenitor cell quality. We found that: (1) there were no significant differences in total nucleated cells (TNC), mononuclear cells (MNC), CFU-GM or BFU-E numbers in paired specimens from the umbilical vein or veins at the base of the placenta. The potential of the CFU-GM to produce secondary colonies from the two sites was similar; (2) storing cord blood at room temperature or at 4°C resulted in a significant reduction in progenitor cell numbers beyond 9 h; and (3) cryopreservation following either controlled rate freezing or passive cooling reduced MNC numbers, viability and CFU-GM survival insignificantly but the potential of CFU-GM to produce secondary colonies was significantly reduced post cryopreservation (P = 0.04). We conclude that the yield of CB progenitor cells is not affected by the site of collection, but is adversely affected by delays between collection and cryopreservation. Furthermore, cryo- preservation reduced the CFU-GM potential to produce secondary colonies. Measures of progenitor cell quality as well as quantity may be relevant to assessing CB blood collections.

Cell biology of CML cells

At the cellular level, expansion of haemopoiesis in chronic myeloid leukaemia (CML) must involve some imbalance in cell production along the myeloid maturation pathway. The relevant kinetic parameters are cell loss by apoptosis and differentiation and cell gain by proliferation (self-renewal). In spite of the predominance of the BCR-ABL-positive leukaemic cells, some BCR-ABL-negative, presumably normal, progenitor cells remain for long periods in chronic phase CML. Thus, understanding the kinetics of CML and normal progenitor cells may lead to therapeutic strategies capable of reducing malignant cell growth and reactivating normal haemopoiesis.

A two-color BCR–ABL probe that greatly reduces the false positive and false negative rates for fluorescence in situ hybridization in chronic myeloid leukemia

The t(9;22) translocation resulting in the fusion of BCRand ABLgenes is pathognomonic in chronic myeloid leukemia (CML) and may be investigated at the molecular level using fluorescence in situ hybridization (FISH). Two‐color BCR–ABL probes visualizing one fusion signal (1F FISH) have high false positive rates (FPR) and false negative rates (FNR). The FPR is a result of the random spatial association of probe signals within normal interphase cells so that some cells appear to contain the BCR–ABLfusion gene. The FNR of 1F FISH probes depends on the distance between the BCR and ABL probes hybridized to the BCR–ABL fusion gene (≤368 kb); the “gap” between the signals causing the cell to be interpreted as normal. To overcome these difficulties, a two‐color probe was used, employing four yeast artificial chromosome (YAC) sequences that span the breakpoint regions of the BCR and ABL genes and that visualize the two fusion signals BCR–ABL and ABL–BCRin CML cells (2F FISH). The FNR for the 2F FISH probes was assessed on clonal Ph+ granulocyte‐macrophage‐colony‐forming cell (CFU‐GM) derived colonies and was reduced to 0.4% (2/450), compared with an FNR of 13.5% (111/823) with 1F FISH. The FPR in normal mononuclear cells for the 2F FISH was 0.19 ± 0.12% (3/1,700), whereas the FPR using 1F FISH was 4.5 ± 2.3% (63/1,294). The 2F FISH can thus be used to evaluate very small frequencies of BCR–ABL‐positive and ‐negative interphase cells and may be of use in the clinical monitoring of CML. Genes Chromosomes Cancer 23:109–115, 1998.

BCR/ABL-negative progenitors are enriched in the adherent fraction of CD34+ cells circulating in the blood of chronic phase chronic myeloid leukemia patients.

Philadelphia chromosome-positive (Ph+) hemopoietic cells predominate in patients with chronic myeloid leukemia (CML) in chronic phase, but some Ph− presumably normal stem cells persist in most patients. Ph− cells are relatively frequent, compared to mature cell populations, in primitive hemopoietic cell populations from CML patients. We have purified CD34+ cells from chronic phase CML blood and separated them into two fractions on the basis of adherence or non-adherence to tissue culture plastic. We also separated CD34+ CML cell populations into HLA-DRhi and HLA-DRlo fractions and CD38hi and CD38lo fractions by flow cytometry. The CD34+ cells that adhered to plastic were predominantly CD33−, CD38− and HLA−-DR; cells with these phenotypic properties were significantly rarer in the CD34+ non-adherent cell population (P = 0.008–0.02). Expression of p210 BCR/ABL mRNA by adherent, non-adherent, HLA-DRhi and HLA-DRloCD34+ cell subpopulations was demonstrated by RT-PCR. Using fluorescence in situ hybridization (FISH) in conjunction with BCR and ABL probes we detected Ph+ and Ph− cells in both adherent and non-adherent CD34+ cell fractions of 15/15 patients studied and in the HLA-DRlo or CD38lo sorted CD34+ cell fractions. The concentration of Ph− cells in the adherent CD34+ cell fraction was three-fold higher than in the non-adherent fraction (P = 0.001). Ph− adherent cells were detected in untreated CML patients and as late as 6 years after diagnosis of CML in patients treated with hydroxyurea (HU) or interferon-α (IFN-α). We conclude that whilst appreciable numbers of Ph− primitive hemopoietic progenitors are present in the circulation in untreated patients and also in treated patients in late chronic phase, the majority of cells expressing CD34 but not CD33, CD38 or HLA-DR antigens, are part of the CML clone.

Peripheral blood progenitor cell mobilisation alters myeloid, but not erythroid, progenitor cell self-renewal kinetics

Transplantation of progenitor cells which have been mobilised into the bloodstream (PBPC) following the administration of G-CSF results in more rapid neutrophil recovery than transplantation of bone marrow (BM). The reasons for the accelerated neutrophil engraftment are not clear, but would be explained by increased self-replication of myeloid progenitor cells (CFU-GM). We have used a CFU-GM replating assay to investigate myeloid progenitor self-replication, and quantification of subcolony formation during erythroid burst formation to quantify erythroid progenitor self-renewal. Secondary colony formation by CFU-GM, grown from PBPC and then replated was increased compared with secondary colony formation by BM CFU-GM (P = 0.0001); erythroid subcolony formation was not altered. There was no difference between the replating abilities of PBPC CFU-GM derived from allogeneic donors (normal individuals) and autologous donors (patients with malignant disease) although differences were found between subgroups of autologous donors. The increased replication of PBPC could not be accounted for by a reduction in progenitor cell apoptosis; PBPC CFU-GM contained slightly fewer apoptotic CD34+ cells than BM CFU-GM. The increased replication by PBPC CFU-GM was reversible because it declined when CFU-GM colonies were passaged through three sequential CFU-GM replating cycles. This decline in self-replication was more rapid than the decline seen in replated BM CFU-GM. The self-replication of PBPC CFU-GM, and subcolony formation by BFU-E could be further enhanced by exposure to cytokines in vitro. We conclude that mobilisation alters the replication kinetics of myeloid, but not of erythroid, progenitor cells, that mobilisation-induced events are of limited duration and that in vitro exposure to cytokines may modify PBPC progenitor cell kinetics. Bone Marrow Transplantation (2001) 27, 241–248.

Combination of interferon alpha with either Ara-C or ATRA in vitro reduces the selective action of interferon against CML CFU-GM

Although interferon (IFN)-α has no specific inhibitory effect on the plating efficiency of granulocyte-macrophage colony- forming cells (CFU-GM) from patients with chronic myeloid leukaemia (CML), it does selectively inhibit the replating ability (secondary colony formation) of CML CFU-GM. Thus, amplification of CFU-GM may be a target for IFN-α and other agents used in the treatment of CML. Here we examined whether cytarabine (Ara-C) or all-trans retinoic acid (ATRA) exert similar effects and whether they might in combination with IFN-α enhance its efficacy. We found that Ara-C preferentially inhibits the formation of CML CFU-GM compared to normal CFU-GM, but this inhibition was not increased by addition of IFN-α. When Ara-C was added to cultures containing IFN-α, the inhibition of replating by CML progenitors was abrogated. ATRA increased significantly the plating efficiency of normal CFU-GM. The addition of IFN-α to ATRA had no effect on CML or normal colony numbers. However, addition of ATRA to cultures containing IFN-α reversed the selective inhibition of CML CFU-GM replating seen in cultures containing IFN-α alone. In four IFN-α/Ara-C experiments, secondary CML patient-derived colonies were examined by fluorescence in situ hybridisation (FISH). All of them were Ph chromosome positive. No significant effects on CFU-GM production were observed when CML primitive haemopoietic progenitor cells were investigated in a delta (Δ) assay. Thus we conclude that combining IFN-α with Ara-C or ATRA neutralises the effect of IFN-α on CML CFU-GM. This observation provides a rationale for treating patients with alternating courses of IFN-α and Ara-C or ATRA, rather than giving either of these two agents in combination with IFN-α.

Discordant erythropoiesis in CML.

1 Cervantes F, Colomer D, Vives-Corrons JL, Rozman C, Montserrat E. Chronic myeloid leukemia with thrombocythemic onset: a CML subtype with distinct hematological and molecular features? Leukemia 1996; 10: 1241–1243. 2 Kwong YL, Chiu EKW, Liang RHS, Chan V, Chan TK. Essential thrombocythemia with BCR/ABL rearrangement. Cancer Genet Cytogenet 1996; 89: 74–76. 3 Michiels JJ, Prins ME, Hagermeijer A, Brederoo P, van der Meulen J, van Vliet HHD, Abels J. Philadelphia chromosome-positive thrombocythemia and megakaryoblast leukemia. Am J Clin Pathol 1987; 88: 645–652. 4 Richards EM, Bloxham DM, Nacheva E, Marcus RE, Green AR.

PI3-Kinase may be involved in cml progenitor cell responses to interferon alfa and STI571

Abstract We investigated whether PI-3 kinase is involved in responses of CML progenitor cells to interferon(IFN)-α and STI571 and/or biological abnormalities in CML. We used a colony (CFU-GM) replating assay, to measure the self-renewal capacity of CFU-GM, expressed as the area-under-the-curve (AUC) of the distribution of secondary CFU-GM numbers. The AUC is greater for CML than for normal CFU-GM (p=0.029). Using an aggregation assay, we found that the aggregation index (AI) of CD34+ cells in the presence of QBEND10 antibody is subnormal in CML IFN-α and STI571 reduce the AUC of CML CFU-GM and increase the AI of CD34+ CML cells. This agrees with our finding that AUC and AI are inversely related. We evaluated the role of PI3-kinase by performing experiments in the presence of 1.0 μM, wortmannin (WM), a PI3-kinase inhibitor. WM reduced the AUC to 77–8% (mean–sem; n=13) of control levels. This result was not significantly different from the effects of IFN-α or STI571 (p=0.87 and 0.38). However, the individual responses to WM correlated better with response to STI571 (r=0.68) than with response to IFN-α (r=0.48). WM increased the AI of CML CD34+ cells 2.5–0.35-fold (n=8). This was not significantly different from the effects of IFN-α or STI571 (p=0.63 and 0.14). The responses to WM and IFN-α in the aggregation assay did not correlate (r=0.0) whilst those to WM and STI571 did (r=0.95). We conclude that (1) IFN-α and STI571 influence the same biological functions in CML but may operate through different biochemical pathways; (2) the PI-3 kinase pathway is more important for responses to STI571 than for responses to IFN-α and (3) the PI-3 kinase pathway can modulate progenitor proliferation and aggregation in CML.

Cml patients with advanced phase disease retain clonogenic progenitor cells which are responsive to interferon alfa and STI571 in vitro

Abstract We investigated if progenitor cells from CML patients with accelerating/transformed (AP) disease differ from chronic phase (CP) cells in their sensitivities to interferon(IFN)-α and STI571. We used a colony replating assay which measures the self-renewal capacity of clonogenic cells (CFU-GM). The results are expressed as the area-under-the-curve (AUC) of the cumulative distribution obtained by plotting the numbers of secondary colonies produced by replating individual primary colonies. The AUC for CP-CML CFU-GM is significantly greater than that for normal marrow CFU-GM (79.6–5.0 (mean–sem), n=90 vs 63–4.4, n=63; p=0.029, t test). CP CFU-GM exposed to pharmacological concentrations of IFN-α (50U/ml) or STI571 (0.1μM) exhibit a reduced AUC (respectively 63–5% and 52–7% of control level; n=33 and 30) but normal CFU-GM show an increase. Moreover, responses to IFN-α and STI571 are closely correlated in samples from individual CP patients (r=0.74). The AUC for CFU-GM from 16 patients with AP disease was increased compared with CP results (114–3.1; p=0.001). It was reduced to 52–7% of control levels by IFN-α and 41–6% by STI571. These results were not significantly different from the corresponding values for CP-CML cells (p=0.4 and 0.6). Finally, the in vitro responses to IFN-α and STI571 of progenitor cells from patients with AP disease were related to one another (r=0.6). Thus: (1) CML patients retain progenitor cells with similar kinetic properties and responses to IFN-α and STI571 throughout the course of their disease; (2) clinical resistance to IFN-α may not be intrinsic to progenitors (3) IFN-α and STI571 may be effective treatment of at least selected AP-CML patients.