Linda Richmond

CD34 positive PBPC expanded ex vivo may not provide durable engraftment following myeloablative chemoradiotherapy regimens

We have previously demonstrated that CD34+ cells, selected from peripheral blood progenitor cells (PBPC), can be expanded in ex vivo culture and can be infused in tandem with unmanipulated PBPC with little or no toxicity. In this study, four patients (two non-Hodgkin’s lymphoma (NHL), two multiple myeloma (MM)) received myeloablative conditioning prior to stem cell rescue using ex vivo expanded cells alone. The two patients with NHL received cyclophosphamide and total body irradiation (CY/TBI) and the two patients with MM, busulphan and melphalan (Bu/M). One case received an inadequate CFU-GM dose, despite expansion, and in one case the expanded cells were contaminated. No definitive conclusions may therefore be drawn concerning engraftment in these two cases. However, the other two cases received high doses of committed progenitors. Following infusion of the expanded material, all four patients failed to show sustained neutrophil engraftment and none showed evidence of platelet engraftment. Back-up, unmanipulated PBPC were therefore infused on days 14, 34, 32 and 28 and subsequently all four cases achieved satisfactory engraftment of both neutrophils and platelets. In conclusion, we feel that, CD34+ cells, expanded ex vivo using the conditions described in this report, may not provide durable engraftment following fully myeloablative conditioning.

Lonafarnib reduces the resistance of primitive quiescent CML cells to imatinib mesylate in vitro.

Recent studies indicate that a rare population of primitive quiescent BCR-ABL+ cells are innately insensitive to imatinib mesylate (IM) and persist after IM therapy of patients with chronic myeloid leukemia (CML). New approaches to the eradication of these cells are therefore likely to be crucial to the development of curative therapies for CML. We have now found that Ara-C, LY294002 (a PI-3 (phosphatidylinositol-3′ kinase) kinase inhibitor), 17AAG (a heat-shock protein (HSP)-90 antagonist) and lonafarnib (a farnesyltransfease inhibitor) all enhance the toxicity of IM on K562 cells and on the total CD34+ leukemic cell population from chronic phase CML patients. However, for quiescent CD34+ leukemic cells, this was achieved only by concomitant exposure of the cells to lonafarnib. Ara-C or LY294002 alone blocked the proliferation of these cells but did not kill them, and Ara-C, LY294002 or 17AAG in combination with IM enhanced the cytostatic effect of IM but did not prevent the subsequent regrowth of the surviving leukemic cells. These studies demonstrate the importance of in vitro testing of novel agents on the subset of primary leukemic cells most likely to determine long-term treatment outcomes in vivo.

Stage-specific alterations in serum levels of G-CSF in chronic myeloid leukaemia

Chronic myeloid leukaemia (CML) is a myeloproliferative disease of stem cell origin, characterised by the presence of the Philadelphia (Ph) chromosome and a BCR–ABL fusion gene that is believed to be causal to the disease. CML cells display changes in many intracellular signalling pathways normally activated by growth factor receptors, suggesting a variety of mechanisms by which these cells might acquire reduced dependence on normal growth factor stimulation or decreased responsiveness to inhibitory factors. This is now known to involve the autocrine production of growth factors, specifically IL-3 and G-CSF, by the CD34 + leukaemic cells in chronic phase (CP) CML patients. Such a mechanism was initially suggested by studies of the effects of enforced expression of p210 BCR–ABL in immortalised growth factor-dependent cell lines. Autocrine production of IL-3 and GM-CSF was later confirmed in p210 BCR–ABL -transduced murine bone marrow cells both before and after their transplantation into irradiated mice. It was therefore of interest to determine whether autocrine growth factor production by primary CML cells in patients also increases their levels in the periphery, and if so, how these change with disease progression. We now describe the interesting results of an analysis of serum samples from a large series of CML patients that show stage-specific changes in circulating levels of G-CSF but not IL-3.

Selection of CD34+ cells from cryopreserved PBPC can be significantly improved by the addition of recombinant human DNase (Pulmozyme).

BACKGROUND It has been reported previously that PBPC can be recovered from cryopreservation and can be efficiently CD34-selected, to provide a product of high purity (> 80% CD34) with good yield (> 50% recovery). METHODS In this study, we have investigated the effects of thawing and CD34-selecting cryopreserved PBPC in the presence of recombinant human deoxyribonuclease (rhDNase; Pulmozyme) and magnesium chloride (MgCl2 injection). RESULTS The addition of Pulmozyme and MgCl2 significantly improves the yield of CD34+ cells, compared with the standard procedure (65.2% and 39.7%, respectively). Following CD34 selection, significantly greater recovery of CFC in the selected fraction can be obtained from Pulmozyme-treated cells, compared with standard cells. The use of recombinant human Pulmozyme and i.v. grade MgCl2 should facilitate the application of this procedure to the clinical setting. CD34+ cells selected from cryopreserved PBPC, can in turn be cryopreserved for a second time. When thawed, these cells still retained good viability (> 80%). DISCUSSION Cells originally processed in the presence of Pulmozyme gave significantly superior yields of CD34+ cells and CFC compared with standard cells. The functional ability of these CD34+ cells was demonstrated further in an ex vivo expansion culture system with extensive proliferation of cells and CFC. In addition, the presence of significant numbers of primitive hemopoietic cells could be readily demonstrated in a cobblestone-area forming assay.

CD34 Selection and EX Vivo Expansion of Haemopoietic Progenitor Cells: A Review of Laboratory Methodology

The CD34 antigen is expressed on haemopoietic stem and progenitor cells. A number of strategies have been developed which allow the selection and purification of CD34(+) cells from bone marrow, peripheral blood, and umbilical cord blood. Transplantation studies have amply demonstrated that rapid and durable engraftment can be achieved following reinfusion of selected CD34(+) cells. More recently, techniques have become available which can produce extensive proliferation of haemopoietic progenitor cells in ex vivo culture systems. The most popular method involves a simple liquid suspension culture system supplemented with a range of cytokines. The degree of expansion and, indeed, the types of cells produced can be significantly influenced by culture conditions like the choice of cytokines, duration of culture, starting cell concentration, and type of culture vessel. Despite many laboratory investigations, there have been few clinical trials using ex vivo expanded cells. Although it has been shown that infusion of ex vivo cultured cells is well tolerated with no associated toxicity, there is no evidence to date that these culture systems sustain sufficient numbers of haemopoietic long-term repopulating cells to secure durable engraftment following myeloablative therapy. Clearly, the major goal is to define culture conditions which will produce true stem cell expansion.

CML leukapheresis products can be enriched for CD34 + cells and simultaneously depleted of CD15 + cells using a simple Ab cocktail

BACKGROUND CML progenitor-cell studies would be greatly facilitated if samples could be repeatedly accessed from a source of well-characterized cells. The present study was designed to develop a simple, inexpensive Ab cocktail that would provide subpopulations of cells enriched for CD34+ cells and simultaneously depleted of CD15+ mature myeloid cells. METHODS Cells from leukapheresis products from CML patients at diagnosis were incubated with each of two Ab cocktails. The standard cocktail (debulking, DB), containing 11 Abs, is recommended for obtaining a highly enriched population of CD34+ cells. The efficacy of an alternative, simpler cocktail (CML custom, CC), containing only four Abs was tested. The recoveries of CD34+ cells, CD15+ cells, colony-forming unit granulocyte-macrophage, and LTCIC were monitored. The samples were then cryopreserved, thawed, and the recoveries remeasured. RESULTS The purity of CD34+ cells was significantly superior using the DB cocktail than with the CC cocktail. Conversely, using the CC cocktail, the yield of CD34+ cells was significantly higher compared to the DB cocktail. These results were maintained even when the amount of Ab was reduced 10-fold. Both Ab cocktails consistently removed > 99% of the CD15+ cells. Consistent with the CD34+ cell-enrichment data, higher colony-forming cell (CFC) frequencies were obtained with the DB cocktail, although superior yields of CFC were obtained with the CC cocktail. After cryopreservation and thawing the yield of CD34+ cells remained high, and a further reduction in the number of CD15+ cells was obtained. DISCUSSION A method is described that allows the rapid and efficient debulking of large CML samples. This strategy will provide a source of well-characterized CML stem/progenitor cells that can be repeatedly accessed.

Detection of IL-3 and g-csf mRNA and protein in CD34+ progenitor cells and serum from cml patients

Abstract Our previous studies demonstrated that primitive subsets of leukaemic cells isolated from patients with chronic phase CML exhibit growth factor independent proliferation, in both single cell and bulk cultures, which is at least partially dependent on autocrine production of bioactive IL-3 and to a lesser degree G-CSF (PNAS 96; 12804, 1999). In those studies ∼ 85% of the cases investigated were CML patients in whom the stem cell compartment (LTC-IC) was already predominantly Ph + . Since such cases represent less than 10% of newly diagnosed patients, it was of interest to examine IL-3 and G-CSF mRNA expression in CD34 + progenitor cells and any corresponding protein production in serum in a larger series of CML patients independent of the Ph status of their LTC-IC compartment. CD34-enriched lineage-negative cells were isolated using STEMSep TM from leucapheresis samples obtained at diagnosis from chronic phase patients and viable, propidium iodide (PI)-negative, annexin V-negative, CD34 + cells then isolated by FACS. Total RNA was extracted from ≥ 5 × 10 4 such cells and c-ABL, BCR-ABL, IL-3 and G-CSF expression examined using a 2-step RT-PCR procedure. 8 of the 10 CML samples tested to date are positive for IL-3 expression. Using sensitive ELISA kits, G-CSF was detected at significantly lower levels in the sera of CML patients (7.2 ± 2.9 pg/ml, n=13) than in normal controls (24.5 ± 3.2 pg/ml, n=9, p=0.001), with levels correlating inversely to the WBC. In untreated patients with elevated WBC (45–166 × 10 9 /L) G-CSF levels proved to be undetectable. In patients on hydroxyurea, in whom the WBC was controlled ( 9 /L), levels were low but above the threshold for detection (2–11 pg/ml). Of interest, in patients who had achieved a complete cytogenetic remission following either allogeneic BMT or interferon therapy, levels were within the normal range. These findings support the proposed negative feedback mechanism between peripheral neutrophil count and serum G-CSF levels. IL-3 was detected in sera from 1 of 9 normal controls and 5 of 29 CMLs (range 1–35 pg/ml), 3 of whom were in accelerated phase. These results confirm that IL-3 mRNA expression in the progenitor compartment is typical of the majority of cases of CML. However, this does not necessarily lead to detectable levels of this growth factor in the circulation suggesting its effects in vivo may be largely confined to extravascular regions of the marrow and other tissues that are infiltrated by CD34 + leukaemic cells.