Re-evaluation of progenitor thresholds and expectations for haematopoietic recovery based on an analysis of 810 autologous transplants: Implications for quality assurance.

Haematological engraftment was assessed in 804 autologous transplants. Neutrophil recovery occurred in over 99% within 14 d but platelet recovery was delayed beyond this time in 14·8%. Time to recovery was dependent on the progenitor cell dose infused. The minimum CD34+ cell threshold adopted in this study (2 × 106 /kg) was safe although recovery was faster with a dose >5 × 106 /kg. CD34+ cell doses of between 1 and 2 × 106 /kg were also acceptable if either the granulocyte-macrophage colony-forming cell dose exceeded 2 × 105 /kg or this dose was due to splitting a higher yield harvest. Prompt neutrophil recovery affords important quality assurance for laboratory processing.

Engraftment defect of cytokine-cultured adult human mobilized CD34(+) cells is related to reduced adhesion to bone marrow niche elements.

In vitro exposure of haematopoietic stem and progenitor cells (HSPC) to cytokines in expansion or gene therapy protocols reduces homing and engraftment in vivo. We have previously reported that this is related in part to altered tissue specificity of short-term homing, leading to loss of cells in non-haematopoietic tissues. Here we demonstrate that defective engraftment persists when cultured HSPC are transplanted by intrabone injection. Changes in engraftment function occur within 24 h of cytokine exposure, and are evident when engraftment is analysed solely in the injected bone. A novel ex vivo model of the bone marrow was developed, in which the attachment of infused HSPC in rodent long bones is reduced following culture with cytokines. Finally, cultured HSPC demonstrated reduced adhesion to N-cadherin, osteopontin and vascular cell-adhesion molecule-1, ligands present in bone marrow niches. These changes in adhesive function occur rapidly, and are not related to downregulation of the relevant receptors. Our findings suggest that cytokine exposure of adult human HSPC results in altered adhesion within bone marrow niches, further leading to reduced engraftment potential in vivo.

High response rate to donor lymphocyte infusion after allogeneic stem cell transplantation for indolent non-Hodgkin lymphoma.

The role of donor lymphocyte infusion (DLI) in the management of lymphoid malignancies after allogeneic stem cell transplantation (SCT) has not been clearly characterized. There is emerging evidence pointing to the effectiveness of this approach, particularly in patients with low-grade disease, although to date this has been reported only in small numbers of patients, and thus the utility of this treatment remains uncertain. A total of 28 patients with low-grade lymphoid malignancies previously treated with allogeneic SCT received a total of 68 infusions of donor lymphocytes. The diagnoses were indolent non-Hodgkin lymphoma (NHL; n = 23) and transformed NHL (n = 5), and the indications for DLI were progressive disease with or without mixed chimerism (MC) (n = 17) and persistent MC alone (n = 11). Escalating doses of cells were administered in the absence of graft-versus-host disease (GVHD) or continued disease progression, until stable full donor chimerism or disease response were achieved. The cumulative response rates after DLI to treat progressive disease and persistent MC were 76.5% and 91.6%, respectively. The major toxicity resulting from the use of donor lymphocytes was GVHD. The cumulative incidence of acute grade II-IV disease was 15%, and that of extensive chronic disease was 31%; there were no deaths resulting from GVHD. Seven patients had graft-versus-lymphoma responses without significant GVHD. These data support the existence of a clinically significant graft-versus-tumor effect in indolent NHL and suggest that this is an effective treatment for progressive disease after allogeneic SCT.

Peripheral blood stem cell yield in 400 normal donors mobilised with granulocyte colony-stimulating factor (G-CSF): impact of age, sex, donor weight and type of G-CSF used.

Mobilised peripheral blood is now the main source of stem cells collected from normal donors. We report our experience of mobilising and collecting 400 normal healthy donors using standardised procedures and techniques. Target recipient doses were reached with one aphaeresis in 63% of donors and with two aphereses in 81% of donors. Approximately 2% of donors yielded such low progenitor values that they were termed 'poor mobilisers'. There were minor effects of donor age, weight and sex and where possible, larger male donors under the age of 55 years should be selected. Two forms of granulocyte colony-stimulating factor (G-CSF) were used at the same dose and no significant difference was seen in the yield of CD34+ cells collected/l of blood processed. However, a greater number of granulocyte-macrophage colony-forming cells were harvested using lenograstim (glycosylated G-CSF) compared with filgrastim (non-glycosylated G-CSF; P = 0.002). CD34+ cell yields were also measured halfway through the aphaeresis procedure. This was found to be highly predictive of final yield and facilitated distribution of the stem cell product to other centres. The observation that CD34+ yields did not decline in the second half compared with the first half of aphaeresis suggests that the circulating cell numbers are not static.

Etoposide, methylprednisolone, cytarabine and cisplatin successfully cytoreduces resistant myeloma patients and mobilizes them for transplant without adverse effects.

Myeloma remains incurable with a median survival of 4 years, but outcome can be improved by the use of high-dose therapy. We used the etoposide, methylprednisolone, cytarabine and cisplatin (ESHAP) regimen as second-line therapy in 42 newly diagnosed myeloma patients who had failed vincristine, adriamycin and dexamethasone (VAD)- type therapy (n = 36), responded to first-line treatment but persisted in having significant residual marrow plasmacytosis (n = 5) or failed prior stem cell harvesting (n = 1), with the dual aim of improving disease response and mobilizing peripheral blood stem cells. Fourteen of 21 (67%) patients with no change or progressive disease after VAD responded to ESHAP; seven of 12 (58%) patients with minor response converted to partial response. Marrow plasmacytosis fell from a median of 52% at diagnosis to 23.5% after primary therapy and to15% after ESHAP. ESHAP chemotherapy was well-tolerated. There were 11 admissions due to febrile neutropenia (n = 7), nausea and vomiting (n = 2), pneumonia (n = 1) and perforated bowel (n = 1). Renal function deteriorated in 13 of 42 patients after ESHAP, but none required renal support. ESHAP mobilization was performed in 32 patients of whom 87% achieved a CD34(+) yield >2 x 10(6)/kg. In all, 38 patients proceeded to high-dose therapy. The overall survival for all patients was 62% at 4 years following ESHAP. We conclude that ESHAP has acceptable toxicity and efficient stem cell mobilizing capability, effectively cytoreduced this chemoresistant group of patients, and did not appear to adversely affect transplant outcome.

Impaired bone marrow homing of cytokine-activated CD34+ cells in the NOD/SCID model.

The reduced engraftment potential of hematopoietic stem/progenitor cells (HSPCs) after exposure to cytokines may be related to the impaired homing ability of actively cycling cells. We tested this hypothesis by quantifying the short-term homing of human adult CD34+ cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) animals. We show that the loss of engraftment ability of cytokine-activated CD34+ cells is associated with a reduction in homing of colony-forming cells (CFCs) to bone marrow (BM) at 24 hours after transplantation (from median 2.8% [range, 1.9%-6.1%] to 0.3% [0.0%-0.7%]; n = 3; P < .01), coincident with an increase in CFC accumulation in the lungs (P < .01). Impaired BM homing of cytokine-activated cells was not restored by using sorted cells in G0G1 or by inducing cell cycle arrest at the G1/S border. Blocking Fas ligation in vivo did not increase the BM homing of cultured cells. Finally, we tested cytokine combinations or culture conditions previously reported to restore the engraftment of cultured cells but did not find that any of these was able to reverse the changes in homing behavior of cytokine-exposed cells. We suggest that these changes in homing and, as a consequence, engraftment result from the increased migratory capacity of infused activated cells, leading to the loss of selectivity of the homing process.

The T-lineage-affiliated CD2 gene lies within an open chromatin environment in acute promyelocytic leukemia cells.

The nature of hemopoietic progenitors subject to leukemic transformation in acute myeloid leukemia (AML) has not been clearly defined. To address this issue, we have used DNase I hypersensitivity assays to study the chromatin structure surrounding the T-lineage-affiliated CD2 gene in the acute promyelocytic subtype of AML (APL). Upstream and downstream flanking regions of CD2 were found to be hypersensitive to DNase I in primary APL blasts, with an identical pattern of hypersensitive sites to those detected in cells of T-lineage. All of the sites were confirmed to be inaccessible to DNase I in B-lineage leukemia cells. The demonstration of T-cell-associated chromatin features in primary APL blasts has implications for the origin of APL that may arise in more primitive progenitors than previously considered to be the case.

Variable product purity and functional capacity after CD34 selection: a direct comparison of the CliniMACS (v2.1) and Isolex 300i (v2.5) clinical scale devices.

The two clinical scale devices currently available for CD34+ cell selection from peripheral blood stem cells (PBSC) apheresis products, the CliniMACS and the Isolex 300i, were compared directly by pooling and splitting two PBSC harvests collected on sequential days from 10 patients and processing half of each pooled harvest on each device. The CliniMACS product had significantly higher median CD34+ purity (90%vs 78%; P = 0.004) and lower median T-cell content (0.06%vs 0.44%; P = 0.003) compared with the Isolex 300i product. The median CD34+ yields were similar (64% and 60% respectively). However, when the functional capacities of the products were compared, the median recovery of colony-forming units was significantly greater from the Isolex 300i product (48%vs 38%; P = 0.035), as was expansion of cells in either erythroid or granulocytic lineage-specific liquid culture (2.1-fold more erythroid and 1.5-fold more granulocytic lineage progenitors on d 9 (P = 0.03 and 0.03 respectively). This was due to a higher proportion of apoptotic cells in the CliniMACS product (28%vs 18%; P = 0.007, annexin V binding). Hence, although the CliniMACS device yielded a higher purity product with fewer T cells, the Isolex 300i product contained fewer apoptotic cells and consequently had greater functional capacity in culture.