L. B. To

Circulating autologous stem cells collected in very early remission from acute non-lymphoblastic leukaemia produce prompt but incomplete haemopoietic reconstitution after high dose melphalan or supralethal chemoradiotherapy.

Summary. Haemopoietic reconstitution (HR) using autologous peripheral blood stem cells (PBSC) was attempted after intensive chemotherapy or chemoradiotherapy in two patients with relapsed acute non‐lymphoblastic leukaemia (ANLL). The PBSC were collected by leukapheresis very early in first remission and cryopreserved in liquid nitrogen. Both patients demonstrated early evidence of trilineage engraftment. The first patient received melphalan 200 mg/m2 followed by rescue with 1·3 × 108 mononuclear cells/kg body weight containing 29 × 104 granulocytemacrophage progenitor cells (CFU‐GM)/kg, and HR was evident by Day 14. The second patient was treated with supralethal chemoradiotherapy followed by rescue with 3·0 × 108 mononuclear cells/kg containing 23 × 104 CFU‐GM/kg. He demonstrated early engraftment with near normal peripheral blood counts by Day 16. There was a subsequent fall in both bone marrow cellularity and peripheral blood counts to a level of low but persistent activity. There was a further phase of haematological recovery from 8 weeks following transplantation with an increase in peripheral blood counts and bone marrow cellularity until final relapse at 13 weeks. This study demonstrates that circulating stem cells have haemopoietic reconstitutive capacity, previously only shown with buffy coat cells from chronic granulocytic leukaemia. The minimum number of PBSC required for satisfactory engraftment remains unknown, although it seems probable that the ratio of pluripotent stem cells to committed progenitor cells is lower in very early remission peripheral blood than in either allogeneic normal bone marrow or autologous bone marrow collected laterin stable remission. The question of leukaemic contamination of the PBSC remains to be answered.

High levels of circulating haemopoietic stem cells in very early remission from acute non-lymphoblastic leukaemia and their collection and cryopreservation

Summary. Circulating myeloid progenitor cells (PB CFU‐GM) were measured in the peripheral blood of 13 patients with acute non‐lymphoblastic leukaemia (ANLL) as they entered first remission. The mean of the recorded peak levels was 2796 × 105 CFU‐GM/1, representing a 2 5‐fold increase above the mean value in normal subjects. These elevated levels of PB CFU‐GM occurred regularly during the very early remission phase when platelet counts rose rapidly. Five of the patients had PB mononuclear cells collected by continuous‐flow leukapheresis during this early recovery phase. CFU‐GM were assayed as a measure of the number of haemopoietic stem cells in each collection. The cells were concentrated and then cryopreserved in liquid nitrogen. Leukapheresis was also performed on five normal subjects for comparison. Low numbers of CFU‐GM were harvested from normal subjects, mean 033 ± 0‐06 × 104 CFU‐GM/kg body weight for each leukapheresis. In ANLL patients entering remission, however, very large numbers of CFU‐GM were regularly harvested. A mean of 11 ± 2 × 104 CFU‐GM/kg body weight were cryopreserved after each leukapheresis, representing 5 times the number of CFU‐GM considered necessary for successful autologous haemopoietic reconstitution. Haemopoietic stem cell viability was assessed after varying periods of cryopreservation. There was no significant stem cell loss after up to 24 months storage. Thus, it is possible to collect and cryopreserve large numbers of CFU‐GM and by inference pluripotent haemopoietic stem cells from the peripheral blood of patients with ANLL during very early remission. The possible biological and therapeutic implications are discussed.

Osteoprotegerin (OPG) is localized to the Weibel-Palade bodies of human vascular endothelial cells and is physically associated with von Willebrand factor†

Recent studies demonstrate roles for osteoprotegerin (OPG) in both skeletal and extra‐skeletal tissues. Although its role in preventing osteoclast (OC) formation and activity is well documented, emerging evidence suggests a role of OPG in endothelial cell survival and the prevention of arterial calcification. In this communication, we show that vascular endothelial cells in situ, and human umbilical vein endothelial cells (HUVEC) in vitro, express abundant OPG. In HUVEC, OPG co‐localizes with P‐selectin and von Willebrand factor (vWF), within the Weibel‐Palade bodies (WPB). Treatment of HUVEC with the pro‐inflammatory cytokines, tumor necrosis factor (TNF)‐α and IL‐1β, resulted in mobilization from the WPBs and subsequent secretion of OPG protein into the culture supernatant. Furthermore, TNF‐α treatment of HUVEC resulted in a sustained increase in OPG mRNA levels and protein secretion over the 24‐h treatment period. Reciprocal immunoprecipitation experiments revealed that while not associated with P‐Selectin, OPG is physically complexed with vWF both within the WPB and following secretion from endothelial cells. Interestingly, this association was also identified in human peripheral blood plasma. In addition to its interaction with vWF, we show that OPG also binds with high avidity to the vWF reductase, thrombospondin (TSP‐1), raising the intriguing possibility that OPG may provide a link between TSP‐1 and vWF. In summary, the intracellular localization of OPG in HUVEC, in association with vWF, together with its rapid and sustained secretory response to inflammatory stimuli, strongly support a modulatory role in vascular injury, inflammation and hemostasis.

The Nitrogen-Containing Bisphosphonate, Zoledronic Acid, Influences RANKL Expression in Human Osteoblast-Like Cells by Activating TNF-α Converting Enzyme (TACE)†

Bisphosphonates are used to prevent osteoclast‐mediated bone loss. Zoledronic acid inhibits osteoclast maturation indirectly by increasing OPG protein secretion and decreasing transmembrane RANKL expression in human osteoblasts. The decreased transmembrane RANKL expression seems to be related to the upregulation of the RANKL sheddase, TACE.

Hypoxia-inducible factor-2 is a novel regulator of aberrant CXCL12 expression in multiple myeloma plasma cells

Background Multiple myeloma is an incurable malignancy of bone marrow plasma cells. Progression of multiple myeloma is accompanied by an increase in bone marrow angiogenesis. Studies from our laboratory suggest a role for the CXCL12 chemokine in this process, with circulating levels of CXCL12 correlating with bone marrow angiogenesis in patients with multiple myeloma. While the mechanisms responsible for aberrant plasma cell expression of CXCL12 remain to be determined, studies in other systems suggest a role for hypoxia and hypoxia-inducible transcription factors. Design and Methods The expression of hypoxia-inducible factor protein was examined in patients’ bone marrow biopsy specimens using immunohistochemistry. The hypoxic regulation of CXCL12 was examined in multiple myeloma plasma cell lines using polymerase chain reaction and western blotting. The role of hypoxia-inducible factors-1 and -2 in the regulation of CXCL12 expression was examined using over-expression and short hairpin RNA knockdown constructs, electrophoretic mobility shift assays and chromatin immunoprecipitation. The contribution of CXCL12 to hypoxia-induced angiogenesis was examined in vivo using a subcutaneous murine model of neovascularization. Results Strong hypoxia-inducible factor-2 protein expression was detected in CD138+ multiple myeloma plasma cells in patients’ biopsy specimens. Prolonged exposure to hypoxia strongly up-regulated CXCL12 expression in multiple myeloma plasma cells and hypoxia-inducible factor-2 was found to play a key role in this response. Promoter analyses revealed increased hypoxia-inducible factor-2 binding to the CXCL12 promoter under hypoxic conditions. Over-expression of hypoxia-inducible factor in multiple myeloma plasma cells strongly induced in vivo angiogenesis, and administration of a CXCL12 antagonist decreased hypoxia-inducible factor-induced angiogenesis. Conclusions Hypoxia-inducible factor-2 is a newly identified regulator of CXCL12 expression in multiple myeloma plasma cells and a major contributor to multiple myeloma plasma cell-induced angiogenesis. Targeting the hypoxic niche, and more specifically hypoxia-inducible factor-2, may represent a viable strategy to inhibit angiogenesis in multiple myeloma and progression of this disease.

Peripheral blood stem cell autografting: a new therapeutic option for AML?

The last annotation on peripheral blood stem cell (PBSC) transplantation in the treatment of malignant disease proposed that clidical trials should be conducted to investigate the haemopoietic reconstitutive capacity of PBSC (Barr & McBride. 1982). A number of recent reports (Juttner et al. 1985. 1986a. b: Korbling et al. 1986: Reiffers et nl. 1986a: Bell et al, 1986a: Tilly et al, 1986: Castaigne et al. 1986: Kessinger et al. 1986: Stiff et nl. 1986) have now shown that PBSC are capable of haemopoietic reconstitution. These and other unreported cases have been summarized in a comprehensive review by Bell et a1 (1986b). PBSC are usually collected by continuous flow leukapheresis during periods of haemopoietic regeneration in very early remission from acute leukaemia or after chemotherapy in lymphoma .and solid tumours (Richman et al, 1976: Lohrmann et al, 1979). The collections contain higher numbers of myeloid pregenitor cells (CFU-GM)/kg BW than bone marrow (To et al, 1984: Bell at a] , 1986b: Gorin. 1986) and the peripheral blood CFUGM yield appears to be greater following intensive chemotherapy than after less myelosuppressive regimes (Reiffers et al. 1986b). There has been increasing interest in autografting using PBSC collected during very early remission of acute myeloid leukaemia (AML) because cells collected during this period of intense haemopoietic recovery may have low or ‘absent leukaemic contamination and give rise to rapid haemopoietic reconstitution (To et al, 1984: Juttner ef al, 1985). Because early animal (Micklem ef al, 1975: Nothdurft et al, 1977: Abrams et al, 198 1 ) and human (Goldman. 1978: Hershko et al, 19 79: Abrams et al, 1980: Korbling et al, 198 1: McCarthy & Goldman, 1984) studies using PBSC for autografting cast doubt on their haemopoietic reconstitutive capacity, the initial autografts in AML were mostly carried out in patients in relapse. The haemopoietic reconstitutive capacity of PBSC has now been established and the next logical step is to consider PBSC autografting in first remission of AML, in accordance with the current strategy in autologous bone marrow transplantation (Gorin et al, 1986: Cahn et al, 1986: Goldstone et al, 1986: Linch & Burnett, 1986). Several questions need to be answered before PBSC autografting during first remission of AML can be recommended as a new therapeutic option: Can the haemopoietic reconstitutive capacity of PBSC be predicted? Is the rate of haemopoietic recovery faster following PBSC autografting than following bone marrow autografting? Is immune reconstitution more rapid and complete following PBSC autografting than following bone marrow autografting? Are there leukaemic cells among PBSC collected during very early Correspondence: Dr C. A. Juttner. Division of Haematology. Institute of Medical and Veterinary Science, PO Box 14. Rundle Street, Adelaide, South Australia, Australia 5000. remission of AML? PBSC may be relatively deficient in stromal activity (To et al, 1986) but no data are yet available.

Distinct evolution and dynamics of epigenetic and genetic heterogeneity in acute myeloid leukemia

Genetic heterogeneity contributes to clinical outcome and progression of most tumors, but little is known about allelic diversity for epigenetic compartments, and almost no data exist for acute myeloid leukemia (AML). We examined epigenetic heterogeneity as assessed by cytosine methylation within defined genomic loci with four CpGs (epialleles), somatic mutations, and transcriptomes of AML patient samples at serial time points. We observed that epigenetic allele burden is linked to inferior outcome and varies considerably during disease progression. Epigenetic and genetic allelic burden and patterning followed different patterns and kinetics during disease progression. We observed a subset of AMLs with high epiallele and low somatic mutation burden at diagnosis, a subset with high somatic mutation and lower epiallele burdens at diagnosis, and a subset with a mixed profile, suggesting distinct modes of tumor heterogeneity. Genes linked to promoter-associated epiallele shifts during tumor progression showed increased single-cell transcriptional variance and differential expression, suggesting functional impact on gene regulation. Thus, genetic and epigenetic heterogeneity can occur with distinct kinetics likely to affect the biological and clinical features of tumors.

Comparison of myeloma cell contamination of bone marrow and peripheral blood stem cell harvests

It could be speculated for patients with myeloma and other lymphoproliferative disorders that peripheral blood stem cells may be preferable to bone marrow for autologous transplantation because they may be less contaminated by neoplastic cells. To test this possibility, the immunoglobulin heavy chain gene rearrangement and limiting dilution polymerase chain reaction were used to sensitively quantify myeloma cells in bone marrow and peripheral blood stem cell collections, taken at a similar time, from eight patients with multiple myeloma. Levels of residual disease in the peripheral blood stem cell harvests were variable and did not reflect the tumour burden in the marrow. Peripheral blood stem cells contained 1.7 to 23 700‐fold fewer myeloma cells compared with the bone marrow and would have resulted in reinfusion of 0.08 to 59 480‐fold fewer myeloma cells based on total reinfused CFU‐GM and 0.24 to 24 700‐fold fewer myeloma cells based on total reinfused nucleated cells. Assuming that the proportion of clonogenic myeloma cells is equivalent, peripheral blood stem cells may be better than bone marrow as a source of haemopoietic stem cells for transplantation in multiple myeloma. The clinical follow‐up suggested that patients transplanted with peripheral blood stem cells containing a low number of myeloma cells had better disease control than those transplanted with peripheral blood stem cells containing a high number.

Blocking cytokine signaling along with intense Bcr-Abl kinase inhibition induces apoptosis in primary CML progenitors

In chronic myeloid leukemia (CML) cell lines, brief exposure to pharmacologically relevant dasatinib concentrations results in apoptosis. In this study, we assess the impact of intensity and duration of Bcr-Abl kinase inhibition on primary CD34+ progenitors of chronic phase CML patients. As CML cells exposed to dasatinib in vivo are in a cytokine-rich environment, we also assessed the effect of cytokines (six growth factors cocktail or granulocyte-macrophage colony-stimulating factor (CSF) or granulocyte-CSF) in combination with dasatinib. In the presence of cytokines, short-term intense Bcr-Abl kinase inhibition (⩾90% p-Crkl inhibition) with 100 nM dasatinib did not reduce CD34+ colony-forming cells (CFCs). In contrast, without cytokines, short-term exposure to dasatinib reduced CML-CD34+ CFCs by 70–80%. When cytokines were added immediately after short-term exposure to dasatinib, CML-CD34+ cells remained viable, suggesting that oncogene dependence of these cells can be overcome by concomitant or subsequent exposure to cytokines. Additional inhibition of Janus tyrosine kinase (Jak) activity re-established the sensitivity of CML progenitors to intense Bcr-Abl kinase inhibition despite the presence of cytokines. These findings support the contention that therapeutic strategies combining intense Bcr-Abl kinase inhibition and blockade of cytokine signaling pathways can be effective for eradication of CML progenitors.

Successful mobilization of peripheral blood stem cells after addition of ancestim (stem cell factor) in patients who had failed a prior mobilization with filgrastim (granulocyte colony-stimulating factor) alone or with chemotherapy plus filgrastim.

Summary:This study assessed the ability of recombinant human stem cell factor (rHuSCF) to mobilize stem cells in 44 patients who had failed a prior mobilization (CD34+ yield 0.5–1.9 × 106/kg BW) with filgrastim-alone or chemotherapy-plus-filgrastim. The same mobilization regimen was used with the addition of rHuSCF. In the filgrastim-alone group (n=13), rHuSCF 20 μg/kg was started 3 days before filgrastim and continued for the duration of filgrastim. In the chemotherapy-plus-filgrastim group (n=31), rHuSCF 20 μg/kg/day plus filgrastim 5–10 μg/kg/day were administered concurrently. Leukaphereses were continued to a maximum of four procedures or a target of ⩾3 × 106 CD34+ cells/kg. In both groups, CD34+ yield (× 106/kg BW) of the study mobilization was higher than that of the prior mobilization (median: 2.42 vs 0.84 P=0.002 and 1.64 vs 0.99 P=<0.001, respectively). In all 54 and 45% of patients in the filgrastim-alone group and chemotherapy-plus-filgrastim group, respectively, reached the threshold yield of 2 × 106/kg. The probability of a successful mobilization was the same in those with a CD34+ yield of 0.5–0.75 × 106/kg BW in the prior mobilization as in those with 0.76–1.99 × 106/kg BW. Downmodulation of c-kit expression and a lower percentage of Thy-1 positivity in the mobilized CD34+ cells were noted in the successful mobilizers compared with those in the poor mobilizers. This study shows that rhuSCF is effective in approximately half the patients who had failed a prior mobilization and allows them to proceed to transplant. It also points to the likely role of the SCF/c-kit ligand pair in mobilization.