Luen To

The University of Adelaide, School of Medical Sciences, Faculty Member

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Papers by Luen To

CD34+ selection of autologous peripheral blood stem cells for transplantation following sequential cycles of high-dose therapy and mobilisation in multiple myeloma

by D. Joshua, P. Dyson, N. Horvath, and Luen To

Bone Marrow Transplantation, 2000

A potential problem of autologous transplantation in the treatment of multiple myeloma (MM) is th... more A potential problem of autologous transplantation in the treatment of multiple myeloma (MM) is the infusion of tumor cells. CD34+ selection has been used to purge autografts in MM and it is also possible to reduce tumour cell contamination of autografts by cytotoxic drug therapy prior to peripheral blood stem cell (PBSC) collection. To evaluate the effectiveness of a protocol combining multiple cycles of high-dose therapy and CD34+ selection to reduce tumour contamination of PBSC autografts, 34 MM patients were entered on a treatment schedule comprising two sequential cycles of mobilisation, CD34+ selection, and transplantation following high-dose therapy. In the second cycle of mobilisation there was a five-fold reduction in tumour contamination of the stem cell harvest (0.5 x 106/kg) compared with the first cycle (2.5 x 106/kg). In the 97 CD34+ selection procedures performed a median of 185 x 108 mononuclear cells (MNC) were processed yielding a median of 0.98 x 108 CD34+-enriched cells. CD34+ cells were enriched 68-fold from 1. 3% to 88.6%. The median yield of CD34+ cells was 42.2%. Following CD34+ selection the tumour cell contamination of the leukapheresis product was reduced by a median of 2.7 logs. This study demonstrates that in multiple myeloma a significant reduction in the malignant contamination of stem cell autografts can be achieved by combining the in vivo purging effect of cytotoxic therapy with in vitro purging by CD34+ selection.

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CD34+ selection of autologous peripheral blood stem cells for transplantation following sequential cycles of high-dose therapy and mobilisation in multiple myeloma

Bone Marrow Transplantation, 2000

A potential problem of autologous transplantation in the treatment of multiple myeloma (MM) is th... more A potential problem of autologous transplantation in the treatment of multiple myeloma (MM) is the infusion of tumor cells. CD34 + selection has been used to purge autografts in MM and it is also possible to reduce tumour cell contamination of autografts by cytotoxic drug therapy prior to peripheral blood stem cell (PBSC) collection. To evaluate the effectiveness of a protocol combining multiple cycles of high-dose therapy and CD34 + selection to reduce tumour contamination of PBSC autografts, 34 MM patients were entered on a treatment schedule comprising two sequential cycles of mobilisation, CD34 + selection, and transplantation following high-dose therapy. In the second cycle of mobilisation there was a five-fold reduction in tumour contamination of the stem cell harvest (0.5 × 10 6 /kg) compared with the first cycle (2.5 × 10 6 /kg). In the 97 CD34 + selection procedures performed a median of 185 × 10 8 mononuclear cells (MNC) were processed yielding a median of 0.98 × 10 8 CD34 +-enriched cells. CD34 + cells were enriched 68-fold from 1.3% to 88.6%. The median yield of CD34 + cells was 42.2%. Following CD34 + selection the tumour cell contamination of the leukapheresis product was reduced by a median of 2.7 logs. This study demonstrates that in multiple myeloma a significant reduction in the malignant contamination of stem cell autografts can be achieved by combining the in vivo purging effect of cytotoxic therapy with in vitro purging by CD34 + selection.

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Relapsed multiple myeloma: who benefits from salvage autografts?

Internal Medicine Journal, 2013

Multiple myeloma is incurable despite the advance of autologous stem cell transplant (ASCT) and n... more Multiple myeloma is incurable despite the advance of autologous stem cell transplant (ASCT) and novel agents (thalidomide, bortezomib, lenalidomide). The role of ASCT as salvage therapy in relapsed myeloma remains unclear. To identify and refine the predictors of survival following salvage ASCT for relapsed multiple myeloma, so that they can be applied clinically for patient selection. Retrospective review of patients treated salvage ASCT for relapsed myeloma at our centre from 1992 to 2011. Following an initial ASCT at diagnosis, 30 patients underwent salvage ASCT for subsequent relapse, with the median time to first relapse/progression being 30.2 months. All patients received reinduction, then melphalan-based conditioning with salvage ASCT. Non-relapse mortality at 100 days following salvage ASCT was 3%. The median overall survival and progression-free survival following salvage ASCT were 45 and 22 months respectively. The progression-free interval (PFI) after initial ASCT predicted survival outcomes in a time-dependent manner. With PFI following initial ASCT of &amp;amp;lt;18, 18-36 and ≥36 months, the median progression-free survival following salvage ASCT was 4.2, 13.8 and 49.1 months respectively (P &amp;amp;lt; 0.0001). The median overall survival was 10.7, 30.9 and 86.1 months respectively (P &amp;amp;lt; 0.0001). Salvage ASCT is an effective and safe treatment option in selected patients and should be considered in patients relapsing ≥36 months after their initial ASCT. The time-dependent relationship between PFI and salvage ASCT outcome is important when stratifying patient groups who may benefit from this procedure.

Effect of peripheral-blood progenitor cells mobilised by filgrastim (G-CSF) on platelet recovery after high-dose chemotherapy

The Lancet, 1992

The haemopoietic growth factor granulocyte colony-stimulating factor (G-CSF; filgrastim) substant... more The haemopoietic growth factor granulocyte colony-stimulating factor (G-CSF; filgrastim) substantially shortens the period of severe neutropenia that follows high-dose chemotherapy and autologous bone-marrow infusion by stimulating granulopoiesis. Filgrastim also increases numbers of circulating progenitor cells. We have studied the ability of filgrastim to mobilise peripheralblood progenitor cells and assessed their efficacy when infused after chemotherapy on recovery of neutrophil and platelet counts. 17 patients with non-myeloid malignant disorders received filgrastim (12 µg/kg daily for 6 days) by continuous subcutaneous infusion. Numbers of granulocyte-macrophage progenitors in peripheral blood increased a median of 58-fold over pretreatment values, and numbers of erythroid progenitors increased a median of 24-fold. Three leucapheresis procedures collected a mean total of 33 (SEM 5·7) × 104 granulocyte-macrophage progenitors per kg body weight. After high-dose chemotherapy in 14 of the patients (busulphan and cyclophosphamide), these cells were used to augment autologous bone-marrow rescue and posttransplant filgrastim treatment. Platelet recovery was significantly faster in these patients than in controls who received the same treatment apart from the infusion of peripheral-blood progenitors; the platelet count reached 50 × 109/l a median of 15 days after infusion of haemopoietic cells in the study patients compared with 39 days in controls (p = 0·0006). The accelerated neutrophil recovery associated with filgrastim treatment after chemotherapy was maintained. This method may be widely applicable to aid both neutrophil and platelet recovery after high-dose chemotherapy; it will allow investigation of peripheral-blood progenitor-cell allotransplantation.

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Circulating N‐cadherin levels are a negative prognostic indicator in patients with multiple myeloma

N-cadherin (cadherin 2, type 1, N-cadherin (neuronal); CDN2) is a homotypic adhesion molecule tha... more N-cadherin (cadherin 2, type 1, N-cadherin (neuronal); CDN2) is a homotypic adhesion molecule that is upregulated in breast, prostate and bladder cancer. Here we investigated the prognostic significance of upregulated N-cadherin expression in multiple myeloma (MM). Our results indicate that N-cadherin protein and gene expression is abnormally increased in trephine biopsies and CD38(++) /CD138(+) plasma cells from MM patients, when compared with those of normal donors. In addition, levels of circulating N-cadherin were elevated in a subset of patients with MM (n = 81; mean: 14·50 ng/ml, range: 0-146·78 ng/ml), relative to age-matched controls (n = 27; mean: 2·66 ng/ml, range: 0-5·96 ng/ml), although this did not reach statistical significance. Notably, patients with abnormally high levels of N-cadherin (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;6 ng/ml) had decreased progression-free survival (P = 0·036; hazard ratio: 1·94) and overall survival (P = 0·002; hazard ratio: 3·15), when compared with patients with normal N-cadherin levels (≤6 ng/ml). Furthermore, multivariate analyses revealed that the combination of N-cadherin levels and International Staging System (ISS) was a more powerful prognostic indicator than using ISS alone. Collectively, our studies demonstrate that circulating N-cadherin levels are a viable prognostic marker for high-risk MM patients.

Comparison of haematological recovery, toxicity and supportive care of autologous PBSC, autologous BM and allogeneic BM transplants

Stem Cells, 1992

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Receptor Activator of Nuclear Factor-B Ligand Expression by Human Myeloma Cells Mediates Osteoclast Formation in Vitro and Correlates with Bone Destruction in Vivo1

Multiple myeloma (MM) is an incurable B-cell malignancy able to mediate massive destruction of th... more Multiple myeloma (MM) is an incurable B-cell malignancy able to mediate massive destruction of the axial skeleton. The aim of this study was to examine the involvement of the tumor necrosis factor-ligand family member, receptor activator of nuclear factor-B ligand (RANKL), and its naturally occurring antagonist, osteoprotegerin (OPG), in MM biology. Using flow cytometry and two independent anti-RANKL anti- bodies, we demonstrate RANKL expression in CD38CD45 and CD38CD45 myeloma plasma cell (MPC) subpopulations derived from patients with osteolytic MM. In addition, highly purified subpopu- lations of MPC express mRNA for both transmembrane and soluble RANKL isoforms but lack expression of OPG mRNA and protein. We also show that RANKL expressed by MPC is functional as in vitro coculture of CD38CD45 and CD38CD45 MPC subpopulations with pe- ripheral blood mononuclear cells resulted in the formation of multinucle- ate, tartrate-resistant acid phosphatase-positive osteoclasts-like cells c...

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The nitrogen-containing bisphosphonate, zoledronic acid, increases mineralisation of human bone-derived cells in vitro

Bone, 2004

Bisphosphonates are used to prevent osteoclast-mediated bone loss. Zoledronic acid inhibits osteo... more 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. Bisphosphonates (BPs) exhibit high affinity for hydroxyapatite mineral in bone and are used extensively to treat malignancy-associated bone disease and postmenopausal bone loss by inhibiting osteoclast (OC)-mediated bone resorption. We examined the effect of the most potent nitrogen-containing BP available, zoledronic acid (ZOL), on the expression of RANKL and osteoprotegerin (OPG), critical factors in the regulation of OC formation and activation, in primary osteoblast (OB)-like cells derived from human bone, using flow cytometry, ELISA, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), in situ immunofluorescence staining, and Western blotting. Our studies show that ZOL, while not significantly affecting RANKL or OPG gene expression, markedly increased OPG protein secretion and reduced transmembrane RANKL protein expression in OB-like cells. The reduction in transmembrane RANKL expression was preceded by a marked increase in the expression of the metalloprotease-disintegrin, TNF-alpha converting enzyme (TACE). In addition, the decreased transmembrane expression of RANKL could be partially reversed by a TACE inhibitor, TAPI-2. Our studies indicate that ZOL, in addition to its direct effects on mature OCs, may inhibit the recruitment and differentiation of OCs by cleavage of transmembrane RANKL in OB-like cells by upregulating the sheddase, TACE.

Dasatinib (Sprycel) inhibits osteoclast activity in vitro and in vivo via a c-Fms-dependent and c-Src-independent mechanism

We have previously shown that the tyrosine kinase inhibitor imatinib mesylate potently inhibits o... more We have previously shown that the tyrosine kinase inhibitor imatinib mesylate potently inhibits osteoclast formation and activity due, in part, to its specificity for the macrophage colony stimulating factor (M-CSF) receptor, c-fms. We have also shown that imatinib therapy is associated with altered bone remodelling and an increase in trabecular bone volume in chronic myeloid leukaemia patients. In the present study, we examined whether a second-generation tyrosine kinase inhibitor, dasatinib, could similarly inhibit c-fms activation and osteoclastogenesis. The effect of dasatinib on c-fms phosphorylation in human peripheral blood CD14+ mononuclear cells (huCD14+) and mouse bone marrow monocytes (mBM) was assessed using Western blotting. M-CSF activation of c-fms phosphorylation was inhibited at pharmacologically-relevant concentrations (5 nM) in huCD14+ and mBM. We next examined the effects of dasatinib treatment on cell proliferation/ survival in M-CSF-dependent FDC-fms and mBM ce...

Ex vivo culture of peripheral blood CD34+ cells: effects of hematopoietic growth factors on production of neutrophilic precursors

by C. Juttner and Luen To

Journal of hematotherapy, 1997

A major potential application for ex vivo culture of hematopoietic progenitor cells is the treatm... more A major potential application for ex vivo culture of hematopoietic progenitor cells is the treatment of cytopenia following high-dose chemotherapy and hematopoietic transplantation. We have previously postulated that infusion of a sufficient number of neutrophil postprogenitor cells generated by ex vivo culture of CD34+ cells may be able to abrogate neutropenia. In this article, we describe further development of an efficient stromal-free, cytokine-dependent, static culture system for generation of these cells. Our previous studies indicated that maximal production of nucleated cells and myeloid progenitor cells from PB CD34+ cells occurred with multiple hematopoietic growth factor (HGF), notably the 6-HGF combination of interleukin (IL)-1, IL-3, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), and stem cell factor (SCF). In the present study, we determine the contribution of each of these 6 HGF in generation of neutrophilic precursors. SCF, ...

The effect of the dual PI3K and mTOR inhibitor BEZ235 on tumour growth and osteolytic bone disease in multiple myeloma

by Andrew Zannettino and Luen To

European journal of haematology, 2015

The plasma cell malignancy multiple myeloma (MM) is unique among haematological malignancies in i... more The plasma cell malignancy multiple myeloma (MM) is unique among haematological malignancies in its capacity to cause osteoclast-mediated skeletal destruction. The PI3K/Akt/mTOR pathway mediates proliferation, survival and drug resistance in MM plasma cells and is also involved in regulating the formation and activity of bone-forming osteoblasts and bone-resorbing osteoclasts. NVP-BEZ235 is a dual pan class I PI3K and mTOR inhibitor that is currently undergoing clinical evaluation in several tumour settings. In this study, we examined the anti-tumorigenic effects of BEZ235 in an immunocompetent mouse model of MM and assessed the effects of BEZ235 on osteoblast and osteoclast formation and function. BEZ235 treatment (50 mg/kg) resulted in a significant decrease in serum paraprotein and tumour burden, and μCT analysis of the proximal tibia revealed a significant reduction in the number of osteolytic bone lesions in BEZ235-treated animals. Levels of the serum osteoblast marker P1NP wer...

c-kit is expressed by primitive human hematopoietic cells that give rise to colony-forming cells in stroma-dependent or cytokine-supplemented culture

by C. Juttner and Luen To

Experimental hematology, 1994

Using monoclonal antibody (MAB) YB5.B8, we have examined the expression of the c-kit protein, the... more Using monoclonal antibody (MAB) YB5.B8, we have examined the expression of the c-kit protein, the receptor for the hematopoietic cytokine stem cell factor (SCF), on primitive hematopoietic cells. Bone marrow mononuclear cells (BMMNC) enriched for immature cells by differential agglutination using the lectin soybean agglutinin (SBA) were subjected to multiparameter fluorescence activated cell sorting (FACS) based on light-scattering properties, the expression of the c-kit protein and the CD34 antigen, and the retention of the vital fluorescent dye, Rhodamine 123 (Rh123). Sorted populations were assayed for their content of directly clonogenic progenitor cells (colony-forming units-granulocyte/macrophage [CFU-GM], burst-forming units-erythroid [BFU-E], and multipotential colony-forming units [CFU-Mix]) and for the presence of more primitive progenitor cells ("pre-CFU"). The latter were assayed by (1) their ability to initiate and sustain hematopoiesis in a standard stromal c...

The tyrosine kinase inhibitor dasatinib decreases osteoclast formation and activity in vitro

ABSTRACT We have previously shown that the tyrosine kinase inhibitor imatinib mesylate potently i... more ABSTRACT We have previously shown that the tyrosine kinase inhibitor imatinib mesylate potently inhibits osteoclast formation and activity due, in part, to its specificity for the macrophage colony stimulating factor (M-CSF) receptor, c-fms. We have also shown that imatinib therapy is associated with altered bone remodelling and an increase in trabecular bone volume in chronic myeloid leukaemia patients. In the present study, we examined whether a second-generation tyrosine kinase inhibitor, dasatinib, could similarly inhibit c-fms activation and osteoclastogenesis. Initially, we assessed whether dasatinib could affect c-fms phosphorylation in human peripheral blood CD14+ mononuclear cells (PBMNC) and mouse bone marrow monocytes (mBM) using Western blotting. We next examined the effects of dasatinib treatment on cell proliferation/survival in M-CSF-dependent (FDC-fms and mBM) and -independent (RAW-264.7) cells. Additionally, we investigated if dasatinib affected osteoclast formation and activity in PBMNC, mBM and RAW-264.7 cells. Our studies show that dasatinib inhibits the activation of c-fms by M-CSF at pharmacologically-relevant concentrations (5 nM) in PBMNC and mBM. Furthermore, dasatinib treatment dose-dependently decreased cell numbers in FDC-fms (IC50 = 57.8 nM) and mBM (IC50 = 5.0 nM) cultured with M-CSF. In contrast, cell numbers were not significantly altered by dasatinib-treatment in M-CSF-independent RAW-264.7 cells (p = 0.4894) or in FDC-fms cells cultured in IL-3 (p = 0.2008). In PBMNC and mBM cultures stimulated with RANKL and M-CSF, the number of TRAP-positive, multinucleated osteoclasts was significantly reduced in the presence of 20 nM dasatinib in PBMNC (p &lt; 0.001; IC50 = 10.5) and 10 nM dasatinib in mBM (p &lt; 0.01; IC50 = 8.0 nM), relative to vehicle controls. In addition, there was a dose-dependent decrease in bone resorption by osteoclasts at 2.5 nM dasatinib and higher (p &lt; 0.05; IC50 = 2.4 nM [PBMNC] and 3.5 nM [mBM]). In RAW-264.7 cells cultured with RANKL alone, osteoclast numbers were significantly decreased at 20 nM dasatinib and higher (p &lt; 0.05) relative to vehicle. These studies show for the first time that dasatinib treatment is associated with a decrease in osteoclast formation and activity in both human and murine systems in vitro. This decrease can partly be attributed to an inhibition of signal transduction through c-fms. These data suggest that decreased bone resorption may be a potential side-effect of dasatinib therapy and these in vitro findings warrant further investigation in vivo.

The effect of the PI3K inhibitor BKM120 on tumour growth and osteolytic bone disease in multiple myeloma

by Luen To and Andrew Zannettino

Leukemia Research, 2015

The plasma cell malignancy multiple myeloma (MM) is unique amongst haematological malignancies in... more The plasma cell malignancy multiple myeloma (MM) is unique amongst haematological malignancies in its capacity to cause osteoclast-mediated skeletal destruction. The PI3K/Akt pathway mediates proliferation, survival and drug resistance in MM plasma cells and is also involved in regulating the formation and activity of bone-forming osteoblasts and bone-resorbing osteoclasts. NVP-BKM120 (Buparlisib, Novartis) is a PI3K inhibitor that is currently undergoing clinical evaluation in several tumour settings. In this study, we have examined the anti-tumorigenic effects of BKM120 in an immunocompetent mouse model of MM and its effects on osteoblast and osteoclast formation and function. BKM120 treatment (40mg/kg) resulted in a significant decrease in serum paraprotein and tumour burden, and μCT analysis of the proximal tibia revealed a significant reduction in the number of osteolytic bone lesions in BKM120-treated animals. BKM120 also mediated a significant increase in serum levels of the osteoblast marker P1NP, and a significant decrease in serum levels of the osteoclast marker TRAcP5. In vitro, BKM120 decreased MM plasma cell proliferation, osteoclast formation and function, and promoted osteoblast formation and function. These findings suggest that, in addition to its anti-tumour properties, BKM120 could be used to treat osteolytic bone disease in MM patients.

SAMSN1 Is a Tumor Suppressor Gene in Multiple Myeloma

by Andrew Zannettino and Luen To

Neoplasia, 2014

Multiple myeloma (MM), a hematological malignancy characterized by the clonal growth of malignant... more Multiple myeloma (MM), a hematological malignancy characterized by the clonal growth of malignant plasma cells (PCs) in the bone marrow, is preceded by the benign asymptomatic condition, monoclonal gammopathy of undetermined significance (MGUS). Several genetic abnormalities have been identified as critical for the development of MM; however, a number of these abnormalities are also found in patients with MGUS, indicating that there are other, as yet unidentified, factors that contribute to the onset of MM disease. In this study, we identify a Samsn1 gene deletion in the 5TGM1/C57BL/KaLwRij murine model of myeloma. In addition, SAMSN1 expression is reduced in the malignant CD138+ PCs of patients with MM and this reduced expression correlates to total PC burden. We identify promoter methylation as a potential mechanism through which SAMSN1 expression is modulated in human myeloma cell lines. Notably, re-expression of Samsn1 in the 5TGM1 murine PC line resulted in complete inhibition of MM disease development in vivo and decreased proliferation in stromal cell-PC co-cultures in vitro. This is the first study to identify deletion of a key gene in the C57BL/KaLwRij mice that also displays reduced gene expression in patients with MM and is therefore likely to play an integral role in MM disease development.

Ex Vivo Hematopoietic Progenitor Cell Expansion

by C. Juttner and Luen To

ImmunoMethods, 1994

The ability to culture and expand hematopoietic progenitor cells ex vivo has major implications f... more The ability to culture and expand hematopoietic progenitor cells ex vivo has major implications for both bone marrow and stem cell support following marrow ablative or subablative high-dose therapy and for improving the efficiency of retroviral transfection in gene marking and gene therapy. This review focuses on methods for the generation of myeloid progenitor and post-progenitor cells from peripheral blood stem cell collections, with particular emphasis on the characterization of these cells and practical issues associated with their expansion.

Non-HLA immunogenetic polymorphisms and the risk of complications after allogeneic hemopoietic stem-cell transplantation