Neil Rabin

Factors that influence short-term homing of human bone marrow-derived mesenchymal stem cells in a xenogeneic animal model

Human mesenchymal stem cells are potential agents for tissue regeneration, enhancing hematopoietic stem cell transplantation and delivering genes of therapeutic interest. This study shows that tissue homing of systemically administered mesenchymal stem cells can be increased by enforced expression of CXCR4, at least in irradiated hosts. Background Human mesenchymal stem cells are potential agents for tissue regeneration, enhancing hematopoietic stem cell transplantation and delivering genes of therapeutic interest. To implement any of these strategies successfully, we need a better understanding of factors that influence the tissue distribution of systemically administered mesenchymal stem cells. Design and Methods The present study was designed to investigate the short-term tissue homing of mesenchymal stem cells in immunodeficient mouse models, exploring the effects of animal age, duration of ex vivo expansion of mesenchymal stem cells, lentiviral transduction and CXCR4 over-expression. Dye-labeled mesenchymal stem cells (1.5–2.0×106/animal) were injected via the tail vein into unconditioned β2m/NOD/SCID animals. Animals were sacrificed 20–24 hours later and cell suspensions from tissues were examined by flow cytometry for the presence of PKH-positive cells. Results PKH-positive cells were readily detected in the bone marrow, spleen, liver and lungs at 20–24 hours after infusion. The homing of systemically infused mesenchymal stem cells to the bone marrow and spleen of unconditioned β2m/NOD/SCID animals was significantly (>2-fold, p<0.001) higher in younger (<10 weeks) animals, and was reduced with increasing passage number. Despite low surface CXCR4 expression, human mesenchymal stem cells migrated to SDF-1 in vitro, and this was enhanced by over-expression of CXCR4 using lentiviral transduction. Over-expression of CXCR4 by lentiviral transduction (>80%) did not alter the bone marrow homing of mesenchymal stem cells in unconditioned animals, but caused a significant (p<0.05) increase in homing to bone marrow and spleen of animals that had received prior irradiation. Conclusions Tissue homing of systemically administered mesenchymal stem cells is influenced by host factors such as age, is diminished by prolonged in vitro culture, and can be increased by enforced expression of CXCR4, at least in irradiated hosts.

A new xenograft model of myeloma bone disease demonstrating the efficacy of human mesenchymal stem cells expressing osteoprotegerin by lentiviral gene transfer

We describe a new model of myeloma bone disease in which β2m NOD/SCID mice injected with KMS-12-BM cells develop medullary disease after tail vein administration. Micro-computed tomography analysis demonstrated significant bone loss in the tibiae and vertebrae of diseased animals compared to controls, with loss of cortical bone (P<0.01), as well as trabecular bone volume, thickness and number (P<0.05 for all). Bone marrow of diseased animals demonstrated an increase in osteoclasts (P<0.01) and reduction in osteoblasts (P<0.01) compared to control animals. Both bone loss and osteoclast increase correlated with the degree of disease involvement. Mesenchymal stem cells (MSCs) were lentivirally transduced to express human osteoprotegerin (hOPG). Systemic administration of OPG expressing MSC reduced osteoclast activation (P<0.01) and trabecular bone loss in the vertebrae (P<0.05) and tibiae of diseased animals, to levels comparable to non-diseased controls. Because of its predominantly medullary involvement and quantifiable parameters of bone disease, the KMS-12-BM xenogeneic model provides unique opportunities to test therapies targeted at the bone marrow microenvironment.

Soluble Rank Ligand Produced by Myeloma Cells Causes Generalised Bone Loss in Multiple Myeloma

Patients with multiple myeloma commonly develop focal osteolytic bone disease, as well as generalised osteoporosis. The mechanisms underlying the development of osteoporosis in patients with myeloma are poorly understood. Although disruption of the RANKL/OPG pathway has been shown to underlie formation of focal osteolytic lesions, its role in the development of osteoporosis in myeloma remains unclear. Increased soluble RANKL in serum from patients with myeloma raises the possibility that this molecule plays a key role. The aim of the present study was to establish whether sRANKL produced by myeloma cells contributes directly to osteoporosis. C57BL/KaLwRij mice were injected with either 5T2MM or 5T33MM murine myeloma cells. 5T2MM-bearing mice developed osteolytic bone lesions (p<0.05) with increased osteoclast surface (p<0.01) and reduced trabecular bone volume (p<0.05). Bone volume was also reduced at sites where 5T2MM cells were not present (p<0.05). In 5T2MM-bearing mice soluble mRANKL was increased (p<0.05), whereas OPG was not altered. In contrast, 5T33MM-bearing mice had no changes in osteoclast surface or trabecular bone volume and did not develop osteolytic lesions. Soluble mRANKL was undetectable in serum from 5T33MM-bearing mice. In separate experiments, RPMI-8226 human myeloma cells were transduced with an human RANKL/eGFP construct, or eGFP alone. RPMI-8226/hRANKL/eGFP cells, but not RPMI-8226/eGFP cells, stimulated osteoclastic bone resorption (p<0.05) in vitro. Sub-cutaneous injection of NOD/SCID mice with RPMI-8226/hRANKL/eGFP or RPMI-8226/eGFP cells resulted in tumour development in all mice. RPMI-8226/hRANKL/eGFP-bearing mice exhibited increased serum soluble hRANKL (p<0.05) and a three-fold increase in osteoclast number (p<0.05) compared to RPMI-8226/eGFP-bearing mice. This was associated with reduced trabecular bone volume (27%, p<0.05), decreased trabecular number (29%, p<0.05) and increased trabecular thickness (8%, p<0.05). Our findings demonstrate that soluble RANKL produced by myeloma cells causes generalised bone loss, suggesting that targeting RANKL may prevent osteoporosis in patients with myeloma.

Bortezomib‐induced inflammatory neuropathy

Dear Editor, Bortezomib is a proteasome inhibitor licensed in the UK for the treatment of patients with untreated or relapsed/refractory multiple myeloma (MM) who are not candidates for stem cell transplantation. Peripheral neuropathy is a significant non-haematological toxicity associated with bortezomib therapy (Richardson et al., 2006). There remain conflicting data about whether bortezomib neuropathy is dose-related (Richardson et al., 2006; Cata et al., 2007; Chaudhry et al., 2008; Lanzani et al., 2008). Treatment emergent neuropathy usually occurs within the first four cycles, and patients who have not experienced this complication by cycle 5 are unlikely to do so (Richardson et al., 2006). This suggests alternative underlying mechanisms besides classical toxic dose-related axonal insult. For example, Ravaglia et al. (2008) presented a series of five cases of patients treated with bortezomib who developed possible inflammatory polyradiculoneuritis. A severe pulmonary reaction, likely vasculitic, has been reported with bortezomib (Miyakoshi et al., 2006). Bortezomib has also been described as causing cutaneous vasculitis in patients with non-Hodgkin lymphoma (Gerecitano et al., 2006). Interestingly, the response rate to treatment in the subgroup of patients who developed skin vasculitis was improved over bortezomib patients who did not develop skin vasculitis. The appearance of skin vasculitis from bortezomib was not dose dependent. The authors suggest there may be a connection between a vasculitic response and an improved response to treatment in this patient group. We report a case of bortezomib-induced inflammatory neuropathy confined to the peripheral nerves in a patient with refractory MM. In 2005, a 59-year-old man noted epistaxis and fatigue. MM was diagnosed based on an IgG kappa paraprotein (61 g/dl), a heavy infiltrate of monoclonal

Functional regulation of D-type cyclins by insulin-like growth factor-I and serum in multiple myeloma cells.

D‐type cyclin genes are universally dysregulated in multiple myeloma (MM), but the functional consequences are unclear as D‐type cyclin gene expression does not correlate with proliferation or disease progression. We examined the protein expression and regulation of D‐type cyclins and other cell cycle regulators in human myeloma cell lines and primary CD138+ plasma cells (PCs). Cyclin D1, cyclin D2, cyclin dependent kinase (CDK) 4, CDK6, p27Kip1 p18INK4C and retinoblastoma protein (pRb) were absent in normal PCs, heterogeneously expressed in primary MM cells and positively correlated with disease activity/progression. Cyclins D1 and D2 complexed with both CDK4 and CDK6, suggesting that both phosphorylate pRb in MM. Furthermore, cyclin D2 expressed via either t(14;16) or t(4;14) IgH translocations was functionally upregulated by fetal calf serum or insulin‐like growth factor‐I, leading to pRb phosphorylation and cell cycle entry/progression, and in some cases inversely correlated with p27Kip1. However, pRb phosphorylation and cell cycle progression mediated by cyclin D1 expressed via t(11;14) was less dependent on exogenous stimuli. These data suggest that the presence or absence of specific IgH translocations underlying aberrant D‐type cyclin expression may influence their response to mitogens in the bone marrow microenvironment. We showed for the first time that D‐type cyclins are functionally regulated in MM, differentially responsive to exogenous growth factors and upregulated with disease progression.

Time to redefine Myeloma

In November 2014 the International Myeloma Working Group (IMWG) revised the definition of multiple myeloma, such that asymptomatic patients with newly diagnosed multiple myeloma without any of the traditional ‘CRAB’ (hypercalcaemia, renal impairment, anaemia, bone disease) end organ damage criteria but with one of three new criteria would be recommended to start treatment. Previously, the standard of care for such patients was expectant management. These three new criteria are: greater than 60% clonal plasma cells on bone marrow biopsy, a serum free light chain (sFLC) ratio of >100 (the involved sFLC must be >100 mg/l) and greater than one unequivocal focal lesion on advanced imaging (low dose whole body computerized tomography, magnetic resonance imaging, 18F fluorodeoxyglucose positron emission tomography). Although this would appear to affect a small number of patients, the impact of these changes are broad, leading to an increased use of advanced imaging, a debate around the management of patients previously diagnosed with smouldering myeloma, changed terminology and clinical trial design and an extension of the use of biomarkers. For the first time the philosophy of treatment in myeloma will change from treatment initiation only being triggered by overt end organ damage to an era where sub clinical risk factors will also be taken into account.

Weekly intravenous bortezomib is effective and well tolerated in relapsed/refractory myeloma.

Bortezomib is an effective antimyeloma therapy, but clinical benefits can be limited by neurotoxicity. In newly diagnosed, older patients, modification of the biweekly dosing schedule to weekly regimens improves tolerability whilst maintaining efficacy. There is less information on the efficacy and tolerability of weekly bortezomib regimens in the relapsed/refractory setting. Here, we report our experience of weekly intravenous bortezomib in clinical practice in relapsed/refractory patients.

Multiple myeloma presenting with spinal cord compression during pregnancy

Dear Editor, Multiple myeloma (MM) is diagnosed before the age of 40 in just 2% of cases [1]. Accordingly, reports of MM in pregnancy are rare, with eight cases arising during pregnancy reported in the literature to date. Here we report the case of a young woman who presented with an aggressive form of MM during the third trimester of pregnancy with spinal cord compression. This is the first report of spinal cord compression secondary to MM presenting during pregnancy. A 39-year-old woman presented with a 3 week history of back pain and a 24 h history of urinary retention and bilateral lower limb weakness at 32 weeks gestation. Emergency MRI spine showed replacement of the T12 vertebral body by abnormal soft tissue with epidural extension, compressing the spinal cord (Fig. 1). She underwent emergency caesarean section delivering a healthy son followed by emergency spinal decompression surgery where a tumour was removed from the T12 vertebral body. She was commenced on dexamethasone and began to make an excellent neurological recovery. Biopsy demonstrated a plasmacytoma, an unusual feature of which was the high proliferation index of 70% Six days later she again developed lower limb weakness and sphincter disturbance. Urgent MRI spine revealed there had been a substantial increase in the volume of an epidural mass lying posterior to the cord from T10 to L1. Note was also made of foci of abnormal signal in many of the other vertebral bodies. She underwent further decompression of the spinal cord followed by radiotherapy to the thoracolumbar spine, (20 Gy) in five fractions. Further investigations confirmed a diagnosis of MM: skeletal survey demonstrated lytic lesions in the pelvis, femora and skull. Serum protein electrophoresis revealed an IgG lambda paraprotein (11 g/l) and Bence Jones proteinuria was also detected of 0.09 g/l. Trephine biopsy demonstrated 5% lambda light-chain restricted plasma cells. Albumin was 35 g/l, beta-2-microglobulin 1.7, and renal function, calcium and full blood count were all normal prior to surgery. She commenced chemotherapy with two cycles of idarubicin and dexamethasone with minimal response and then received second line chemotherapy, ESHAP (etoposide, cisplatin, cytarabine, methylprednisolone). Repeat MRI after the second cycle showed a partial response. Allogeneic bone marrow-transplantation was not considered at this time in view of her poor performance status and lack of a HLA-matched sibling, so she proceeded to highdose therapy with peripheral blood stem cell rescue. Three Ann Hematol (2009) 88:181–182 DOI 10.1007/s00277-008-0558-9

Myeloma impairs mature osteoblast function but causes early expansion of osteo-progenitors: temporal changes in bone physiology and gene expression in the KMS12BM model

Myeloma bone disease results from an uncoupling of osteoclastic resorption and osteoblastic bone formation, but early changes in osteogenic function remain poorly defined. We used the KMS12BM xenograft model to investigate cellular and molecular events at early and late stages of disease. Lytic lesions and changes in osteoblast and osteoclast numbers occur late (8 weeks), however, micro‐computed tomography of femora revealed significant reduction in bone volume at earlier disease stages (3 weeks) when tumour burden is low. Calcein labelling demonstrated reduced mineralization and bone formation at 3 weeks, suggesting functional impairment despite preserved osteoblast numbers. Osteo‐progenitors from compact bone increased early (1 week), but fell at 3 weeks and were profoundly suppressed by 8 weeks. Exposure of osteoblast progenitors to multiple myeloma (MM) cells in vitro induced cell cycling, suggesting a mechanistic basis for early expansion of osteo‐progenitors. We observed temporal changes in chemokine, osteogenic and osteoclastogenic genes in the stromal compartment. Notably, an early rise in CCL3 may underlie functional changes in mature osteoblasts at 3 weeks. Our data indicate that MM has distinct effects on mature osteoblasts and immature osteo‐progenitors. Our findings argue for early clinical intervention to prevent bone changes that ultimately lead to the development of osteolytic disease.

Proof-of-Principle for Immune Control of Global HIV-1 Reactivation In Vivo

It is unclear whether the human immune response is sufficiently potent to clear human immunodeficiency virus (HIV) type 1 latently infected cells globally reactivated by drug treatment. We report an elite controller who, following myeloablation and full HIV reactivation, achieved sustained control of viremia.