Osteoblastic Lesions Research Articles

Beyond Skeletal-Related Events

Objectives Zoledronic acid may have potential antitumor activity; it is the most active agent among the bisphosphonates in blocking the prenylation (activation) of small signaling proteins required for cell function and survival. This presentation discusses the results of preclinical studies evaluating the antitumor activity of zoledronic acid and discusses initiated clinical studies. Methods Experimental models included nude mice bearing human prostate cancer cells, castrated rats, and in vitro endothelial cells. Results Zoledronic acid inhibited osteolytic and osteoblastic bone lesions in mice bearing human prostate cancer cells. Zoledronic acid also inhibited endothelial cell proliferation and capillary-like tube formation in vitro and testosterone-induced revascularization of the prostate in castrated rats without altering testosterone activity directly. The ZEUS study is one of several studies investigating whether zoledronic acid may prevent bone metastases. Conclusions The preclinical data on the antitumor effects of zoledronic acid were encouraging. Therefore, the role of zoledronic acid in earlier-stage prostate cancer is being investigated in clinical trials. The results of these trials are awaited to evaluate the effects in patients with prostate cancer.

Prostate-Specific Antigen Modulates Genes Involved in Bone Remodeling and Induces Osteoblast Differentiation of Human Osteosarcoma Cell Line SaOS-2

The high prevalence of osteoblastic bone metastases in prostate cancer involves the production of osteoblast-stimulating factors by prostate cancer cells. Prostate-specific antigen (PSA) is a serine protease uniquely produced by prostate cancer cells and is an important serologic marker for prostate cancer. In this study, we examined the role of PSA in the induction of osteoblast differentiation. Human cDNA containing a coding region for PSA was transfected into human osteosarcoma SaOS-2 cells. SaOS-2 cells were also treated with exogenously added PSA. We evaluated changes in global gene expression using cDNA arrays and Northern blot analysis resulting from expression of PSA in human osteosarcoma SaOS-2 cells. SaOS-2 cells expressing PSA had markedly up-regulated expression of genes associated with osteoblast differentiation including runx-2 and osteocalcin compared with the controls. Consistent with these results, the stable clones expressing PSA showed increased mineralization and increased activity of alkaline phosphatase in vitro compared with controls, suggesting that these cells undergo osteoblast differentiation. We also found that osteoprotegerin expression was down-regulated and that the receptor activator of NF-kappaB ligand expression was up-regulated in cells expressing PSA compared with controls. Modulation of the expression of osteogenic genes and alteration of the balance between osteoprotegerin-receptor activator of NF-kappaB ligand by PSA suggests that PSA produced by metastatic prostate cancer cells may participate in bone remodeling in favor of the development of osteoblastic metastases in the heterogeneous mixture of osteolytic and osteoblastic lesions. These findings provide a molecular basis for understanding the high prevalence of osteoblastic bone metastases in prostate cancer.

IGF‐I secretion by prostate carcinoma cells does not alter tumor‐bone cell interactions in vitro or in vivo

IGF-I is an important growth and differentiative factor for osteoblasts and may have a role in defining prostate cancer risk and skeletal metastases. Conditioned media (CM) from human prostate cancer (PC), C4-2 and C4-2B, which produce osteoblastic lesions, and PC-3, which causes osteolysis, was added to MC3T3-E1 bone cultures. SCID mice were injected intratibially with these engineered cells. Tumor bearing tibiae were analyzed by microCT and pQCT. CM from PC cells increased osteoblast proliferation and differentiation and was unaltered by the type of PC cell, IGF-I antibodies, or exogenous IGF-I and IGFBP2. Study of intratibial PC tumors in SCID mice showed that C4-2 cells grew slowly preserving bone structure, while PC-3 tumors caused rapid osteolysis. Overexpression of IGF-I did not change either tumor progression or skeletal response. IGF-I is neither necessary nor sufficient for the osteoblastic response to PC metastases.

Percutaneous Vertebroplasty for Spinal Metastases: Complications

To retrospectively evaluate complications of percutaneous vertebroplasty (PV) performed with polymethylmethacrylate cement to treat pain in patients with metastases to the spine. This study had institutional review board approval; patient informed consent for the review of records and images was not required. In 2 years, 117 patients (38 men [32.5%] and 79 women [67.5%]; mean age, 58.2 years) underwent 159 fluoroscopy-guided PV procedures to treat 304 vertebrae. Spinal metastases included osteolytic, osteoblastic, and mixed lesions. Complications were characterized as local or systemic. Evaluated data included immediate imaging findings (on radiographs and computed tomographic scans) and clinical findings at 30-day follow-up. Chi2 or Fisher exact testing was performed for univariate analysis of variables. The primary cancers were breast cancers (45.3%), lung cancers (14.5%), myeloma (7.7%), or other cancers (32.5%). Among the 423 cement leakages identified, 332 (78.5%) were vascular and 91 (21.5%) were nonvascular. Vascular leaks were classified as venous epidural leaks, paravertebral and foraminal plexus leaks, and leaks to the vena cava, while nonvascular leaks included puncture trajectory leaks, paravertebral soft tissue leaks, and diskal leaks. Patients with nonvascular leaks were asymptomatic. Eight (6.8%) patients experienced complications, and seven of these complications were symptomatic. Among these eight patients, six (5.1%) had local complications (puncture site hematoma in two patients and radicular pain [successfully treated with nonsteroidal anti-inflammatory drugs or corticosteroids] in four patients), and two (1.7%) had systemic complications (pulmonary embolism resulting from cement migration through the vena cava). One of the latter patients died. Univariate analyses revealed a significant association between cement migration through the vena cava and pulmonary embolism (P = .001) but not between foraminal venous leakage and radicular pain (P = .123). Despite numerous technical incidents (leaks), PV-induced complications were rare, leading to the hypothesis that systemic complications are a consequence of intravascular leakage while local complications are a consequence of cement-related irritation, compression and/or ischemia, and/or needle-induced trauma.

Role of wnts in prostate cancer bone metastases

Prostate cancer (CaP) is unique among all cancers in that when it metastasizes to bone, it typically forms osteoblastic lesions (characterized by increased bone production). CaP cells produce many factors, including Wnts that are implicated in tumor-induced osteoblastic activity. In this prospectus, we describe our research on Wnt and the CaP bone phenotype. Wnts are cysteine-rich glycoproteins that mediate bone development in the embryo and promote bone production in the adult. Wnts have been shown to have autocrine tumor effects, such as enhancing proliferation and protecting against apoptosis. In addition, we have recently identified that CaP-produced Wnts act in a paracrine fashion to induce osteoblastic activity in CaP bone metastases. In addition to Wnts, CaP cells express the soluble Wnt inhibitor dickkopf-1 (DKK-1). It appears that DKK-1 production occurs early in the development of skeletal metastases, which results in masking of osteogenic Wnts, thus favoring osteolysis at the metastatic site. As metastases progress, DKK-1 expression decreases allowing for unmasking of Wnt's osteoblastic activity and ultimately resulting in osteosclerosis at the metastatic site. We believe that DKK-1 is one of the switches that transitions the CaP bone metastasis activity from osteolytic to osteoblastic. Wnt/DKK-1 activity fits a model of CaP-induced bone remodeling occurring in a continuum composed of an osteolytic phase, mediated by receptor activator of NFkB ligand (RANKL), parathyroid hormone-related protein (PTHRP) and DKK-1; a transitional phase, where environmental alterations promote expression of osteoblastic factors (Wnts) and decreases osteolytic factors (i.e., DKK-1); and an osteoblastic phase, in which tumor growth-associated hypoxia results in production of vascular endothelial growth factor and endothelin-1, which have osteoblastic activity. This model suggests that targeting both osteolytic activity and osteoblastic activity will provide efficacy for therapy of CaP bone metastases.

Influence of BMPs on the Formation of Osteoblastic Lesions in Metastatic Prostate Cancer

The purpose of this study was to evaluate the role of BMPs on the formation of metastatic prostate cancer lesions to bone. Our results show that BMPs influence the development and progression of osteoblastic lesions and suggest that therapies that inhibit BMP activity may reduce the formation and progression of osteoblastic lesions. Prostate adenocarcinoma is the leading cause of cancer in North American men. The formation of skeletal metastases affects approximately 70% of patients with advanced disease, and a majority of these patients have osteoblastic lesions. Although BMPs have been found to be expressed in multiple oncogenic cell lines, their role in the formation of metastatic osteoblastic lesions remains uncharacterized. We hypothesized that BMPs influence the development of metastatic osteoblastic lesions associated with prostate cancer. Western blot analysis and RT-PCR was used to determine BMP receptor expression on osteoblastic prostate cancer cell lines LAPC-4 and LAPC-9. Migration, invasion, and cellular proliferation assays were used to quantify the effects of BMP-2, -4, and -7 on LAPC-4 cells in vitro. LAPC-9 cells alone or transfected with a retrovirus overexpressing noggin were injected into the tibias of SCID mice, and the animals were followed for 8 weeks. Tumor size was determined by radiographs and direct measurement. Histology was performed at the time of death. We determined that BMP receptor mRNA and protein was expressed on osteoblastic prostate cancer cell lines LAPC-4 and LAPC-9. In vitro studies showed that BMP-2 and -7 stimulated cellular migration and invasion of prostate cancer cells in a dose-dependent fashion, although BMP-4 had no effect. Noggin inhibited cellular migration and invasion of BMP-2- and -7-stimulated LAPC-4 cells. LAPC-9 cells implanted into immunodeficient mouse tibias formed an osteoblastic lesion with sclerotic bone at 8 weeks. Formation of osteoblastic lesions was inhibited by overexpression of noggin by prostate cancer cells transduced with a retrovirus containing the cDNA for noggin. BMPs are critical in the formation of the osteoblastic lesions associated with prostate cancer metastases, and future treatment strategies that inhibit local BMP activity may reduce the formation and progression of osteoblastic lesions.

Rat Models of Bone Metastases

Bone metastases occur frequently in patients with advanced breast or prostate cancer. Bone metastases can be predominantly osteolytic, osteoblastic or mixed. Studies with animal models allow advances in understanding the molecular basis for bone metastases and provide new targets for therapy. Several animal models have been developed in rat with different pathophysiologies; they required injection or implantation of neoplastic cells into orthotopic locations, bones or the left ventricle of the heart. Several specific strains of rat have an increased incidence of spontaneous tumors. Carcinomas can be induced by either chemicals or physical agents. However, the most used and convenient way to induce bone metastases is a syngeneic transmission. MAT-Ly-Lu cells have been used in several models using Copenhagen rats to induce osteoblastic bone lesions. PA-III cells derived from Pollard tumors can also produce a combination of osteolytic and osteoblastic reactions at the site of transplantation. Osteolytic bone lesions can be obtained with an injection of Walker cells. The use of 13762 or c-SST2 cells allows also leads to osteolysis. Human xenografts can only be used in nude animals. It is essential to validate and correctly interpret the lesions in several models of bone metastasis. No animal model is sufficient by itself to represent the clinical findings observed in humans. The use of models developed in different species should be more predictive and bring a beam of arguments for a better knowledge of pathophysiological and therapeutic mechanisms.

Vascular Endothelial Growth Factor Contributes to Prostate Cancer–Mediated Osteoblastic Activity

Prostate cancer frequently metastasizes to bone resulting in the formation of osteoblastic metastases through unknown mechanisms. Vascular endothelial growth factor (VEGF) has been shown recently to promote osteoblast activity. Accordingly, we tested if VEGF contributes to the ability of prostate cancer to induce osteoblast activity. PC-3, LNCaP, and C4-2B prostate cancer cell lines expressed both VEGF-165 and VEGF-189 mRNA isoforms and VEGF protein. Prostate cancer cells expressed the mRNA for VEGF receptor (VEGFR) neuropilin-1 but not the VEGFRs Flt-1 or KDR. In contrast, mouse pre-osteoblastic cells (MC3T3-E1) expressed Flt-1 and neuropilin-1 mRNA but not KDR. PTK787, a VEGFR tyrosine kinase inhibitor, inhibited the proliferation of human microvascular endothelial cells but not prostate cancer proliferation in vitro. C4-2B conditioned medium induced osteoblast differentiation as measured by production of alkaline phosphatase and osteocalcin and mineralization of MC3T3-E1. PTK787 blocked the C4-2B conditioned medium-induced osteoblastic activity. VEGF directly induced alkaline phosphatase and osteocalcin but not mineralization of MC3T3-E1. These results suggest that VEGF induces initial differentiation of osteoblasts but requires other factors, present in C4-2B, to induce mineralization. To determine if VEGF influences the ability of prostate cancer to develop osteoblastic lesions, we injected C4-2B cells into the tibia of mice and, after the tumors grew for 6 weeks, administered PTK787 for 4 weeks. PTK787 decreased both intratibial tumor burden and C4-2B-induced osteoblastic activity as measured by bone mineral density and serum osteocalcin. These results show that VEGF contributes to prostate cancer-induced osteoblastic activity in vivo.

The effects of RANK blockade and osteoclast depletion in a model of pure osteoblastic prostate cancer metastasis in bone

Adenocarcinoma of the prostate exhibits a clear propensity for bone and is associated with the formation of osteoblastic metastases. It has previously been suggested that osteoclast activity may be necessary for the development of these osteoblastic metastases based on data from lytic and mixed lytic-blastic tumors. Here we investigate the effects of complete in vivo osteoclast depletion via the blockade of receptor activator of NF:kappaB (RANK) on the establishment and progression of purely osteoblastic (LAPC-9 cells) bone lesions induced by human prostate cancer cells using a SCID mouse intratibial injection model. The subcutaneous administration of the RANK antagonist (15 mg/kg) RANK:Fc did not prevent the formation of purely osteoblastic lesions, indicating that osteoclasts may not be essential to the initial development of osteoblastic metastases. However, RANK:Fc protein appeared to inhibit the progression of established osteoblastic lesions, suggesting that osteoclasts may be involved in the subsequent growth of these tumors once they are already present. In contrast, RANK:Fc treatment effectively blocked the establishment and progression of purely osteolytic lesions formed by PC-3 cells, which served as a positive control. These results indicate that in vivo RANK blockade may not be effective for the prevention of osteoblastic metastasis but may potentially represent a novel therapy that limits the growth of established metastatic CaP lesions in bone.

Mast cell leukaemia

In May 2003, a 65-year-old white male presented with weight loss, night sweats, pruritus, diarrhoea, and joint pain. Radiographic studies showed hepatosplenomegaly, abdominal lymphadenopathy and both osteoblastic and osteolytic bone lesions. Bone marrow biopsy showed extensive mast cell infiltration and effacement of marrow structure with reduction of fat cells and marked osteomyelosclerosis (top, Giemsa stain). We administered six courses of therapy with cladribine (0AE1 mg/kg body weight for 5 d, monthly), which reduced mast cell infiltration and restored marrow structure to a nearnormal state (bottom, Giemsa stain). Mediator-related symptoms (night sweats, pruritus, arthralgia, diarrhoea) disappeared completely. Two years after the diagnosis of mast cell leukaemia the patient has a 90–100% Karnofsky performance status.

Gemcitabine Radiosensitization after High-Dose Samarium for Osteoblastic Osteosarcoma

Osteoblastic metastases and osteosarcoma can avidly concentrate bone-seeking radiopharmaceuticals. We sought to increase effectiveness of high-dose (153)Samarium ethylenediaminetetramethylenephosphonate (153Sm-EDTMP, Quadramet) on osteosarcomas using a radiosensitizer, gemcitabine. Fourteen patients with osteoblastic lesions were treated with 30 mCi/kg 153Sm-EDTMP. Gemcitabine was administered 1 day after samarium infusion. Residual total body radioactivity was within the safe range of <3.6 mCi on day +14 (1.1 +/- 0.4 mCi; range, 0.67-1.8 mCi). All patients received autologous stem cell reinfusion 2 weeks after 153Sm to correct expected grade 4 hematopoietic toxicity. Peripheral blood progenitor cells were infused in 11 patients; three patients had marrow infused. Blood count recovery was uneventful after peripheral blood progenitor cells in 11 of 11 patients. Toxicity from a single infusion of gemcitabine (1,500 mg/m2) in combination with 153Sm-EDTMP was minimal (pancytopenia). However, toxicity from a daily gemcitabine regimen (250 mg/m2/d x 4-5 days) was excessive (grade 3 mucositis) in one of two patients. There were no reported episodes of hemorrhagic cystitis (hematuria) or nephrotoxicity. At the 6- to 8-week follow-up, there were six partial remissions, two mixed responses, and six patients with progressive disease. In the 12 patients followed >1 year, there have been no durable responses. Thus, although high-dose 153Sm-EDTMP + gemcitabine has moderate palliative activity (improved pain; radiologic responses) in this poor-risk population, additional measures of local and systemic control are required for durable control of relapsed osteosarcoma with osteoblastic lesions. The strategy of radioactive drug binding to a target followed by a radiosensitizer may provide synergy and improved response rate.

Bone Morphogenetic Protein-6 Promotes Osteoblastic Prostate Cancer Bone Metastases through a Dual Mechanism

Prostate cancer frequently metastasizes to bone where it forms osteoblastic lesions through unknown mechanisms. Bone morphogenetic proteins (BMP) are mediators of skeletal formation. Prostate cancer produces a variety of BMPs, including BMP-6. We tested the hypothesis that BMP-6 contributes to prostate cancer-induced osteosclerosis at bone metastatic sites. Prostate cancer cells and clinical tissues produced BMP-6 that increased with aggressiveness of the tumor. Prostate cancer-conditioned medium induced SMAD phosphorylation in the preosteoblast MC3T3 cells, and phosphorylation was diminished by anti-BMP-6 antibody. Prostate cancer-conditioned medium induced mineralization of MC3T3 cells, which was blocked by both the BMP inhibitor noggin and anti-BMP-6. Human fetal bones were implanted in severe combined immunodeficient mice and after 4 weeks, LuCaP 23.1 prostate cancer cells were injected both s.c. and into the bone implants. Anti-BMP-6 or isotype antibody administration was then initiated. Anti-BMP-6 reduced LuCaP 23.1-induced osteoblastic activity, but had no effect on its osteolytic activity. This was associated with increased osteoblast numbers and osteoblast activity based on bone histomorphometric evaluation. As endothelin-1 has been implicated in bone metastases, we measured serum endothelin-1 levels but found they were not different among the treatment groups. In addition to decreased bone production, anti-BMP-6 reduced intraosseous, but not s.c., tumor size. We found that BMP-2, BMP-4, BMP-6, and BMP-7 had no direct effect on prostate cancer cell growth, but BMP-2 and BMP-6 increased the in vitro invasive ability of prostate cancer cell. These data show that prostate cancer promotes osteoblastic activity through BMP-6 and that, in addition to its bone effects, suggest that BMPs promote the ability of the prostate cancer cells to invade the bone microenvironment.

Hypercalcaemia of malignancy and basic research on mechanisms responsible for osteolytic and osteoblastic metastasis to bone

Calcium homeostasis is a tightly regulated process involving the co-ordinated efforts of the skeleton, kidney, parathyroid glands and intestine. Neoplasms can alter this homeostasis indirectly through the production of endocrine factors resulting in humoral hypercalcaemia of malignancy. Relatively common with breast and lung cancer, this paraneoplastic condition is most often due to tumour production of parathyroid hormone-related protein and ensuing increased osteoclastic bone resorption. Although control of hypercalcaemia is generally successful, the development of this complication is associated with a poor prognosis. The metastasis of tumour cells to bone represents another skeletal complication of malignancy. As explained in the 'seed and soil' hypothesis, bone represents a fertile ground for cancer cells to flourish. The molecular mechanisms of this mutually beneficial relationship between bone and cancer cells are beginning to be understood. In the case of osteolytic bone disease, tumour-produced parathyroid hormone-related protein stimulates osteoclasts that in turn secrete tumour-activating transforming growth factor-beta that further stimulates local cancer cells. This 'vicious cycle' of bone metastases represents reciprocal bone/cancer cellular signals that likely modulate osteoblastic bone metastatic lesions as well. The development of targeted therapies to either block initial cancer cell chemotaxis, invasion and adhesion or to break the 'vicious cycle' is dependent on a more complete understanding of bone metastases. Although bisphosphonates delay progression of skeletal metastases, it is clear that more effective therapies are needed. Cancer-associated bone morbidity remains a major public health problem, and to improve therapy and prevention it is important to understand the pathophysiology of the effects of cancer on bone. This review will detail scientific advances regarding this area.

Overexpression of noggin inhibits BMP-mediated growth of osteolytic prostate cancer lesions

Introduction Although a majority of metastatic prostate cancer lesions are osteoblastic in nature, some are mixed or lytic; and, osteoblastic lesions require osteolytic activity in order to progress. The role of BMPs in the formation of prostate cancer metastases to bone remains unknown. We hypothesized that BMPs influence the development and progression of osteolytic prostate cancer lesions. Methods RT-PCR and Western blot analysis were used to determine BMP receptor expression on the osteolytic prostate cancer cell line PC-3. Migration, invasion, and cellular proliferation assays were performed on PC-3 cells to quantify the effects of BMP-2, -4, and -7. In vivo, PC-3 cells were injected alone, with an empty retroviral vector, or with a retroviral vector overexpressing noggin, into the tibias of SCID mice. The animals were followed for 8 weeks, and histologic and radiographic analysis were performed at 2, 4, 6, and 8 weeks. Results BMP receptors are expressed on PC-3 cells, suggesting that they would be responsive to host BMP secretion. BMP-2, and to a lesser extent, BMP-4, stimulated PC-3 cell migration and invasion in a dose-dependent fashion. Noggin inhibited cellular migration and invasion of BMP-2 and -4 stimulated PC-3 cells. BMP-2 alone stimulated PC-3 cell proliferation, but BMP-4 had no effect. BMP-7 had no effect on proliferation, migration, or invasion. PC-3 cells implanted into SCID mouse tibias formed osteolytic lesions as early as 2 weeks and completely destroyed the proximal tibia by 8 weeks. Overexpression of noggin in PC-3 cells inhibited the expansion of the lesion in vivo. Conclusions BMPs influence the formation of the osteolytic prostate cancer metastases, and treatment modalities that inhibit BMP activity may limit the progression of the lytic component of prostate cancer metastases.

Inhibition of prostate cancer-meditated osteoblastic bone lesions by the low-calcemic analog 1α-hydroxymethyl-16-ene-26,27-bishomo-25-hydroxy vitamin D 3

Prostate cancer metastasizes almost exclusively into the bone whereby it induces primarily an osteoblastic response. Non-calcemic vitamin D analogs have been shown to inhibit proliferation of prostate cancer cells in culture and inhibit their growth as subcutaneous xenografts in mice. However, their effect on prostate cancer cell growth in the bone has not been examined. In the present study, we inoculated the osteoblastic prostate cancer cell line MDA-PCa 2b into the bone of male SCID mice and examined the effect of the low-calcemic hybrid analog 1α-hydroxymethyl-16-ene-26,27-bishomo-25-hydroxy vitamin D 3 (JK-1626-2) on their ability to induce bone lesions. We found that 7 weeks after inoculation of MDA-PCa 2b cells, 90% of the mice in the vehicle-treated group had significant bone lesions that were detectable by micro-computed tomography and characterized by thickening of the cortical bone and ossification of the epiphysis. Only 30% of the mice in the analog-treated group (daily injections of 4 μg/kg, 5 days/week for up to 7 weeks) had detectable bone lesions. Histological examination of the decalcified tumor-bearing bones has shown that tumor cells completely replaced the bone marrow in the diaphysis, and destroyed the trabecular bone in the metaphysis in 90% of the vehicle-treated mice. In contrast, the metaphysis of 60% of analog-treated mice appeared normal, although tumor cells were still found in the diaphysis of 70% of the bones in the analog-treated group. There was no evidence of hypercalcemia in any of the analog-treated mice. In a co-culture, MDA-PCa 2b cells induced a profound mitogenic response in osteoblasts followed by enhanced differentiation. However, in the presence of the analog the mitogenic response of the osteoblasts to the malignant cells was significantly attenuated. These experiments led to the hypothesis that, in vivo, JK-1626-2 prevented the metastatic bone lesions by inhibiting the mitogenic response of osteoblasts to growth factors produced by MDA-PCa 2b cells.

Spiculated periosteal response induced by intraosseous injection of 22Rv1 prostate cancer cells resembles subset of bone metastases in prostate cancer patients

Prostate cancer bone metastasis is distinguished by the predominance of osteoblastic lesions. This phenotype has been difficult to reproduce in animal models. Here, we describe a model utilizing the 22Rv1 human prostate cancer cell line that generates osteolytic lesions and a prominent spiculated periosteal osteoblastic response following intraosseous injection in scid mice. We injected 22Rv1-luciferase prostate cancer cells directly into the tibiae of C.B-17 scid mice. We analyzed tumor growth and pathology every 2 weeks using radiographic and histologic techniques. X-ray analysis revealed that 22Rv1 tumors elicit a mixed-type lesion including some osteolysis and a robust induction of periosteal bone formation, in contrast to PC3M-luciferase intraosseous tumors which induce only extensive osteolysis. Micro-computerized tomographic imaging shows that 22Rv1 tumors exhibit both osteolytic and osteoblastic features which become apparent between 4 and 6 weeks post injection. There is initial disruption of the cortex and corresponding invasion of the periosteum which is associated with a vigorous osteoblastic response. Histological analysis of late stage tumors shows that the tumor has grown outside of the medullary cavity and surrounds the tibia underneath the periosteum and intermixed with spicules of woven bone which is detected in the radiographic analysis. The overall pattern of this model is suggestive of clinical cases of prostate cancer metastasis in which periosteal responses are noted, often in association with rapidly progressive disease. We expect that intraosseous injection of 22Rv1 cells will provide a new experimental model for the study of osteoblastic prostate cancer metastasis.

The effects of RANK blockade and osteoclast depletion in a model of pure osteoblastic prostate cancer metastasis in bone

Adenocarcinoma of the prostate exhibits a clear propensity for bone and is associated with the formation of osteoblastic metastases. It has previously been suggested that osteoclast activity may be necessary for the development of these osteoblastic metastases based on data from lytic and mixed lytic–blastic tumors. Here we investigate the effects of complete in vivo osteoclast depletion via the blockade of receptor activator of NF:κB (RANK) on the establishment and progression of purely osteoblastic (LAPC-9 cells) bone lesions induced by human prostate cancer cells using a SCID mouse intratibial injection model. The subcutaneous administration of the RANK antagonist (15 mg/kg) RANK:Fc did not prevent the formation of purely osteoblastic lesions, indicating that osteoclasts may not be essential to the initial development of osteoblastic metastases. However, RANK:Fc protein appeared to inhibit the progression of established osteoblastic lesions, suggesting that osteoclasts may be involved in the subsequent growth of these tumors once they are already present. In contrast, RANK:Fc treatment effectively blocked the establishment and progression of purely osteolytic lesions formed by PC-3 cells, which served as a positive control. These results indicate that in vivo RANK blockade may not be effective for the prevention of osteoblastic metastasis but may potentially represent a novel therapy that limits the growth of established metastatic CaP lesions in bone.

Gemcitabine radiosensitization after 153Sm-EDTMP (quadramet) bone-seeking radioisotope therapy for osteoblastic lesions of osteosarcoma: Activity and principles

8534 Background: Osteoblastic metastases can avidly concentrate bone-seeking radiopharmaceuticals. We sought to increase effectiveness of high-dose 153Samarium ethylenediaminetetramethylenephosphonate (153Sm-EDTMP; Quadramet) on osteosarcomas using a radiosensitizer, gemcitabine. Methods: Fourteen patients with osteoblastic lesions of osteosarcoma were treated. With high-dose 153Sm-EDTMP (30 mCi/kg) and stem cell support. Gemcitabine was administered 1 day after samarium. Imaging studies used to assess response. Results: The residual total body radioactivity was within the safe range of <3.6 mCi on day +14 (1.1 +/− 0.4 mCi; range 0.67–1.8 mCi). Expected cytopenias from skeletal binding of the radiopharmaceutical occurred within 14 days. Peripheral blood progenitor cells (PBPC) were infused 2 weeks after samarium in 11 patients; three patients had marrow infused. Blood count recovery was uneventful after PBPC in 11/11 patients. Toxicity from the daily gemcitabine regimen (250 mg/M2/day x 5) was excessive (grade 3 mucositis; N=1). Toxicity from a single infusion of gemcitabine (1500 mg/M2) in combination with 153Sm-EDTMP was minimal (pancytopenia). There were no reported episodes of hemorrhagic cystitis (hematuria) or nephrotoxicity. At the 6–8 week follow-up there were 6 PR, 2 mixed responses, and 6 patients with progressive disease. In the 12 patients followed >1 year, there have been no durable responses. Conclusions: High-dose 153Sm-EDTMP + gemcitabine has activity in osteoblastic osteosarcoma lesions, but recurrence is very common. Other strategies such as repeated standard doses of samarium with gemcitabine and/or use of additional measures of local and systemic control should be considered for more prolonged control of osteoblastic metastases. No significant financial relationships to disclose.

Cladribine therapy in a patient with an aleukemic subvariant of mast cell leukemia

Dear Sir, We present the case of a patient with an aleukemic subvariant of mast cell leukemia (MCL) with a very good partial response to cladribine therapy. MCL is very rare and is characterized by neoplastic mast cell (MC) proliferation that may lead to myelofibrosis with hemopoietic insufficiency, organ dysfunction, bleeding and death after a median survival time of 6–7 months. Imatinib is ineffective in cases where the activating mutation of the tyrosine kinase receptor c-kit affects its enzymatic site (D816V) [2, 3, 6]. Recently, promising treatment results were published using cladribine (2-CDA) in patients with systemic mastocytosis [1, 4, 5]. However, there are no reports on 2-CDA in patients with MCL. A 65-year-old white male presented in his usual symptom-free state of health in September 2002 for assessment of hepatosplenomegaly, mild normochromic–normocytic anemia (hemoglobin level 110 g/l) and thrombocytopenia (125,000/mm), with normal total and differential white blood cell counts. Radiographic studies revealed abdominal lymphadenopathy as well as osteoblastic and osteolytic bone lesions. Bone marrow biopsy showed osteomyelosclerosis and infiltration with atypical MC accounting for 30% of all nucleated cells. The level of the serum tryptase was 10.1 ng/ml (normal 200 ng/ ml), chymase, CD2, CD25, and CD117 (KIT). The characteristic point mutation of the c-kit proto-oncogene in codon 816 (Asp-816-Val) was detected in DNA from bone marrow, while the karyotype was normal. However, circulating MCs were not found in the peripheral blood. An aleukemic subvariant of MCL was diagnosed according to the WHO classification of tumors [7]. Pegylated interferon α-2b given initially had no therapeutic effect whatsoever. Due to the patient’s deteriorating condition, we started continuous intravenous infusion of 2-CDA (0.1 mg/kg body weight) for 5 days and repeated monthly. This led to only a moderate reduction of absolute CD4 lymphocyte counts, and prophylaxis with cotrimoxazole was thus not considered necessary. All six courses of 2-CDA therapy were given on time without severe (grades 3–4) hematological toxicity or infectious complications. Mediator-related symptoms (night sweats, pruritus, arthralgia, diarrhea) began to improve after 1 week and disappeared completely within 2 months. The spleen size had decreased from 23 cm (maximum diameter) before 2-CDA therapy to 12 cm post-treatment. The serum tryptase level dropped to 29 ng/ml and the degree of bone marrow MC infiltration was reduced to 10–15%. The patient regained a 90–100% Karnofsky performance status, reflected by his ability to hike 10 miles/day. However, radiological evaluations were performed after treatment and revealed no significant changes in bone lesions. In contrast to the other 15 cases of proven MCL in the literature, this patient does not show a progressive course on 2-CDA 30 months after the diagnosis of MCL. In conclusion, 2-CDA has demonstrated an excellent therapeutic index in this patient with MCL and warrants further investigations of this infrequent entity. O. Penack (*) . E. Thiel . M. Notter Department of Hematology, Oncology, and Transfusion Medicine, Charite-Campus Benjamin Franklin, Berlin, Germany e-mail: olaf.penack@charite.de

Expression of recombinant MDA-BF-1 with a kinase recognition site and a 7-histidine tag for receptor binding and purification

Prostate cancer metastasizes predominantly to bone, where it induces osteoblastic lesions. Paracrine factors secreted by the metastatic cancer cells are thought to mediate these events. We previously isolated a novel bone metastasis-related factor (MDA-BF-1) from bone marrow aspirate samples from patients with prostate cancer and bone metastasis, and found that this factor stimulated osteoblast differentiation, possibly by interacting with a receptor on the osteoblasts. Identifying this putative MDA-BF-1 receptor biochemically requires the expression of MDA-BF-1 for receptor binding assays and for the preparation of a ligand-affinity column. We tagged MDA-BF-1 with a peptide containing a protein kinase A phosphorylation site plus a 7-histidine sequence to facilitate the labeling of MDA-BF-1 for receptor binding assay and the binding of MDA-BF-1 to an immobilized metal affinity column. The recombinant MDA-BF-1 protein (MDA-BF1-kinase-his) was expressed in Sf9 cells using a baculovirus expression system. About 0.8 mg of purified MDA-BF1-kinase-his protein was obtained from 4 × 10 8 Sf9 cells. MDA-BF1-kinase-his can be phosphorylated by PKA with a specific activity around 10 5 cpm/μg protein. Receptor binding assays using this 32P-labeled MDA-BF-1 showed that MDA-BF-1 bound to membranes prepared from Saos-2, an osteosarcoma cell line, and C2C12, a mouse pluripotent mesenchymal precursor cell line that can be induced to become osteoblast by BMP-2. In contrast, MDA-BF-1 did not bind to membranes from PC-3 human prostate cancer cells or HEK293 human embryonic kidney cells. These observations suggest that the MDA-BF-1 receptor is expressed in cells of osteoblastic lineage. In addition to its use as a ligand for receptor binding assays, a ligand affinity column can be prepared by binding MDA-BF1-kinase-his to an IMAC for the purification of MDA-BF-1 receptor.