Development Of Bone Lesions Research Articles

Highlights of This Issue

The MYC oncogene family is amplified and aberrantly expressed in many cancers. Despite MYC1s unquestioned relevance to human cancer and decades of research, there are no therapies that target MYC. Here, Moser and colleagues showed that WRN, a DNA helicase involved in DNA repair and ageing, is required for proliferation of MYC-overexpressing cancer cells but not normal cells. Blocking WRN expression or function causes regression of MYC-associated lung cancers and increases survival of mice with MYC-driven lymphomas. As WRN is a nonessential, but druggable gene, targeting its enzymatic activity could be effective treatment for MYC-associated cancers.Nearly 70% of prostate cancer patients have lost expression of TGF-β type II receptor in the stroma, contributing to cancer progression. Li and colleagues evaluated whether TβRII loss in the primary environment affected prostate cancer bone metastasis, and found that TβRII expression was lost in prostate cancer cancer-associated fibroblasts in bone metastatic tissues. Using conditional stromal Tgfbr2 knockout mice, they showed that loss of TβRII in prostatic fibroblasts contributes to cell adhesion, and early establishment and subsequent bone lesion development through upregulation of CXCL16 and CXCL1. Thus, changes in the primary tumor microenvironment can be predictive of and therapeutic targets for prostate cancer metastasis.Altered expression of miRNAs is associated with various human cancers, including colorectal cancer (CRC). Reid and colleagues analyzed miRNA profiles of 40 CRCs and their paired normal tissues and searched for putative targets of the differentially expressed miRNAs in gene expression datasets of normal and tumor samples. They identified 121 genes involved in key pathways of CRC progression, adding a novel layer of regulation in the Vogelstein multistep model of CRC pathogenesis. One pair, miR-1 and MET, was studied and miR-1 emerged to have a tumor suppressor function in CRC by directly downregulating MET oncogene.Early life environmental exposures can increase risk for cancer later in life. For developmental exposures to endocrine disruptors, such as environmental xenoestrogens, reprogramming of the epigenome is thought to underlie this increased cancer risk. Greathouse and colleagues showed that the histone methyltransferase EZH2 is a target for xenoestrogens that induce this type of developmental reprogramming. They showed that activation of PI3K/AKT signaling by xenoestrogens phosphorylates and inactivates the methyltransferase EZH2. As a result, levels of the repressive H3K27 epigenetic methyl mark are reduced, reprogramming genes to become hyperresponsive to estrogen and promoting the development of hormone-dependent tumors. These data emphasize the importance of a life-course approach to identifying environmental causes of cancer, and provide a direct mechanism by which early life environmental exposures can disrupt the epigenome and increase susceptibility to tumorigenesis later in life.

Osteochondral Destruction in Pigmented Villonodular Synovitis During the Clinical Course

In pigmented villonodular synovitis (PVNS), some cases recur and progress to osteochondral destruction. The aim of our study was to clarify the occurrence of osteochondral destruction according to the location of PVNS during the clinical course. Seventy-two patients with PVNS (43 female, 29 male) with a mean age of 40 years (range 3-87 yrs) had been referred to our institutions. Factors influencing the occurrence of osteochondral destruction were investigated. Mean followup was 60 months (range 12-190 mo). Adjacent bone change occurred in 24 (42%) of 57 patients, who were evaluated at the time of the first consultation. Eight (89%) of 9 patients with hip lesions initially had bone lesions, significantly more frequently than those with other lesions (p = 0.038). Duration of symptoms was significantly correlated with the occurrence of bone lesions in diffuse knee lesions (p = 0.005). During followup, patients with location in the knee had a significantly higher incidence of osteoarthritic change (73%) compared to those with foot and ankle involvement (p = 0.027). Re-operation was more frequently required for knee lesions due to the high recurrence rate (32%). Patients who required re-operation had significantly more marked osteoarthritic change in knees (p = 0.001) during followup than those who did not. For PVNS arising in knees, repeated recurrences followed by re-operation resulted in the progression of osteoarthritic change. PVNS arising in hips, feet, and ankles developed bone lesions initially, probably due to the limited volume of these joints. The indications for re-operation for recurrent knee lesions require careful consideration regarding progression of osteoarthritic change.

Extensive Bone Marrow Infiltration and Abnormal Free Light Chain Ratio Identifies Patients with Smoldering Myeloma At High Risk for Progression to Symptomatic Disease,

Abstract 3926Asymptomatic/smoldering multiple myeloma (SMM) is a proliferative plasma cell disorder characterized by a substantial risk of progression to symptomatic myeloma. According to current recommendations, patients with SMM should be followed without treatment until they develop symptomatic disease. However, the risk of progression to symptomatic myeloma varies between different series and for individual patients; thus, significant effort is needed in order to identify factors that could discriminate those who are at high risk for progression. Such patients should be followed closer and should be considered candidates for clinical trials. In order to evaluate previously recognized risk factors and study patterns of progression we analyzed our series of patients with SMM, who have been diagnosed and followed in the Department of Clinical Therapeutics in Athens, Greece.SMM was defined as serum monoclonal (M) protein (IgG or IgA) level of ≥3 g/dL and/or bone marrow plasma cells ≥10%, absence of end-organ damage, such as lytic bone lesions, anemia, hypercalcemia, or renal failure, that can attributed to a plasma cell proliferative disorder (IMWG criteria, Br J Haematol 2003;121:749–57). Progression to symptomatic myeloma was defined as per the IMWG proposed criteria.We analyzed 95 patients with SMM, 53% of whom were females, 70% had IgG heavy chain, 22% had IgA, 5% had a biclonal SMM and 3% had light chain only SMM, while 65% had a kappa light chain and 35% a lambda light chain. Median infiltration by clonal plasma cells in BM trephine biopsy was 20% (range 10–90%), 10% of patients had ≥60% clonal plasma cells in BM biopsy. Fifty patients had MRI of the spine at the time of diagnosis of SMM and 19.5% had an abnormal pattern of BM infiltration (diffuse, focal or variegated pattern). In patients with available bone marrow immunohistochemistry data, 61% had clonal plasma positive for CD56, 17% for CD20 and 19% for cyclin D1.The median follow up of the cohort was 27 months (range 1–253 months) and 23 (24%) patients have progressed to symptomatic myeloma. The one-year, 2-year and 3-year cumulative probability of progression was 7%, 12% and 20% respectively. Nine patients (9.5%) progressed within the first two years from the diagnosis of SMM. All these patients had an M-protein of ≥1 g/dl (10 g/L), 67% had bone marrow plasma cells >60% and 80% had an abnormal MRI pattern of BM infiltration. The 3-year probability of progression to symptomatic myeloma was 4%, 18% and 87% for patients with <20%, 20–59% and ≥60% clonal plasma cells in bone marrow biopsy (P<0.001). The 2-year probability of progression to symptomatic myeloma was 0%, 13% and 60% for patients with <20%, 20–59% and ≥60% clonal plasma cells in BM biopsy (P<0.001). Patients with significantly abnormal free light chain ratio (either kappa/lambda ≥8 or kappa/lambda ≤0.125, according to Dispenzieri et al, Blood 2008;111:785–9) had a 3-year probability of progression to symptomatic MM of 41% vs. 15% (p=0.07). There was no significant difference in the risk of progression to symptomatic MM for patients with IgA vs. IgG myeloma. In multivariate analysis, abnormal FLC ratio less than 0.125 or more than 8 (HR: 6.4, 95% CI 1.3–34.5 p=0.032) and BM clonal plasma cells infiltration ≥60% (HR: 23, 95% CI 5–125, p<0.001) were independent risk factors for progression to symptomatic myeloma.Progression to symptomatic MM was manifested by the development of anemia in 52% of patients who progressed to symptomatic MM, development of lytic bone lesions or pathologic fracture in 48%, an increase of serum creatinine to ≥2 mg/dl in 13%, development of a soft tissue plasmacytoma in 4% and development of hypercalcemia in 4%.In conclusion, in our series of patients the 3-year probability of progression to symptomatic myeloma is about 20%, but there is a subgroup of patients with extensive bone marrow infiltration (≥60%) and highly abnormal FLC ratio, who have a substantial risk of progression to symptomatic disease within the first two years from the diagnosis of SMM. These high risk patients may also have other features such as abnormal MRI of the spine. Patients at high risk for progression should be considered for clinical trials evaluating the role of treatment before the development of symptomatic disease, which in most cases is manifested with anemia and/or lytic bone disease or pathologic fractures. Disclosures:No relevant conflicts of interest to declare.

Smoldering Multiple Myeloma (SMM) At High-Risk of Progression to Symptomatic Disease: A Phase III, Randomized, Multicenter Trial Based On Lenalidomide-Dexamethasone (Len-Dex) As Induction Therapy Followed by Maintenance Therapy with Len Alone Vs No Treatment

Abstract 991▪▪This icon denotes a clinically relevant abstractSmoldering Multiple Myeloma (SMM) is an asymptomatic proliferative disorder of plasma cells (PCs) defined by a serum monoclonal component (MC) of 30 g/L or higher and/or 10% or more plasma cells in the bone marrow (BM). There are several risk factors predicting high-risk of progression to symptomatic disease: >10% of PCs in BM, serum MC >30g/L, >95% aberrant PCs by immunophenotyping, or abnormal free-light chains. Standard of care of SMM is no treatment until progression disease.In this phase III trial, SMM patients at high-risk of progression were randomized to receive Len-dex as induction followed by Len alone as maintenance vs no treatment in order to evaluate whether the early treatment prolongs the time to progression (TTP) to symptomatic disease. The high-risk population was defined by the presence of both >PC 10% and MC >30g/L or if only one criterion was present, patients must have a proportion of aberrant PCs within the total PCsBM compartment by immunophenotyping of 95% plus immunoparesis.Len-dex arm received an induction treatment consisting on nine four-weeks cycles of lenalidomide at dose of 25 mg daily on days 1–21 plus dexamethasone at dose of 20 mg daily on days 1–4 and 12–15 (total dose: 160mg), followed by maintenance until progression disease with Lenalidomide at dose of 10 mg on days 1–21 every two months (amended in May 2010 into monthly).The 124 planned patients were already recruited, and 118 were evaluable (six patients didn't meet inclusion criteria).According to baseline characteristics, both groups were well balanced. On an ITT analysis (n=57), based on IMWG criteria, the overall response rate during induction therapy was 81%, including 56% PR, 11% VGPR, 7% CR and 7% sCR. 51 patients have completed the nine induction cycles, and the ORR was 87%, including 12% VGPR, 8% CR and 8% sCR. After a median of 7 cycles of maintenance therapy (1-21), the sCR increased to 12%.After a median follow-up of 22 months (range: 5–42), six patients progressed to symptomatic disease in the Len-dex arm: four of them during maintenance therapy and the other two progressed 3 and 8 months after early discontinuation of the trial due to personal reasons. In addition, twelve patients have developed biological progression during maintenance, and dex was added according to the protocol. In nine of them, the addition of dex was able to control again the disease without CRAB symptoms (median of 11 months). In the therapeutic abstention arm, 28 out of 61 patients (46%) progressed to active MM. The estimated hazard ratio was 6·2 (95%CI= 2·6-15), corresponding to a median TTP from inclusion of 25 months for the not treatment arm vs median not reached in the treatment arm (p<0.0001). It should be noted that 13 out of these 28 patients developed bone lesions as a symptom of active MM. Deaths in the Len-dex and no treatment arms were 1 and 2, respectively (p=0·6). Estimated 3-years overall survival (OS) from the inclusion in the trial was 98% for Len-dex arm and 82% for no treatment arm (p=0·05) and this difference was more evident if we evaluate the OS from the moment of diagnosis (HR: 6.7; 95% IC (0.7–57); p=0.03).As far as toxicity is concerned, during induction therapy, no G4 adverse events (AEs) were reported with Len-dex; 1 pt developed G3 anemia, 4 patients G3 asthenia 2 patients G3 diarrhea and 1 patient G3 skin rash; 3 patients developed G2 DVT. During maintenance, no G4 AEs were reported and only 1 patient developed G3 infection. Two patients in the Len-dex arm developed second primary malignancies (SPM): one developed polycythemia vera JAK2+, but the analysis of a frozen DNA sample obtained at the moment of inclusion in the trial demonstrated that JAK2 was already positive. The second-one was a prostate cancer in a patient with previous history of prostate enlargement plus elevated prostate specific antigen (PSA) who was closely followed by the urologist.In conclusion, this analysis shows that in high-risk SMM patients, delayed treatment resulted in early progression to symptomatic disease (median 25 months), while Len-dex as induction followed by Len as maintenance significantly prolonged the TTP (HR: 6·2), with a trend to improve the overall survival; in addition, tolerability is acceptable and concerning SPM, no safety warnings are at the present time. Moreover, biological progressions occurring under maintenance have remained controlled over a prolonged period of time. Disclosures:Mateos:Celgene: Honoraria; Janssen: Honoraria. Off Label Use: lenalidomide is not approved for smoldering myeloma. Rosiñol:Janssen: Honoraria; Celgene: Honoraria. Baquero:Celgene: Employment. Quintana:Celgene: Employment. García:Celgene: Employment.

Low-grade Bone Lesions in Survivors of Childhood Medulloblastoma/Primitive Neuroectodermal Tumor

Medulloblastoma/primitive neuroectodermal tumor (MB/PNET) is the most common malignant tumor of the central nervous system (CNS) in children. MB/PNET survivors are at an increased risk for developing second malignancies. Little has been reported on development of low-grade lesions of the calvarium in the radiation field in MB/PNET survivors. The purpose of this study was to assess the frequency of the low-grade bone lesion development in the radiotherapy field in pediatric MB/PNET survivors and describe the imaging characteristics of these lesions. Institutional review board approval was obtained for this retrospective review which was compliant with Health Insurance Portability and Accountability Act. Forty-one MB/PNET patients (29 male) who survived for at least 2 years after initiation of radiation therapy were included. The medical records were reviewed. The most recent available brain magnetic resonance imaging studies were evaluated. Three patients (7.3%) developed low-grade calvarial lesions and underwent resection and/or biopsy of the lesions. There were one Langerhans cell histiocytosis, one benign spindle cell lesion with myxoid change, and one fibrous dysplasia. Development of low-grade bone lesions of calvarium is not very rare in pediatric PNET/MB survivors. Bones in the radiation therapy field need to be carefully examined for assessment of secondary lesions.

Gaucher’s Disease in Lithuania: Its Diagnosis and Treatment

Gaucher's disease is a lysosomal storage disease caused by the lack of beta-glucocerebrosidase enzyme, leading to the accumulation of glucocerebroside. Gaucher's disease is the most frequent type of sphingolipidosis as well as the most frequent lysosomal disease. Clinically, two forms of Gaucher's disease are defined: nonneuronopathic form, so-called type 1, characterized by hepatosplenomegaly, thrombocytopenia, anemia, and osteopenia, and neuronopathic form, known as types 2 and 3, which are also characterized by hepatosplenomegaly, hematological and bone changes; however, involvement of the central nervous system dominates in the clinical picture. Severe deficiency of beta-glucocerebrosidase activity allows confirming the diagnosis based on the clinical picture or the findings of bone marrow examination. Treatment with human glucocerebrosidase was introduced in 1991. Clinically good results are achieved: not only accumulation of glucocerebroside is stopped, but also positive changes in the reticuloendothelial system and an improvement in development and hematological parameters of children are observed as well as the development of bone lesions is reduced. To date, Gaucher's disease has been diagnosed in 8 patients in Lithuania: 3 persons have type 3 and 5 have type 1 disease. Enzyme replacement therapy was started in 2001, and currently 6 persons are being treated. In majority of patients, Gaucher's disease was suspected after exclusion of other possible proliferative diseases. All patients within the first or second year of treatment achieved the therapeutic goals, namely: normalization of hematological parameters, reduction in liver and spleen volumes, and bone pain relief.

Cysteamine Toxicity in Patients with Cystinosis

To report new adverse effects of cysteamine. Detailed clinical information was obtained from the patients' physicians. New adverse events were reported in 8 of 550 patients with cystinosis treated with cysteamine in Europe during the last 5 years. Detailed clinical information was not available for 2 of these patients, 1 of whom died from cerebral ischemia. The 6 evaluable patients developed vascular elbow lesions (6/6), neurologic symptoms (1/6), bone and muscle pain (2/6), and/or skin striae (2/6). Analysis of biopsy specimens from the elbow lesions demonstrated angioendotheliomatosis with irregular collagen fibers. In 3 of the 6 patients, the daily cysteamine dose exceeded the recommended maximum of 1.95 g/m(2)/day. Dose reduction led to improvement of signs and symptoms in all 6 patients, suggesting a causal relationship with cysteamine administration. Cysteamine administration can be complicated by the development of skin, vascular, neurologic, muscular, and bone lesions. These lesions improve after cysteamine dose reduction. Doses >1.95 g/m(2)/day should be prescribed with great caution, but underdosing is not advocated.

Abstract 527: Loss of TGF-β responsiveness in stromal cells of the prostate facilitates early prostate cancer bone lesion development

Abstract The death of late stage prostate cancer patients is commonly due to incurable bone metastasis. Prostate cancer associated fibroblasts (CAF) play a role in cancer progression at the primary site, however it is unclear whether they are significant players at the bone metastatic site. We hypothesize that the primary CAF facilitate prostate tumor cell establishment in the bone environment and initial bone lesion development. Around two thirds of human prostate cancer tissues show loss of stromal TGFbeta type II receptor (Tgfbr2) expression at the primary site. We observed Tgfbr2 expression was also lost in bone marrow adjacent to prostate cancer cells. In addition, prostate cancer bone metastases expressed androgen receptor (AR) in the cancer cells and alpha-smooth muscle actin (SMA) in the adjacent CAF. Neither AR nor SMA was seen in the bone marrow environment when no prostate cancer cells were present. A mouse model of conditional Tgfbr2 knock-out in the stromal cells was used in this study. Since there is no evidence, so far, of CAF metastasis along with cancer cells, we focused on the paracrine effects of CAFs from primary site on prostate cancer bone metastasis. GFP labeled prostate cancer C4-2B cells, were incubated with conditioned medium from primary cultured Tgfbr2-flox or Tgfbr2-KO mouse prostate stromal cells for 48hrs. The C4-2B cells were then injected into the tibiae of SCID mice. Bone lesion was monitored weekly and the tumor growth was imaged by Maestro when the tibiae were harvested. It was found that the conditioned medium from Tgfbr2-KO mice prostate stromal cells promoted prostate cancer growth in the bone and mixed bone lesion development early at week 4 and week 5. The incidence and area of the bone lesions were not significant different between week 6 and 10. The tumor growth in the tibia was not different week 10. After 48 hr of incubation with the conditioned medium from Tgfbr2-KO mice prostate stromal cells, the adhesion of C4-2B cells to collagen I was increased by 5.8 fold compared to the cells without previous conditioned media incubation, and 1.5 fold compared to incubation with stromal conditioned medium from Tgfbr2-Flox mice. To examine the secreted factors from the Tgfbr2-KO prostate stromal cells we performed cytokine antibody array analysis. GCSF, KC(Cxcl1), Cxcl16, LIX(Cxcl5) and Decorin were found to be increased more than 2 fold in the medium conditioned by prostate stromal cells from Tgfbr2-KO mice compared to that conditioned by Tgfbr2-flox mice. In conclusion, loss of TGF-β responsiveness in prostate stromal cells resulted in up-regulation of some cytokines. These changes are proposed to increase prostate cancer cell adhesion in the bone microenvironment promoting early establishment of prostate cancer cells in the bone and subsequent bone lesion development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 527. doi:10.1158/1538-7445.AM2011-527

Inhibiting activin-A signaling stimulates bone formation and prevents cancer-induced bone destruction in vivo

Cancers that grow in bone, such as myeloma and breast cancer metastases, cause devastating osteolytic bone destruction. These cancers hijack bone remodeling by stimulating osteoclastic bone resorption and suppressing bone formation. Currently, treatment is targeted primarily at blocking bone resorption, but this approach has achieved only limited success. Stimulating osteoblastic bone formation to promote repair is a novel alternative approach. We show that a soluble activin receptor type IIA fusion protein (ActRIIA.muFc) stimulates osteoblastogenesis (p < .01), promotes bone formation (p < .01) and increases bone mass in vivo (p < .001). We show that the development of osteolytic bone lesions in mice bearing murine myeloma cells is caused by both increased resorption (p < .05) and suppression of bone formation (p < .01). ActRIIA.muFc treatment stimulates osteoblastogenesis (p < .01), prevents myeloma-induced suppression of bone formation (p < .05), blocks the development of osteolytic bone lesions (p < .05), and increases survival (p < .05). We also show, in a murine model of breast cancer bone metastasis, that ActRIIA.muFc again prevents bone destruction (p < .001) and inhibits bone metastases (p < .05). These findings show that stimulating osteoblastic bone formation with ActRIIA.muFc blocks the formation of osteolytic bone lesions and bone metastases in models of myeloma and breast cancer and paves the way for new approaches to treating this debilitating aspect of cancer.

Brain-Derived Neurotrophic Factor Is a Potential Osteoclast Stimulatory Factor In Multiple Myeloma

AbstractAbstract 1917 Background and Objective:Multiple myeloma (MM) is characterized by accumulation of monoclonal plasma cells in the bone marrow and progression of lytic bone lesions. The mechanisms of enhanced bone resorption in patients with myeloma are not fully defined. We have previously identified the role of brain-derived neurotrophic factor (BDNF) in proliferation and migration of MM cells. In the present study, we investigated whether BDNF was present in marrow from patients with MM and possibly involved in MM cell-induced osteolysis. Methods and Results:Levels of bone marrow plasma BDNF was measured by ELISA in a cohort of individuals with MM and controls. The concentration of BDNF was found to be significantly elevated in patients with MM (879 ± 93) pg/ml when compared with bone marrow plasma derived from normal control subjects (186 ± 52) pg/ml (p < 0.001). Moreover, bone marrow plasma levels of BDNF positively correlated with plasma cell burden and extent of bone disease in MM patients. In osteoclast formation assay, bone marrow plasma from 31 of 37 patients with MM tested significantly stimulated the formation of osteoclast when compared to controls (61.8 ± 7 [mean ± SEM for the 31 patients] versus 25.2 ± 6 TRAP+ multinucleated cells/well [mean ± SEM for the 12 controls]; p < 0.01). The effect was significantly blocked by a neutralizing antibody to BDNF (p < 0.05), suggesting a critical role for BDNF in osteoclast activation. Furthermore, BDNF was found to dose-dependently increased the formation of multinucleated, TRAP+ osteoclast. The direct effects of recombinant BDNF on osteoclast formation and bone resorption support the potential role of BDNF in the MM bone disease. Using reverse-transcriptase polymerase chain reaction analysis and western blotting assay, we demonstrated that BDNF receptor TrkB was expressed by human osteoclast precursors and a Trk inhibitor K252a markedly inhibited osteoclast formation stimulated with BDNF. These data suggested that TrkB is the functional receptor mediating BDNF's effect on osteoclast formation. Finally, bone marrow plasma BDNF level positively correlated with macrophage inflammatory protein (MIP)-1α (r = 0.45, p < 0.005) and receptor activator of nuclear factor-κB ligand (RANKL) (r = 0.68, p < 0.0001), two major osteoclast stimulatory factors in MM. Conclusion:Taken together, our results demonstrate the ability of MM cells to secret BDNF correlates with the severity of osteoclastic bone resorption, and provide evidence that BDNF play a causal role in the development of MM bone lesions through TrkB receptor. Disclosures:No relevant conflicts of interest to declare.

Smoldering Multiple Myeloma (SMM) at High-Risk of Progression to Symptomatic Disease: A Phase III, Randomized, Multicenter Trial Based On Lenalidomide-Dexamethasone (Len-Dex) as Induction Therapy Followed by Maintenance Therapy with Len Alone Vs No Treatment

AbstractAbstract 1935Smoldering Multiple Myeloma (SMM) is an asymptomatic proliferative disorder of plasma cells (PCs) defined by a serum monoclonal component (MC) of 30 g/L or higher and/or 10% or more plasma cells in the bone marrow (BM), but no evidence of end-organ damage. There are several risk factors predicting high-risk of progression to symptomatic disease (>50% at 2 years): >10% of PCs in BM, serum MC >30g/L, >95% aberrant PCs by immunophenotyping, or abnormal free-light chains. Standard of care of SMM is close follow-up without treatment until progression disease. Several trials have evaluated the role of early treatment with convencional agents (melphalan), bisphosphonates and novel agents (thalidomide, anti-IL1a), with no clear benefit, but they didn't focus on high-risk patients.In this phase III trial, SMM patients at high-risk of progression were randomized to receive Len-dex as induction followed by Len alone as maintenance vs no treatment in order to evaluate whether the early treatment prolongs the time to progresioÃŒn (TTP) to symptomatic disease. The high risk population was defined by the presence of both >PC 10% and MC >30g/L or if only one criterion was present, patients must have a proportion of aberrants PCs within the total PCsBM compartment by immunophenotyping of 95% plus immunoparesis.Len-dex arm received an induction treatment consisting on nine four-weeks cycles of lenalidomide at dose of 25 mg daily on days 1–21 plus dexamethasone at dose of 20 mg daily on days 1–4 and 12–15 (total dose: 160mg), followed by maintenance until progression disease with Lenalidomide at dose of 10 mg on days 1–21 every two months (ammended in May 2010 into monthly).The 124 planned patients were recruited between October 2006 and June 2010, and 118 were evaluables (three in Len-dex and three in therapeutic abstention arm didn't meet inclusion criteria). This second interim analysis was planned when all patients were recruited.According to baseline characteristics, both groups were well balanced. On an ITT analysis (n=57), based on IMWG criteria, the overall response rate during induction therapy was 75%, including 51% PR, 12% VGPR, 5% CR and 7% sCR. If we select the group of 33 patients who completed the nine induction cycles, the ORR was 91%, including 15% VGPR, 9% CR and 9% sCR. After a median of 8 cycles of maintenance therapy (1-15), the sCR increased to 16%.After a median follow-up of 16 months (range:1-33), four patients progressed to symptomatic disease in the Len-dex arm: two of them during maintenance therapy after 24 and 28 months from inclusion and the other two progressed 3 and 8 months after early discontinuation of the trial due to personal reasons. In addition, nine patients have developed biological progression during maintenance, but in all but one of these, Len has been able to control the disease without CRAB symptoms (median of 9·5 months (1-18)). In the therapeutic abstention arm, 21 out of 61 patients progressed to active MM. The estimated hazard ratio was 6·7 (95%CI= 2·3-19·9), corresponding to a median TTP from inclusion of 25 months for the not treatment arm vs median not reached in the treatment arm (p<0.0001). It should be noted that 10 out of these 21 patients developed bone lesions as a symptom of active MM. Deaths in the Len-dex and no treatment arms were 1 and 2, respectively (p=0·6).As far as toxicity is concerned, during induction therapy, no G4 adverse events (AEs) were reported with Len-dex; 1 pt developed G3 anemia, 4 patients G3 asthenia 2 patients G3 diarrhea and 1 patient G3 skin rash; 3 patients developped G2 DVT. During maintenance, no G4 AEs were reported and only 1 patient developed G3 infection.In conclusion, this second interim analysis shows that in high-risk SMM patients, delayed treatment resulted in early progression to symptomatic disease (median 25 months), while Len-dex as induction followed by Len as maintenance significantly prolonged the TTP (HR: 6·7), with excelent tolerability; moreover, biological progressions occurring under maintenance have remained controlled over a prolonged period of time. Disclosures:Mateos:Celgene: Honoraria. Off Label Use: Lenalidomide is not approved for the treatment of smoldering multiple myeloma. De La Rubia:Celgene: Honoraria. Rosiñol:Celgene: Honoraria. Lahuerta:Celgene: Honoraria. Palomera:Celgene: Honoraria. Oriol:Celgene: Honoraria. Garcia-Laraña:Celgene: Honoraria. Hernández:Celgene: Honoraria. Leal-da-Costa:Celgene: Honoraria. Alegre:Celgene: Honoraria. Quintana:Celgene: Employment. Baquero:Celgene: Employment. García:Celgene: Honoraria. San Miguel:Celgene: Honoraria.

Combined inhibition of the BMP pathway and the RANK-RANKL axis in a mixed lytic/blastic prostate cancer lesion

The purpose of this study was to investigate the influence of combined inhibition of receptor activator of nuclear factor kappa-B ligand (RANKL) and bone morphogenetic protein (BMP) activity in a mixed lytic/blastic prostate cancer lesion in bone. Human prostate cancer cells (C4 2b) were injected into immunocompromised mice using an intratibial injection model to create mixed lytic/blastic lesions. RANK-Fc, a recombinant RANKL antagonist, was injected subcutaneously three times a week (10 mg/kg) to inhibit RANKL and subsequent formation, function and survival of osteoclasts. Inhibition of BMP activity was achieved by transducing prostate cancer cells ex vivo with a retroviral vector expressing noggin (retronoggin; RN). There were three treatment groups (RANK-Fc treatment, RN treatment and combined RN and RANK-Fc treatment) and two control groups (untreated control and empty vector control for the RN treatment group). The progression of bone lesion and tumor growth was evaluated using plain radiographs, hindlimb tumor size, 18F-Fluorodeoxyglucose and 18F-fluoride micro PET-CT, histology and histomorphometry. Treatment with RANK-Fc alone inhibited osteolysis and transformed a mixed lytic/blastic lesion into an osteoblastic phenotype. Treatment with RN alone inhibited the osteoblastic component in a mixed lytic/blastic lesion and resulted in formation of smaller osteolytic bone lesion with smaller soft tissue size. The animals treated with both RN and RANK-Fc demonstrated delayed development of bone lesions, inhibition of osteolysis, small soft tissue tumors and preservation of bone architecture with less tumor induced new bone formation. This study suggests that combined inhibition of the RANKL and the BMP pathway may be an effective biologic therapy to inhibit the progression of established mixed lytic/blastic prostate cancer lesions in bone.

A novel role of IL-17–producing lymphocytes in mediating lytic bone disease in multiple myeloma

AbstractOsteoclast (OC)–mediated lytic bone disease remains a cause of major morbidity in multiple myeloma. Here we demonstrate the critical role of interleukin-17–producing marrow infiltrating lymphocytes (MILs) in OC activation and development of bone lesions in myeloma patients. Unlike MILs from normal bone marrow, myeloma MILs possess few regulatory T cells (Tregs) and demonstrate an interleukin-17 phenotype that enhances OC activation. In univariate analyses of factors mediating bone destruction, levels of cytokines that selectively induce and maintain the Th17 phenotype tightly correlated with the extent of bone disease in myeloma. In contrast, MILs activated under conditions that skew toward a Th1 phenotype significantly reduced formation of mature OC. These findings demonstrate that interleukin-17 T cells are critical to the genesis of myeloma bone disease and that immunologic manipulations shifting MILs from a Th17 to a Th1 phenotype may profoundly diminish lytic bone lesions in multiple myeloma.

Expression of a protein involved in bone resorption, Dkk1, is activated by HTLV-1 bZIP factor through its activation domain

BackgroundHuman T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia, a malignancy characterized by uncontrolled proliferation of virally-infected CD4+ T-cells. Hypercalcemia and bone lesions due to osteoclast-mediated bone resorption are frequently associated with more aggressive forms of the disease. The HTLV-1 provirus contains a unique antisense gene that expresses HTLV-1 basic leucine zipper (bZIP) factor (HBZ). HBZ is localized to the nucleus where it regulates levels of transcription by binding to certain cellular transcriptional regulators. Among its protein targets, HBZ forms a stable complex with the homologous cellular coactivators, p300 and CBP, which is modulated through two N-terminal LXXLL motifs in the viral protein and the conserved KIX domain in the coactivators.ResultsTo determine the effects of these interactions on transcription, we performed a preliminary microarray analysis, comparing levels of gene expression in cells with wild-type HBZ versus cells with HBZ mutated in its LXXLL motifs. DKK1, which encodes the secreted Wnt signaling inhibitor, Dickkopf-1 (Dkk1), was confirmed to be transcriptionally activated by HBZ, but not its mutant. Dkk1 plays a major role in the development of bone lesions caused by multiple myeloma. In parallel with the initial findings, activation of Dkk1 expression by HBZ was abrogated by siRNA-mediated knockdown of p300/CBP or by a truncated form of p300 containing the KIX domain. Among HTLV-1-infected T-cell lines tested, the detection of Dkk1 mRNA partially correlated with a threshold level of HBZ mRNA. In addition, an uninfected and an HTLV-1-infected T-cell line transfected with an HBZ expression vector exhibited de novo and increased DKK1 transcription, respectively. In contrast to HBZ, The HTLV-1 Tax protein repressed Dkk1 expression.ConclusionsThese data indicate that HBZ activates Dkk1 expression through its interaction with p300/CBP. However, this effect is limited in HTLV-1-infected T-cell lines, which in part, may be due to suppression of Dkk1 expression by Tax. Consequently, the ability of HBZ to regulate expression of Dkk1 and possibly other cellular genes may only be significant during late stages of ATL, when Tax expression is repressed.

Alternate protein kinase A activity identifies a unique population of stromal cells in adult bone

A population of stromal cells that retains osteogenic capacity in adult bone (adult bone stromal cells or aBSCs) exists and is under intense investigation. Mice heterozygous for a null allele of prkar1a (Prkar1a(+/-)), the primary receptor for cyclic adenosine monophosphate (cAMP) and regulator of protein kinase A (PKA) activity, developed bone lesions that were derived from cAMP-responsive osteogenic cells and resembled fibrous dysplasia (FD). Prkar1a(+/-) mice were crossed with mice that were heterozygous for catalytic subunit Calpha (Prkaca(+/-)), the main PKA activity-mediating molecule, to generate a mouse model with double heterozygosity for prkar1a and prkaca (Prkar1a(+/-)Prkaca(+/-)). Unexpectedly, Prkar1a(+/-)Prkaca(+/-) mice developed a greater number of osseous lesions starting at 3 months of age that varied from the rare chondromas in the long bones and the ubiquitous osteochondrodysplasia of vertebral bodies to the occasional sarcoma in older animals. Cells from these lesions originated from an area proximal to the growth plate, expressed osteogenic cell markers, and showed higher PKA activity that was mostly type II (PKA-II) mediated by an alternate pattern of catalytic subunit expression. Gene expression profiling confirmed a preosteoblastic nature for these cells but also showed a signature that was indicative of mesenchymal-to-epithelial transition and increased Wnt signaling. These studies show that a specific subpopulation of aBSCs can be stimulated in adult bone by alternate PKA and catalytic subunit activity; abnormal proliferation of these cells leads to skeletal lesions that have similarities to human FD and bone tumors.

Abstract 4321: A four-pronged imaging approach to characterization of bone metastasis in mouse models

Abstract Introduction: The process of bone metastasis involves both osteoblastic and resorptive components and is a common feature of both prostate and breast cancers. The ability to visualize and quantify the early stages of bone involvement in mouse models of bone metastasis would provide a platform for development of new agents targeted at inhibition or treatment of bone metastases. Two approaches that stand to enable this are 18F-NaF PET and optical imaging using biphosphonate fluorescent probes such as Osteosense, through targeting of hydroxyapatite (HA), a biomarker for osteoblastic activity. Additionally, bioluminescence imaging using luciferase expressing tumor lines and micro-CT imaging of the skeleton can enable anatomic characterization of tumor burden and development of bone lesion in bone metastasis models. Methods: MDA-MB-231-luc-D3H2LN human mammary adenocarcinoma cells (105 cells in 100µl) were injected into the left ventricle of female nu/nu mice on Day 0 approximately 6-7min after IP injection of luciferin (150mg/kg). Bioluminescence scans were used to determine successful injection by distribution of light throughout the body. On Day 14, all tumor pool mice were imaged using bioluminescence and enrolled on study based on incidence of luciferase signals at bone sites. After staging, serial bioluminescence imaging was used to localize tumor signals and determine incidence and growth of bone metastases and micro-CT was used to image the extent of bone lesions associated with the metastases. Additionally, whole body PET scans were used at multiple time points after 90min uptake of 18F-NaF. Images were reconstructed using a 2D OSEM method and localized bone sites segmented and analyzed for standardized uptake value (SUV). Fluorescence imaging was also used 24h after administration of Osteosense 750 to characterize localization of the tracer at the bone sites of interest. After background correction, the bone sites of interest were segmented and analyzed for average efficiency. The 18F-NaF and Osteosense endpoints were correlated with bioluminescence determined tumor burden and location. Results and Discussion: Both 18F-NaF PET imaging and fluorescent imaging using Osteosense highlighted localized bone signals that could be correlated with bioluminescent signals and micro-CT visualized bone lesions from approximately Day 17. Both the PET and fluorescence imaging approaches showed early indication of bone involvement, presumably through HA, indicating osteoblastic activity. This multi-endpoint approach enabled non-invasive determination of both soft tumor and bone components of metastatic bone disease. An approach like this may enable more quantitative characterization of the early stages of bone metastasis in mouse models, and facilitate development of bone targeted treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4321.

Abstract 5394: Sequential treatment with zoledronic acid and doxorubicin has no additive effects in an established mouse model of bone metastasis

Abstract Bone metastases still present an unmet need for effective treatment. One preclinical study has suggested a beneficial effect with sequential treatment of doxorubicin (DOX) and zoledronic acid (ZOL). Furthermore, bisphosphonates are used as adjuvant therapy, but controversy of their anti-tumor effects remains. We tested clinically relevant doses of DOX, ZOL and their sequential combination in a treatment setting in a widely used nude mouse model of breast cancer bone metastasis. GFP-transfected MDA-MB-231(SA) human breast cancer cells were inoculated into five-week-old female nude mice via left cardiac ventricle. Subsequent tumor growth and development of osteolytic bone lesions were quantitated by fluorescence imaging and radiography, respectively, on days 14 and 25. The mice were randomized to four groups (n=8/group) on day 14 according to body weight and the presence of osteolytic lesions. Starting on day 15, groups received either weekly dose of vehicle (control group), DOX 2.5 mg/kg i.p. weekly, ZOL 0.1 mg/kg s.c. on day 15 and vehicle weekly, or weekly dose of DOX and a single injection of ZOL 24 hrs after the first dose of DOX (combination group). Bone resorption marker tartrate-resistant acid phosphatase 5b (TRACP 5b) was determined in serum samples collected on days 1, 9, 17 and 24. Bone samples were collected on day 25, and tumor area, trabecular bone area and apoptotic index (TUNEL and apoptotic morphology) were quantitated by histomorphometry. Statistical analysis was performed using One-way ANOVA followed by Dunnett's test or Kruskal-Wallis test followed by Mann-Whitney U-test. All animals in the control and DOX groups had lesions at sacrifice, whereas 12.5% of the mice in the ZOL group and 42.9% in the combination group had lesions. Osteolytic lesion area at sacrifice was significantly lower in both groups treated with ZOL compared to the control group, with no increase from day 14. Lesion area in the DOX-treated group was not different from control. Whole body tumor burden or the tumor area in hind limbs did not differ between the groups, indicating no inhibition on tumor growth. Apoptotic index was increased in groups treated with DOX. ZOL inhibited the increase in serum TRACP 5b, although less effectively in the combination group. Taken together, clinically relevant doses of DOX, ZOL or a sequential combination treatment of these two were not able to inhibit tumor growth in the established disease in this aggressive model of breast cancer bone metastasis. However, DOX increased the apoptosis of cancer cells and ZOL potently inhibited cancer-induced osteolysis, but the sequential treatment did not have additive affects on either tumor growth or osteolytic lesion area. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5394.

Abstract 1418: Stromal cells of prostate, not the bone marrow, facilitate prostate cancer osteoblastic tumor growth in bone

Abstract Stromal cells promote prostate cancer initiation and progression. Metastasized prostate cancers form osteoblastic bone lesions. The prostate cancer associated stroma expresses alpha-smooth muscle actin (alpha-SMA). Additionally, 69% of human prostate cancer tissues lost stromal TGFbeta type II receptor (Tgfbr2) expression. Conditional stromal knockout of the Tgfbr2 mouse model developed prostate adenocarcinoma. In parallel to the primary site, we observed human prostate cancer associated stroma in bone metastasis gained the expression of androgen receptor and alpha-SMA, which were negative in the normal bone marrow. Tgfbr2 expression was also lost in prostate cancer associated stroma in bone marrow, compared to its high expression in the normal bone marrow. This led us to question whether the stromal cells from the primary site or the bone marrow contribute the prostate cancer osteoblastic bone lesion development. In vitro, the conditioned media from Tgfbr2-flox or Tgfbr2-KO mouse prostate stromal cells (PSC) or bone marrow stromal cells (BMSC) were incubated on the C42B prostate cancer epithelia. Expressions of PTHrP, RANKL osteolytic factors and ET1, RUNX2, BMP2 osteoblastic factors by C42B cells were analyzed by qRT-PCR. The Tgfbr2-flox PSC conditioned media significantly increased the C42B cell expression of all the osteolytic and osteoblastic factors examined compared to C42B cells without conditioned media. However, the Tgfbr2-KO PSC conditioned media decreased the expression of PTHrP and RANKL, while further increased the expression of osteoblastic factors, ET1, RUNX2 and BMP2 by C42B cells by at least 2-fold. Conditioned media of Tgfbr2-flox and Tgfbr2-KO BMSC increased the expression of C42B cells on osteolytic factors (10-fold PTHrP and 2-fold RANKL), but neither has significant effect on expression of osteoblastic factors. Importantly, Tgfbr2-KO PSC and BMSC conditioned media increased C42B proliferation similarly by 2-fold over that of respective Tgfbr2-Tgfbr2-flox conditioned media. We found that the reduced PTHrP and RANKL expression was associated with respective promoter methylation, when C42B cells were incubated with Tgfbr2-KO PSC, but not BMSC. Epigenetic silencing of such osteolytic genes, would suggest for the first time, that the prostate stroma could influence osteoblastic tumor at a distant site. So, GFP labeled C42B cells, following pre-incubation with PSC or BMSC conditioned media, were injected into the tibia of SCID mice. X-rays of the bone lesions and fluorescence imaging of GFP-expressing tumor growth were measured. The data suggested, that the stromal cells from primary prostates, but not from the second metastasis site of bone marrow, promotes prostate cancer growth in the bone and osteoblastic bone lesion development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1418.

Bisphosphonates as Treatment of Bone Metastases

Accelerated bone loss is a common clinical feature of advanced breast cancer, and anti-resorptive bisphosphonates are the current standard therapy used to reduce the number and frequency of skeletal-related complications experienced by patients. Bisphosphonates are potent inhibitors of bone resorption, acting by inducing osteoclast apoptosis and thereby preventing the development of cancer-induced bone lesions. In clinical use bisphosphonates are mainly considered to be bone-specific agents, but anti-tumour effects have been reported in a number of in vitro and in vivo studies. By combining bisphosphonates with chemotherapy agents, growth and progression of breast cancer bone metastases can be virtually eliminated in model systems. Recent clinical trials have indicated that there may be additional benefits from bisphosphonate treatment, including positive effects on recurrence and survival when added to standard endocrine therapy. Whereas the ability of bisphosphonates to reduce cancer-induced bone disease is well established, their potential direct anti-tumour effect remain controversial. Ongoing clinical trials will establish whether bisphosphonates can inhibit the development of bone metastases in high-risk breast cancer patients. This review summarizes the main studies that have investigated the effects of bisphosphonates, alone and in combination with other anti-cancer agents, using in vivo model systems of breast cancer bone metastases. We also give an overview of the use of bisphosphonates in the treatment of breast cancer, including examples of key clinical trials. The potential side effects and future clinical applications of bisphosphonates will be outlined.

Pathogenesis of myeloma bone disease

Multiple myeloma (MM) is the most common cancer to involve bone with up to 90% of patients developing bone lesions. The bone lesions are purely osteolytic in nature and do not heal in the vast majority of patients. Up to 60% of patients develop pathologic fractures over the course of their disease. Bone disease is a hallmark of MM, and myeloma bone disease differs from bone metastasis caused by other tumors. Although myeloma and other osteolytic metastases induce increased osteoclastic bone destruction, in contrast to other tumors, once myeloma tumor burden exceeds 50% in a local area, osteoblast activity is either severely depressed or absent. The basis for this severe imbalance between increased osteoclastic bone resorption and decreased bone formation has been the topic of intensive investigation over the last several years. These studies have helped to identify novel targets for treating myeloma bone disease and will be discussed in this chapter.