OC15. Potential influence of zoledronic acid on primary tumour response during neoadjuvant chemotherapy for breast cancer

Abstracts: VII International meeting on cancer induced bone disease Phase III. Translation and content validation took place in nine countries: Argentina, Australia, Canada, China, Germany, Greece, the Netherlands, Spain, and the United Kingdom. One hundred and fifty-five patients participated. There were 60 males (43%) and the median age was 59 years (range 29–92). The BM22 was well-received in all nine countries requiring minimal changes. The BM 22 will be presented at the conference. Conclusion: The establishment of a QOL assessment tool relevant for bone metastasis patients for use in future clinical trials is essential. The BM22 will facilitate better overall patient management through the identification of effective treatments and comprehensive follow-up assessment of patients in a clinical setting. doi:10.1016/j.ctrv.2008.03.039

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zation of the VBRC wall. In conclusion, the VBRC is a micro-anatomical structure that is important for the integrity of the bone remodeling process and its collapse is a key event in the development of MM bone disease. The VBRC is disrupted directly by MM cells, favoring the development of osteolytic lesions and the formation of myeloma-osteoclast hybrids.

doi:10.1016/j.ctrv.2008.03.040

OC15. Potential influence of zoledronic acid on primary tumour response during neoadjuvant chemotherapy for breast cancer Matthew Winter a, Helen Thorpe b, Roger Burkinshaw a, Samantha Beevers b, Robert Coleman a

OC14. Osteolysis and the generation of osteoclastmyeloma hybrid cells are related to the myeloma cell-induced collapse of the vascular bone remodeling compartments Thomas Levin Andersen a, Kent Søe a, Teis Esben Sondergaard a, Katarzyna Kupisiewicz a, Torben Plesner b, Jean-Marie Delaisse a a

Department of Clinical Cell Biology (KCB), Vejle Hospital, IRS-CSFU, Southern Denmark University, Vejle, Denmark b Department of Hematology, Vejle Hospital, IRS-CSFU, Southern Denmark University, Vejle, Denmark Bone remodeling is a tightly coupled process where bone formation follows bone resorption to maintain the integrity of the skeleton throughout life. Bone remodeling occurs in a specialized vascular bone remodeling compartment (VBRC), separated from the bone marrow by a cell wall. In myeloma (MM) bone disease, bone formation does not compensate for increased bone resorption, and is responsible for the formation of characteristic osteolytic lesions. Here, we report that VBRCs are often disrupted in MM and that this destabilization correlates with a number of typical features of myeloma bone disease. (1) Uncoupling of bone resorption and bone formation correlated with disruption of VBRCs as we showed earlier. (2) Forteen out of 16 patients with more than 5 osteolytic lesions showed disrupted VBRCs, whereas 12 out of 14 patients without osteolytic lesions showed intact VBRCs. (3) Disruption correlated with a high density of MM cells positioned focally, and not with interstitially positioned MM cells. (4) Disruption led to a direct cell contact between MM cells and osteoclasts. (5) Finally, we discovered that the disruption is a critical event for generating osteoclast-myeloma hybrids, which were characterized as osteoclasts containing transcriptional active myeloma nuclei and identified by interface FISH for MM clone-specific chromosomal translocations as previously published. As an in vitro model of the VBRC wall, we established a confluent G0-arrested mono-layer of MC3T3 cells. Co-culturing MM cells with these MC3T3 cells, disrupted the confluent layer of MC3T3 cells mimicking the destabili-

a Academic Unit of Clinical Oncology, University of Sheffield, Weston Park Hospital, Sheffield, United Kingdom b Clinical Trials Research Unit, University of Leeds, Leeds, United Kingdom

Background: Pre-clinical studies have demonstrated sequence-dependent synergistic anti-tumour effects of chemotherapy (CT) and zoledronic acid (Z) in bone and soft tissues. The AZURE trial recruited 3360 women with stage II/III breast cancer to determine whether treatment with zoledronic acid in addition to (neo)adjuvant therapy improves disease related outcomes. We have performed a retrospective exploratory evaluation of the subgroup of AZURE patients who received neoadjuvant CT to determine if the addition of Z to CT influences pathological response in the primary tumour. Methods: Eligible patients received neoadjuvant CT according to local guidelines and were randomised to receive no additional treatment or Z 4 mg i.v. (3–4 weekly for 6 months in the neoadjuvant period). The primary surrogate endpoint for response was pathologically assessed residual invasive tumour size (RITS,mm) at surgery. Secondary endpoints included number of positive axillary nodes. Outcome differences between the groups were compared using multivariate analysis, adjusting for T stage, ER and PR status, CT type (anthracycline/taxane), treatment duration, and menopausal status. Results: Two hundred and six (6.1%) patients received neoadjuvant CT (104 CT, 102 CT+Z). Baseline characteristics and treatments were well balanced, and median number of CT cycles and treatment duration the same in both groups. Median RITS was 40 mm (IQR 19–60) in the CT group and 28 mm (IQR 16–50) in the CT+Z group. In a multivariate analysis (of 130 patients with complete data) the adjusted mean RITS in CT and CT+Z groups were 41.4 mm and 30.0 mm respectively, the difference being statistically significant ( 11.4 mm, 95%CI 23.0 mm, 0.2 mm; p = 0.054). Median number of positive lymph nodes was 3 (IQR 0–8) in the CT group and 2 (IQR 0–5) in the CT+Z group. There was no statistical significant difference in pathological axillary nodal status. Conclusion: These data suggest a possible direct anti-tumour effect of Z in combination with neoadju-

A. Chantry et al.

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vant CT and warrants formal evaluation in prospective studies.

doi:10.1016/j.ctrv.2008.03.041

Poster Presentations PreClinical Studies P1. Breast cancer cells cause a reduction in osteoblast number – An early event in the development of osteolytic bone lesions Penelope Ottewell, Peter Croucher, Ingunn Holen University of Sheffield, Sheffield, United Kingdom Advanced breast cancers preferentially metastasise to bone. Breast cancer cells promote osteoclast formation and activation, resulting in osteolytic lesions. The extent to which breast cancer cells reduce osteoblast activity and their contribution to osteolysis is yet to be established. We have investigated the effects of breast cancer cells on the number and distribution of osteoclasts and osteoblasts at different stages in osteolytic tumour progression. MDA-MB-436-GFP cells were injected into tibiae of nude mice. Mice were sacrificed on day 0, 2, 9, 12, 15,19, 23 and 28. Tumour growth was visualised by GFP imaging, and tumour size measured on histological sections. Osteoblasts and osteoclasts were counted following H& E and TRAP staining. Areas of endocortical bone in contact with tumour were scored separately from areas not in contact with tumour. Effects on bone were analysed following Xray and lCT analysis. Tumours were detected in the intra-tibial bone marrow cavity by histological analysis on day 2; by day 19 tumours could be visualised by GFP imaging. Osteolytic lesions were visible on X-rays by day 23. There were reduced numbers of osteoblasts lining the endocortical bone on day 2, and osteoblast numbers reduced further from day 9 to 28. In contrast, numbers of osteoclasts in contact with endocortical bone increased with tumour size from day 2 to 28. Greater changes in osteoblast and osteoclast numbers were seen at sites where the tumour was in contact with bone, although decreased numbers of osteoblasts and increased numbers of osteoclasts were also observed along endocortical bone not in contact with tumour cells compared with control tibiae. MDA-MB-436 cells growing in bone reduce numbers of osteoblasts and increase numbers of osteoclasts resulting in osteolytic lesions. Disrupted balance between bone formation and resorption in favour of osteolysis is an early event, detectable 2 days following the introduction of tumour cells.

P2. Proteasome inhibition enhances anti-myeloma and anti-osteoclastic effects of glucocorticoids and weakens the anti-osteoblastic effects Kent Soe a, Thomas L. Andersen a, Torben Plesner b, Jean-Marie Delaisse a a Department of Clinical Cellbiology, Vejle Hospital, IRS/ CSFU, Southern Denmark University, Vejle, Denmark b Department of Hematology, Vejle Hospital, IRS/CSFU, Southern Denmark University, Vejle, Denmark

In multiple myeloma, malignant plasma cells accumulate in the bone marrow. The accumulation of these cancer cells in the marrow trigger enhanced bone destruction by osteoclasts and a limited bone repair by osteoblasts. Bortezomib and glucocorticoids are both powerful drugs that are used to kill myeloma cells in cancer patients. Besides the anti-tumor effect bortezomib was described to exert direct anti-osteoclastic and pro-osteoblastic effects that may contribute to bone protection in multiple myeloma. However, glucocorticoids have more ambiguous effects on these bone cells and are clearly anti-osteoblastic. Recent clinical trials based on the combination of bortezomib and glucocorticoids drew the attention on the very promising antimyeloma efficiency of this combination. However, the effect of this combination on bone cells has not been tested. In order to address this question, we performed an in vitro study with a model that takes into account the pharmacokinetics of bortezomib and glucocorticoid in patients. Our results show that bortezomib as a single treatment was very efficient in killing sensitive myeloma cells whereas more resistant cells were more efficiently killed in combination with prednisolone. Under the same experimental conditions bortezomib alone and in combination was effective in reducing osteoclastic bone resorption and TRACP activity. Most importantly however, bortezomib protected the osteoblasts against the detrimental effects of glucocorticoids. MC3T3 cells showed an increased resistance to prednisolone induced cytotoxicity when pretreated with bortezomib. Furthermore, Q-PCR analyses showed that genes such as vitamin D3 receptor, collagen type I, osteopontin and osteocalcin were significantly up-regulated in bortezomib and prednisolone treated cells as compared to prednisolone alone. Thus, the combination of bortezomib and glucocorticoid is not only a powerful treatment of multiple myeloma itself, but also shows promise for treating bone disease.

doi:10.1016/j.ctrv.2008.03.043

P3. A soluble activin type II receptor prevents the development of myeloma bone disease Andrew Chantry a, Deborah Heath a, Les Coulton a, Holly Evans a, Nicole Abdul a, Jas Seehra c, Karin Vanderkerken b, Peter Croucher a a

doi:10.1016/j.ctrv.2008.03.042

University of Sheffield, Sheffield, United Kingdom Vrije Universiteit Brussel (VUB), Brussels, Belgium c Acceleron Pharma, Cambridge, MA, United States b