Sajida Piperdi

Platelet-derived growth factor receptor as a prognostic marker and a therapeutic target for imatinib mesylate therapy in osteosarcoma.

The purpose of this review was to determine whether imatinib mesylate (STI571, Gleevec) has a role in the treatment of osteosarcoma. The expression of platelet‐derived growth factor (PDGF) receptor and its ligand was examined in a panel of surgical specimens obtained from 54 osteosarcoma patients, and then the expression was compared with prognosis. The effects of imatinib mesylate on growth and molecular events in 10 patient‐derived osteosarcoma cell cultures were investigated. Immunohistochemical studies demonstrated frequent expression of PDGF‐AA (80.4%) and PDGF‐α receptor (79.6%) and their correlation with inferior event‐free survival (P < .05). PDGF‐B–B and PDGF‐β–receptor expressions were also frequent (75.4% and 86%, respectively); however, statistically significant inferior event‐free survival was not demonstrated (P = .15). In vitro studies demonstrated that imatinib mesylate had a variable cytotoxic effect on various osteosarcoma primary cultures, with an IC50 of 5.6 μM to 9.5 μM, and blocked the PDGF‐induced intracellular signal transduction as well as inhibition of downstream Akt phosphorylation. Mitogen‐activated protein kinase (MAPK) was constitutively activated despite PDGF stimulation and imatinib mesylate treatment in 7 of 10 osteosarcoma cultures, perhaps explaining uncontrolled proliferation and relative unresponsiveness to imatinib. Imatinib mesylate could not be viewed as having a role as a single agent at current conventional doses for the treatment of osteosarcoma. These findings predicted activity in osteosarcoma clinical trials and suggested that in vitro model systems predict clinical behavior and that PDGF and its receptor expression could potentially be used for determining prognosis of osteosarcoma. Cancer 2008. ©2008 American Cancer Society.

Ganglioside GD2 as a therapeutic target for antibody‐mediated therapy in patients with osteosarcoma

Survival outcomes for patients with osteosarcoma have remained stagnant over the past 30 years. Targeting of ganglioside GD2, a glycosphingolipid on the cell surface of some tumors, with immunotherapy has resulted in improved outcomes for patients with neuroblastoma. In the current study, the expression pattern of GD2 was examined in osteosarcoma.

Cell surface receptor expression patterns in osteosarcoma

Although the presence of numerous cell signaling receptors in osteosarcoma is known, their simultaneous characterization has not been performed to date. The current study sought to characterize and quantify the expression of cell surface receptors across a variety of osteosarcoma cell lines.

Insulin-like growth factor 1 receptor and response to anti-IGF1R antibody therapy in osteosarcoma

Background Survival outcomes for patients with osteosarcoma (OS) have remained stagnant over the past three decades. Insulin-like growth factor 1 receptor (IGF1R) is over-expressed in a number of malignancies, and anti-IGF1R antibodies have and are currently being studied in clinical trials. Understanding the molecular aberrations which result in increased tumor response to anti-IGF1R therapy could allow for the selection of patients most likely to benefit from IGF1R targeted therapy. Methods IGF1R mRNA expression was assessed by RT PCR in OS patient primary tumors, cell lines, and xenograft tumors. IGF1R copy number was assessed by 3 approaches: PCR, FISH, and dot blot analysis. Exons 1–20 of IGF1R were sequenced in xenograft tumors and 87 primary OS tumors, and surface expression of IGF1R was assessed by flow cytometry. Levels of mRNA and protein expression, copy number, and mutation status were compared with tumor response to anti-IGF1R antibody therapy in 4 OS xenograft models. Results IGF1R mRNA is expressed in OS. Primary patient samples and xenograft samples had higher mRNA expression and copy number compared with corresponding cell lines. IGF1R mRNA expression, cell surface expression, copy number, and mutation status were not associated with tumor responsiveness to anti-IGF1R antibody therapy. Conclusions IGF1R is expressed in OS, however, no clear molecular markers predict response to IGF1R antibody-mediated therapy. Additional pre-clinical studies assessing potential predictive biomarkers and investigating targetable molecular pathways critical to the proliferation of OS cells are needed.

Preclinical activity of palifosfamide lysine (ZIO-201) in pediatric sarcomas including oxazaphosphorine-resistant osteosarcoma

PurposeOxazaphosphorines, such as ifosfamide (IFA), are frequently used in the treatment of pediatric sarcomas. They are pro-drugs and undergo hepatic metabolism into the active moiety and potentially toxic by-products such as acrolein and chloracetaldehyde, which may cause hemorrhagic cystitis and encephalopathy, respectively. In addition, resistance to oxazaphosphorines can be mediated by overexpression of enzymes involved in their catabolism. Isophosphoramide mustard (IPM, palifosfamide) is the active moiety of IFA. In the current study, the activity of palifosfamide lysine (ZIO-201), a stable form of palifosfamide, was evaluated in a panel of sarcoma cell lines and tumor xenografts including oxazaphosphorine-resistant xenografts.MethodsThe cytotoxic effect of palifosfamide lysine was studied in osteosarcoma (OS), Ewing’s sarcoma (ES) and rhabdomyosarcoma (RMS) cell lines using the MTT assay. In vivo, the maximum tolerated dose (MTD) of palifosfamide lysine was determined in SCID mice based on a 3-day intravenous (IV) administration schedule. The effect on tumor growth and event-free survival was assessed at the MTD in all three sarcoma xenografts. In OS, cyclophosphamide (CPA)-resistant and -sensitive xenografts (OS31 and OS33, respectively) were evaluated for palifosfamide lysine activity. ALDH1A1 and ALDH3A1 gene expression data for the OS xenografts were mined from the Pediatric Preclinical Testing Program gene expression data. ALDH3A1 enzyme levels were compared between the CPA-resistant and -sensitive xenografts.ResultsPalifosfamide lysine was cytotoxic against all the cell lines tested with the IC50 ranging from 0.5 to 1.5xa0μg/ml except for OS222, which had an IC50 of 7xa0μg/ml. The IV MTD of palifosfamide lysine in mice was 100xa0mg/kg per day for three consecutive days. Tumor growth inhibition was seen in both OS31 and OS33 xenografts and the RMS xenograft resulting in a significant difference in event-free survival between the control and the treated groups. Differential gene expression of ALDH3A1 but not ALDH1A1 was noted in the OS31 xenograft. This was confirmed by RT-PCR and the ALDH3A1 enzyme assay. ALDH3A1 enzyme activity was measured at 100xa0mIU/mg of protein in OS31 xenograft but no significant activity was seen in the OS33 xenograft.ConclusionsWe conclude that palifosfamide lysine has broad activity in a panel of sarcoma cell lines. It inhibits tumor growth in OS and RMS xenografts. Furthermore, it is active against the CPA-resistant, ALDH3A1 overexpressing, OS xenograft suggesting that it might have the potential of overcoming this resistance mechanism against oxazaphosphorines and may be an active agent in resistant/relapsed sarcomas in patients.

Targeting Glycoprotein NMB With Antibody-Drug Conjugate, Glembatumumab Vedotin, for the Treatment of Osteosarcoma.

Cure rates for children and young adults with osteosarcoma have remained stagnant over the past three decades. Targeting glycoprotein non‐metastatic b (GPNMB) with the antibody‐drug conjugate glembatumumab vedotin has improved outcomes for patients with melanoma and breast cancer. The potential utility of targeting GPNMB in osteosarcoma was explored.

Ganglioside GD2 expression is maintained upon recurrence in patients with osteosarcoma.

BackgroundOsteosarcoma is the most common primary malignant bone tumor in children and young adults. Ganglioside GD2 has been previously found on the cell surface in various tumor types, including osteosarcomas.FindingsIn this study, forty-nine additional osteosarcoma samples from 14 individual patients were assessed for GD2 expression via immunohistochemistry, of which 47 samples were found to express GD2. In matched samples from patients, GD2 expression seen at initial biopsy was found to persist in 100% of tissues taken at recurrence.ConclusionsGD2 expression was found to persist upon recurrence. These results suggest a phase 2 trial in children with recurrent osteosarcoma should provide an appropriate read out on the efficacy of anti-GD2 antibody.

Down-regulation of Skp2 expression inhibits invasion and lung metastasis in osteosarcoma

Osteosarcoma (OS), the most common primary cancer of bone, exhibits a high propensity for local invasion and distant metastasis. This study sought to elucidate the role of S phase kinase-associated protein (Skp2) in osteosarcoma invasion and metastasis and to explore flavokawain A (FKA), a natural chalcone from kava extract, as a potential Skp2 targeting agent for preventing osteosarcoma progression. Skp2 was found to be overexpressed in multiple osteosarcoma cell lines, including 5 standard and 8 primary patient-derived cell lines. Patients whose tumors expressed high levels of Skp2 sustained a significantly worse metastasis-free (pu2009=u20090.0095) and overall survival (pu2009=u20090.0013) than those with low Skp2. Skp2 knockdown markedly reduced in vitro cellular invasion and in vivo lung metastasis in an orthotopic mouse model of osteosarcoma. Similar to Skp2 knockdown, treatment with FKA also reduced Skp2 expression in osteosarcoma cell lines and blocked the invasion of osteosarcoma cells in vitro and lung metastasis in vivo. Together, our findings suggest that Skp2 is a promising therapeutic target in osteosarcoma, and that FKA may be an effective Skp2-targeted therapy to reduce osteosarcoma metastasis.

Abstract 4134: Immune infiltration and PD-L1 expression in the tumor microenvironment are prognostic in osteosarcoma

Purpose: Over the past four decades, osteosarcoma (OS) survival rates have remained stagnant. There is a need to identify novel therapies to target OS. In this study we examined the expression of Programed Death Ligand 1 (PD-L1) and defined the tumor microenvironment in OS in order to assess the feasibility of utilizing immune checkpoint inhibitors as a treatment modality. Experimental Design: PD-L1 expression in OS was examined in two patient cohorts using immunohistochemistry (IHC) (n = 48, n = 59) and expression was validated using quantitative real time PCR (qRT-PCR) (n = 21) and western blotting (n = 9). IHC was used to determine presence of tumor infiltrating lymphocytes and antigen presenting cells (APCs) in the tumor. Expression of PD-L1 was correlated with the presence of immune cells and with event free survival in OS. Results: PD-L1 is expressed in up to 25% of primary OS tumors. Of the PD-L1 positive tumors the majority were also PD-1 positive (92% vs 8%, p = 0.002). In addition, the presence of immune cells in the tumor mass was significantly associated with PD-L1 expression. Although all immune cell types examined were present in OS, only infiltration by APCs, specifically CD1a positive dendritic cells (48.6% vs. 51.4%, p = 0.0010) and CD68 positive macrophages (70.3% vs. 29.7%, p = 0.0316), was associated with worse outcomes. PD-L1 expression was significantly associated with worsened survival (21.6% pos. vs. 78.4% neg., p = 0.0146). Conclusions: For the first time we have identified PD-L1 expression or presence of CD1a or CD68 positive antigen presenting cells as prognostic markers for worsened outcome in OS. Furthermore, we show that IHC is a valid detection method for PD-L1 in OS. With up to 25% of OS patients expressing PD-L1, this study provides rational for targeting the PD-L1:PD-1 axis for immunotherapy in OS. Citation Format: Pratistha Koirala, Michael Roth, Jonathan Gill, Jordan Chinai, Sajida Piperdi, David Geller, Bang Hoang, Vincent Poon, Xingxing Zang, Richard Gorlick. Immune infiltration and PD-L1 expression in the tumor microenvironment are prognostic in osteosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4134.

Abstract 645: The effect of rhBMP-2 on in vitro osteosarcoma tumorigenesis and on pulmonary growth and development

Introduction: Complete surgical resection is a critical component of care for patients with osteosarcoma (OS). Limb-salvage surgery is performed in over 90% of cases. Non-union is a well-recognized complication of allograft reconstructions, frequently performed following tumor extirpation. Bone Morphogenetic Protein-2 (BMP-2) has been shown to be osteoinductive and could theoretically improve allograft-host bone union. The purpose of this investigation is to evaluate the effect of exogenous recombinant human BMP-2 (rhBMP-2) on osteosarcoma. Methods: Functional assays were performed using five standard osteosarcoma cell lines and three patient derived xenograft cell lines. Random migration motility was measured via wound healing scratch assay. Migration (haptotaxis) was quantitatively measured using Boyden Chamber assay. Invasion was assessed using the Biocoat Matrigel Invasion Kit. Cell proliferation rate was measured by using MTT reagent. Metastatic disease was assessed using a xenograft murine model. Results: Findings suggest that exogenous rhBMP-2 does not meaningfully affect osteosarcoma migration, invasion or proliferation across all tested lines. Average corrected lung weight in the single-dose rhBMP-2 experiment was 1.56 g in the experimental group versus 1.67 g in the control group (p = 0.69). Average corrected lung weight in the triple-dose rhBMP-2 experiment was 1.23 g in the experimental group versus 1.06 g in the control group (p = 0.07). Conclusions and Future Directions: Results support the contention that exposure of osteosarcoma to exogenous rhBMP-2 does not increase either in vitro tumorigenesis or pulmonary disease growth and development within this experimental model. While results are compelling, additional characterization and confirmation of these findings are necessary safety prerequisites prior to proposing clinical application. Citation Format: David S. Geller, So Hak Chung, Wendong Zhang, Sajida Piperdi, Carrie Freeman, Bang Hoang, Rui Yang, Michael Roth, Jonathan Gill, Richard Gorlick. The effect of rhBMP-2 on in vitro osteosarcoma tumorigenesis and on pulmonary growth and development. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 645.