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Although hematogenous spread of osteosarcoma is well known, the imaging findings of cardiovascular involvement by osteosarcoma are seldom reported and can be difficult to recognize. The enhanced resolution of modern CT and MRI scanners may lead to better detection of cardiovascular involvement. To describe the key imaging findings and clinical behavior of cardiovascular involvement by osteosarcoma. We retrospectively reviewed the imaging findings and clinical characteristics of 20 patients with cardiovascular involvement by osteosarcoma identified by two pediatric radiologists from a review of imaging studies at our institution from 2007 to 2013. At initial diagnosis, the median age of the patients was 15.1 years (range 4.8–24.6 years), and 7 (35%) patients had detectable metastases. Median time to detection of cardiovascular metastases was 1.8 years (range 0–7.3 years). Sixteen patients died of disease; 4 have survived a median of 7.4 years since initial diagnosis. The sites of cardiovascular involvement were the systemic veins draining the primary and metastatic osteosarcoma, pulmonary arteries, pulmonary veins draining the pulmonary metastases, and heart. A dilated and mineralized terminal pulmonary arteriole is an early sign of metastatic osteosarcoma in the lung. Unfamiliarity with the imaging features resulted in under-recognition and misinterpretation of intravascular tumor thrombus as bland thrombus. Knowledge of imaging findings in the era of modern imaging modalities has enhanced our ability to detect cardiovascular involvement and lung metastases early and avoid misinterpreting tumor thrombus in draining systemic veins or pulmonary arteries as bland thrombus.

The pediatric bone sarcomas osteosarcoma and Ewing sarcoma represent a tremendous challenge for the clinician. Though less common than acute lymphoblastic leukemia or brain tumors, these aggressive cancers account for a disproportionate amount of the cancer morbidity and mortality in children, and have seen few advances in survival in the past decade, despite many large, complicated, and expensive trials of various chemotherapy combinations. To improve the outcomes of children with bone sarcomas, a better understanding of the biology of these cancers is needed, together with informed use of targeted therapies that exploit the unique biology of each disease. Here we summarize the current state of knowledge regarding the contribution of receptor tyrosine kinases, intracellular signaling pathways, bone biology and physiology, the immune system, and the tumor microenvironment in promoting and maintaining the malignant phenotype. These observations are coupled with a review of the therapies that target each of these mechanisms, focusing on recent or ongoing clinical trials if such information is available. It is our hope that, by better understanding the biology of osteosarcoma and Ewing sarcoma, rational combination therapies can be designed and systematically tested, leading to improved outcomes for a group of children who desperately need them.

PURPOSE Survival of patients with osteosarcoma lung metastases has not improved in 20 years. We evaluated the efficacy of combining natural killer [NK] cells with aerosol interleukin-2 [IL-2] to achieve organ-specific NK cell migration and expansion in the metastatic organ, and to decrease toxicity associated with systemic IL-2.

Purpose The purpose of this study was to characterize the complications associated with surgical treatment of pediatric melanoma. Methods We retrospectively reviewed all pediatric patients who received surgical treatment for melanoma at our institution between 1992 and 2010. We compared complications between three groups: wide local excision only (WLE), WLE and sentinel lymph node biopsy (SLNB), and WLE and completion lymph node dissection (CLND). Results One hundred twenty-five patients were identified: 37 patients received WLE only, 47 received WLE and SLNB, and 41 patients had WLE and CLND. Complication rates differed between the three groups: 19% in WLE, 11% in WLE+SLNB, and 39% in WLE+CLND ( P =.006). The risk of complications was significantly lower among patients having WLE+SLNB versus WLE+CLND (OR 0.19, 95% CI 0.06–0.57, P =.0032). Lymphedema was a common complication with a higher incidence in the CLND group compared to the SLNB group (19.5% vs. 2.1%, P =.01). Complications were more frequent in inguinal compared to axillary dissections (52.0% vs. 17.1%, P =.006). Conclusions In the surgical treatment of pediatric melanoma, the addition of a completion lymph node dissection significantly increases complication risk. Thus, it is critical to determine which patients truly benefit from this procedure.

BACKGROUND: The UBE4B gene, which is located on chromosome 1p36, encodes a ubiquitin ligase that interacts with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a protein involved in epidermal growth factor receptor (EGFR) trafficking, suggesting a link between EGFR trafficking and neuroblastoma pathogenesis. The authors analyzed the roles of UBE4B in the outcomes of patients with neuroblastoma and in neuroblastoma tumor cell proliferation, EGFR trafficking, and response to EGFR inhibition. METHODS: The association between UBE4B expression and the survival of patients with neuroblastoma was examined using available microarray data sets. UBE4B and EGFR protein levels were measured in patient tumor samples, EGFR degradation rates were measured in neuroblastoma cell lines, and the effects of UBE4B on neuroblastoma tumor cell growth were analyzed. The effects of the EGFR inhibitor cetuximab were examined in neuroblastoma cells that expressed wild-type and mutant UBE4B. RESULTS: Low UBE4B gene expression is associated with poor outcomes in patients with neuroblastoma. UBE4B overexpression reduced neuroblastoma tumor cell proliferation, and UBE4B expression was inversely related to EGFR expression in tumor samples. EGFR degradation rates correlated with cellular UBE4B levels. Enhanced expression of catalytically active UBE4B resulted in reduced sensitivity to EGFR inhibition. CONCLUSIONS: The current study demonstrates associations between UBE4B expression and the outcomes of patients with neuroblastoma and between UBE4B and EGFR expression in neuroblastoma tumor samples. Moreover, levels of UBE4B influence neuroblastoma tumor cell proliferation, EGFR degradation, and response to EGFR inhibition. These results suggest UBE4B-mediated growth factor receptor trafficking may contribute to the poor prognosis of patients who have neuroblastoma tumors with 1p36 deletions and that UBE4B expression may be a marker that can predict responses of neuroblastoma tumors to treatment. Cancer 2013. © 2012 American Cancer Society.

To improve cancer outcomes, investigators are turning increasingly to small molecule medicines that disrupt vital signaling cascades, inhibit malignant growth, or induce apoptosis. One vital signaling molecule is Ras, and a key step in Ras activation is membrane anchoring of Ras through prenylation, the C-terminal addition of a lipid anchor. Small molecule inhibitors of farnesyltransferase (FTI), the enzyme most often responsible for prenylating Ras, showed clinical promise, but development of FTIs such as tipifarnib has been stalled by uncertainty about their mechanism of action, because Ras seemed unimpeded in tipifarnib-treated samples. Interpretation was further complicated by the numerous proteins that may be farnesylated, as well as availability of an alternate prenylation pathway, geranylgeranylation. Our initial observations of varied response by cancer cell lines to tipifarnib led us to evaluate the role of FTI in Ras signal alteration using various tumor models. We describe our novel counterintuitive finding that endogenous Ras activity increases in cancer cell lines with low endogenous Ras activity when farnesyltransferase is inhibited by either tipifarnib or short hairpin RNA. In response to tipifarnib, variable growth arrest and/or cell death correlated with levels of activated extracellular signal–regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Sensitivity to tipifarnib treatment was shown by growth inhibition and by an increase in subdiploid cell numbers; cells with such sensitivity had increased activation of ERK and p38 MAPK. Because Ras must be prenylated to be active, our findings suggest that geranylgeranylated N-Ras or K-Ras B interacts differently with downstream effector proteins in sensitive cancer cells responding to tipifarnib, switching the balance from cell proliferation to growth inhibition. Mol Cancer Ther; 9(5); 1111–9. ©2010 AACR.

BACKGROUND: ERBB receptor tyrosine kinases can mediate proliferation, migration, adhesion, differentiation, and survival in many types of cells and play critical roles in many malignancies. Recent reports suggest a role for EGFR signaling in proliferation and survival of neuroblastoma, a common form of pediatric cancer that often has an extremely poor outcome. METHODS: The authors examined ERBB family expression in neuroblastoma cell lines and patient samples by flow cytometry, western blot, and quantitative real time polymerase chain reaction (Q-PCR). Response to ERBB inhibition was assessed in vitro by cell-cycle analysis and western blot and in vivo by serial tumor-size measurements. RESULTS: A panel of neuroblastoma cell lines and primary patient tumors expressed EGFR, HER-3, and HER-4, with HER-2 in some tumors. HER-4 mRNA was expressed predominantly in cleavable isoforms. Whereas EGFR inhibition with erlotinib and pan-ERBB inhibition with CI-1033 inhibited EGF-induced phosphorylation of EGFR, AKT, and ERK1/2, only CI-1033 induced growth inhibition and dose-dependent apoptosis in vitro. Both CI-1033 and erlotinib treatment of neuroblastoma xenograft tumors resulted in decreased tumor growth in vivo, although CI-1033 was more effective. In vivo expression of EGFR was observed predominantly in vascular endothelial cells. CONCLUSIONS: Pan-ERBB inhibition is required for ERBB-related neuroblastoma apoptosis in vitro, although EGFR contributes indirectly to tumor growth in vivo. Inhibition of EGFR in endothelial cells may be an important aspect of erlotinib's impact on neuroblastoma growth in vivo. Our results suggest that non-EGFR ERBB family members contribute directly to neuroblastoma growth and survival, and pan-ERBB inhibition represents a potential therapeutic target for treating neuroblastoma. Cancer 2010. © 2010 American Cancer Society.

PURPOSE OF REVIEW The survival curves for many pediatric sarcomas have remained flat for the past 2 decades or more and novel therapeutics - those small molecule medicines that selectively inhibit specific signaling molecules - have been slow to enter into pediatric practice. The preclinical basis for their use is reviewed here. RECENT FINDINGS Preclinical and phase I studies showing efficacy of antiinsulin-like growth factor receptor 1 therapies for Ewing sarcoma have led to numerous ongoing clinical trials using these agents for Ewing and other sarcomas. Early studies of ERBB signaling as a target in sarcoma therapy have been tantalizing, but progress in this area has been controversial. In-vitro analysis of Src inhibitors suggested that these agents would prevent metastasis in osteosarcoma, whereas in-vivo analysis showed no effect on metastasis, underscoring the need for thorough preclinical investigations of promising new therapies to guide future clinical trials. Antiangiogenic and immunomodulatory therapies are gaining momentum in the pediatric arena and should be tested in combination with traditional cytotoxic agents for recurrent and high-risk primary pediatric sarcomas. SUMMARY Pediatric sarcomas have diverse biology and distinct signaling pathways, making detailed preclinical evaluation of small molecule inhibitors essential to guiding the design of further clinical investigations.

Cetuximab, which blocks ligand binding to epidermal growth factor receptor (EGFR), is currently being studied as a novel treatment for non–small cell lung cancer (NSCLC). However, its mechanisms of action toward metastasis, and markers of drug sensitivity, have not been fully elucidated. This study was conducted to (a) determine the effect of Cetuximab on invasion and NSCLC-metastasis; (b) investigate urokinase-type plasminogen activator receptor (u-PAR), a major molecule promoting invasion and metastasis, as a target molecule; (c) delineate molecular mediators of Cetuximab-induced metastasis inhibition; and (d) identify biomarkers of drug sensitivity in NSCLC. Cetuximab treatment resulted in reduced growth and Matrigel invasion of H1395 and A549 NSCLC cell lines, in parallel with reduced u-PAR mRNA and protein. u-PAR down-regulation was brought about by suppressing the binding of JunD and c-Jun to u-PAR promoter motif −190/−171 in vivo, and an inhibition of MAP/ERK kinase signaling. Furthermore, Cetuximab inhibited NSCLC proliferation and metastasis to distant organs in vivo as indicated by the chicken embryo metastasis assay. Low E-cadherin and high u-PAR, but not EGFR, was associated with resistance to Cetuximab in seven NSCLC cell lines. Furthermore, siRNA knockdown of u-PAR led to a resensitization to Cetuximab. Moreover, low E-cadherin and high u-PAR was found in 63% of resected tumor tissues of NSCLC patients progressing under Cetuximab therapy. This is the first study to show u-PAR as a target and marker of sensitivity to Cetuximab, and to delineate novel mechanisms leading to metastasis suppression of NSCLC by Cetuximab. [Cancer Res 2009;69(6):2461–70]

New investigational agents and chemotherapy regimens including cyclophosphamide + topotecan, temozolomide + irinotecan, and anti-IGF-1R antibodies in Ewing's sarcoma (ES) and liposomal muramyltripeptide phosphatidylethanolamine (L-MTP-PE), aerosol therapy, and bone-specific agents in osteosarcoma (OS) may improve survival and/or quality of life on 'continuation' therapy.Review of investigational approaches and control paradigms for recurrent or metastatic primary bone tumors.Analyze temozolomide + irinotecan data and review in the context of other newer approaches including antiangiogenesis, anti-IGF-1R antibodies and bisphosphonates for ES. Review some current state-of-the-art approaches for OS including L-MTP-PE, anti-IGF-1R inhibition, aerosol therapies and bone specific agents.L-MTP-PE with chemotherapy in OS has been shown to improve survival; compassionate access is available for recurrence and/or metastases. Aerosol therapy (granulocyte-macrophage colony stimulating factor, cisplatin, gemcitabine) for lung metastases is a promising approach to reduce systemic toxicity. The bone-specific agents including denosumab (anti-receptor activator of NF-kappaB ligand antibody) and bisphosphonates may have benefit against giant cell tumor, ES and OS. Anti-IGF-1R antibody SCH717454 has preclinical activity in OS but best effectiveness will most likely be in combination with chemotherapy earlier in therapy. Both temozolomide + irinotecan and cyclophosphamide + topotecan combinations are very active in ES and are likely to be tested with anti-IGF-1R antibodies against ES.

Although sarcoma surgery is very important for cancer control, it is not always possible or practical to offer in some situations, including sarcoma recurrences, metastatic disease and/or unacceptable loss of function. We review some pragmatic approaches and examples of how to balance indications, risks and alternatives to control cancer in young people with sarcomas that are no longer using ‘front-line’ therapy. Radiotherapy combined with chemotherapy and outpatient ‘continuation’ chemotherapy regimens using drugs that cause less alopecia can improve function and quality of life. Some effective strategies to help cope when cure is not possible may include tumor ablation techniques performed in interventional radiology and percutaneous nerve blocks. Family centered care and effective problem solving of difficult issues can be greatly facilitated by consultation with a multidisciplinary team experienced in the management of very difficult cases. Treatment of young people with recurrent, relapsed and/or metastatic sarcoma still remains an art very much in the realm of compassion not protocol and persistent advocacy is required for the young person for whom cure may not be possible. A reduction of suffering and assistance in writing more chapters of a rich life narrative is the goal.

Purpose: Notch signaling is an important mediator of growth and survival in several cancer types, with Notch pathway genes functioning as oncogenes or tumor suppressors in different cancers. However, the role of Notch in osteosarcoma is unknown. Experimental Design: We assessed the expression of Notch pathway genes in human osteosarcoma cell lines and patient samples. We then used pharmacologic and retroviral manipulation of the Notch pathway and studied the effect on osteosarcoma cell proliferation, survival, anchorage-independent growth, invasion, and metastasis in vitro and in vivo. Results: Notch pathway genes, including Notch ligand DLL1, Notch1 and Notch2, and the Notch target gene HES1, were expressed in osteosarcoma cells, and expression of HES1 was associated with invasive and metastatic potential. Blockade of Notch pathway signaling with a small molecule inhibitor of γ secretase eliminated invasion in Matrigel without affecting cell proliferation, survival, or anchorage-independent growth. Manipulation of Notch and HES1 signaling showed a crucial role for HES1 in osteosarcoma invasiveness and metastasis in vivo. Conclusion: These studies identify a new invasion and metastasis-regulating pathway in osteosarcoma and define a novel function for the Notch pathway: regulation of metastasis. Because the Notch pathway can be inhibited pharmacologically, these findings point toward possible new treatments to reduce invasion and metastasis in osteosarcoma.

Purpose Chemotherapy during radiation and/or bone-seeking radioisotope therapy (153-samarium; 1 mCi/kg) during radiation may improve osteosarcoma cancer control. Patients and Methods We analyzed our preliminary radiation experience in high-risk, metastatic, and/or recurrent patients during a consecutive period of 20 months (May 2005–December 2006). Results Thirty-nine high-risk osteosarcoma patients had radiotherapy; 119 sites were irradiated. A median four sites were irradiated per patient (range 1–14). The median radiation dose and number of fractions of radiation was 30 Gy in 10 fractions (range 10–70 Gy in 4–35 fractions). Chemotherapy, most commonly ifosfamide or methotrexate, was used in 80% (100/119) radiotherapy courses. Of 38 painful sites, 29 had improvement (76%), 4 had no change (10%), and 5 had more pain (13%). Objective and potentially durable responses were documented using PET-CT and bone scans with persistent and sustained reduction of standard uptake values (SUVs; initial SUV of indication lesion 9.5 became

Causes of lytic bone lesions in children include benign, malignant, and infectious processes. Here, we present the case of a 3-year-old boy presenting with a lytic bone lesion and surrounding soft tissue mass sent for evaluation of possible malignancy versus osteomyelitis. Biopsy revealed granulomatous osteomyelitis, and subsequent purified protein derivative resulted in 20-mm induration. Lesion cultures eventually identified pan-sensitive Mycobacterium tuberculosis. We emphasize that tuberculosis can cause primary lytic bone lesions in children in the United States, even in the absence of pulmonary symptoms or known exposure, and advise clinicians to include mycobacterial cultures when analyzing biopsies of lytic bone lesions.

To evaluate the impact of treated extra-pulmonary metastatic disease on overall (OS) and event-free survival (EFS) for pediatric osteosarcoma patients undergoing pulmonary metastatectomy.We retrospectively reviewed pediatric patients who were treated for osteosarcoma at our institution from 2001 to 2011 and received pulmonary metastatectomy (n=76). We compared OS and EFS between patients with metastases limited to the lungs (Group A, n=58) to those with treated extra-pulmonary metastases (Group B, n=18) at the time of first pulmonary metastatectomy.The estimated median OS and EFS from first pulmonary metastatectomy were 2.0years (95% CI 1.5-2.8years) and 5.5months (95% CI 3.0-8.1months), respectively. Median OS was significantly greater for Group A (2.6years, 95% CI 1.9-3.8) compared to Group B (0.9years, 95% CI 0.6-1.5) (log rank p=0.0001). Median EFS was significantly greater for Group A (7.9months, 95% CI 5.0-10.7) compared to Group B (1.6months, 95% CI 0.8-2.7) (log rank p0.0001). Independent predictors of OS included extra-pulmonary metastatic disease at the time of first thoracotomy, bilateral pulmonary metastases, and4 nodules resected at first thoracotomy (all p0.001).Osteosarcoma patients with treated extra-pulmonary metastatic disease at the time of pulmonary metastatectomy have significantly worse survival compared to those with disease limited to the lungs.

The purpose of this study was to identify health disparities in children with non-CNS solid tumor malignancies and examine their impact on disease presentation and outcome.We examined the records of all children (age≤18years) diagnosed with a non-CNS solid tumor malignancy and enrolled in the Texas Cancer Registry between 1995 and 2009 (n=4603). The primary outcome measures were disease stage and overall survival (OS). Covariates included gender, age, race/ethnicity, year of diagnosis, socioeconomic status (SES), and driving distance to the nearest pediatric cancer treatment facility. Statistical analyses included life table methods, logistic, and Cox regression. Statistical significance was defined as p0.05.Children with advanced-stage disease were more likely to be male,10years old, and Hispanic or non-Hispanic Blacks (all p0.05). Distance to treatment and SES did not impact stage of disease at presentation. However, Hispanic and non-Hispanic Blacks and patients in the lowest SES quartile had the worst 1- and 5-year survival (all p0.05). The adjusted OS differed by age, race, and stage, but not SES or distance to the nearest treatment facility.Race/ethnicity plays an important role in survival for children with non-CNS solid tumor malignancies. Future work should better define these differences to establish mechanisms to decrease their impact.

The Notch pathway has been described as an oncogene in osteosarcoma, but the myriad functions of all the members of this complex signaling pathway, both in malignant cells and nonmalignant components of tumors, make it more difficult to define Notch as simply an oncogene or a tumor suppressor. The cell-autonomous behaviors caused by Notch pathway manipulation may vary between cell lines but can include changes in proliferation, migration, invasiveness, oxidative stress resistance, and expression of markers associated with stemness or tumor-initiating cells. Beyond these roles, Notch signaling also plays a vital role in regulating tumor angiogenesis and vasculogenesis, which are vital aspects of osteosarcoma growth and behavior in vivo. Further, osteosarcoma cells themselves express relatively low levels of Notch ligand, making it likely that nonmalignant cells, especially endothelial cells and pericytes, are the major source of Notch activation in osteosarcoma tumors in vivo and in patients. As a result, Notch pathway expression is not expected to be uniform across a tumor but likely to be highest in those areas immediately adjacent to blood vessels. Therapeutic targeting of the Notch pathway is likewise expected to be complicated. Most pharmacologic approaches thus far have focused on inhibition of gamma secretase, a protease of the presenilin complex. This enzyme, however, has numerous other target proteins that would be expected to affect osteosarcoma behavior, including CD44, the WNT/β-catenin pathway, and Her-4. In addition, Notch plays a vital role in tissue and organ homeostasis in numerous systems, and toxicities, especially GI intolerance, have limited the effectiveness of gamma secretase inhibitors. New approaches are in development, and the downstream targets of Notch pathway signaling also may turn out to be good targets for therapy. In summary, a full understanding of the complex functions of Notch in osteosarcoma is only now unfolding, and this deeper knowledge will help position the field to better utilize novel therapies as they are developed.

Cancer of any type often can be described by an arrest, alteration or disruption in the normal development of a tissue or organ, and understanding of the normal counterpart’s development can aid in understanding the malignant state. This is certainly true for osteosarcoma and the normal developmental pathways that guide osteoblast development that are changed in the genesis of osteogenic sarcoma. A carefully regulated crescendo–decrescendo expression of RUNX2 accompanies the transition from mesenchymal stem cell to immature osteoblast to mature osteoblast. This pivotal role is controlled by several pathways, including bone morphogenic protein (BMP), Wnt/β-catenin, fibroblast growth factor (FGF), and protein kinase C (PKC). The HIPPO pathway and its downstream target YAP help to regulate proliferation of immature osteoblasts and their maturation into non-proliferating mature osteoblasts. This pathway also helps regulate expression of the mature osteoblast protein osteocalcin. YAP also regulates expression of MT1-MMP, a membrane-bound matrix metalloprotease responsible for remodeling the extracellular matrix surrounding the osteoblasts. YAP, in turn, can be regulated by the ERBB family protein Her-4. Osteosarcoma may be thought of as a cell held at the immature osteoblast stage, retaining some of the characteristics of that developmental stage. Disruptions of several of these pathways have been described in osteosarcoma, including BMP, Wnt/b-catenin, RUNX2, HIPPO/YAP, and Her-4. Further, PKC can be activated by several receptor tyrosine kinases implicated in osteosarcoma, including the ERBB family (EGFR, Her-2 and Her-4 in osteosarcoma), IGF1R, FGF, and others. Understanding these functions may aid in the understanding the mechanisms underpinning clinical observations in osteosarcoma, including both the lytic and blastic phenotypes of tumors, the invasiveness of the disease, and the tendency for treated tumors to ossify rather than shrink. Through a better understanding of the relationship between normal osteoblast development and osteosarcoma, we may gain insights into novel therapeutic avenues and improved outcomes.

The two most common primary bone malignancies, osteosarcoma and Ewing sarcoma, are both aggressive, highly metastatic cancers that most often strike teens, though both can be found in younger children and adults. Despite distinct origins and pathogenesis, both diseases share several mechanisms of progression and metastasis, including neovascularization, invasion, anoikis resistance, chemoresistance and evasion of the immune response. Some of these processes are well-studies in more common carcinoma models, and the observation from adult diseases may be readily applied to pediatric bone sarcomas. Neovascularization, which includes angiogenesis and vasculogenesis, is a clear example of a process that is likely to be similar between carcinomas and sarcomas, since the responding cells are the same in each case. Chemoresistance mechanisms also may be similar between other cancers and the bone sarcomas. Since osteosarcoma and Ewing sarcoma are mesenchymal in origin, the process of epithelial-to-mesenchymal transformation is largely absent in bone sarcomas, necessitating different approaches to study progression and metastasis in these diseases. One process that is less well-studied in bone sarcomas is dormancy, which allows micrometastatic disease to remain viable but not growing in distant sites – typically the lungs – for months or years before renewing growth to become overt metastatic disease. By understanding the basic biology of these processes, novel therapeutic strategies may be developed that could improve survival in children with osteosarcoma or Ewing sarcoma.

Gastrointestinal stromal tumors (GISTs) are driven by gain-of-function mutations of KIT or PDGFRa. The introduction of imatinib has significantly extended survival for patients. However, most patients develop resistances. Notch signaling is a conserved developmental pathway known to play a critical role in the development of several cancers, functioning as a tumor promoter or a tumor suppressor. Given that the normal progenitor cell for GIST, the interstitial cell of Cajal, has characteristics similar to those of cells of neuroendocrine origin, we hypothesized that Notch pathway impacts the biology of GIST cells. In this study, we retrovirally and pharmacologically manipulated the Notch pathway in human GIST cells. We also performed a retrospective analysis of a cohort on 15 primary tumors to determine the role of Hes1, a major target gene of Notch, as a prognostic marker for GIST. Constitutively, active intracellular domain of Notch1 (ICN1) expression potently induced growth arrest and downregulated KIT expression in vitro. Additionally, treatment with the histone deacetylase inhibitor suberoylanilide hydroxamic acid caused dose-dependent upregulation of Notch1 expression and a parallel decrease in viability in these cells. Retroviral silencing of downstream targets of Notch (dominant-negative Hes1) and pharmacological inhibition of Notch activation (γ-secretase inhibition) partially rescued GIST cells from suberoylanilide hydroxamic acid treatment. GIST patients with high Hes1 mRNA levels have a significantly longer relapse-free survival. These results identify a novel anti-tumor effect of Notch1 and cross talk between the Notch and KIT pathways. Thus, activation of this pathway by treatment with histone deacetylase inhibitors is an appealing potential therapeutic strategy for GISTs. Précis: This study is the first report of the tumor suppressor effects of Notch pathway in gastrointestinal stromal tumors via a negative feedback with the oncogene KIT and may lead the development of new therapeutic strategies for GISTs patients.

BACKGROUND: Neuroblastoma (NBL) is a common pediatric solid tumor, and outcomes for patients with advanced neuroblastoma remain poor despite extremely aggressive treatment. Chemotherapy resistance at relapse contributes heavily to treatment failure. The poor survival of patients with high-risk NBL prompted this investigation into novel treatment options with the objective of gaining a better understanding of resistance mechanisms. On the basis of previous work and on data from publicly available studies, the authors hypothesized that human epidermal growth factor receptor 4 (Her4) contributes to resistance. METHODS: Her4 expression was reduced with small-hairpin RNA (shRNA) to over express intracellular HER4, and the authors tested its impact on tumor cell survival under various culture conditions. The resulting changes in gene expression after HER4 knockdown were measured by using a messenger RNA (mRNA) array. RESULTS: HER4 expression was up-regulated in tumor spheres compared with the expression in monolayer culture. With HER4 knockdown, NBL cells became less resistant to anoikis and serum starvation. Moreover, HER4 knockdown increased the chemosensitivity of NBL cells to cisplatin, doxorubicin, etoposide, and activated ifosfamide. In mRNA array analysis, HER4 knockdown predominately altered genes related to cell cycle regulation. In NBL spheres compared with monolayers, cell proliferation was decreased, and cyclin D expression was reduced. HER4 knockdown reversed cyclin D suppression. Overexpressed intracellular HER4 slowed the cell cycle and induced chemoresistance. CONCLUSIONS: The current results indicated that HER4 protects NBL cells from multiple exogenous apoptotic stimuli, including anoikis, nutrient deficiency, and cytotoxic chemotherapy. The intracellular fragment of HER4 was sufficient to confer this phenotype. HER4 functions as a cell cycle suppressor, maintaining resistance to cellular stress. The current findings indicate that HER4 overexpression may be associated with refractory disease, and HER4 may be an important therapeutic target. Cancer 2012. © 2012 American Cancer Society.

The ability of osteosarcoma cells to form lung metastases has been inversely correlated to cell surface Fas expression. Downregulation of Fas allows osteosarcoma cells to circumvent FasL-mediated apoptosis upon entrance into the FasL+ lung microenvironment. However, the mechanism of Fas regulation remains unclear. Here, we show that miRNA plays a role in the downregulation of Fas expression in osteosarcoma. Expression levels of several members of the miR-17–92 cluster including miR-20a and miR-19a were found to be higher in metastatic low-Fas–expressing LM7 cells than in the parental nonmetastatic high-Fas–expressing SAOS-2 cells. We also found an inverse correlation between Fas and miR-20a expression in all 8 cell lines derived from patient samples. Overexpression of miR-20a consistently resulted in the downregulation of Fas expression in SAOS-2 cells and thus in decreased sensitivity to FasL. Conversely, inhibiting miR-20a in LM7 cells increased Fas expression and their sensitivity to FasL. Mice injected with LM7 stably transfected with anti-miR-20a had fewer metastases than those with control plasmids. Taken together, our findings suggest that miR-20a, encoded by miR-17–92, downregulates Fas expression in osteosarcoma, thus contributing to the metastatic potential of osteosarcoma cells by altering the phenotype and allowing survival in the FasL+ lung microenvironment. Cancer Res; 72(4); 908–16. ©2011 AACR.

Bone sarcomas cause disproportionate morbidity and mortality and desperately need new therapies as there has been little improvement in outcomes in 20 years. Identification of critical signaling pathways, including type 1 insulin-like growth factor receptor (IGF-1R) for Ewing sarcoma and possibly osteosarcoma, and the ERBB and the Wnt signaling pathways for osteosarcoma, have emerged as receptors mediating vital signals for bone sarcoma. Akt, mammalian target of rapamycin (mTOR), phosphoinositide 3-kinases, mitogen-activated protein kinase kinase, extracellular signal-regulated kinases, and Ras pathway play key roles in at least some tumors, and inhibition of mTOR in particular will likely lead to improved survival, although clinical trials are still underway. The Notch pathway and ezrin are essential for osteosarcoma metastasis, and Fas down-regulation is necessary for survival of metastases in lungs. As little is known about chondrosarcoma signaling, more preclinical work is needed. By defining vital signaling pathways in bone sarcomas, small molecule inhibitors can be applied rationally, leading to longer survival and reducing morbidity and late effects from intensive chemotherapy.

Background: Conventional therapy for osteosarcoma has reached a plateau of 60 – 70%, a 5-year survival rate that has changed little in two decades, highlighting the need for new approaches. Objective: To review the alternative means of delivering effective therapy for osteosarcoma that reach beyond the central venous catheter. Methods: Drawing on the author's own experiences providing care to high-risk osteosarcoma patients and reviewing the last two decades of literature describing sarcoma therapy, available information is summarized about potential osteosarcoma treatments that deliver therapy by a less conventional route. Results/conclusions: Intra-arterial chemotherapy has a limited impact on survival, but may help to achieve a better limb salvage. Intrapleural chemotherapy is important for managing malignant effusions. The development of inhalation therapies, treatments that target new bone formation such as bisphosphonates, chemically targeted radiation and antibody-based therapies all have potential...

BACKGROUND. Approximately 30% to 40% of all patients with osteosarcomas ultimately experience recurrence. The study investigated the hypothesis that the resistance of osteosarcoma to chemotherapy may be related to the expression of a pregnane xenobiotic receptor (PXR) variant protein and its role as the major inducer of P450 3A4 in these tumors. METHODS. Polymerase chain reaction (PCR) and Western blot analysis were used to determine PXR mRNA and protein expression, respectively. Real-time PCR and CYP3A catalytic activity using 7-benzyl-trifluoromethyl coumarin (BFC) as the probe substrate were used to measure the induction of P450 3A4 or MDR1. siRNA transfections were performed for PXR and cytotoxicity determined by a colorimetric based assay or Annexin v-Fitc staining. RESULTS. Differences were observed in the molecular size of the PXR protein expressed in sarcoma cell lines when compared with the wildtype PXR expressed in normal liver, kidney, or small intestine. A polyclonal PXR antibody raised against the N-terminus of the wildtype PXR did not detect PXR expressed in these sarcoma cell lines. In the osteosarcoma cell lines, etoposide and doxorubicin were better inducers of P450 3A4 and MDR1 than rifampin. siRNA against PXR down-regulated P450 3A4 expression only in the osteosarcoma cell line. Cytotoxicity assays showed that the resistance of the osteosarcoma cell lines to etoposide correlated with PXR protein expression levels and activation of P450 3A4 and could be prevented by ketoconazole. CONCLUSION. The results suggest that PXR plays a critical role in the regulation of P450 3A4 expression in osteosarcoma and that its expression and activation in these tumors may influence the effect of chemotherapeutic agents on the induction of target genes implicated in drug resistance. Cancer 2007. © 2007 American Cancer Society.

Osteosarcoma (OS) is the most common primary bone cancer in children, and despite more than 20 years of clinical trials, the 5-year survival for OS patients remains essentially unchanged (∼70% for non-metastatic patients and 30% for metastatic patients), with the vast majority deaths arising from metastasis to lungs or bones. We wanted to identify signaling pathways that mediate tumor survival and metastasis in OS, and we focused on Her-4 (ERBB4), which promoted survival on neuroblastoma cells in our previous work. Purpose: To evaluate the role of Her-4 in OS cell survival, metastasis and “stemness”. Methods: Her-4 expression was measured by flow cytometry, western blot and Q-PCR. Validated OS cell lines were manipulated with shRNA to knock-down Her-4 (compared to scrambled control). In vitro responses were measured by varying culture conditions (normal, high-density or anchorage-independent growth), serum starvation, hypoxia or chemotherapy (methotrexate, cisplatin, doxorubicin and 4-OH-ifosfamide) and measuring proliferation, cell cycle, and apoptosis (by both flow cytometry and PARP cleavage). A sarcosphere assay assessed expression of “stemness” markers, or tumor initiating cells (TICs), and Her-4 expression. The luciferase-labeled CCH-OS-O xenograft model was used to assess the impact of Her-4 knock-down on metastatic potential, with weekly luciferase measures (IVIS 100, Xenogen) assessing tumor burden. Immunohistochemistry (IHC) assessed Her-4 expression in archival material and murine xenografts. Results: Most OS cells express Her-4 which upregulated with increased culture density and with serum starvation. RT-PCR showed that the proportion of cleavable isoforms increased with increasing cell density (p < .05 with One-Way ANOVA analysis), while non-cleavable isoforms decreased (p < .05). Hypoxia increased the cleavage of Her-4 to the 80KD intracellular fragment without increasing Her-4 expression. IHC analysis of OS tissue microarrays showed that Her-4 expression is higher in metastatic lesions relative to primary tumors (p>0.01). Knock-down of Her-4 in OS cell lines reduced proliferation and increased apoptosis (by sub-diploid DNA and PARP cleavage) for cells grown in conventional culture, high density or spheres. Her-4 knock-down increased sensitivity to methotrexate (60% increase in apoptosis) but not other chemotherapies. To assess the role of Her-4 in metastasis, NOD/SCID/IL2Rg-/- mice were injected in the tibia with CCH-OD-O cells expressing luciferase and either Her-4 shRNA or scrambled control. With this model, a “primary” tumor arises in the tibia, with spontaneous metastasis to the lungs. Non-invasive imaging showed reduced growth of primary tumors in knock-down mice and complete elimination of tumor signal from lungs. Xenograft lungs showed a significant reduction in metastasis in knock-down mice. IHC for Her-4 showed that, while tibial tumors of knock-down mice still expressed no Her-4, the tumors present in these mice had re-expressed Her-4 despite the shRNA. Stress resistance, chemotherapy resistance and metastatic potential are TIC-associated behaviors, so we examined the association of Her-4 with other described TIC markers for OS. Sarcosphere culture induced expression of both Her-4 and stemness markers CD117 and Stro1 over a period of 2-7 days. Her-4 expression preceded upregulation of other markers by 1-2 days. Conclusions: Expression of Her-4 confers a survival benefit to OS cells under stressed conditions, including anoikis, serum starvation and methotrexate exposure. Her-4 is necessary for OS metastasis. Her-4 expression increases in sarcophere cultures and may be a mediator of stemness. Therapies targeting Her-4 in OS should be evaluated in further preclinical models. Citation Format: Rocio K. Rivera-Valentin, Yingqi Hua, Yi Zhang, Nupur Lala, Yanwen Yang, Dennis P.M. Hughes. Her-4 mediates anoikis resistance, chemoresistance, and metastatic potential in osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A71.

Osteosarcoma is a highly invasive bone malignancy in which metastasis accounts for the vast majority of death and morbidity in patients. Understanding the mechanisms controlling metastasis is essential for improving patient survival in this disease. In order to improve the clinical outcomes for patients with poor prognosis, it is urgent to find new therapeutic targets to block metastasis in this disease. Recent studies have shown that Metadherin (MTDH) plays an essential role in mediating tumorigenesis and metastasis in a variety of human cancers. Our study assessed the role of MTDH in osteosarcoma metastasis and elucidated the mechanisms underlying its metastasis-promoting activity. To evaluate the expression of MTDH in primary and metastatic lesions of osteosarcoma, two tissue microarrays containing patient-derived primary and metastatic tumor specimens were examined by immunohistochemical staining with anti-MTDH antibody. We also examined MTDH in a cDNA array expression database made from pretreatment diagnostic biopsies of high-grade osteosarcoma patients to further assess the correlation between MTDH expression and clinical outcome. We used western blot, qPCR, and flow cytometry to measure the expression of MTDH in a panel of osteosarcoma cell lines. In parallel experiments we used MTDH-specific shRNA to reduce endogenous MTDH expression, and blocked cell surface MTDH by anti-MTDH antibodies. The impact of MTDH inhibition was assessed in vitro using transwell migration assays and matrigel invasion assays. In addition, we developed an orthotopic xenograft mouse model to study the relationship between MTDH expression and osteosarcoma pulmonary metastasis. To investigate the role of MTDH in cell-extracellular matrix (ECM) interaction and to identify the extracellular binding partner for cell surface MTDH, a series of adhesion assays were performed, followed by bidirectional co-immunoprecipitation. We have demonstrated that MTDH is upregulated in human osteosarcoma cell lines and patient-derived specimens compared with normal human osteoblasts. Overexpression of MTDH is more profound in metastatic lesions compared to primary tumors and is correlated with poor clinical outcomes in osteosarcoma patients. MTDH knockdown and blockade of cell surface MTDH significantly reduced migration and invasion in osteosarcoma cells. In the in vivo experiments, downregulation of MTDH in osteosarcoma cells delayed primary tumor growth and prohibited pulmonary metastasis. Both in vitro and in vivo studies confirmed the critical role of MTDH in the invasive and metastatic capacity of osteosarcoma cells. More importantly, we have identified the significance of cell surface localization of MTDH in mediating osteosarcoma motility and invasiveness. We showed that MTDH exists as a type II membrane protein in osteosarcoma cells and its expression on cell surface is facilitating cell invasion by means of modulating cell adhesion to the ECM through interaction with laminin. In total, these observations establish MTDH as a promising target for therapeutic interventions in metastatic osteosarcoma. The novel connection between MTDH and extracellular laminin also establishes a new paradigm for the function of MTDH in mediating tumor cell metastasis. Citation Format: Limin Zhu, Pingyu Zhang, Yanwen Yang, Adrianna S. Buford, Wei-Lien Wang, Dafydd G. Thomas, Dennis P.M. Hughes. Metadherin functions as a laminin receptor that is essential for metastasis and is associated with poor survival in osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A53.

Osteosarcoma is the most common primary tumor of bones. In the past three decades treatment paradigms and survival rates have not improved. While osteosarcoma clinical phenotype has been well characterized, the underlying tumor molecular biology remains unclear. We have previously demonstrated that IL-11Rα is overexpressed in primary and metastatic osteosarcoma. Recently investigators identified that IL-11 and IL-11Rα play a role in the development of metastasis in breast and gastrointestinal cancer. However the role of IL-11/IL-11Rα signaling in osteosarcoma is unknown. Using in vitro and in vivo osteosarcoma models we investigated the role of IL-11/IL-11Rα in osteosarcoma metastasis. The purpose of our study is to determine the effect of the knockdown of IL-11Rα in osteosarcoma. We stably knocked down IL-11Rα expression in a highly metastatic osteosarcoma cell line, using a lentiviral short hair-pin (shRNA) based approach. Knockdown of IL-11Rα led to inhibition of STAT-3(Tyr705) protein phosphorylation, cell growth, migration, invasion and colony formation in vitro. To determine the role of IL-11Rα in osteosarcoma tumorigeniesis and metastasis, we used an orthotopic metastatic model. In this model, mice injected with KRIB-IL-11Rα shRNA cells developed significantly less metastasis than control mice injected with KRIB control shRNA (P Citation Format: Valerae O. Lewis, Eswaran Devarajan, Dennis PM Hughes. Targeting IL-11Rα inhibits osteosarcoma pulmonary metastasis in an orthotopic xenograft mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1261. doi:10.1158/1538-7445.AM2014-1261

Osteosarcoma is the most common primary bone tumor in children and adolescents, with recurring disease or lung metastases being the primary cause of death in patients. Expression of EGFR, Her-2, and Her-4 (ERBB1, 2 and 4) have been reported in human primary osteosarcoma, suggesting that they may be contributing to osteosarcoma pathogenesis, but the correlation of expression levels with patient outcomes has been controversial. We have shown that a pan-ERBB small molecule kinase inhibitor causes growth inhibition and apoptosis of osteosarcoma cell in vitro, suggesting a potential therapeutic benefit of using this drug in vivo. In the present study, we show that protein expression levels of EGFR, Her-2 and Her-4 are higher in osteosarcoma lung metastases than in primary tumors by TMA, and that these levels are significantly higher for Her-2 and Her-4. We created eight new osteosarcoma cell lines from primary patient tissues and found that all eight, in addition to six commonly used osteosarcoma cell lines, express detectable levels of EGFR and Her-2, and several express detectable Her-4. EGF ligand stimulation in vitro produces robust phosphorylation of EGFR, Her-2 and Her-4 in most cell lines and moderate activation in others. EGF stimulation also produces phosphorylation of Akt, MAPK and p70S6K in most cell lines and of STAT5 in six cell lines, suggesting that EGF has growth and survival stimulation effects. When adding a pan-ERBB kinase inhibitor, Pf-299804, to cells pre-stimulated with EGF, phosphorylation of EGFR, Her-2, Her-4, Akt, etc. are inhibited in a dose-dependent manner. Reverse phase protein array also shows an increase in pro-apoptotic proteins Bim and cleaved PARP with Pf-299804 treatment, confirmed by Western blots. Mouse xenograft experiments, where cells injected in the tibia spontaneously form lung metastases, showed a significant decrease in three categories of Her-4-positive lung metastases in drug-treated mice based on size: 1-4 cell oligomets (p=0.0055); 5 cell-200 micron micromets (p=0.0276); and greater than 200 micron macromets (p=0.0055). These data suggest that EGF produces mitogenic and survival stimulation in osteosarcoma cells and that pan ERBB-family kinase inhibition causes growth inhibition and apoptosis in vitro and reduction of lung metastases in a mouse model in vivo. This provides evidence that pan-ERBB kinase inhibition has therapeutic potential for osteosarcoma lung metastases. Citation Format: Laura D. Nelson, Tiffany N. Lynch, Yanwen Yang, Wei-Lien Wang, Diane Liu, Dafydd G. Thomas, Dennis P. M. Hughes. EGF has stimulatory and survival effects in osteosarcoma in vitro and pan-ERBB kinase inhibition causes cell growth inhibition in vitro and reduces lung metastases in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3970. doi:10.1158/1538-7445.AM2014-3970

Osteosarcoma is a highly invasive bone malignancy in which metastasis accounts for the vast majority of death and morbidity in patients. Understanding the mechanisms controlling metastasis is essential for improving patient survival in this disease. Recent studies have shown that Metadherin (MTDH) plays an essential role in mediating tumorigenesis and metastasis in a variety of human cancers. Our study assessed the role of MTDH in osteosarcoma metastasis and elucidated the mechanisms underlying its metastasis-promoting activity. We used western blot, qPCR, and flow cytometry to measure the expression of MTDH in a panel of osteosarcoma cell lines, and used immunohistochemistry to confirm expression in patient-derived material. In parallel experiments we used MTDH-specific shRNA to reduce endogenous MTDH expression, and blocked cell surface MTDH by anti-MTDH antibodies. The impact of MTDH inhibition was assessed in vitro using transwell migration assays and matrigel invasion assays. We also developed an orthotopic xenograft mouse model to study the relationship between MTDH expression and osteosarcoma pulmonary metastasis. To investigate the role of MTDH in cell-extracellular matrix (ECM) interaction, a series of adhesion assays were performed to assess the adhesion ability of osteosarcoma cells with or without MTDH inhibition to major components of ECM. Compared to normal human osteoblasts, all osteosarcoma cell lines tested displayed increased MTDH expression. MTDH knockdown significantly reduced migration and invasion in osteosarcoma cells. Blockade of cell surface MTDH with an anti-MTDH antibody targeting the extracellular domain of MTDH also profoundly impeded osteosarcoma cell motility and invasiveness. In the in vivo experiments, down-regulation of MTDH in osteosarcoma cells delayed primary tumor growth and prohibited pulmonary metastasis. Immunohistochemical analysis of tumors samples isolated from the mice showed that lung metastases had elevated MTDH expression level compared to primary tumors. Altogether, these data indicated an important role for Metadherin in osteosarcoma metastasis. Further, knockdown of MTDH by shRNA and inhibition of cell surface MTDH by anti-MTDH antibodies both profoundly decreased cell adhesion to matrigel and specific ECM proteins, which suggested that MTDH, particularly the proportion localized at the cell surface, may be involved in the interaction between tumor cells and the non-cellular matrix proteins that constitute tissue stroma. The interaction will be confirmed by future experiments. By identifying the ECM protein binding partners for MTDH, we may establish a new paradigm for the function of MTDH in mediating tumor cell migration and invasion in osteosarcoma. Citation Format: Limin Zhu, Adrianna Buford, Yanwen Yang, Pingyu Zhang, Wei-Lien Wang, Dennis P. Hughes. Metadherin mediates tumor invasion and interaction with extracellular matrix proteins in osteosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2683. doi:10.1158/1538-7445.AM2013-2683

Osteosarcoma is the most common type of bone malignancy in children. It is a highly invasive mesenchymal tumor in which metastasis accounts for the vast majority of mortality and morbidity in patients. Understanding the mechanisms controlling metastasis in osteosarcoma and developing novel therapeutic options targeting metastasis are essential for improving patient survival in this disease. Recent studies have shown that Metadherin (MTDH) plays a key role in mediating metastasis in a variety of human cancers. This study examined the role of MTDH in osteosarcoma metastasis and elucidated the mechanisms underlying its oncogenic activity. We used western blot, qPCR, and flow cytometry to measure the expression of MTDH in a panel of osteosarcoma cell lines. Patient-derived osteosarcoma samples were also examined for MTDH expression using immunohistochemistry. In parallel experiments we used MTDH-specific shRNA to down-regulate endogenous MTDH expression, and blocked cell surface MTDH by anti-MTDH antibodies. The impact of MTDH inhibition was assessed in vitro using migration assays and matrigel invasion assays. We also developed an osteosarcoma orthotopic xenograft model of NOD/SCID/IL2Rγ-deficient mice through intratibial injection of osteosarcoma cells with or without MTDH knockdown to study the relationship between MTDH expression and pulmonary metastatic potential of osteosarcoma cells. Compared to normal human osteoblasts, all osteosarcoma cell lines tested displayed elevated MTDH expression as shown by western blot, qPCR and flow cytometry analyses. MTDH knockdown reduced migration and decreased invasion in matrigel. Inhibition of cell surface MTDH with an anti-MTDH antibody targeting the extracellular domain of MTDH also profoundly impeded osteosarcoma cell motility and invasiveness in vitro. In an orthotopic xenograft mouse model using CCHD cells, inhibition of MTDH delayed primary tumor growth and prohibited pulmonary metastasis. All mice in the control group developed lung metastases while fewer than 20% of the mice in the MTDH-knockdown group showed sign of metastasis at six weeks after inoculation. MTDH knockdown also reduced mean lung weight and mean number of pulmonary metastasis. Immunohistochemical analysis of tumor samples showed that lung metastases had elevated MTDH expression level compared to primary tumors. Altogether these studies suggested that MTDH plays a critical role in promoting pulmonary metastasis of osteosarcoma and may serve as an attractive target for therapeutic intervention. Citation Format: Limin Zhu, Adrianna S. Buford, Yanwen Yang, Pingyu Zhang, Wei-Lien Wang, Dennis P. Hughes. Metadherin mediates osteosarcoma invasion and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B67.

Small molecule kinase inhibitors have dramatically improved the survival of patients with Gastrointestinal Stromal Tumors (GIST), yet most patients develop resistance, urging us to understand better the molecular mechanisms underlying this disease and identify new therapeutic strategies. Notch signaling is a conserved developmental pathway known to play a critical role in the development of several cancers, functioning as a tumor promoter or a tumor suppressor. Given that the normal progenitor cell for GIST, the Interstitial Cell of Cajal, has characteristics similar to those of cells of neuroendocrine origin, we hypothesized that Notch pathway impacts the biology of GIST cells. In a previous study, we demonstrated that constitutively active intracellular domain of Notch1 (ICN-1) induced growth arrest in GIST cell lines. Here we report that ICN-1 downregulated KIT protein by a flow cytometric analysis in the three cell lines analyzed (GIST-T1, GIST882, GIST48IM). In parallel, inhibitions of Hes1, a major target gene of ICN-1, with a dominant negative construct, upregulated KIT protein expression in vitro (60% increase on western blot). This anti-tumor effect of Notch1 in GIST leads to a therapeutic opportunity as we showed in the present study that treatment with the histone deacetylase inhibitor, SAHA, caused a dose-dependent upregulation of Notch1 (6 fold increase of the mRNA with SAHA 2 µm/L on GIST-T1, P80 months for the high group; P=0.005). These results identify a novel anti-tumor effect of Notch1 via a crosstalk between the Notch1 and KIT pathways in GIST cells. This finding may lead to new therapeutic strategies for GIST patients, in particular after failure of tyrosine kinase inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3239. doi:1538-7445.AM2012-3239

Farnesyltransferase inhibitors (FTIs), small molecules that inhibit prenylation of multiple proteins such as Ras and Rho, have clinical promise for cancer. FTIs block malignant growth and promote cell death in a wide variety of tumor types in vitro and in vivo, but the mechanisms underlying the antitumor activity of FTIs are not completely understood. We have observed that colon cancer cell line OS187 responds to the FTI tipifarnib with a senescent phenotype, demonstrated as altered cell morphology, elevated senescence-associated β-galactosidase activity, upregulated protein and mRNA levels of senescence markers, and long-term cell cycle arrest. Our previous results also indicated that cells retaining intact p53 are more sensitive to tipifarnib than are cells with absent/inactive p53, suggesting a possible role for p53 in the senescent response to tipifarnib. To test this hypothesis, we used shRNA to reduce p53 expression in OS187 cells. Loss of p53 reversed the swollen and enlarged senescent-like cellular phenotype induced by tipifarnib. In addition, p53 knockdown cells showed less senescence-associated β-galactosidase activity than did scrambled control cells after 7-day treatment with tipifarnib. Cell viability assay suggested that p53 knockdown partially rescued the cytotoxic effects of tipifarnib at low concentrations. Western blot also demonstrated that cell cycle regulatory proteins such as p27, p21, and p15 were significantly upregulated in scrambled control cells while only a slight increase of these proteins were observed in p53 knockdown cells after tipifarnib treatment. Flow cytometry analysis showed that cells with decreased p53 level had a reduced percentage of cells in sub G1 phase when treated with a high concentration of tipifarnib, and fewer cells arresting in G2/M phase when treated with low concentration of tipifarnib compared to the scrambled control cells. This observation suggested that p53 may involve in different pathways depending on the strength of the stress signal imposed by different concentration of tipifarnib. In conclusion, these results confirm that p53 is important for inducing senescence when farnesylation is inhibited in OS187 cells. However, since p53 knockdown has very limited impact on the anti-proliferation effects of tipifarnib at high concentrations, other pathways are expected to play a role in mediating cell susceptibility to tipifarnib. Future experiments will focus on investigating the p53-independent pathways that are responsible for tipifarnib-induced senescence and apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2876. doi:1538-7445.AM2012-2876

Abstract 1397 The ERBB family of receptor tyrosine kinases (EGFR, Her-2, Her-3 and Her-4) are receptor tyrosine kinases that, through mutation or aberrant expression, serve as oncogenes by promoting hallmark behaviors of cancer in many solid tumors. Previous work has suggested that HER2 is expressed in as much as 30% of B-ALL patients, and correlates with chemoresistance. We therefore hypothesized that HER2 signaling in Ph+ ALL may augment growth signaling and promote other malignant behaviors, such as resistance to cell death and independence from growth factors. Western blot and flow cytometric analyses of two human Ph+ ALL cell lines, Z119 and Z181, revealed cell surface expression of HER2, but not other family members. To determine the role of HER2 signaling in Ph+ ALL cell lines, the pan-HER family small molecule kinase inhibitor canertinib was used, and reverse phase protein array (RPPA) was conducted in Z119 and Z181 cell lines. Briefly, lysates from canertinib treated cells were spotted using a GeneTAC™ G3 arrayer onto nitrocellulose-coated FAST® slides. Incubation of the slides was performed with forty-three antibodies directed towards various cell signaling proteins followed by colorimetric detection and results were subsequently validated by western blotting. RPPA analyses revealed that treatment with canertinib effectively diminished HER2 phosphorylation in both cell lines. Additionally, we found decreased phosphorylation of the pro-survival molecules ribosomal protein S6, p70S6kinase, and c-Src, as well as increased expression of the pro-apoptotic molecules BIM and cleaved-PARP in both Ph+ ALL cell lines. Congruent with these findings, elevated activity of the executioner caspase 3 and increased DNA fragmentation, two distinct biochemical markers of apoptosis, were present after canertinib treatment in Z181 and Z119 cells, suggesting that inhibition of HER2 signaling results in programmed cell death of Ph+ ALL cell lines. This induction of apoptosis paralleled a decrease in overall proliferation of these cell lines, further implicating HER2 signaling in proliferation of Ph+ ALL. Next, we analyzed if clinically approved inhibitors of HER2 function could be utilized to produce the same biological consequence as canertinib in Ph+ ALL cell lines. Lapatinib (Tykerb) is a dual EGFR/HER2 small molecule kinase inhibitor approved by the FDA for the treatment of breast cancer. Consistent with our results utilizing canertinib, lapatinib was capable of inhibiting proliferation of both Z119 and Z181 cell lines. Interestingly, the FDA approved monoclonal antibody HER2 inhibitor trastuzumab (Herceptin) did not inhibit proliferation of these cell lines. Similarly, trimerized herceptin conjugates, which improve internalization of HER2 receptor, also had no effect on Ph+ ALL cell line proliferation. These results highlight an important distinction between the effects of the intracellular small molecule inhibitors of HER2 and monoclonal HER2 antibodies. In particular, extracellular engagement of the HER2 receptor by monoclonal antibodies may not be effective in targeting the HER2 signaling pathways required for proliferation and survival of Ph+ ALL. Taken together, our studies suggest that HER2 may play an important role in growth and survival signaling of Ph+ ALL cell lines and inhibition of HER2 with small molecule kinase inhibitors may improve treatment regimens. Thus, additional studies are warranted to determine the importance of HER2 in clinical specimens and the potential benefit of combining HER2 inhibitor therapy with imatinib treatment for Ph+ ALL. Disclosures: Mills: Glaxosmithkline: Research Funding; Pfizer: Research Funding.

Multi-cellular tumor spheroids (MCTS) model is an in vitro model holding an intermediate complexity between monolayer culture and solid tumor. It is cell-cell adhesion dependent and closely resembles avascular tumor regions and micrometastasis in pro-angiogenesis stage. Osteosarcoma (OS) is the most common malignant bone tumor with high incidence of early lung metastasis; therefore, multi-cellular tumor spheroid can be used to model its in vivo behaviors. Our group previously reported the expression profile of ERBB family in osteosarcoma tissue samples and cell lines. To further study ERBB pathways in osteosarcoma tumor spheroids, we generated OS tumor spheroids by plating cells on non-adherent poly-HEMA pre-coated dishes. Under anchorage-independent conditions, OS cells spontaneously generate multi-cellular spheroids to acquired anoikis resistant by cell-cell adhesion; blocking the formation of spheroids will result rapid cell death and increased PARP cleavage. MCTSs showed lower proliferation rate than monolayer culture, and they held a chemo-resistant phenotype, as shown by proliferation assay and subdiploid DNA content. Surprisingly, ERBB4 expression was up-regulated in non-adherent tumor spheroids compared with monolayer culture (1.2, 2.9, 6.3 fold respectively in 3 cell lines) while EGFR and ERBB2 did not. With knockdown of ERBB4 expression by shRNA, OS cells became less tolerant to anoikis and serum starvation, as shown by PARP cleavage and subdiploid DNA content. Moreover, knockdown of ERBB4 increased the chemo-sensitivity of OS cells against multiple chemotherapy including cisplatin, doxorubicin, and methotrexate (71%, 71%, 60% in survival compared to scramble control). In conclusion, our data showed that ERBB4 contributes to the tumorigenicity of osteosarcoma and plays a protective role against multiple exogenous apoptotic stimuli including matrix detachment, nutrition insufficiency and cytotoxic reagents. Although wildly expressed in osteosarcoma, the role of ERBB4 in tumor biology is largely unknown. These findings indicate that ERBB4 is a key factor regulating survival pathway of OS under various cellular stress condition. Thus, in primary osteosarcoma and metastatic lesion, ERBB4 might be a potential therapeutic target to facilitate multi-modal therapies. 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 197. doi:10.1158/1538-7445.AM2011-197

Farnesyltransferase inhibitors (FTIs), small molecules that inhibit prenylation of multiple proteins such as Ras and Rho, have clinical promise for cancer. FTIs block malignant growth and promote cell death in a wide variety of tumor types in vitro and in vivo, but the mechanisms underlying the antitumor activity of FTIs are not completely understood. We have observed that osteosarcoma cell lines respond to the FTI tipifarnib with growth arrest, cellular enlargement, and flattened morphology, all changes consistent with the senescent phenotype (Hall et al., 2010). These observations led us to evaluate the ability of FTIs to induce senescence in osteosarcoma. Further, it was clear that cells retaining intact p53 responded at much lower concentrations of FTIs than did cells with absent/inactive p53, suggesting a possible role for p53 in this response. We measured endogenous β-galactosidase activity by in situ chromatography and quantified expression levels of common senescence markers including p53, p21Cip1, p16INK4a and DEC1 by densitometry analysis of western blot and qPCR in two human osteosarcoma cell lines, OS187 and Saos2. Cell growth was measured by direct counting of nuclei, and cell cycle was analyzed by PI staining and flow cytometry. Apoptosis was assessed by PARP cleavage, annexin V staining and flow cytometry. Variable responses to tipifarnib correlated with p53 status. In p53 proficient OS187 cells, 24h exposure to very low concentration of tipifarnib (10−9 M) induced upregulation of DEC1 and a 45-fold increase in p21Cip1 protein, accompanied by increased expression and phosphorylation of p53. This event also correlated with transient reduction of cyclin A, B1, and E proteins in a time-dependent manner, leading to cell cycle arrest which is comparable to tipifarnib-treated 293T cells. OS187 cells also exhibited elevated senescence-associated β-galactosidase activity in response to low-dose tipifarnib treatment after 24h. Meanwhile, Saos2 cells (p53-null) gave no response at 10−9 M tipifarnib. At higher concentrations (10−7 M), modest induction of p21Cip1 (10-fold increase) and p16INK4a (2-fold increase) proteins and mild inhibition of cell cycle progression were observed. Augmented senescence-associated β-galactosidase activity became detectable after Saos2 cells were exposed to high concentration of tipifarnib (10−6 M) for at least 48h. In addition to senescence, tipifarnib increased PARP cleavage and annexin V staining in OS187 cells, indicating that apoptotic cell death may also play a role in the anti-tumor activity of FTIs. These findings suggest that tipifarnib could efficiently promote senescence through a p53-dependent pathway at low concentrations, and via p53 independent pathways at higher concentrations. Induction of both cellular senescence and apoptosis may contribute to the growth inhibition induced by FTIs in osteosarcoma. 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 2640. doi:10.1158/1538-7445.AM2011-2640

Gastrointestinal Stromal Tumors (GISTs) are the most common sarcomas of the GI tract and harbor a gain-of-function mutation of the KIT receptor tyrosine kinase. The introduction of imatinib, a specific inhibitor of KIT, has significantly extended survival of many GIST patients. Unfortunately, patients inevitably develop resistance, prompting interest in understanding GIST biology and developing new therapeutic options. Notch signaling may act as an oncogene or a tumor suppressor depending cellular context. In neuroendocrine tumors Notch signaling acts constantly as a tumor suppressor. Additionally, histone deacetylase inhibitors (HDACi) were recently shown to induce Notch signaling in neuroendocrine neoplasms. Given that GIST is of neuroectodermal origin, we hypothesized that inhibition of growth in GIST cells by HDAC inhibitors is mediated by activation of Notch signaling. To characterize Notch signaling in GIST, we assessed the expression of Notch-1, and its effector human hairy enhancer of split-1 (HES1) in human GIST cell lines (GIST-T1 and GIST 882) by quantitative RT-PCR. To determine the activity of Notch signaling in GIST, we assayed the effect of Notch activation in GIST cells by transduction with retroviral vectors to overexpress active forms of Notch1 and HES1 in GIST cells. The proportion of GFP-positive cells in unselected culture was assessed to determine the transgene's effect on cell growth. To study the effect of HDAC inhibition, we quantified viability, caspase activity, cell cycle analysis and expression of Notch pathway genes after HDAC inhibition with suberoylanilide hydroxamic acid (SAHA). Notch signaling genes (Notch1, HES1) were weakly expressed at the basal level in cells compared to other sarcomas. Notch signaling with a constitutively active, truncated form of the receptor (ICN-1) and HES1 potently induced growth arrest in GIST cell lines. The proportion of ICN-1 GFP+ cells dramatically declined after transduction compared to GFP- cells, (decrease of 69% at day 7 and 78% at day 9 after transduction). Vector empty-transduced GFP+ cells gave no change in the proportion of positive cells. SAHA caused dose dependent decrease in viability, activation of caspase 3 and 7, increase of sub-G1 population (6 fold increase at 1µM, p These results suggest that Notch signaling is a tumor suppressor in GIST cells and the activation of Notch signaling by HDAC inhibitors may represent a new strategy for treating GIST patients. 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 618. doi:10.1158/1538-7445.AM2011-618

The human epidermal growth factor receptor 2 (Her2) is overexpressed in multiple types of tumors, including breast tumors and osteosarcoma. Tumor-targeted antibody therapy is one of the safest therapeutics available, but it is often ineffective in treating primary and acquired antibody-resistant tumor cells via indirect tumor cell death and inducing direct tumor cell death. This study reveals that Herceptin conjugates, if generated via the crosslinker EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodimide hydrochloride), is capable of causing Her2 positive tumor cell death. Using an HPLC system, two peaks with estimated molecular weights of antibody dimers and trimers were isolated. The Herceptin dimer and trimer separated by HPLC, especially the trimer, induces significant levels of necrotic tumor cell death. Unlike SMCC conjugated Herceptin, this study indicates that the EDC-conjugated Herceptin overcomes the acquired resistance. The Herceptin trimer is more effective than the dimer in enhancing Herceptin-resistant tumor cell death. Surprisingly different from the known cell death mechanism often resulting from antibody treatment, the Herceptin trimer causes effective and direct tumor cell death via a novel cell death mechanism: programmed cell necrosis. Inhibition of RIP, a critical regulator of programmed necrotic cell death, reversed the Herceptin trimer-induced cell death. In summary, the Herceptin conjugate especially trimer linked by EDC could overcome Her2 positive breast cancer or osteosarcoma cells as well as the resistant cell lines via program cell necrosis cell death. This conjugate has great potential for overcoming the tumor cell resistance to the Herceptin treatment. 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 4559. doi:10.1158/1538-7445.AM2011-4559

This study is to determine if triplex DNA-binding proteins in human cells play an important role in processes that lead to cancer such as genetic instability or growth deregulation. Cytoplasmic and nuclear extracts from 60 colorectal cancer patients’ tumor (T) and normal (N) tissues were isolated and examined by gel shifts (EMSA) for triplex DNA-binding proteins. Total tumor extracts showed significantly higher triplex DNA-binding activities of the major H3 complex than normal tissue extracts (Wilcoxon test). Neither T-staging nor M-staging at diagnosis was associated with any of the factors, but N-Stage was significantly associated with the ratio of tumor/normal (T/N) for the total of cytoplasmic and nuclear extracts. This means all patients without lymph node affectation had decreased binding ratios (T/N) in both cytoplasmic and nuclear extracts. Kaplan-Meier survival analysis using a cut-off of 1.5 (rounded-up median) for the nuclear binding activity ratio (T/N) showed significantly poorer survival for those patients whose nuclear binding was greater than 1.5. This suggests that although the major triplex DNA-binding protein is present in normal tissue extracts, it is more abundant in tumor extracts, and that a specific abundance of the major triplex-binding protein (H3) in the tumor nucleus is associated with poorer patient survival. Biotinylated triplex DNA-protein complexes were isolated from the RKO colorectal cancer cell line with streptavidin-agarose and analyzed by nano-HPLC ESI-MS-MS. We identified 100 and 60 kDa proteins from nuclear extracts as PSF (polypyrimidine tract binding-associated splicing factor) and p54nrb (NonO), known to function as RNA polymerase II-associated splicing factors, bind as heterodimers, and implicated in the regulation of expression of the myc family of oncoproteins and COX2. We also identified a 75 kDa protein from cytoplasmic extracts as U2AF65 (U2 small nuclear RNA auxiliary factor 2 isoform b), also a splicing factor that can directly interact with p54 nrb and shuttle continuously between the nucleus and cytoplasm. To examine differences between tumor and normal tissues in multiple signaling proteins and proteins known to be relevant in colorectal cancer, reverse phase protein array (RPPA), Pearson correlation and cluster analysis of tumor nuclear lysates ranked from lowest to highest H3 EMSA values shows a highly significant correlation between decreased β-catenin expression in tissues that have the lowest H3 EMSA values and increased β-catenin expression in tissues that have the highest H3 EMSA values. We found this pattern in all tissue lysate types: normal-cytoplasm, normal-nucleus, tumor-cytoplasm, and tumor-nucleus. We believe these splicing factors are overexpressed in tumor tissues and could cause deregulated growth of cells by influencing other proteins associated with tumorigenesis. 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 4566.

Introduction Osteosarcoma (OS) is the most common bone cancer and often has poor outcomes: despite surgery and chemotherapy, only 30% of patients with metastases at diagnosis survive their disease. This stagnant survival curve generates interest in finding novel therapeutic targets and new treatment options to improve outcomes. Tipifarnib (TF) (ZARNESTRA®, R115777) is a small molecule farnesyltransferase inhibitor (FTI). Inhibition of farnesylation causes relocalization of Ras in responsive OS cells, resulting in alternate activation of downstream effectors of Ras in sensitive vs. resistant lines in response to FTI. Markers for susceptibility and pathway(s) affected by FTI in OS need to be determined to identify patients who might benefit from FTI. Methods For Immunofluorescence (IF), OS cells were treated with 1 µM TF or no drug for 24 hours, fixed and stained for pan Ras, and analyzed by standard IF techniques. OS187 cells were treated with 0, 0.01 or 1 µM TF with or without z-vad (a pan caspase inhibitor) for up to 4 days, and cell yield determined daily by automated counting (ViCell). OS cells were treated with 0, 0.01 or 1 µM TF for 24 hours and lysates assessed by western blot for downstream targets of RAS, including MEK, p38 and ERK. OS187 was treated with SB 203580 (p38 MAPK inhibitor), U0126 (MEK inhibitor) TF, or all possible combinations for 72 hours. Cell yield was determined as above. Results OS cells demonstrate variable growth inhibition from FTI, with a 2-log difference between highly responsive (OS187) and highly resistant OS lines (Saos-2). In OS187, response to FTI was associated with redistribution of Ras from a diffuse, perimembranous pattern in untreated cells to a punctuate and perinuclear pattern with FTI as assessed by IF. Neither z-vad, U0126 or SB 203580 rescued cells from FTI-induced growth inhibition induced. Ras activity increased with FTI. Activation of Ras effectors differs between sensitive and resistant cell lines: p38, ERK, MEK, MKK3/6, ATF2 and MSK-1 are activated in sensitive cells in response to FTI. Conclusion Subcellular relocalization of Ras in response to FTI suggests that altering prenylation changes Ras’ interaction with effector molecules. Though we have shown that FTI induces cell death and sub-G1 DNA content in responding cells (Hall et al., 2010), the fact that z-vad does not rescue OS cells from growth inhibition and death suggests that FTI-induced death in OS is caspase independent. While multiple members of the MAPK cascade (especially p38) are activated in response to FTI, neither a p38 nor a MEK inhibitor rescued OS cells from FTI-induced growth inhibition. This suggests that either multiple MAPK components need to be inhibited to rescue cells, or that growth inhibition and cell death are independent of MAPK pathway activation. Regardless of mechanism, the strong response of some OS cells to low concentrations of TF suggests that some patients may benefit from this class of drugs. 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 5269.

10060 Background: Sarcoma metastasizing to the heart is a difficult and complicated clinical problem, yet local control is feasible and effective for select patients with cardiac metastasis. Diligence is required to detect these lesions while still amenable to treatment, and echocardiogram remains the best tool for detecting cardiac disease. Methods: We reviewed all echocardiograms (1330) performed on 307 pediatric sarcoma patients treated at UT M. D. Anderson Cancer Center between 1997 and 2008. Measures of cardiac function and pathology (including size of ventricles and atria, function of ventricles and valves, presence of metastasis, and presence of pericardial effusion) were assigned numerical values. Fisher's exact test and the Wilcoxon rank-sum test compared clinical characteristics of patients with and without cardiac metastases. Results: The prevalence of cardiac metastases was 1.6% (5/307) with 95% confidence interval 0.5% to 3.8%. The presence of cardiac metastasis positively correlated with pericardial effusion (p = 0.001) and tricuspid valve insufficiency (p = 0.014). The probability of a patient with pericardial effusion having a cardiac metastasis was 28.6% (95% CI: 3.7%-71%). Of the 5 patients with documented sarcoma metastasis to the heart, 3 had widespread refractory disease, were given no cardiac-specific therapy, and rapidly died from disease. One patient who had widespread disease controlled with chemotherapy and radiation had open resection of 2 cardiac metastases which resulted in site-specific disease control for 6 months before succumbing to progressive extra-cardiac disease. Another patient had isolated cardiac metastasis, treated with open resection of 2 metastases followed by adjuvant chemotherapy, and has been without recurrent cardiac metastases for 5 years and 9 months. Conclusions: Durable local control for sarcoma metastasizing to the heart is possible and effective for select patients. Pericardial effusion merits evaluation for potential associated cardiac metastasis. Echocardiogram monitoring of sarcoma patients remains important, and should include screening for possible cardiac metastasis. No significant financial relationships to disclose.

10036 Background: Extrapulmonary osteosarcoma (OS) metastases portend poor outcome and short survival. Local control is essential for durable remission. Intracardiac, mediastinal and CNS metastases...

20006 Background: Epithelial salivary gland neoplasms (ESGN) are uncommon tumors that mainly occur in adults, only 5 percent of ESGNs occur in children. Data regarding differences in clinical presentation and biologic behavior of these tumors between adult and pediatric patients remain unclear. There is less data concerning the role of radiation therapy in the treatment of ESGNs in pediatric patients. Methods: Retrospective review of medical records of patients presenting under age 18 years from January 1988 to August 2006. Results: 45 patients were evaluable. 26 patients were female. Median age at diagnosis was 14.7 years. Mucoepidermoid carcinoma (MEC) was seen in 22 cases (49%), acinic cell carcinoma 10 cases (22%), adenocystic carcinoma (ACC) 8 cases, and pleomorphic adenoma (PA) 5 cases. The parotid area was the most common involved site (25/45; 56%) with MEC and acinic cell noted in 80% of parotid cases. 76% of MEC cases (16/21) were grade 2 or 3 tumors. Initial complete resection was achieved in 20 ESGN cases, while the remaining cases required re-excision or adjuvant therapy. Thirteen patients received postoperative radiation. Median age was 15.8 years. Radiation therapy was used in 6 ACC cases, 4 MEC and 3 adenoid cystic. Primary site and first echelon nodes dose range was 54–64 Gy, and supraclavicular dose was 50 Gy in those in whom regional nodes were treated. Positive margin status and local invasion (perineural, lymphatic, or nodal) were the most common indications. Mean follow-up for 35 patients was 60.3 months (2–175 months). Nine patients were lost to follow- up, and one patient received therapy at another institution. Two patients died from progressive disease, and both had received radiation therapy. Conclusions: MEC is the most common ESGN in pediatrics, and the parotid is the most common involved site. Complete surgical resection remains the treatment of choice. Radiation therapy may be used as adjuvant therapy, but more study is required. No significant financial relationships to disclose.

19502 Background: It has been suggested that sedentary behavior during cancer treatment or effects of the treatment itself may decrease cardiorespiratory fitness in cancer survivors. However, few studies have tested whether cancer survivors actually have impaired cardiorespiratory fitness compared to similarly aged individuals who have not had cancer. We investigated whether endometrial cancer survivors had poorer fitness than women of similar age and activity level with no cancer history. Methods: The sample included endometrial cancer survivors in a pilot exercise study; all had been diagnosed with Stage I-IIIa disease and were at least 6 months post- treatment. Controls were recruited from a local multi-speciality clinic. Both groups had no contraindications to exercise and were sedentary. Cardiorespiratory fitness was measured by predicted maximal myocardial oxygen carrying capacity (MV02 max) on a submaximal ramped cycle ergometer test. Quality of life was measured using the SF-36 (physical and mental component scores). Results: The study accrued 20 survivors (Mean age=58.6, SD=7.9; Mean BMI=30.5, SD=7.7) and 13 controls (Mean age=55.8, SD=7.6; Mean BMI=30.3, SD=7.0). In t-tests comparing survivors and controls, there were significant differences in predicted MV02 max (p=0.044) and physical aspects of quality of life (p=0.004), but not mental aspects of quality of life. After adjusting for age and BMI using ANCOVA, the p value for predicted MV02 max increased to 0.068, but differences in physical quality of life remained significant (see Table 1 ). Conclusions: Endometrial cancer survivors have diminished quality of life in the physical domain compared to women who have not had cancer; similar results were found for cardiorespiratory fitness, although the effect was attenuated after adjusting for BMI and age. The results suggest endometrial cancer survivors could benefit from exercise interventions to improve fitness and physical functioning. Table 1 : Fitness and quality of life of endometrial cancer survivors and controls. [Table: see text] No significant financial relationships to disclose.

Philadelphia chromosome-positive ALL (Ph+ALL) strikes older children and adults, is difficult to treat and often has poor outcomes with conventional therapies. Tremendous advances have been made in other cancers through identification of essential signaling molecules that can be inhibited by small molecule medicines. ERBB proteins are such targets for several adult carcinomas, including breast and lung cancer. Expression of one member of the ERBB family, Her-2, is associated with chemotherapy resistance in one report of adult B-ALL. We examined the expression, activation status and contribution to growth and survival of ERBB receptors in Ph+ALL cell lines Z33, Z119 and Z181. Phosphorylated Her-2 was detected by western blot in all 3 lines, as was trace amounts of Her-3. Western-blot identified expression of the p80 form of Her-4 in Z119, which correlated with flow cytometry assessment of Her-4. Expression in Z33 of EGFR, detected by western blot, correlated with down-regulation of both EGFR and Her-2 from the cell surface, as assessed by flow cytometry. Internalization of EGFR homo- and hetero-dimers is associated with receptor phosphorylation and active signaling. To assess the physiologic importance of ERBB signaling in Ph+ALL, cells were cultured in the pan-ERBB inhibitor CI-1033 (Pfizer Global Research and Development), which is specific for ERBB family receptors in tissue culture up to 20 μM concentrations. All lines demonstrated reduced cell yield after CI-1033 exposure, with average IC50 of 0.783 for Z119, 1.178 for Z181 and 1.737 for Z33. The increased sensitivity of Z119 to CI-1033 correlated with an increased rate of apoptosis: 3 μM CI-1033 caused a 26% increase in the fraction of sub-diploid Z119 cells at 24 hours, compared to 13% for Z181 and 16% for Z33. Higher sensitivity to ERBB inhibition also correlated with growth factor resistance, since Z33 and Z181 are dependent upon autocrine GM-CSF for growth while Z119 grows independent of known autocrine secretion of hematopoietic growth factors. These data suggest that Ph+ALL cells may acquire GM-CSF independence by relying more on ERBB-derived signals, since these receptors are capable of activating similar second messenger pathways. Our data suggest that, surprisingly, the ERBB family of receptor tyrosine kinases are intact and functioning in Ph+ALL, and inhibition of these signals may provide benefit to Ph+ALL patients, either in combination with imatinib or in patients who develop imatinib resistance.