Leslie Kurtzberg

Endosialin Protein Expression and Therapeutic Target Potential in Human Solid Tumors: Sarcoma versus Carcinoma

Purpose: Endosialin/CD248/tumor endothelial marker 1 is expressed in stromal cells, endothelial cells, and pericytes in various tumors; however, few studies have focused on expression in malignant cells. Experimental Design: We studied expression of endosialin in clinical specimens, cell culture, and animal models and designed an anti-endosialin therapeutic prototype. Results: Fifty human tumor cell lines and 6 normal cell types in culture were assayed by reverse transcription-PCR and/or flow cytometry for endosialin. Cell surface protein was found on 7 sarcoma lines, 1 neuroblastoma, and 4 normal cell types in culture. A fully human anti-endosialin antibody bound to human A-673 Ewings sarcoma cells and SK-N-AS neuroblastoma cells but not HT-1080 cells. Exposure of cells to an anti-human IgG conjugated to saporin resulted in growth inhibition only of endosialin-expressing cells. Endosialin expression was assessed by immunohistochemistry in 250 clinical specimens of human cancer including 20 cancer subtypes. Endosialin is frequently found in human cancers. Endosialin expression is mainly a perivascular feature in carcinomas, with some expression in stromal cells. In sarcomas, endosialin is expressed by malignant cells, perivascular cells, and stromal cells. Development and characterization of experimental models for studying endosialin biology in sarcomas and evaluating anti-endosialin therapies is presented. Conclusions: Findings suggest that an anti-endosialin immunotoxin might be a promising therapeutic approach for endosialin-positive neoplasia, especially synovial sarcoma, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, and osteosarcoma. Thus, a diagnostic/therapeutic targeted therapeutic approach to treatment of endosialin-expressing tumors may be possible.

Placental Growth Factor Upregulation Is a Host Response to Antiangiogenic Therapy

Purpose: Placental growth factor (PlGF) is an angiogenic protein. Upregulation of PlGF has been observed in the clinic following antiangiogenic regimens targeting the VEGF pathway. PlGF has been proposed as a therapeutic target for oncology. sFLT01 is a novel fusion protein that neutralizes mouse and human PlGF (mPlGF, hPlGF) and mouse and human VEGF-A (mVEGF-A, hVEGF-A). It was tested in syngeneic and xenograft tumor models to evaluate the effects of simultaneously neutralizing PlGF and VEGF-A and to investigate changes observed in the clinic in preclinical models. Experimental Design: Production of PlGF and VEGF-A by B16F10 and A673 cancer cells in vitro was assessed. Mice with subcutaneous B16F10 melanoma or A673 sarcoma tumors were treated with sFLT01. Tumor volumes and microvessel density (MVD) were measured to assess efficacy. Serum levels of hVEGF-A, hPlGF, and mPlGF at early and late time points were determined by ELISA. Results: Exposure of cancer cell lines to sFLT01 caused a decrease in VEGF secretion. sFLT01 inhibited tumor growth, prolonged survival, and decreased MVD. Analysis of serum collected from treated mice showed that sFLT01 administration caused a marked increase in circulating mPlGF but not hPlGF or hVEGF. sFLT01 treatment also increased circulating mPlGF levels in non–tumor-bearing mice. Conclusion: With the tumor cell lines and mouse models we used, antiangiogenic therapies that target both PlGF and VEGF may elicit a host response rather than, or in addition to, a malignant cell response that contribute to therapeutic resistance and tumor escape as suggested by others. Clin Cancer Res; 17(5); 976–88. ©2011 AACR.

Bone marrow and tumor cell colony-forming units and human tumor xenograft efficacy of noncamptothecin and camptothecin topoisomerase I inhibitors

Topoisomerase I (TopoI), an established anticancer target, is an enzyme producing a single-strand DNA break during transcription. Several noncamptothecin TopoI inhibitors have been identified. One of these, ARC-111, was compared with two clinically used camptothecins, topotecan and irinotecan/SN-38. In mouse and human bone marrow colony formation [colony-forming units granulocyte-macrophage (CFU-GM)] assays, the IC90 values were 519 and 331 nmol/L for topotecan and SN-38 mouse CFU-GM and were 19 and 26 nmol/L for human CFU-GM, giving mouse to human differentials of 28- and 13-fold. ARC-111 produced IC90 values of 28 nmol/L in mouse and 6.2 nmol/L in human CFU-GM, thus only a 4.5-fold differential between species. Human bone marrow CFU-GM was more sensitive to topotecan than were several human cancer cell lines, but ARC-111 cytotoxicity was similar for human bone marrow CFU-GM and the seven human tumor cell lines tested. In HCT-116 xenografts, tumor growth delays (TGD) were 17 days for irinotecan and 20 days for ARC-111. In HT-29 xenografts, the TGD was 9 days for both irinotecan and ARC-111. Both ARC-111 and docetaxel had a TGD of 21 days in NCI-H460 xenografts, and both ARC-111 and gemcitabine had a TGD of 7 days in MiaPaCa2 xenograft. Current TopoI inhibitors have broad antitumor activity in human tumor xenografts that is not achieved in the clinic. This may be due to greater sensitivity of human bone marrow than mouse to the cytotoxicity of these agents. It may be possible to achieve similar levels of ARC-111 in patients as in mice allowing improved antitumor activity. [Mol Cancer Ther 2008;7(10):3212–22]

Genz-644282, a Novel Non-Camptothecin Topoisomerase I Inhibitor for Cancer Treatment

Purpose: Genz-644282 [8,9-dimethoxy-5-(2-N-methylaminoethyl)-2,3-methylenedioxy-5H-dibenzo[c,h][1,6]naphthyridin-6-one] has emerged as a promising candidate for antitumor agents. This report describes the bone marrow colony-forming unit, granulocyte macrophage (CFU-GM) and tumor cell CFU activity of topoisomerase I (Top1) inhibitors, such as Genz-644282, topotecan, irinotecan/SN-38, and ARC-111, and examines their activity in several human tumor xenograft models. Experimental Design: Colony-forming assays were conducted with mouse and human bone marrow and eight human tumor cell lines. In addition, 29 human tumor cell lines representing a range of histology and potential resistance mechanisms were assayed for sensitivity to Genz-644282 in a 72-hour exposure assay. The efficacy of Genz-644282 was compared with standard anticancer drugs (i.e., irinotecan, docetaxel, and dacarbazine) in human tumor xenografts of colon cancer, renal cell carcinoma, non–small cell lung cancer, and melanoma. Results: Human bone marrow CFU-GM was more sensitive to the Top1 inhibitors than was mouse bone marrow CFU-GM. The ratio of mouse to human IC90 values was more than 10 for the camptothecins and less than 10 for Genz-644282, which had more potency as a cytotoxic agent toward human tumor cells in culture than the camptothecins in the colony-forming and 72-hour proliferation assays. Genz-644282 has superior or equal antitumor activity in the human tumor xenografts than the standard drug comparators. Conclusions: On the basis of preclinical activity and safety, Genz-644282 was selected for development and is currently undergoing phase 1 clinical trial. Clin Cancer Res; 17(9); 2777–87. ©2011 AACR.

Erufosine, an alkylphosphocholine, with differential toxicity to human cancer cells and bone marrow cells

PurposeTo investigate the activity and myeloprotective properties of erufosine, a novel alkylphosphocholine (APC), on human malignant cells and normal bone marrow cells.MethodsHuman or mouse bone marrow cells were exposed to erufosine, miltefosine, perifosine, or edelfosine in CFU-GM assays. Human MDA-MB-231 breast carcinoma, Panc-1 pancreatic carcinoma, and RPMI8226 multiple myeloma cells were exposed to erufosine in colony formation assays. Colony formation of Panc-1 tumor cells and mouse bone marrow cells ex vivo were quantified following intravenous administration of erufosine to tumor-bearing mice. Western blotting methods were applied to human U87 glioblastoma cells exposed to erufosine to investigate Akt inhibition.ResultsErufosine was less toxic to human and mouse bone marrow cells than perifosine, miltefosine, and edelfosine and was equally toxic to human and mouse CFU-GM. The human cancer cells MDA-MB-231 breast, Panc-1 pancreatic, and RPMI8226 MM cells were more sensitive to erufosine in a colony formation assay than were human bone marrow cells generating an approximately tenfold differential in IC90 values. Erufosine injected intravenously significantly reduced Panc-1 tumor cell colony formation ex vivo but not mouse bone marrow CFU-GM. Erufosine inhibited Akt phosphorylation in human U87 glioblastoma cells.ConclusionsErufosine offers potential as a novel therapeutic for cancer with a reduced toxicity profile to bone marrow cells compared with other agents in this class. Human cancer cells were more sensitive to erufosine than human or mouse bone marrow cells indicating a favorable therapeutic window for erufosine.

Characteristics of human Ewing/PNET sarcoma models.

Ewing/PNET (peripheral neuroepithelioma) tumors are rare aggressive bone sarcomas occurring in young people. Rare-disease clinical trials can require global collaborations and many years. In vivo models that as accurately as possible reflect the clinical disease are helpful in selecting therapeutics with the most promise of positive clinical impact. Human Ewing/PNET sarcoma cell lines developed over the past 45 years are described. Several of these have undergone genetic analysis and have been confirmed to be those of Ewing/PNET sarcoma. The A673 Ewing sarcoma line has proven to be particularly useful in understanding the biology of this disease in the mouse. The chromosomal translocation producing the EWS/FLI1 fusion transcript characterizes clinical Ewing sarcoma. Cell lines that express this genetic profile are confirmed to be those of Ewing sarcoma. The A673 Ewing sarcoma line grows in culture and as a xenograft in immunodeficient mice. The A673 model has been used to study Ewing sarcoma angiogenesis and response to antiangiogenic agents. Many Ewing sarcoma clinical specimens express the cell surface protein endosialin. Several Ewing sarcoma cell lines, including the A673 line, also express cell surface endosialin when grown as subcutaneous tumor nodules and as disseminated disease; thus the A673 is a useful model for the study of endosialin biology and endosialin-directed therapies. With the advent of tools that allow characterization of clinical disease to facilitate optimal treatment, it becomes imperative, especially for rare tumors, to develop preclinical models reflecting disease subsets. Ewing PNET sarcomas are a rare disease where models are available.

Endosialin: A novel malignant cell therapeutic target for neuroblastoma

Endosialin emerged recently as a potential therapeutic target for sarcoma. Since some sarcoma subtypes, such as Ewings sarcoma, show characteristics of neuroendocrine differentiation, we wondered whether cancers with neuro-endocrine properties and/or neuroectodermal origin, such as neuroblastoma, small cell lung cancer and melanoma, may express endosialin. Endosialin protein expression was surveyed in neuroblastoma, small cell lung cancer and melanoma in human clinical specimens by immunohistochemistry (IHC) and in human cell lines by flow cytometry. Side population cells were examined to determine whether cancer stem cells can express endosialin. Endosialin-expressing neuroblastoma cell lines were implanted in immunodeficient mice and allowed to grow. The xenograft tumors were resected and tested for endosialin expression by IHC. In human clinical specimens, vascular endosialin staining was observed in neuroblastoma, small cell lung cancer and melanoma. Malignant cell staining was strongest in neuroblastoma, weak in melanoma and rare in small cell lung cancer. In human cell lines, endosialin was detected in neuroblastoma cell lines, including cancer stem cell-like side population (SP) cells, but was absent in melanoma and was both rare and weak in small cell lung cancer. Human neuroblastoma xenograft tumors were found to be positive for endosialin. Our work suggests that endosialin may be a suitable therapeutic target for neuroblastoma.

Human choriocarcinomas: Placental growth factor-dependent preclinical tumor models

Choriocarcinomas are a rare form of cancer that develops in the uterus from tissue which would normally become the placenta. Choriocarcinomas are a trophoblastic gestational disease and have been studied largely to investigate conditions related to pregnancy such as preeclampsia. Choriocarcinomas are highly angiogenic and produce high levels of placental growth factor (PlGF) to promote the development of blood vessels. Upregulation of PlGF expression also occurs during the development of other human malignancies such as breast cancer and melanoma. Both tumor specimens and plasma samples have higher levels of PlGF than normal tissues. Hence, PlGF has emerged as a valid target for anti-angiogenic therapy. The cell lines BeWo, JAR and JEG-3, derived from human choriocarcinomas, were investigated in vitro and in vivo for suitability as PlGF-dependent models. BeWo, JAR and JEG-3 cells were characterized in culture and were implanted into immunodeficient mice to generate subcutaneous tumors. The PlGF and VEGF angiogenic profiles of the choriocarcinoma cells and tumors were investigated by ELISA and by immunohistochemical methods. Double immunofluorescence methods were applied to choriocarcinoma xenograft sections to characterize the cellular components of the blood vessels. sFLT01, a fusion protein that neutralizes PlGF, was assessed in cell culture experiments and xenograft studies. The novel results presented here validate the importance of human choriocarcinoma cell lines and xenografts in further exploring the role of PlGF in tumor angiogenesis, for evaluating PlGF as an anti-angiogenic target, and for developing therapies that may provide clinical benefit.

Abstract 1388: sFLT01: An anti-angiogenic fusion protein that neutralizes placental growth factor (PlGF) and vascular endothelial growth factor (VEGF)

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: PlGF and VEGF stimulate angiogenesis and promote the growth of tumor vasculature. PlGF is a member of the VEGF family and binds to VEGFR1. sFLT01 is a novel fusion protein comprised of the Fc portion of human IgG1 and the PlGF- and VEGF-binding domain of VEGFR1/Flt-1. The properties of sFLT01 and the potential of sFLT01 as an anti-angiogenic agent to inhibit tumor growth were investigated in several in vitro assays and in multiple xenograft tumor models. Methods: The binding kinetics of sFLT01 for both the human and murine homologues of PlGF and VEGF were assessed by Biacore. The abilities of recombinant human PlGF and VEGF to induce endothelial cell and pericyte proliferation and of sFLT01 to inhibit this stimulation were investigated in cell-based assays. The secretion of human VEGF and PlGF in culture by the HT29 colon carcinoma, H460 lung carcinoma, and A673 sarcoma human cell lines was quantified by ELISA. In efficacy studies, sFLT01 was administered by intraperitoneal injection twice per week to immunodeficient mice bearing HT29, H460, or A673 subcutaneous tumors. Antibodies specific for human IgG and VEGR2 were applied to A673 sarcoma tumor sections from mice treated with sFLT01 to visualize sFLT01 in the tumors and determine VEGFR2 expression in the cellular components. Pericytes and endothelial cells were identified with antibodies against NG2 and CD31. Results: The Biacore results indicated that sFLT01 has high affinity for human and murine PlGF and VEGF. Human recombinant PlGF and VEGF each induced the proliferation of human pericytes and endothelial cells in culture. This stimulation was inhibited by sFLT01. A673 sarcoma, HT29 colon and H460 lung carcinoma cells secreted higher levels of VEGF than PlGF in culture. In vivo, 10 mg/kg sFLT01 was effective at significantly slowing the growth of HT29 colon carcinoma and A673 sarcoma tumors compared to controls. Further analysis of the A673 sarcoma tumors in sFLT01-treated mice by immunohistochemistry revealed that sFLT01 penetrated multiple areas of the tumor. sFLT01 was detected in the vasculature, stroma, necrotic areas, and adjacent to malignant cells. sFLT01 treatment in the A673 model disrupted vessel integrity with a lack of association between endothelial cells and pericytes. A673 sarcoma cells expressed VEGFR2 in vivo. Conclusion: sFLT01 neutralizes the angiogenic activity of multiple vasculogenic VEGF family members in vitro and inhibited the proliferation of cells that form blood vessels, endothelial cells and pericytes. In vivo, sFLT01 treatment resulted in disorganized tumor vasculature thereby slowing the growth of xenografts tumors. The expression of VEGFR2 in A673 sarcoma tumors suggests that VEGF may play a role in autocrine signaling in some malignant cells. sFLT01 has antitumor and antiangiogenic activity in several human tumor xenografts and may offer therapeutic benefit. 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 1388.

Abstract 5251: Endosialin: A novel cell surface therapeutic target for early-stage and late-stage neuroblastoma

Hypothesis: Endosialin emerged recently as a potential marker and therapeutic target for adult and pediatric sarcoma. Given evidence of a possible common progenitor cell for mesenchymal and neural cell lineages, we wondered whether expression of endosialin may be shared by sarcomas and neuroblastomas, which are cancers of mesenchymal origin and neural crest origin, respectively. Methods: Endosialin protein expression was studied in live human neuroblastoma cell lines by flow cytometry using a fully human monoclonal antibody against human endosialin. Endosialin-positive human neuroblastoma cells were subsequently implanted at different anatomic sites in nu/nu mice and allowed to grow. Tumors were collected, formalin fixed and subjected to immunohistochemistry using a fully human monoclonal antibody against human endosialin. Using the same IHC assay, endosialin protein expression was also studied in formalin-fixed paraffin-embedded human clinical specimens of neuroblastoma. Results: We tested 10 human neuroblastoma cell lines for endosialin protein expression by flow cytometry and found that 9/10 expressed endosialin. Several of the positive cell lines were derived from bone marrow metastases, suggesting that endosialin expression is maintained in advanced disease. We modeled endosialin-positive neuroblastoma in vivo by implanting SK-N-AS cells in mice subcutaneously and in the subrenal capsule, kidney being a site where neuroblastoma sometimes originates. Immunohistochemical analysis revealed that SK-N-AS cells, which are positive for endosialin in vitro and derived from the bone marrow metastasis of an adrenal primary tumor, formed endosialin-positive subcutaneous tumors and endosialin-positive renal tumors, demonstrating that endosialin expression is supported by different microenvironments in various anatomic locations. Immunohistochemistry of human clinical specimens of neuroblastoma showed expression of endosialin. All specimens of neuroblastoma were bone marrow metastases, demonstrating that endosialin is expressed in advanced disseminated neuroblastoma. Conclusions: Our work demonstrates that endosialin expression is shared by sarcomas and neuroblastomas. The expression of endosialin in neuroblastoma potentially opens up a new therapeutic horizon for neuroblastoma patients, including those suffering from advanced disease. 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 5251.