Robert Smale

Folate receptor alpha (FRA) expression in breast cancer: identification of a new molecular subtype and association with triple negative disease

Given that several targeted therapies directed towards folate receptor alpha (FRA) are in late stage clinical development, the sensitive and robust detection of FRA in tissues is of paramount importance relative to patient selection, prognosis and prediction. In the present study we undertook an immunohistochemical evaluation of expression of FRA in breast cancer samples using formalin-fixed, paraffin-embedded (FFPE) tissues, primarily invasive ductal carcinomas, using a newly described monoclonal antibody, 26B3. Samples assessed included both tissue microarrays (TMA) and whole tissue sections from archival tissue blocks. Normal breast shows a highly restricted expression of FRA to luminal membrane staining of secretory ductal cells, consistent with FRA secretion into milk. In early stage (stages I-III) invasive ductal carcinomas, FRA staining was observed in approximately 30% of all samples, independent of molecular subtype (estrogen receptor (ER)/progesterone receptor (PR)/human epidermal growth factor receptor type 2 (Her2)). However, FRA expression was shown to associate with ER/PR negative tumors relative to ER/PR positive tumors (p = 0.012) and perhaps more importantly, with triple negative breast cancers (TNBC; p < 0.0001). FRA immunoreactivity was also shown to be retained in stage IV metastatic breast cancer samples from diverse anatomic sites including lymph node and bone. In metastatic breast cancer, FRA was shown to be expressed in 86% of TNBC patients. Taken together, these data suggest that FRA expressing breast cancer represents a novel molecular subtype and, further, may represent a new therapeutic target for this devastating disease.

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.

Endosialin is expressed in high grade and advanced sarcomas: Evidence from clinical specimens and preclinical modeling

We previously surveyed the expression of endosialin/ CD248/TEM-1 by immunohistochemistry in human clinical specimens of sarcomas and documented expression in tumor cells, stromal cells and vasculature. In the present study, we completed a retrospective analysis of the diagnostic reports available for these same samples in order to identify high-grade and metastatic disease. Our results show that endosialin can be detected in advanced disease. We screened human sarcoma cell lines in vitro for endosialin expression and developed preclinical human xenograft models of disseminated sarcoma. We found that 22 out of 42 human sarcoma cell lines were positive for endosialin with a positive correlation between mRNA and protein levels. When implanted in vivo, endosialin was expressed at all sites of dissemination. These data provide clinical and preclinical evidence that endosialin can be detected in advanced sarcoma. These results demonstrate for the first time that endosialin is a suitable therapeutic target for poor prognosis and advanced disease.

Tumor endothelial marker 7 (TEM-7): A novel target for antiangiogenic therapy

Antiangiogenesis has been validated as a therapeutic strategy to treat cancer, however, a need remains to identify new targets and therapies for specific diseases and to improve clinical benefit from antiangiogenic agents. Tumor endothelial marker 7 (TEM-7) was investigated as a possible target for therapeutic antiangiogenic intervention in cancer. TEM-7 expression was assessed by in situ hybridization or by immunohistochemistry (IHC) in 130 formalin-fixed paraffin-embedded (FFPE) and 410 frozen human clinical specimens of cancer plus 301 normal tissue samples. In vitro TEM-7 expression was evaluated in 4 human endothelial cell models and in 32 human cancer cell lines by RT-PCR and flow cytometry. An anti-TEM-7 antibody was tested in vitro on human SKOV3 ovarian and MDA-MB-231 breast carcinoma cells that expressed TEM-7 in antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis assays. In frozen tumor tissues, TEM-7 mRNA and protein was detected in all but one of the cancer types tested and was infrequently expressed in normal frozen tissues. In FFPE tumor tissues, TEM-7 protein was detected by IHC in colon, breast, lung, bladder, ovarian and endometrial cancers and in sarcomas. TEM-7 protein was not detected in head and neck, prostate or liver cancers. TEM-7 expression was restricted to the vasculature and was absent from tumor cells. In vitro, TEM-7 was not detected in human microvascular endothelial cells (HMVEC) or human umbilical vein endothelial cells (HUVEC) but was induced in endothelial precursor/progenitor cells (EPC) in the presence of the mitogen phorbol ester PMA. An anti-TEM-7 antibody mediated ADCC and phagocytosis in SKOV3 and MDA-MB-231 cell lines infected with an adenovirus expressing TEM-7. These data demonstrate that TEM-7 is a vascular protein associated with angiogenic states. TEM-7 is a novel and attractive target for antiangiogenic therapy.

Anti-Endosialin Antibody–Drug Conjugate: Potential in Sarcoma and Other Malignancies

Endosialin/TEM1/CD248 is a cell surface protein expressed at high levels by the malignant cells of about 50% of sarcomas and neuroblastomas. The antibody–drug conjugate (ADC) anti-endosialin-MC-VC-PABC-MMAE was selectively cytotoxic to endosialin-positive cells in vitro and achieved profound and durable antitumor efficacy in preclinical human tumor xenograft models of endosialin-positive disease. MC-VC-PABC-MMAE was conjugated with anti-endosialin with 3–4 MMAE molecules per ADC. The anti-endosialin-MC-VC-PABC-MMAE conjugate was tested for activity in four human cell lines with varied endosialin levels. The HT-1080 fibrosarcoma cells do not express endosialin, A-673 Ewing sarcoma cells and SK-N-AS neuroblastoma cells are moderate expressers of endosialin, and SJSA-1 osteosarcoma cells express very high levels of endosialin. To determine whether endosialin expression was maintained in vivo, A-673 Ewing sarcoma, SK-N-AS neuroblastoma, and SJSA-1 osteosarcoma cells were grown as xenograft tumors in nude mice. The SK-N-AS neuroblastoma and the A-673 Ewing sarcoma lines were selected for in vivo efficacy testing of the anti-endosialin-MC-VC-PABC-MMAE conjugate. The treatment groups included a vehicle control, unconjugated anti-endosialin, an admix control consisting of anti-endosialin and a dose of free MMAE equivalent to the dose administered as the ADC, and the anti-endosialin-MC-VC-PABC-MMAE conjugate. The unconjugated anti-endosialin had no antitumor activity and resulted in similar tumor growth as the vehicle control. The admix control produced a modest tumor growth delay. Administration of the anti-endosialin-MC-VC-PABC-MMAE conjugate resulted in a marked prolonged tumor response of both xenograts. These proof-of-concept results break new ground and open a promising drug discovery approach to these rare and neglected tumors. Mol Cancer Ther; 14(9); 2081–9. ©2015 AACR.

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.

Abstract C80: Characterizing models for personalized medicine: Endosialin in sarcoma from cultured cells to mice to humans

A survey of endosialin by IHC was previously conducted in 250 clinical specimens of cancer, revealing a key difference between sarcomas and carcinomas: in sarcomas, endosialin was expressed in tumor cells, stromal fibroblasts and perivascular cells, while in carcinomas tumor cells were negative. Endosialin expression was frequent among sarcoma patients and reached high levels: 70 of 86 sarcomas (81%) were positive for endosialin, with 44 (51%) displaying at least 50% coverage of all 3 immunoreactive cell types as a whole. Staining intensity was scored on the scale 0, 1+, 2+, 3+: all 9 sarcoma subtypes surveyed included specimens reaching 2+ and 3+. Endosialin emerged from that study as a potential molecular target for sarcomas. However, despite frequent expression and high levels observed in patients, some sarcomas are endosialin‐negative and some express low levels. Therefore, targeting endosialin would mandate a personalized medicine approach where patients are tested for endosialin expression in order to determine eligibility for endosialin‐directed therapy. We characterized the expression of endosialin in human tumor cell lines in vitro and in vivo, from mRNA and protein in cell culture to protein in xenograft tumors. Thirty‐seven human sarcoma cell lines and one neuroblastoma cell line were assayed for endosialin by real‐time PCR and flow cytometry. Eighteen cell lines were positive for the protein at various levels. There was a positive correlation between transcript and protein expression, with a few exceptions: 3 cell lines expressed very low levels of the transcript yet clearly detectable protein and one cell line expressed detectable transcript yet no protein. Fifteen of the 18 cell lines positive for the protein in vitro were implanted in mice subcutaneously. Eight of the 15 cell lines implanted failed to form tumors. Seven formed tumor nodules that were excised and tested for endosialin by IHC using the same assay as that used for clinical specimens. Protein expression was not always as robust in vivo as the in vitro expression might have suggested. Staining in the xenograft tumors was scored on the same scale as the clinical specimens. Some models showed robust and homogeneous expression throughout the tumor tissue (SK‐N‐AS neuroblastoma), while others showed highly heterogeneous staining. In one model representing extreme heterogeneity (SJSA‐1 osteosarcoma), endosialin was highly regional, with areas staining positive at the 3+ intensity level and adjacent areas being negative. Both homogeneous and heterogeneous staining was seen in human clinical specimens. Our work demonstrates the importance of characterizing disease models to ascertain target expression at all stages of testing from in vitro to mice and to humans, since expression in vivo cannot be assumed from expression in cell culture and expression patterns in mice may or may not be similar to expression patterns in humans. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C80.

Abstract 2516: Immunohistochemical characterization of a novel, highly sensitive monoclonal antibody to folate receptor α

Background: Folate receptor ≤ (FRα) has restricted expression in normal tissues but high-level expression in a subset of epithelial-derived tumors, including non-mucinous ovarian carcinomas and non-small cell lung (NSCL) adenocarcinomas. FRα has shown significant potential for targeted cancer therapy and several promising agents are in late-stage clinical development. As such, robust reagents are needed that can be used across diverse diagnostic platforms to identify patients who have FRα-expressing tumors. The immunohistochemical (IHC) characterization of a novel, high-affinity monoclonal antibody (MAb) against FRα is described. Methods: MAbs were generated to modified full-length FRα protein using standard methods. One MAb, 26B3.F2, displayed high affinity and the ability to bind FRα on both fresh/frozen and formalin-fixed, paraffin-embedded tissue sections. An optimized IHC protocol was developed and FRα expression was assessed on commercial tissue microarrays (US Biomax). Data were analyzed using an M-score, a newly introduced, weighted-intensity score that incorporates both the proportion of FRα-positive cells and the staining intensity. A board-certified pathologist scored the level of FRα expression. Results: Normal tissue staining for MAb 26B3.F2 demonstrated a limited distribution on focal epithelial surfaces of pancreas, lung, salivary gland, kidney, hypophysis, thyroid, and breast samples. Staining was mainly located in the lumen. The tissue distribution is consistent with the published literature. Strong staining was observed in 100% of serous ovarian samples and a significant proportion of ovarian endometrioid adenocarcinoma samples. FRα expression, as determined by MAb 26B3.F2 in NSCL cancer samples, demonstrated high discrimination between adenocarcinoma (positive) and squamous cell carcinomas (negative) (P 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 2516. doi:1538-7445.AM2012-2516

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.