Search

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 Ewing's 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.

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

Context A histologic classification of lung cancer subtypes is essential in guiding therapeutic management. Objective To complement morphology-based classification of lung tumors, a previously developed lung subtyping panel (LSP) of 57 genes was tested using multiple public fresh-frozen gene-expression data sets and a prospectively collected set of formalin-fixed, paraffin-embedded lung tumor samples. Design The LSP gene-expression signature was evaluated in multiple lung cancer gene-expression data sets totaling 2177 patients collected from 4 platforms: Illumina RNAseq (San Diego, California), Agilent (Santa Clara, California) and Affymetrix (Santa Clara) microarrays, and quantitative reverse transcription–polymerase chain reaction. Gene centroids were calculated for each of 3 genomic-defined subtypes: adenocarcinoma, squamous cell carcinoma, and neuroendocrine, the latter of which encompassed both small cell carcinoma and carcinoid. Classification by LSP into 3 subtypes was evaluated in both fresh-frozen and formalin-fixed, paraffin-embedded tumor samples, and agreement with the original morphology-based diagnosis was determined. Results The LSP-based classifications demonstrated overall agreement with the original clinical diagnosis ranging from 78% (251 of 322) to 91% (492 of 538 and 869 of 951) in the fresh-frozen public data sets and 84% (65 of 77) in the formalin-fixed, paraffin-embedded data set. The LSP performance was independent of tissue-preservation method and gene-expression platform. Secondary, blinded pathology review of formalin-fixed, paraffin-embedded samples demonstrated concordance of 82% (63 of 77) with the original morphology diagnosis. Conclusions The LSP gene-expression signature is a reproducible and objective method for classifying lung tumors and demonstrates good concordance with morphology-based classification across multiple data sets. The LSP panel can supplement morphologic assessment of lung cancers, particularly when classification by standard methods is challenging.

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 Ewing's 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/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.

HER2 gene amplification and receptor overexpression by tumors seems to be associated with poorer prognosis and may be predictive of response to certain anticancer therapies. Furthermore, and paramount to the clinician and patient, a positive HER2 status is a prerequisite eligibility requirement for Herceptin® (trastuzumab) therapy in women with metastatic breast cancer. As a consequence, issues relating to accurate and reliable laboratory assessment of HER2 status are a matter of significant debate to pathologists and oncologists. Out of a wide range of techniques that have been used in research for the detection of HER2 status, two technologies are now predominant in the routine clinical pathology laboratory: determination of HER2 protein overexpression by immunohistochemistry (IHC) and HER2 gene amplification by fluorescence in-situ hybridization (FISH). This article discusses some of the recent experiences, guidelines, and opinions of pathologists and clinicians concerning aspects of HER2 testing with respect to when to test (at initial diagnosis or pretreatment), the relative advantages/disadvantages of IHC and FISH, and where to test (local or centralized laboratories).

Three different PD-L1 immunohistochemical (IHC) assays have recently been approved to help guide treatment decisions regarding anti-PD-1 (Keytruda®, Opdivo®) and anti-PD-L1 (Tecentriq®) immuno-oncology based therapies. The three assays differ in the primary monoclonal antibodies used, the assay platform (Dako, Ventana), scoring and interpretation criteria, and intended use. Our clinical laboratories currently offer all 3 assays (pharmDx™ 22C3, pharmDx™ 28-8, and SP142) because they are associated with different intended uses, companion and complementary diagnostics for non-small-cell lung cancer (NSCLC), and complementary diagnostic for urothelial carcinomas (UC). In the NSCLC samples evaluated in the first year since the approval of the two pharmDx™ assays, we have noted a higher percentage of positive samples with the pharmDx™ 28-8 assay (50%) compared to the pharmDx™ 22C3 assay (30%). This difference is mainly due to the use of different assay cutoffs associated with the two assays, as there is good correlation between the overall staining patterns, including the distribution of the percentage of tumor cells exhibiting positive staining. For the SP142 assay tested on UC samples, 22% of the cases were considered positive. All 3 assays are robust, with a limited number of samples considered inconclusive ( Citation Format: Steven M. Anderson, Bruce Horten, Bryan McCune, Ronald Paler. Clinical performance and utilization of 3 PD-L1 immunohistochemical assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 663. doi:10.1158/1538-7445.AM2017-663

RET rearrangement represents a unique molecular subset of lung cancer. The identification of specific clinicopathologic characteristics and RET gene status would provide critical information on targeted therapeutics. In this study, we investigated the patterns of RET gene in a series of lung carcinomas. Of one hundred and sixteen tumors, a low frequency (1.7%) of RET translocation was identified. Only two specimens of lung adenocarcinomas displayed the rearrangement of RET in 54% and 78% of tumor cells respectively. A high incidence of gain of copy number (3-4 copies) and amplification (≥ 5 copies) of the RET gene was observed in 52% and 12% of all 116 samples. An association between increased copy number of RET and EGFR mutation was statistically significant (p < 0.05) in these lung carcinomas. This study sheds light on the unique molecular characteristics of the RET gene in lung carcinomas.

11612Background: Recently two different immunohistochemical assays for PD-L1 expression in non-small cell lung cancers have been approved in conjunction with immune checkpoint inhibitors. One assay...

Disseminated Acanthamoeba infections and bacillary angiomatosis are among the un usual opportunistic infections encountered in patients with AIDS. We report a fatal case of disseminated Acanthamoeba palestinensis infection involving the skin, palate, brain, lungs, and testes in a patient with AIDS in whom the mucocutaneous lesions were initially interpreted clinically and pathologically as bacillary angiomatosis. Int J Surg Pathol 2(1):11-16, 1994

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.

Pancreatic cancer is characterized by aggressive growth and resistance to treatment. Identification of unique biomarkers for diagnosis and prognosis is important for treatment of this disease. We investigated the expression patterns of mucin 1 (MUC1), mucin 2 (MUC2) and cytokeratin 17 (CK17) in both normal tissues and metastatic adenocarcinomas using immunohistochemistry (IHC). We have shown that MUC1 (pan-epithelial membrane mucin), MUC2 (intestinal-type secretory mucin) and CK17 can be used as a panel of markers to distinguish collectively pancreatobiliary carcinoma from other primary site carcinomas. Tumors originating in the pancreatobiliary system showed an expression pattern of MUC1 (+), MUC2 (-) and CK17 (+). By contrast, tumors arising from the colorectal region were MUC1 (-), MUC2 (+) and CK17 (-), while tumors originating from non-pancreatobiliary system tissue expressed a MUC1 (+), MUC2 (-) and CK17 (-) profile. More importantly, the MUC1 (+), MUC2 (-) and CK17 (+) result showed greater sensitivity than CA19-9 by IHC, which is the currently accepted and widely used pancreatic tumor marker for diagnosing pancreatic cancer. Thirteen of 51 cases (25%) of pancreatobiliary adenocarcinomas with the pattern MUC1 (+), MUC2 (-) and CK17 (+) showed no immunoreactivity for CA19-9, while 34/51 (67%) cases having MUC1 (+), MUC2 (-) and CK17 (+) were correlated with positive CA19-9 staining. Our data support using an antibody panel of MUC1, MUC2 and CK17 to enhance current methods for pancreatic cancer diagnosis by identifying specifically the primary tissue of origin.

Endosialin emerged recently as a potential therapeutic target for sarcoma. Since some sarcoma subtypes, such as Ewing's 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.

Pancreatic cancer is characterized by aggressive growth and treatment resistance. Identification of unique biomarkers for diagnosis and prognosis is important in the treatment of this disease. In this study, we investigated the expression patterns of MUC1, MUC2 and CK17 in normal tissues as well as metastatic adenocarcinomas by immunohistochemical analysis. Our results clearly show that MUC1 (pan-epithelial membrane mucin), MUC2 (intestinal-type secretory mucin) and CK17 can be utilized as a panel of markers to collectively distinguish pancreatobiliary carcinoma from other primary site carcinomas. That is, tumors originating in the pancreatobiliary system showed an expression pattern of MUC1 (+), MUC2 (-) and CK17 (+). In contrast, tumors that arose from the colorectal region were MUC1 (-), MUC2 (+) and CK17 (-), while those originating from the lung and kidney had a MUC1 (+), MUC2 (-) and CK17 (-) expression profile. More importantly, the MUC1 (+), MUC2 (-) and CK17 (+) result displayed greater sensitivity and specificity than CA19-9, which is the currently accepted and widely used pancreatic tumor marker for the diagnosis of pancreatic cancer. Taken together, our data support an antibody panel that can enhance the current methods for pancreatic cancer diagnosis by specifically identifying the primary tissue of origin from which pancreatic carcinomas are derived. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 LB-274.

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.

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.

Two patients with cancer, one with Hodgkin's disease and the other with a granulosa cell tumor of the ovary, developed a progressive, eventually fatal infection of the central nervous system exhibiting multifocal symptoms and signs. Pathologically, gross abnormalities of the brain resembled those in progressive multifocal leukoencephalopathy (PML), with discrete and confluent plaque-like lesions concentrated in the white matter, particularly along the gray-white junction. Microscopically, pathological changes differed distinctly from those associated with PML; in addition to confluent foci of white matter injury characterized by early demyelination and subsequent necrosis, prominent Cowdry type A eosinophilic intranuclear inclusions were noted in oligodendrocytes, astrocytes, and neurons. By electron microscopy, intranuclear spherical particles consistent in size and appearance with herpesvirus nucleocapsids were found within the lesions. Immunoperoxidase studies detected varicella-zoster virus (VZV) antigens in infected cells, implicating this virus as the responsible agent despite a lapse of many months between the cutaneous herpes zoster and onset of cerebral symptoms in both patients.

Four children with medulloblastoma had massive supratentorial recurrences in the region of the cribriform plate after adequate craniospinal irradiation. The pathogenesis of these recurrences is probably related to underdosage to this region by shielding of the eyes. This hypothesis was corroborated by autopsy findings in two other patients in whom subfrontal implants were histologically different from recurrences elsewhere. Two possible solutions to avoid this problem in the future are suggested.

A patient with Hodgkin's disease developed progressive multifocal leukoencephalopathy (PML), documented by brain biopsy to be associated with JC virus infection. His disease progressed over several months, resulting in severe neurological deficit, but then stabilized with little or no further clinical progression during the remaining year of his life. Histopathological evaluation of the brain at autopsy supported the clinical impression that brain infection was arrested. Whereas the brain biopsy exhibited the histological features of active PML including giant bizarre astrocytes, at postmortem examination brain lesions appeared inactive, with regression of astrocytic changes and elimination of oligodendroglial inclusions. Similarly, JC virus antigen, present in the brain biopsy, was not detected in the autopsied brain. This case provides further evidence that PML is not invariably fatal and that clinical and cytological remission can occur.

Although numerous "cures# have been reported following surgical extirpation of symptomatic foci of cerebral radiation nerosis, delayed progressive white matter destruction and neurological deterioration may occur in some patients who survive for prolonged periods after operation. The postoperative appearance on CT scans of hypodensity within heavily irradiated white matter structures at a distance from the initial radionecrotic focus or operative site suggests continuing radiation-induced tissue injury and a poor prognosis. Anticipated survival as well as administered radiation dose must be taken into account when "safe# radiation thresholds are calculated.

Sixteen of 21 human malignant glial tumors were successfully heterotransplanted into the brains of nude mice, and one other was transplanted into the brain after prior subcutaneous heterotransplantation. Most xenografts grew preferentially as diffusely infiltrating tumors within hemispheric white matter, generally sparing cortex and deep gray matter. The heterogeneity of most in vivo human tumors gave way to a tumor of generally uniform cell type while growing in nude mice. From six human tumors, all glioblastomas, there emerged histologic patterns or cell forms that were not evident in the original tumor. Tumors from 15 patients were treated with standard chemotherapeutic agents while growing in nude mouse brains. The most common morphologic change induced in tumors by several agents was a distinctive giant cell change characterized by large bizarre nuclei and abundant cytoplasm. It is concluded that the human brain-tumor-nude-mouse xenograft model offers morphological parallels with the clinical situation, but selects for growth only some of the many subpopulations of the human tumor. Such selection imposes restriction on the clinical inferences that may be drawn from this model.