Expression of the coxsackievirus and adenovirus receptor in musculoskeletal tumors and mesenchymal tissues: efficacy of adenoviral gene therapy for osteosarcoma

Recombinant adenovirus is used as a competent vector in a wide spectrum of cancer gene therapies. Adenovirus infection depends on coxsackievirus and adenovirus receptor (CAR)-mediated virus attachment to the cell surface. However, the expression levels of CAR and the efficiency of adenoviral gene transduction in musculoskeletal tumors have not been systematically investigated. To study the feasibility of gene therapy in musculoskeletal tumors, the expression levels of CAR and the antiproliferative effect of an adenovirally transduced wild-type p53 tumor suppressor gene were examined in 15 distinct musculoskeletal tumor cell lines, 19 tumor tissue samples, and the corresponding pathologically unremarkable mesenchymal tissues. The expression levels of the CAR gene were significantly higher in six of seven osteosarcoma cell lines and two of five osteosarcoma tissue samples than in the other cell lines, musculoskeletal tumors, and mesenchymal tissues. CAR expression levels were closely correlated with adenoviral gene transduction efficiency and the antiproliferative effect of a transduced adenoviral p53 gene in the tested cell lines. In addition, an immunocytochemical study confirmed that transfected green fluorescent protein (GFP) borne by Ad-CAG-GFP was expressed at the cell surface of CAR-positive cells. These results indicate that CAR expression is a critical determinant of transduction efficiency in adenovirus-based gene therapy. Most osteosarcomas appeared to express high levels of CAR, and thus adenovirus-mediated p53 gene therapy is likely to be suitable for the treatment of such tumors. (Cancer Sci 2003; 94: 70–75)     

Estrogen sulfotransferase (SULT1E1) expression in benign and malignant human bone tumors

BACKGROUND AND OBJECTIVES: 17beta-estradiol regulates growth and differentiation in normal and malignant bone. E2 is inactivated to 17beta-estradiol-sulfate through estrogen sulfotransferase (SULT1E1). RESULTS: In an explorative study, SULT1E1 mRNA expression was assessed in a broad range of samples from benign, primary and secondary malignant bone tumors. We detected SULT1E1 mRNA in 31/50 tumor samples (10/19 malignant, 6/13 benign tumors; 15/18 metastases). Significantly more SULT1E1-positive samples were found in metastases than in primary bone tumors (P = 0.019). Yet, there was no difference between malignant and benign primary tumors (P = 0.718). SULT1E1 mRNA levels were not related to patients' age, gender, tumor location, stage, grading, and chemotherapy pretreatment. Relative SULT1E1 mRNA levels did not correlate with that of estrogen-receptor alpha (ERalpha) as assessed by quantitative TaqMan PCR (10 malignant, 8 benign tissue samples). In the latter, ERalpha mRNA, but not SULT1E1 mRNA levels were significantly lower than in the malignant samples (P = 0.006 and P = 0.71, respectively). Also, pronounced expression of SULT1E1 mRNA but not of ERalpha mRNA was observed in osteosarcoma (MG-63, HOS) and Ewing's sarcoma (TC-71) cells, while human osteoblasts and BMSC contained ERalpha but not SULT1E1 mRNA. CONCLUSION: Frequent expression of SULT1E1 mRNA in various human bone tumors suggests that sulfonation might be important to control E2 levels and activity.     

The role of radiation treatment in the contemporary management of bone tumors

Radiotherapy is integral in the multidisciplinary approach to patients with musculoskeletal neoplasms. Multiple studies have established a role for radiotherapy as a definitive local treatment of unresectable lesions or when surgery might yield unacceptable functional outcomes, such as in Ewing's tumor or base of skull chondrosarcoma. Radiotherapy is also used as an adjuvant treatment after surgery with close or positive margins. In the metastatic setting, external beam radiotherapy and bone-seeking intravenous radioisotopes are used on a case-by-case basis for palliation. As radiotherapy and its delivery techniques have evolved, so has its role in treating tumors such as Ewing's sarcoma, chordoma and chondrosarcoma, osteosarcoma, primary lymphoma of bone, malignant fibrous histiocytoma of bone, and vascular tumors. Radiation can also be successfully used to treat unresectable or recurrent benign tumors, such as giant cell tumor and aneurysmal bone cyst. This article reviews the indications for radiotherapy for various bone tumors and summarizes some of the important data supporting its use.     

Monoclonal antibody identification and characterization of two human sarcoma-associated antigens

Two murine monoclonal antibodies, 29-13 (IgG1) and 29-2 (IgG2a), generated against malignant fibrous histiocytoma plasma membranes immunoprecipitated a Mr 200,000 protein (p200), with an isoelectric point between 6.3 and 7.5. Two additional antibodies, 35-16 (IgG1) and 30-40 (IgG2a), generated against Ewing's sarcoma membranes, immunoprecipitated an acidic protein of Mr 160,000 (p160), with an isoelectric point between 5.8 and 6.7. Monoclonal antibodies 29-13 and 29-2 recognize a similar determinant(s) on p200 while 35-16 and 30-40 recognize different determinants on p160. Monoclonal antibody 29-13 exhibited significant binding to membranes isolated from fibrosarcoma and aggressive fibromatosis; moderate binding to osteosarcoma, hemangiopericytoma, and malignant fibrous histiocytoma; and minimal to no binding to other soft tissue sarcoma plasma membranes. The p200 protein was not expressed in 16 other malignant tumors and in only 3 of 35 normal human tissue specimens. High levels of p200 were selectively expressed by leiomyosarcoma, Ewing's sarcoma, and fibrosarcoma cells as well as neonatal fibroblasts in vitro, but not by other carcinoma cell lines or B-lymphoblasts. The p160 protein appeared to be selectively expressed by Ewing's sarcoma with little or no expression on other sarcomas, carcinomas, or normal tissues. However, the p160 antigen was expressed in Ewing's sarcoma, leiomyosarcoma, melanoma, 4 of 9 carcinomas, and neonatal fibroblasts in vitro. The affinity of MoAbs 29-13, 29-2, 35-16, and 30-40 ranged from 5.3 x 10(8) to 4.7 x 10(9) M-1 for sarcoma membranes with approximately 5 x 10(4) binding sites/sarcoma cell.     

Expression of the coxsackie and adenovirus receptor in human astrocytic tumors and xenografts

The sensitivity of human tissues and tumors to infection with type C adenoviruses correlates with the expression of the human coxsackie B- and adenovirus receptor, hCAR. HCAR is heterogeneously expressed in various tissues and types of human cancer cells, which has implications for the use of adenoviruses as vectors in cancer gene therapy. Using immunoblotting, real-time PCR, FACS-analysis and sensitivity to infection with adenovirus-lacZ, we analyzed the expression level of hCAR in glioma Grade IV cell lines. With real-time PCR, we also analyzed hCAR expression in primary human astrocytomas of different malignancy grades, as well as in their xenograft derivatives. Analysis of a set of 10 cell lines showed great variation in hCAR expression. Susceptibility to Ad5lacZ correlated well with hCAR expression, whereas no correlation was observed with the expression of alphavbeta3/alphavbeta5 integrins, proposed to function as co-receptors for adenoviruses. A great variation of CAR expression was also observed in primary astrocytomas of different malignancy grades. The mean value of CAR expression was significantly lower in 22 Grade IV tumors as compared to the values for 6 Grade II (p = 0.01) and 6 Grade III (p = 0.01) tumors. When the hCAR expression in 11 xenografts derived from Grade IV gliomas were compared to the levels detected in the original parental tumors, a mean 12-fold higher expression was seen in the xenografts (P = 0.01). Two xenografts with low hCAR expression grew considerably faster than the hCAR-expressing cells. Our results have relevance for the use of adenoviruses in gene therapy against astrocytomas.     

Expression of the coxsackievirus- and adenovirus receptor in gastrointestinal cancer correlates with tumor differentiation

Modified adenoviruses represent a new approach to treatment of gastrointestinal cancer. However, their uptake by cells in many cases requires the major receptor for adenoviruses, the coxsackievirus and adenovirus receptor (CAR). Thus, lack of CAR expression is a potential cause of intrinsic resistance of tumor cells to this type of treatment. To evaluate this, we studied the localization of CAR protein in normal and malignant gastrointestinal tissues. In normal tissues, CAR was concentrated at sites of cell-cell interaction, in particular at the apico-lateral cellular surface. Expression was particularly strong around bile and pancreatic ducts, which is in agreement with CAR's physiological function as a tight-junction protein. In GI malignancies (esophageal, pancreatic, colorectal and liver cancer), expression of the receptor varied substantially. Loss of CAR expression at cell-cell junction was evident in many samples. A significant correlation between CAR expression and histological grade was found, with moderately to poorly differentiated tumors most frequently demonstrating loss or reduction of CAR expression. These data indicate that CAR expression is frequently altered in gastrointestinal malignancy, potentially reducing the efficacy of adenovirus-based therapies.     

Enhanced antitumor efficacy of fiber‐modified,midkine promoter‐regulated oncolytic adenovirus in human malignant mesothelioma

Oncolytic virotherapy using adenoviruses has potential for therapeutic benefits in malignant mesothelioma. However, the downregulation of coxsackie virus/adenovirus receptor (CAR) expression is frequently a critical rate‐limiting factor that impedes the effectiveness of adenovirus serotype 5 (Ad5)‐based vectors in many cancer types. We evaluated CAR (Ad5 receptor) and CD46 (adenovirus serotype 35 [Ad35] receptor) expression in six human malignant mesothelioma cell lines. Very low CAR expression was observed in MSTO‐211H and NCI‐H2052 cells, whereas the other cell lines showed strong expression. In contrast, CD46 was highly expressed in all mesothelioma cell lines. On this basis, we replaced the CAR binding sequence of Ad5 with the CD46 binding sequence of Ad35 in the replication‐defective adenoviruses and the tumor‐specific midkine promoter‐regulated oncolytic adenoviruses. By this fiber modification, the infectivity, virus progeny production, and in vitro cytocidal effects of the adenoviruses were significantly enhanced in low CAR‐expressing MSTO‐211H and NCI‐H2052 cells, also resulting in similar or even higher levels in high CAR‐expressing mesothelioma cell lines. In MSTO‐211H xenograft models, the fiber‐modified oncolytic adenovirus significantly enhanced antitumor effect compared to its equivalent Ad5‐based vector. Our data demonstrate that Ad35 fiber modification of binding tropism in a midkine promoter‐regulated oncolytic Ad5 vector confers transductional targeting to oncolytic adenoviruses, thereby facilitating more effective treatment of malignant mesothelioma.     

Preoperative adjuvant therapy for primary malignant bone tumors

In primary bone sarcomas, the efficacy of chemotherapy varies according to the histological types. Prognoses are poor in patients with osteosarcoma or Ewing's sarcoma, when surgery alone is performed. However, because these sarcomas are chemosensitive, their prognoses have been improved with adjuvant chemotherapy. Nowadays, in highgrade bone sarcomas, especially in osteosarcoma, Ewing.s sarcoma and malignant fibrous histiocytoma of bone, adjuvant chemotherapy including neoadjuvant or preoperative chemotherapy is usually performed. The purpose of the neoadjuvant chemotherapy is (I) to prevent distant metastases, (II) to reduce the size of the primary tumor and (III) to evaluate the efficacy of the chemotherapeutic agents. Reducing the tumor size facilitates easier excision with less risk of local recurrence. In addition, not only limb-saving but also function-preserving surgery is made possible. Evaluating the efficacy of the chemotherapeutic agents in preoperative chemotherapy facilitates rational selection of postoperative chemotherapeutic agents. Several kinds of anticancer agents are used, and many authors have reported various kinds of protocols and their clinical results. Commonly used drugs include adriamycin, ifosfamide, cisplatin, methotrexate and vincristine in osteosarcoma, and vincristine, adriamycin, cyclophosphamide, ifosfamide, actinomycin-D and etoposide in Ewing's sarcoma. In contrast, chondrosarcomas are chemoresistant, and chemotherapy is rarely performed. Low-grade bone sarcomas, e. g., parosteal osteosarcoma, central low-grade osteosarcoma, are well cured only by surgical excision, and adjuvant chemotherapy is not performed for these low-grade sarcomas. To enhance the efficacy of preoperative chemotherapy, various modalities have been used e. g., intraarterial infusion, caffeine-assisted chemotherapy, and local perfusion with hyperthermia. Good clinical results have been reported.     

Secondary tumors in bone sarcomas after treatment with chemotherapy.

New oncologic treatments have improved survival in osteosarcoma and Ewing's sarcoma. However, these treatments may cause secondary malignancies after radiotherapy. This study evaluated the incidence of secondary malignancies after neoadjuvant chemotherapy. Between April 1972 and December 1990, 518 osteosarcoma and 299 Ewing's sarcoma patients entered neoadjuvant chemotherapy protocols. Follow-up records of all patients were analyzed and malignant tumors were reported. Nine patients developed another malignancy, including 5 leukemias, 1 astrocytoma, 1 liposarcoma, 1 parotid, and 1 breast carcinoma. Four leukemias were found in patients treated for osteosarcoma with chemotherapy, but not radiotherapy. Only one leukemia developed after Ewing's sarcoma treated with chemotherapy and radiotherapy. The incidence of leukemias is high, while the other tumors can be explained as unrelated cases. Incidence densities for leukemia were calculated for both groups of patients. Treated osteosarcoma patients seem to have a predisposition to develop leukemias, but whether this is chemotherapy induced needs to be investigated.     

Inhibition of the Raf/MEK/ERK pathway up-regulates expression of the coxsackievirus and adenovirus receptor in cancer cells

Recombinant adenoviruses are presently being tested clinically as a new strategy for the treatment of cancer. An important determining factor for the successful entry of such adenoviruses into target cells is expression of the coxsackievirus and adenovirus receptor (CAR) at the cell surface. Recent observations suggest that expression of this receptor, which physiologically participates in formation of cell-cell adhesions, is frequently reduced in highly malignant cancer cells. This raises the possibility that those tumors representing the greatest therapeutic challenge might be the least susceptible to infection with therapeutic adenoviruses. We explored the role of the Raf-MEK-ERK pathway on CAR expression in a panel of cancer cells because this pathway is frequently up-regulated in cancer cells and is known to down-regulate cell-cell adhesion molecules. We found that disruption of signaling through the Raf-MEK-ERK pathway by inhibition of MEK up-regulated CAR expression, which was accompanied by increased representation of the protein at the cell surface. After Raf-MEK-ERK inhibition, adenovirus entry into cells was increased and cell killing by replication competent adenoviruses was enhanced in a CAR-dependent manner. Conversely, induction of Raf-1 resulted in reduction and disruption of CAR expression at the cell surface. We conclude that loss of CAR expression in cancer cells is, at least in part, mediated through the Raf-MEK-ERK signal transduction pathway and that pharmacological restoration of CAR at the cell surface could improve adenovirus-based treatments of cancer.     

柯萨奇-腺病毒受体在肿瘤发生发展机制中的研究进展

柯萨奇-腺病毒受体（Coxsackie and adenovirus receptor,CAR）最早作为2型和5型腺病毒的受体而被人们发现和认识。大量研究发现CAR可以影响肿瘤细胞的生长、细胞骨架的变化、细胞间的粘附等,从而在肿瘤侵袭转移过程中起非常重要的作用。而且,CAR的表达与肿瘤的预后和腺病毒介导的减瘤效应也有密切的关系。基于CAR的重要作用,CAR已逐渐成为肿瘤发生发展机制及治疗领域研究中新的“热点”,本文结合CAR的基本结构及功能对以上研究的进展作一综述。     

Blood serum somatomedin activity in children with bone tumors

Blood somatomedin levels were assayed biologically in 10 children with Ewing's sarcoma and osteosarcoma and 11 children with other benign tumors. The patients were of the same age. The basal level of somatomedin in malignant bone sarcoma was similar to that in controls. However, after glucose loading, the patterns of somatomedin level curve in children and adults with normal lipo-carbohydrate metabolism were different. The difference may be due either to the specific features of child's organism or a rise in the level of blood somatomedin inhibitors.     

Limb salvage management of pathologic fractures of primary malignant bone tumors.

BACKGROUND: Little is known about oncologic outcomes of patients with primary bone tumors complicated by a pathologic fracture and treated by limb salvage. METHODS: Our study included 17 men and 14 women aged 6 to 61 years (average age 17 years). All 31 patients had primary bone tumors complicated by a pathologic fracture. Diagnoses included osteosarcoma (17 patients), Ewing's sarcoma (10), malignant fibrous histiocytoma (3), and lymphoma (1). All received preoperative chemotherapy. The distal femur was affected in 13 patients, the proximal femur in 6, mid shaft femur in 4, the proximal humerus in 4, the proximal tibia in 3, and the fibula in 1. All patients underwent limb salvage and achieved a wide resection margin. RESULTS: The average follow-up period was 18 months (range 8 to 51 months). Two patients required amputation due to local recurrence. Six patients developed pulmonary metastases and eventually died. CONCLUSIONS: A pathologic fracture of primary bone tumor is not always a contraindication for limb salvage since the oncologic outcome appears acceptable.     

Antitumor activity of the insulin-like growth factor-I receptor kinase inhibitor NVP-AEW541 in musculoskeletal tumors

Identification of new drugs is strongly needed for sarcomas. Insulin-like growth factor-I receptor (IGF-IR) was found to provide a major contribution to the malignant behavior of these tumors, therefore representing a very promising therapeutic target. In this study, we analyzed the therapeutic potential of a novel kinase inhibitor of IGF-IR, NVP-AEW541, in Ewing's sarcoma, osteosarcoma, and rhabdomyosarcoma, the three most frequent solid tumors in children and adolescents. NVP-AEW541 inhibits IGF-I-mediated receptor activation and downstream signaling. Ewing's sarcoma cells were generally found to be more sensitive to the effects of this drug compared with rhabdomyosarcoma and osteosarcoma, in agreement with the high dependency of this neoplasm to IGF-IR signaling. NVP-AEW541 induced a G1 cell cycle block in all cells tested, whereas apoptosis was observed only in those cells that show a high level of sensitivity. Concurrent exposure of cells to NVP-AEW541 and other chemotherapeutic agents resulted in positive interactions with vincristine, actinomycin D, and ifosfamide and subadditive effects with doxorubicin and cisplatin. Accordingly, combined treatment with NVP-AEW541 and vincristine significantly inhibited tumor growth of Ewing's sarcoma xenografts in nude mice. Therefore, results encourage inclusion of this drug especially in the treatment of patients with Ewing's sarcoma. For the broadest applicability and best efficacy in sarcomas, NVP-AEW541 may be combined with vincristine, actinomycin D, and ifosfamide, three major drugs in the treatment of sarcomas.     

Periosteal Ewing's sarcoma

Ewing's sarcoma is a small cell malignant tumor that usually arises in the medullary cavity of bone. Less frequently, it originates in soft tissue and may secondarily invade underlying bone. The origin of Ewing's sarcoma in a periosteal location without extension into either the bone or adjacent soft tissue has not been clearly documented. Other malignant tumors of bone (e.g., osteosarcoma) appear to have a somewhat better prognosis when confined between periosteum and bone. The case of a patient with a periosteal Ewing's sarcoma who received a radical excision and postoperative chemotherapy and who is without evidence of disease with over 2 years follow-up is reported.     

Regional chemotherapy with the use of cisplatin and doxorubicin as primary treatment for advanced sarcomas in shoulder, pelvis, and thigh

Eight patients who had large sarcomas in the hip, thigh, or shoulder girdle have been described. Three had osteogenic sarcomas, and one each had Ewing's sarcoma, biphasic synovial sarcoma, pleomorphic liposarcoma, undifferentiated spindling sarcoma, and malignant fibrous histiocytoma. All eight tumors showed evidence of regression after intraarterial infusion of cisplatin and Adriamycin (doxorubicin) given over 48 hours at 3-week intervals, for a total of between three and seven courses. Tru-cut needle biopsy specimens of five of the lesions were normal after chemotherapy. However, after resection of the regressed fibrotic tumor in seven of the patients, four contained foci of probably viable malignant cells. These cell foci were intraosseous in three cases and in the wall of a cyst in one case. In the remaining case, tumor in the distribution of the infused artery regressed, but tumor in a region supplied by an artery that was not infused continued to enlarge. In one patient with osteogenic sarcoma in the pelvis, despite a good response to intraarterial chemotherapy that was followed by surgical resection and radiotherapy, tumor recurred in an adjacent area in tissues supplied by an artery not infused. A hindquarter amputation subsequently was required. With the exception of the two cases in which adequate tumor arterial infusion was not achieved, local primary tumor control was accomplished by intraarterial infusion chemotherapy followed by local resection or radiotherapy and local resection in all patients. Four patients are well without evidence of residual or metastatic sarcoma 3.5 years after presentation in the case of an osteogenic sarcoma of shoulder, 2.5 years after presentation in the case of a large pleomorphic liposarcoma of thigh and groin, 20 months after presentation in the case of lower-thigh malignant fibrous histiocytoma, and 1 year after presentation in a child with an osteogenic sarcoma of lower femur.     

膝关节周围恶性骨肿瘤的保肢治疗（附48例随访报告）

目的总结膝关节周围恶性骨肿瘤的保肢治疗经验。方法回顾分析1999年2月～2007年2月48例接受保肢治疗的膝关节周围恶性骨肿瘤患者的临床资料。男30例,女18例。平均年龄27.3岁（11～67岁）。病理证实：高恶性肿瘤32例（A组）,包括骨肉瘤23例,恶性纤维组织细胞瘤5例,尤文肉瘤2例,恶性淋巴瘤2例;低度恶性肿瘤16例（B组）,包括侵袭性骨母细胞瘤1例,骨巨细胞瘤15例。手术方式包括：瘤段切除假体置换术或灭活再植术、异体骨移植术、异体骨复合假体移植术、病灶刮除充填术保肢。A组术前、术后给予化疗。保留肢体功能按Enneking肌肉骨骼肿瘤外科治疗重建后功能评估标准评估。结果平均随访3.2年（0.5～8年）。A组中因局部肿瘤复发、感染等并发症截肢11例（34.4%）,死亡13例,存活19例,3年存活率59.3%（19/32）。B组中因局部肿瘤复发和感染截肢2例（12.5%,2/16）,死亡1例。肢体功能优良率A组71%,B组81%。结论膝关节周围恶性骨肿瘤保肢应掌握个体化原则,假体置换术及异体骨复合假体移植术保肢功能最佳,高恶性肿瘤患者若无法承受强力的辅助化疗则不宜行保肢术。     

Virus-associated RNA I-deleted adenovirus, a potential oncolytic agent targeting EBV-associated tumors

Given the growing number of tumor types recognizably associated with EBV infection, it is critically important that therapeutic strategies are developed to treat such tumors. Replication-selective oncolytic adenoviruses represent a promising new platform for anticancer therapy. Virus-associated I (VAI) RNAs of adenoviruses are required for efficient translation of viral mRNAs. When the VAI gene is deleted, adenovirus replication is impeded in most cells (including HEK 293 cells). EBV-encoded small RNA1 is uniformly expressed in most EBV-associated human tumors and can functionally substitute for the VAI RNAs of adenovirus. It enables replication to proceed through complementation of VAI-deletion mutants. We hypothesized that VAI-deleted adenovirus would selectively replicate in EBV-positive tumor cells due to the presence of EBV-encoded small RNA1 with no (or poor) replication in normal or EBV-negative tumor cells. In this report, we show that high levels of replication occurred in the VAI-deleted mutant in the EBV-positive tumor cells compared with low (or negligible) levels in EBV-negative and normal human primary cells. Correspondingly, high toxicity levels were observed in EBV-positive tumor cells but not in EBV-negative tumor or normal human primary cells. In vivo, VAI-deleted adenovirus showed superior antitumoral efficacy to wild-type adenovirus in EBV-positive tumor xenografts, with lower hepatotoxicity than wild-type adenovirus. Our data suggest that VAI-deleted adenovirus is a promising replication-selective oncolytic virus with targeting specificity for EBV-associated tumors.