Telomerase activity correlates with growth of transplantable osteosarcomas in rats treated with cis-diammine dichloroplatinum or the angiogenesis inhibitor AGM-1470

To determine the role of telomerase activity in the growth of tumors in rats undergoing chemotherapy, a comparison of the volumes of telomerase-positive transplantable osteosarcomas was made in rats treated with the antineoplastic agent cis-diammine dichloroplatinum (CDDP) or the angiogenesis inhibitor O-(chloroacetylcarbamoyl)fumagillol (AGM-1470). Male F344 rats, 8 weeks old, received transplants of macroscopic lung metastatic nodules into the subcutaneous back space and treatment was started on day 14 thereafter. CDDP was injected i.v. at doses of 0, 0.625, 1.25 and 2.5 mg/kg body weight (b.w.) and AGM-1470 was administered at total doses of 0, 2.5, 5 and 10 mg/kg b.w. over 2 weeks by osmotic pumps, also implanted into the subcutaneous back space, but remote from the transplanted tumors. On day 28, all animals were killed for measurement of transplanted tumor size and determination of telomerase activities by telomeric repeat amplification protocol (TRAP) assay. The results showed telomerase activity to be highly correlated with the treated/non-treated (T/C) tumor size ratio (r = 0.96, P < 0.0001). In a second experiment, CDDP at 2.5 mg/kg b.w. and AGM-1470 at 10 mg/kg b.w., these being the most effective doses, were given as in the first experiment, and animals were serially killed on days 14, 21, 28, 35 and 42. Tumors in rats treated with CDDP and AGM-1470 showed 18.2% and 20.5% of the control telomerase activity on days 35 and 21, respectively, when tumor growth was inhibited. However, on day 42, the activities increased to 46.5% and 92.5%, this correlating with re-growth (r = 0.73, P < 0.0001). These results suggest that decline of telomerase activity may be involved in tumor growth retardation induced by chemotherapeutic agents. This possibility clearly warrants further mechanistic studies.     

Comparative immunoreactivity of CD-68 and HMB-45 in malignant melanoma, neural tumors and nevi

The monoclonal antibody CD 68 (KP 1) reacts with fibrohistiocytic and some epithelial neoplasms; its reactivity compared with that of HMB 45 in malignant melanoma (MM) and neural tumors needs further elucidation. Using a streptavidin-biotin-immunoperoxidase procedure, we examined the reactivity of 65 MM (46 conventional, 1 polypoid, 6 desmoplastic [DMM], and 12 metastatic), 21 neurofibromas, 1 neurofibrosarcoma, 10 schwannomas, 1 perineurioma, 2 neurothekeomas, and 14 blue and 26 other nevi for CD-68, HMB-45-defined antigen, S 100 and neurofilament protein. A positive staining for CD 68 was observed in 38 of 42 primary, 5 of 6 DMM, and 11 of 12 metastatic melanomas; 6 of 10 schwannomas; 5 of 10 nevi with junctional component and all 14 blue nevi. All 21 neurofibromas, 1 each neurofibrosarcoma and perineurioma, both neurothekeomas, and all 12 nevi with dermal component were CD 68-negative. HBM 45 was expressed by all 44 primary, none of 6 DMM, and 7 of 12 metastatic melanomas; by none of 10 schwannomas, 6 neurofibromas, 1 neurofibrosarcoma, 1 perineurioma and 2 neurothekeomas. Both junctional nevi, 8 of 10 nevi with junctional components, 1 of 10 dermal components of junctional nevi, and 11 of 13 blue nevi were also HMB 45 positive. Except for 1 perineurioma, S 100 decorated all tumors examined. NF was immunoreactive in 1 of 45 conventional melanomas, 2 of 21 neurofibromas, 2 of 10 schwannomas, and 3 of 10 blue nevi; it was non-reactive in all polypoid, desmoplastic and metastatic melanomas; neurofibrosarcoma, perineurioma, neurothekeoma and other nevi. We conclude that the CD-68-reactivity in primary melanomas, neurofibromas, neurofibrosarcomas, perineuriomas, and nevi was similar to that of HMB 45. The significantly higher CD 68-positivity than of HMB 45 in metastatic and desmoplastic melanomas and schwannomas may be of diagnostic value.     

Human vestibular schwannoma growth in the nude mouse: evaluation of a modified subcutaneous implantation model

HYPOTHESIS: Based on the hypothesis that vestibular schwannomas can be successfully implanted and grown in the nude mouse model, an in vivo experiment was designed for subcutaneous implantation of solid vestibular schwannoma tissue. BACKGROUND: Vestibular schwannomas are benign tumors arising from Schwann cells of cranial nerve VIII. Little in vivo research has been carried out with these tumors, due in part to the difficulty to grow cells in culture or maintain tumor in an animal model. Recently, vestibular schwannomas have been implanted in nude mice with moderate success. The current study evaluates a modification of prior techniques in an effort to establish a dependable research model. METHODS: Thirty-six nude mice were implanted with variable-sized vestibular schwannoma tissue from three human subjects. Volumes implanted ranged from 14-170 mm3. Mice were observed for 28 days and individual volumes recalculated. Eleven of the mice were observed for a total of 56 days with volumes re-evaluated, and tumors subsequently were removed for assessment of viability and vascularity. RESULTS: At 28 days, 36 tumors (100%) showed take with 34 tumors (94%) showing macroscopic growth. The 11 tumors observed for 56 days showed a trend of stable or decreased size at 56 days compared with that of the 28-day measurement. Overall growth from time of implantation to measurements at 56 days was noted in 8 (73%) of 11 tumors when measured at the skin and in 10 (91%) of 11 tumors when direct tumor volume was measured. One hundred percent of tumors evaluated microscopically at 56 days was viable. All tumors at the time of removal had significant vascularity with a mean of 70.68% (SD = 23.42) of surface covered with vessels. There were no significant differences in take and growth for the larger tumor specimens compared with those of smaller sizes. CONCLUSION: Human vestibular schwannomas successfully can be implanted and maintained in the subcutaneous pocket of the nude mouse. This in vivo tumor model provides a reliable, accessible base for further research with vestibular schwannomas.     

Vascular endothelial growth factor increased by pulmonary surgery accelerates the growth of micrometastases in metastatic lung cancer

BACKGROUND: The use of surgery for metastatic lung cancer has been established recently and the indications have been extended to multiple and bilateral lung metastases. However, in some patients, secondary lung metastasis appears soon after the first pulmonary surgery, making curative treatment very difficult. Postoperative weakness of tumor angiogenesis suppression mechanisms seems to play an important role in the recurrence of lung metastases. To verify this hypothesis, we performed a clinical and an experimental study. RESULTS AND CONCLUSION: The clinical study revealed that serum vascular endothelial growth factor (VEGF), also known as vascular permeability factor, increased after pulmonary surgery. The experimental study showed that VEGF played an important role in the rapid growth of dormant micrometastases of the lung. These results suggested that the postoperative increase of VEGF disrupted angiogenesis suppression and induced the growth of dormant micrometastases early in the postoperative period. It was also demonstrated that this effect of VEGF on micrometastases was abolished by AGM-1470, an angiogenesis inhibitor. In conclusion, postoperative treatment with AGM-1470 might inhibit the early recurrence of malignant tumors.     

The angiogenesis inhibitor AGM-1470 selectively increases E-selectin

The levels of E-selectin mRNA and protein were analyzed in bovine capillary cells treated with or without the angiogenesis inhibitor AGM-1470 (also known as TNP-470). Cells treated with AGM-1470 had a two- to sevenfold (median fivefold) increase in E-selectin mRNA compared with little or no increase in P-selectin, PECAM-1 and VCAM-1 mRNA. E-selectin protein was also significantly increased after exposure to AGM-1470. In contrast, there was no detectable effect on PECAM-1 protein. The increase in E-selectin mRNA and protein was always greater with subconfluent growing cells than with confluent cells. This apparent resistance of confluent endothelial cells to AGM-1470 may be relevant to its specificity in vivo. The fact that the effect of AGM-1470 on E-selectin is relatively selective for subconfluent growing cells may provide a clue as to how AGM-1470 is able to both reversibly inhibit endothelial cell proliferation in vitro and inhibit tumor growth in vivo without apparent effects to quiescent endothelium.     

Suppression of collagen-induced arthritis using an angiogenesis inhibitor, AGM-1470, and a microtubule stabilizer, taxol

Collagen-induced arthritis (CIA) is a T-cell-dependent rat model of rheumatoid arthritis (RA) that is induced by injection of collagen type II in incomplete Freund's adjuvant. Neovascularization within the synovium is a prominent feature of CIA and RA. The novel angiogenesis inhibitor AGM-1470 and the microtubule-stabilizing agent Taxol represent two new classes of agents with specific mechanisms of action. AGM-1470 inhibits fibroblast growth factor-induced stimulation of endothelial cell migration, endothelial cell proliferation, and capillary tube formation, resulting in effective suppression of new blood vessel formation. By enhancing microtubule polymerization, Taxol interferes with normal microtubule function in cell mitosis, migration, chemotaxis, and intracellular transport. Using a suppression protocol in established CIA, the effects of AGM-1470 and Taxol as single agents and in combination were evaluated. Combination therapy significantly reduced clinical arthritis compared to control rats (P < 0.00001). The combination therapy group also experienced earlier and significantly greater reduction of clinical arthritis compared to either single agent-treated groups (P < 0.05). Blinded radiographic scores at the end of the study demonstrated less soft tissue swelling and joint destruction using combination therapy than either single agent. This is the first use of AGM-1470 and Taxol in combination therapy. Further study of agents with distinct mechanisms of action may lead to more effective treatment options in chronic inflammatory arthritis and to a better understanding of the pathophysiologic processes of pannus formation.     

Structural investigation of proteasome inhibition

TNP-470 (AGM-1470), a synthetic analog of fumagillin (6-chloroacetyl-carbamoyloxy-4-(1,2-epoxy-1,5-dimethyl- 4-hexenyl)-5-methoxy-1-oxaspiro [2,5] octane 1, has been reported to reduce the supply of nutrients to experimental tumors by inhibiting angiogenesis. In this study, we investigated anti-tumor activity of TNP-470 against human thyroid anaplastic carcinoma with a view to developing a new treatment for this thyroid tumor. A transplantable tumor was established from thyroid anaplastic carcinoma of a 78-year-old woman, as a xenograft in nude mice (BALB/c, nu/nu, male). This transplantable tumor, with chromosomal abnormality was shown to be non-functional in excretory hormones and to preserve morphological characteristics of the original anaplastic tumor tissue. TNP-470 was given at a dose of 50 mg/kg b.w. to nude mice transplanted with human thyroid anaplastic carcinoma by different routes of administration: intratumoral, peritumoral, subcutaneous and intraperitoneal. Intratumoral and peritumoral administration were effective, and especially the TNP-470 administered by the former route completely inhibited tumor growth. Immunohistochemical analysis using anti-factor VIII antibody revealed the density of microvessels to be significantly decreased by local administration of TNP-470 (intratumoral administration, 7.8 +/- 2.9/mm2, control, 27.0 +/- 6.3/mm2; peritumoral administration, 9.7 +/- 2.6/mm2, control, 21.1 +/- 5.1/mm2). Our findings suggested the possibility of clinical application of TNP-470 to control the growth of human anaplastic thyroid carcinoma.     

Direct injection analysis of chlorzoxazone and its major metabolite 6-hydroxychlorzoxazone in human serum using a semipermeable surface (SPS) HPLC column

The efficacy of combination therapy with cis-diammine-dichloroplatinum (II) (CDDP) and o-(chloroacetyl-carbamoyl) fumagillol (AGM-1470) was evaluated experimentally using a transplantable rat osteosarcoma line, previously established in our laboratory, with a high potential for metastasis. Tumor-bearing male Fischer 344 rats were administered CDDP (2.5 mg/kg) together with, or after discontinuation of, AGM-1470 treatment (10 mg/kg/body weight/week). When CDDP was administered three days after discontinuation of AGM-1470 the most pronounced antimetastatic effects were observed, although the antitumor effect was approximately the same.     

Screening flexible sigmoidoscopy by nonphysician endoscopists: it's here to stay, but is it the right test to do?

Vestibular schwannomas have been noted to have increased frequency and aggressivity in female patients, suggesting a possible role of estrogen. This study evaluated the effects of estrogen and tamoxifen on the growth of human vestibular schwannoma tissue implanted in subcutaneous pockets of nude mice. Animals were implanted with 1 of 3 human vestibular schwannomas and observed for 28 days. Mice were then separated into 3 treatment groups: controls, estrogen (receiving 1.7 mg of 17B-estradiol), and estrogen + tamoxifen (receiving 1.7 mg of 17B-estradiol + 10 mg of tamoxifen), and treated for 28 days. Mice treated with estrogen showed increased growth that was statistically significant (P < 0.05) when compared with that of both the controls and the animals treated with estrogen + tamoxifen. Controls and animals treated with estrogen + tamoxifen showed a general trend of decreased volume during the treatment period. These early results support the hypothesis that estrogen modulates the growth of vestibular schwannomas in the nude mouse model and that these effects can be blocked by tamoxifen administration.     

Estrogenic effects of genistein on the growth of estrogen receptor-positive human breast cancer (MCF-7) cells in vitro and in vivo

Genistein, found in soy products, is a phytochemical with several biological activities. In the current study, our research focused on the estrogenic and proliferation-inducing activity of genistein. We have demonstrated that genistein enhanced the proliferation of estrogen-dependent human breast cancer (MCF-7) cells in vitro at concentrations as low as 10 nM, with a concentration of 100 nM achieving proliferative effects similar to those of 1 nM estradiol. Expression of the estrogen-responsive gene pS2 was also induced in MCF-7 cells in response to treatment with a concentration of genistein as low as 1 microM. At higher concentrations (above 20 microM), genistein inhibits MCF-7 cell growth. In vivo, we have shown that dietary treatment with genistein (750 ppm) for 5 days enhanced mammary gland growth in 28-day-old ovariectomized athymic mice, indicating that genistein acts as an estrogen in normal mammary tissue. To evaluate whether the estrogenic effects observed in vitro with MCF-7 cells could be reproduced in vivo, MCF-7 cells were implanted s.c. in ovariectomized athymic mice, and the growth of the estrogen-dependent tumors was measured weekly. Negative control animals received the American Institute of Nutrition (AIN)-93G diet, the positive control group received a new s.c. estradiol (2 mg) pellet plus the AIN-93G diet, and the third group received genistein at 750 ppm in the AIN-93G diet. Tumors were larger in the genistein (750 ppm)-treated group than they were in the negative control group, demonstrating that dietary genistein was able to enhance the growth of MCF-7 cell tumors in vivo. Increased uterine weights were also observed in the genistein-treated groups. In summary, genistein can act as an estrogen agonist in vivo and in vitro, resulting in the proliferation of cultured human breast cancer cells (MCF-7) and the induction of pS2 gene expression. Here we present new information that dietary genistein stimulates mammary gland growth and enhances the growth of MCF-7 cell tumors in ovariectomized athymic mice.     

CT imaging in adults with neurofibromatosis-1: frequent asymptomatic plexiform lesions

OBJECTIVE: The authors examined the incidence and radiologic characteristics of plexiform neurofibromas in neurofibromatosis-1 (NF-1) to define a cohort at greatest risk for malignant nerve-sheath tumors. BACKGROUND: Plexiform neurofibromas are a frequent complication of NF-1. They can impair function, produce disfigurement, and be the site for the development of malignant nerve-sheath tumors. The incidence and natural history of plexiform neurofibromas is unknown. METHODS: CT imaging of the chest, abdomen, and pelvis was performed in 91 of 125 consecutive adults (age, > or = 16 years) with NF-1. RESULTS: Twenty percent of patients had plexiform neurofibromas of the chest in the paraspinal, mediastinal, or supraclavicular area. Approximately 40% of patients had abnormal abdominal/pelvic scans. The paraspinal, sacral plexus, sciatic notch, and perirectal regions were the most common sites. Most plexiform neurofibromas were asymptomatic. Imaging also revealed a number of tumors, including malignant nerve-sheath tumors, adrenal tumors, carcinoids, and schwannomas. CONCLUSIONS: The frequency of plexiform lesions and other tumors in NF-1 indicates that clinicians should monitor young adults carefully; however, imaging characteristics alone cannot reliably distinguish benign from malignant lesions.     

CD44 expression is aberrant in benign Schwann cell tumors possessing mutations in the neurofibromatosis type 2, but not type 1, gene

Atypical expression of CD44 splice variants has been implicated in the progression of numerous tumors. This abnormal CD44 expression is presumed to result from gene alterations that cause tumorigenic transformation. Two tumor types that have been linked to specific gene alterations are schwannomas, which have mutations in the neurofibromatosis (NF) type 2 (NF2) gene, and neurofibromas, which characteristically possess NF type 1 (NF1) gene mutations. We examined CD44 expression in normal sciatic nerves, in schwannomas with confirmed NF2 mutations, and in neurofibromas and malignant peripheral nerve sheath tumor tissue and cell lines from NF1 patients. Compared to normal nerves, schwannomas express higher total levels of CD44 and additional splice variants, whereas CD44 expression in neurofibromas is unaltered. Malignant peripheral nerve sheath tumor tissue and cell lines express the CD44v6 epitope, which is not expressed by normal Schwann cells or by other Schwann cell tumors. These data indicate that altered CD44 expression correlates strictly with mutations in the NF2 but not NF1 gene and suggest that CD44v6 might be a marker for the malignant transformation of Schwann cells.     

Adhesion formation is reduced after laparoscopic surgery

BACKGROUND: Adhesion formation after abdominal operations causes significant morbidity. METHODS: Adhesion formation in pigs was compared after placement of prosthetic mesh during celiotomy (group 1), laparoscopy with large incision (group 2), and laparoscopy (group 3). After peritoneum was excised, polypropylene mesh was fixed to the abdominal wall, then to the opposite abdominal wall in the preperitoneal space followed by peritoneal closure. Adhesion area, grade, and vascularity were measured. RESULTS: More adhesions (p < 0.02) covered intraperitoneal mesh (7.57 +/- 1.89 cm2) than covered reperitonealized mesh (2.16 +/- 1.13 cm2), and adhesion grade was significantly greater (p < 0.02). Adhesion areas were significantly greater in groups 1 and 2 than in group 3 (p = 0.001 and 0.03, respectively). Adhesion grade was significantly greater in groups 1 and 2 than in group 3 (p = 0.02 and p = 0.04, respectively). Groups 1 and 2 had more vascular adhesions than group 3 (p < 0.01 and p = 0.02, respectively) CONCLUSIONS: A foreign body within the peritoneum stimulates more numerous and denser adhesions. Tissue trauma distant from the site of adhesions increases their formation. A major advantage of laparoscopic surgery is decreased adhesion formation.     

Involvement of serotonin and dopamine in the mechanism of action of novel antidepressant drugs: a review

BACKGROUND: By the time patients are diagnosed with ovarian carcinoma, peritoneal dissemination of the tumor often has occurred. The progressive growth and spread of ovarian carcinoma depend, in part, on the formation of an adequate blood supply. We determined whether the expression of genes that regulate distinct steps in angiogenesis (i.e., the formation of new blood vessels) was associated with the pattern and progressive growth of human ovarian carcinomas implanted in the peritoneal cavity of nude mice. METHODS: Five different human ovarian carcinomas were injected individually into the peritoneal cavity of female NCr-nu/nu nude mice. The expression of basic fibroblast growth factor, vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), interleukin 8 (IL-8), and collagenase type IV (MMP-2 [matrix metalloproteinase-2] and MMP-9) was determined by northern blot analysis, in situ hybridization of messenger RNA, and immunohistochemical analysis. Blood vessel distribution and density, macrophage infiltration pattern, and stromal reaction were determined by immunohistochemical analysis with specific antibodies. RESULTS: Three of the carcinomas produced both solid lesions and ascitic tumors, whereas the remaining two produced only solid lesions. Two of the carcinomas produced rapidly progressive disease, two produced slow disease, and one produced intermediate disease. The formation of ascites was directly associated with expression of VEGF/ VPF, and survival was inversely associated with expression of IL-8. In rapidly growing tumors, the number of blood vessels was high throughout the lesion; in contrast, in slow-growing tumors, most vessels (and infiltrating macrophages) were located at the periphery. CONCLUSIONS: The expression of various genes that regulate angiogenesis in human ovarian carcinomas is associated with the pattern of the disease and its progression. Therefore, targeting specific genes that regulate angiogenesis could offer new approaches to the treatment of ovarian cancer.     

Role of vascular endothelial growth factor in ovarian cancer: inhibition of ascites formation by immunoneutralization

Ovarian cancer is characterized by the rapid growth of solid intraperitoneal tumors and large volumes of ascitic fluid. Vascular endothelial growth factor (VEGF) augments tumor growth by inducing neovascularization and may stimulate ascites formation by increasing vascular permeability. We examined the role of VEGF in ovarian carcinoma using in vivo models in which intraperitoneal or subcutaneous tumors were induced in immunodeficient mice using the human ovarian carcinoma cell line SKOV-3. After tumor engraftment (7 to 10 days), some mice were treated with a function-blocking VEGF antibody (A4.6.1) specific for human VEGF. A4.6.1 significantly (P < 0.05) inhibited subcutaneous SKOV-3 tumor growth compared with controls. However, tumor growth resumed when A4.6.1 treatment was discontinued. In mice bearing intraperitoneal tumors (IP mice), ascites production and intraperitoneal carcinomatosis were detected 3 to 7 weeks after SKOV-3 inoculation. Importantly, A4.6.1 completely inhibited ascites production in IP mice, although it only partially inhibited intraperitoneal tumor growth. Tumor burden was variable in A4.6.1-treated IP mice; some had minimal tumor, whereas in others tumor burden was similar to that of controls. When A4.6.1 treatment was stopped, IP mice rapidly (within 2 weeks) developed ascites and became cachectic. These data suggest that in ovarian cancer, tumor-derived VEGF is obligatory for ascites formation but not for intraperitoneal tumor growth. Neutralization of VEGF activity may have clinical application in inhibiting malignant ascites formation in ovarian cancer.     

Delayed vascular changes after antiangiogenic therapy with antivascular endothelial growth factor antibodies in human glioma xenografts in nude mice

OBJECTIVE: The purpose of this study was to examine the delayed effects of antivascular endothelial growth factor treatment on tumor growth and vascularity in a subcutaneous mouse tumor model of human glioblastoma. METHODS: Antivascular endothelial growth factor antibody treatment was administered for a period of 6 weeks, to suppress tumor growth. To detect late vascular effects, tumor vascular parameters for treated tumors and control tumors were analyzed 4 weeks thereafter. By that time, tumors had grown to adequate sizes (diameter, 8-10 mm) for comparison with untreated control tumors. Vascular parameters were quantified by using an image-analysis system. RESULTS: Vascular density was significantly lower in antivascular endothelial growth factor antibody-treated tumors, compared with control tumors of similar size. The vascular architecture of treated tumors was also distinctly different, compared with control tumors, showing larger but sparser vessel structures. CONCLUSION: These findings suggest that antiangiogenic therapy may have a prolonged effect on the vascular architecture of certain tumors, resulting in enduring changes in the tumor vessels. Because tumor vasculature plays an important role in the sensitivity to various treatment modalities, these changes are likely to influence the responses of these tumors to further therapy.     

Extensive polymorphism of the FUT2 gene in an African (Xhosa) population of South Africa

OBJECTIVE: Nephroureterectomy is the standard treatment for tumors of the renal pelvis and ureter. Conservative management or indication of adjuvant treatment in these neoplasms is based mainly in histological grade and stage. The aim of this study is to assess the relation of Ki67 index with other established prognostic factors and to define its predictive value for long term survival, which could be useful in selecting the best treatment for each individual case. METHODS: 81 patients with urothelial tumors of the renal pelvis and ureter, diagnosed and treated between 1975 and 1993, comprised the present study. Ki67 immunostaining was performed in paraffin-embedded tissue. A cut-off limit of 20% was chosen. Tumor location, histological grade, histological pattern, local (T), nodal (N), vascular and perineural invasion and stage (TNM) were assessed in relation to the proliferation index and as prognostic criteria for survival in both univariate and multivariate analysis. RESULTS: The Ki67 proliferation index was found to be related to grade (p < 0.001), T (T0 vs T1-4; p < 0.01), N (p < 0.038), TNM categories (stage 0 vs I-IV; p < 0.048) and perineural invasion (p < 0.01). There was a marginal relation to vascular invasion (p < 0.11). Survival was better for the patients with low proliferating tumors (90%) than for high proliferating ones (67%) (p < 0.02). In the multivariate analysis only T stage was statistically significant (p < 0.01) but a highly suggestive trend was found for the Ki67 index (p < 0.07). CONCLUSIONS: Tumor proliferation assessed by Ki67 immunostaining is related to the progression of the disease and proved to be of predictive value for long-term survival in tumors of the renal pelvis and ureter. The Ki67 index is able to detect high-risk patients that could not be cured by radical surgery alone, raising the need for some type of aduvant treatment in these cases. The treatment predictive effect observed in low grade-low stage cases suggests its possible utility in patients managed conservatively.     

Ras activation in astrocytomas and neurofibromas

Oncogenic mutations resulting in activated Ras Guanosine Triphosphate (GTP) are prevalent in 30% of all human cancers, but not primary nervous system tumors. Several growth factors/receptors are implicated in the pathogenesis of malignant astrocytomas including epidermal growth factor (EGFR) and platelet derived growth factor (PDGF-R) receptors, plus the highly potent and specific angiogenic vascular endothelial growth factor (VEGF). A significant proportion of these tumors also express a truncated EGFR, which is constitutively activated. Our work demonstrates that the mitogenic signals from both the normal PDGF-R and EGFR and the truncated EGFR activate Ras. Inhibition of Ras by genetic or pharmacological strategies leads to decreased astrocytoma tumorgenic growth in vitro and decreased expression of VEGF. This suggests that these agents may be potentially important as novel anti-proliferative and anti-angiogenic therapies for human malignant astrocytomas. In contrast to astrocytomas, where increased levels of activated Ras GTP results from transmitted signals from activated growth factor receptors, the loss of neurofibromin is postulated to lead to functional up-regulation of the Ras pathway in neurofibromatosis-1(NF-1). We have demonstrated that NF-1 neurofibromas and neurogenic sarcomas, compared to non-NF-1 Schwannomas, have markedly elevated levels of activated Ras GTP. Increased Ras GTP was associated with increased tumor vascularity in the NF-1 neurogenic sarcomas, perhaps related to increased VEGF secretion. The role of Ras inhibitors as potential therapy in this tumor is also under study.     

Regulation of local host-mediated anti-tumor mechanisms by cytokines: direct and indirect effects on leukocyte recruitment and angiogenesis

The regulation of tumor growth by cytokine-induced alterations in host effector cell recruitment and activation is intimately associated with leukocyte adhesion and angiogenic modulation. In the present study, we have developed a novel tumor model to investigate this complex series of events in response to cytokine administration. Gelatin sponges containing recombinant human basic fibroblast growth factor (rhFGFb) and B16F10 melanoma cells were implanted onto the serosal surface of the left lateral hepatic lobe in syngeneic C57BL/6 mice. The tumor model was characterized by progressive tumor growth initially localized within the sponge and the subsequent development of peritoneal carcinomatosis. Microscopic examination of the sponge matrix revealed well developed tumor-associated vascular structures and areas of endothelial cell activation as evidenced by leukocyte margination. Treatment of mice 3 days after sponge implantation with a therapeutic regimen consisting of pulse recombinant human interleukin-2 (rhIL-2) combined with recombinant murine interleukin-12 (rmIL-12) resulted in a marked hepatic mononuclear infiltrate and inhibition of tumor growth. In contrast to the control group, sponges from mice treated with rhIL-2/rmIL-12 demonstrated an overall lack of cellularity and vascular structure. The regimen of rhIL-2 in combination with rmIL-12 was equally effective against gelatin sponge implants of rhFGFb/B16F10 melanoma in SCID mice treated with anti-asialo-GM1 in the absence of a mononuclear infiltration, suggesting that T, B, and/or NK cells were not the principal mediators of the anti-tumor response in this tumor model. The absence of vascularity within the sponge after treatment suggests that a potential mechanism of rhIL-2/rmIL-12 anti-tumor activity is the inhibition of neovascular growth associated with the establishment of tumor lesions. This potential mechanism could be dissociated from the known activities of these two cytokines to induce the recruitment and activation of host effector cells. Moreover, this model provides a unique opportunity to study the cellular and molecular mechanism(s) underlying both tumor angiogenesis and leukocyte recruitment to metastatic lesions.     

Consistent hepatic metastasis of human colorectal cancer in severe combined immunodeficient mice

A major stumbling block in the study of human colorectal cancer metastasis has been the lack of an effective in vivo model producing liver metastasis on a consistent basis. In this study surgical specimens of colorectal carcinoma were implanted in scid mice and studied for engraftment, growth, and the capacity to produce hepatic metastases. Human colorectal cancers would engraft and propagate in the subcutis and intraperitoneally. Sporadic metastasis to the liver occurred in 3 of 54 (6%) animals with cancer implanted subcutaneously. Liver metastasis occurred in 24 of 25 (96%) mice with cancer implanted in the gonad fat pad. Tumor growth to extremely large volumes subcutaneously did not enhance metastatic potential, and neither did longer term growth in the subcutaneous space. Tumor placed in the gonad fat required no special manipulation and in most cases a single piece of solid tumor was implanted. In situ hybridization confirmed the persistence of the human tissue in these metastasizing tumors. Our model will allow for the study of the processes involved in metastasis of solid tumors, characterization of differences between the primary tumor and the metastatic one, and evaluation of possible therapeutic modalities.