Endothelial specific YY1 deletion restricts tumor angiogenesis and tumor growth.

Angiogenesis is a physiological process for the formation of new blood vessels from the pre-existing vessels and it has a vital role in the survival and growth of neoplasms. During tumor angiogenesis, the activation of the gene transcriptions in vascular endothelial cells (ECs) plays an essential role in the promotion of EC proliferation, migration, and vascular network development. However, the molecular mechanisms underlying transcriptional regulation of EC and tumor angiogenesis remains to be fully elucidated. Here we report that the transcription factor Yin Yang 1 (YY1) in ECs is critically involved in tumor angiogenesis. First, we utilized a tamoxifen-inducible EC-specific YY1 deficient mouse model and showed that YY1 deletion in ECs inhibited the tumor growth and tumor angiogenesis. Using the in vivo matrigel plug assay, we then found that EC-specific YY1 ablation inhibited growth factor-induced angiogenesis. Furthermore, vascular endothelial growth factor (VEGF)-induced EC migration was diminished in YY1-depleted human umbilical vein endothelial cells (HUVECs). Finally, a rescue experiment revealed that YY1-regulated BMP6 expression in ECs was involved in EC migration. Collectively, our results demonstrate that endothelial YY1 has a crucial role in tumor angiogenesis and suggest that targeting endothelial YY1 could be a potential therapeutic strategy for cancer treatment.

Gambogic acid exhibits anti-metastatic activity on malignant melanoma mainly through inhibition of PI3K/Akt and ERK signaling pathways.

Gambogic acid (GA) is a potential anti-cancer compound that is extracted from the resin of Garciania hanburyi. The present study was designed to evaluate the anti-metastatic effect of GA on melanoma cell lines in vitro and to explore the underlying mechanism. The anti-proliferative activity of GA on melanoma cells was assessed by CCK-8 assay. The Wound-healing, transwell, adhesion, and tube formation assays were performed to examine the inhibition of GA on the cell's migration, invasion, adhesion, and angiogenesis capacities, respectively. Enzymatic activity of MMP-2 and MMP-9 were detected by gelatin zymography assay. Protein expressions regulated by GA treatment were tested by Western blot assay. The present results showed that GA significantly inhibited the proliferation of highly metastatic melanoma A375, B16-F10 cells and human umbilical vein endothelial cells (HUVECs) in time- and doses-dependent manners. Furthermore, GA significantly inhibited the migratory, invasive and adhesive properties of A375 and B16-F10 cells, and tube-forming potential of HUVECs at sub-IC50 concentrations, where no significant cytotoxicity was observed. Mechanistically, GA treatment suppressed the EMT and angiogenesis processes and reduced the enzymatic activity of MMP-2 and MMP-9. Moreover, abnormal PI3K/Akt and ERK signaling pathways in A375 and B16-F10 cells and HUVECs were notably suppressed by GA treatment. Collectively, our results suggest that GA exerts anti-metastasis activity in melanoma cells by suppressing the EMT and angiogenesis through the PI3K/Akt and ERK signaling pathways, and might be used as a phytomedicine against metastatic melanoma.

A Decade of Experience in Developing Preclinical Models of Advanced- or Early-Stage Spontaneous Metastasis to Study Antiangiogenic Drugs, Metronomic Chemotherapy, and the Tumor Microenvironment.

The clinical circumstance of treating spontaneous metastatic disease, after resection of primary tumors, whether advanced/overt or microscopic in nature, is seldom modeled in mice and may be a major factor in explaining the frequent discordance between preclinical and clinical therapeutic outcomes where the trend is "overprediction" of positive results in preclinical mouse model studies. To evaluate this hypothesis, a research program was initiated a decade ago to develop multiple models of metastasis in mice, using variants of human tumor cell lines selected in vivo for enhanced spontaneous metastatic aggressiveness after surgical resection of established orthotopic primary tumors. These models have included breast, renal, and colorectal carcinomas; ovarian cancer (but without prior surgery); and malignant melanoma. They have been used primarily for experimental therapeutic investigations involving various antiangiogenic drugs alone or with chemotherapy, especially "metronomic" low-dose chemotherapy. The various translational studies undertaken have revealed a number of clinically relevant findings. These include the following: (i) the potential of metronomic chemotherapy, especially when combined with a vascular endothelial growth factor pathway targeting drug to successfully treat advanced metastatic disease; (ii) the development of relapsed spontaneous brain metastases in mice with melanoma or breast cancer whose systemic metastatic disease is successfully controlled for a period with a given therapy; (iii) foreshadowing the failure of adjuvant antiangiogenic drug-based phase III trials; (iv) recapitulating the failure of oral antiangiogenic tyrosine kinase inhibitors plus standard chemotherapy in contrast to the modest successes of antiangiogenic antibodies plus chemotherapy in metastatic breast cancer; and (v) revealing "vessel co-option" and absence of angiogenesis as a determinant of intrinsic resistance or minimal responsiveness to antiangiogenic therapy in lung metastases. Developing similar models of metastatic disease but involving mouse tumors grown in syngeneic immunocompetent mice may also prove useful for future translational studies of immune therapy-based treatments.

Millimeter waves as a source of selective heating of skin.

This study demonstrates that 20-100 GHz range can be used for spatially-accurate focusing of heating inside the skin achieved by varying frequency and exposure beam size, as well as by enforcing air convection. The latter is also used to reduce overheating of skin surface. Heating at different skin depths depending on these parameters is investigated in detail using the hybrid bio-heat equation. In particular, it is shown that decreasing frequency and/or increasing exposure beam size at forced airflow result in elevation of heating of deeper layers of tissue and decrease of skin surface temperature. Changes of water content within 15%, which exceed those due to aging and presence of tumors, only slightly affect heating. Exposure intensity necessary to reach a target temperature significantly increases in different areas of body with elevated blood flow. Dependence on exposure intensity and hyperthermia treatment duration is also investigated and discussed. Results of this study suggest that the lower part of the millimeter-wave range is an attractive alternative for non-invasive thermal treatment of skin cancer with a high spatial resolution.

Stable tumor vessel normalization with pO2 increase and endothelial PTEN activation by inositol trispyrophosphate brings novel tumor treatment.

Tumor hypoxia is a characteristic of cancer cell growth and invasion, promoting angiogenesis, which facilitates metastasis. Oxygen delivery remains impaired because tumor vessels are anarchic and leaky, contributing to tumor cell dissemination. Counteracting hypoxia by normalizing tumor vessels in order to improve drug and radio therapy efficacy and avoid cancer stem-like cell selection is a highly challenging issue. We show here that inositol trispyrophosphate (ITPP) treatment stably increases oxygen tension and blood flow in melanoma and breast cancer syngeneic models. It suppresses hypoxia-inducible factors (HIFs) and proangiogenic/glycolysis genes and proteins cascade. It selectively activates the tumor suppressor phosphatase and tensin homolog (PTEN) in vitro and in vivo at the endothelial cell (EC) level thus inhibiting PI3K and reducing tumor AKT phosphorylation. These mechanisms normalize tumor vessels by EC reorganization, maturation, pericytes attraction, and lowering progenitor cells recruitment in the tumor. It strongly reduces vascular leakage, tumor growth, drug resistance, and metastasis. ITPP treatment avoids cancer stem-like cell selection, multidrug resistance (MDR) activation and efficiently enhances chemotherapeutic drugs activity. These data show that counteracting tumor hypoxia by stably restoring healthy vasculature is achieved by ITPP treatment, which opens new therapeutic options overcoming hypoxia-related limitations of antiangiogenesis-restricted therapies. By achieving long-term vessels normalization, ITPP should provide the adjuvant treatment required in order to overcome the subtle definition of therapeutic windows for in vivo treatments aimed by the current strategies against angiogenesis-dependent tumors.

Microcirculation patterns in indocyanine green angiography and the results of plaque therapy in choroidal melanoma.

PURPOSE: To determine the effect of radioactive plaque therapy on blood vessel behaviour in choroidal melanomas using indocyanine green (ICG) angiography. MATERIAL AND METHODS: Fifty-five patients with choroidal melanoma were studied. Ruthenium-106 plaques were used in 30 eyes, in 11 the "sandwich method" (Ruthenium-106 plaque with transpupillary thermotherapy), was applied and 14 tumours were treated with Iodine-125. In all cases ICG angiography was performed prior to treatment and 12 months after, and at different time afterwards. Baseline tumour microcirculation patterns (MCPs) were studied prior to treatment and post-treatment blood vessels changes were evaluated. Total follow-up period ranged from 14-22 months (mean: 16 months). RESULTS: Pre-treatment ICG angiography revealed complex MCPs, combining parallel with cross-linking, arcs with branching, loops and networks patterns in 23 (41.8%) and non-complex MCPs, including straight, parallel without cross-linking and arcs without branching patterns in 32 (58.2%) melanomas. Twelve months after treatment, 38 tumours (69.1%) showed a significant changes in their MCPs. The mean ultrasonographic regression rate in tumours with complex MCPs was 57.4% as opposed to 36.2% in the group with non-complex MCPs (p = 0.01). No statistically significant correlation in the height regression rate was found among the various methods of therapy, however a significant difference between the type of therapy and MCPs changes was observed (p < 0.001). Melanomas treated with Ruthenium-106 and TTT demonstrated slight or no MCPs changes, while tumours treated with Ruthenium-106 and Iodine-125 plaques alone showed a significant MCPs changes (p < 0.001). The statistical analysis showed the correlation between the type of baseline MCPs and the degree of their changes after treatment (p < 0.001). Tumours with networks, loops, arcs with branching and parallel with crossing showed an increased regression as compared to other MCPs. Twelve patients whose tumours contained complex MCPs developed metastatic disease. CONCLUSIONS: This study suggests that the response of choroidal melanoma to irradiation is related to MCPs as identified by ICG angiography; the presence of complex MCPs is associated with a high regression rate after plaque therapy and a high risk of development of systemic metastatic disease.

Dual targeting of tumor and endothelial cells by gonadotropin-releasing hormone agonists to reduce melanoma angiogenesis.

We showed previously that GnRH receptors are expressed in melanoma cells; their activation reduces cell growth and metastatic behavior. Here, we investigated whether GnRH agonists might affect the expression of genes involved in melanoma progression. By genome-wide transcriptomic and real-time PCR analysis, we first observed that GnRH agonists decrease the expression of the pro-angiogenic factor vascular endothelial growth factor (VEGF) (all isoforms) in BLM melanoma cells. Then, we demonstrated that GnRH agonists specifically decrease the expression of the VEGF165 isoform as well as its secretion from BLM cells. These data suggested that activation of GnRH receptors might reduce the pro-angiogenic behavior of melanoma cells. To verify this hypothesis, we treated BLM cells with a GnRH agonist; the conditioned medium from these cells was tested to assess its capability to stimulate human umbilical vein endothelial cell (HUVEC) motility. The migration of HUVECs towards the conditioned medium of GnRH agonist-treated BLM cells was significantly lower than the migration of HUVECs toward the conditioned medium of untreated cells. Thus, GnRH agonists reduce the pro-angiogenic behavior of melanoma cells through a decreased production of bioactive VEGF. We then found that GnRH receptors are also expressed on HUVECs and that GnRH agonists reduce their ability to proliferate and to form capillary-like tubes when stimulated by VEGF. These findings suggest that GnRH agonists exert an anti-angiogenic activity indirectly by decreasing VEGF secretion from tumor cells and directly by counteracting the pro-angiogenic activity of the growth factor. These data might lead to the development of novel targeted approaches for melanoma.

Intravascular presence of tumor cells as prognostic parameter in uveal melanoma: a 35-year survey.

PURPOSE: Invasion of tumor cells into blood vessels is essential for metastasis of uveal melanoma. The occurrence of ingrowth of tumor cells in blood vessels in uveal melanoma was analyzed, and this parameter was compared with the survival of the patients. METHODS: Between 1972 and 2007, 643 eyes primarily enucleated for uveal melanoma were evaluated histopathologically. Survival data were obtained from charts and from the Integral Cancer Center patient registry. RESULTS: No vascular ingrowth of tumor cells occurred in 59% of the eyes, whereas 18% had tumor cell ingrowth in vessels inside the tumor, 10% in vessels outside the tumor, and 8% in vessels inside as well as outside the tumor. The presence of any intravascular ingrowth of tumor cells correlated significantly with the diameter (P < 0.01) and prominence of the tumor (P < 0.01), as well as with non-spindle-cell type (P = 0.03) and intrascleral ingrowth (P < 0.01), and was associated with a worse survival. When extravascular matrix patterns were not included in the multivariate analysis, intravascular ingrowth came out as an independent prognostic factor, but this was not the case when extravascular matrix patterns were included in the multivariate model. CONCLUSIONS: Intravascular ingrowth of tumor cells in uveal melanoma occurs frequently in combination with well-known histopathologic factors such as large tumor size, epithelioid cell type, and intrascleral ingrowth.

Combination therapy targeting the tumor microenvironment is effective in a model of human ocular melanoma.

BACKGROUND: Ocular melanoma is the leading intraocular malignancy. There is no effective treatment for metastatic ocular melanoma. We sought a treatment targeting the tumor microenvironment as well as the tumor cells. METHODS: Migration of HUVEC cells, the ability of HUVEC cells to form tubes, and proliferative capacity of a human ocular melanoma cell line were tested in the presence of lenalidomide and sorafenib alone and in combination. The compounds were also tested in a rat aortic ring assay and were tested in a highly aggressive human ocular melanoma xenograft model. RESULTS: Lenalidomide and Sorafenib inhibit HUVEC ability to migrate and form tubes and when used in combination the inhibition is increased. The agents alone and in combination inhibit outgrowth in the rat aortic ring model. The combination of the agents improved the inhibition over either single agent. In a xenograft model, combination therapy inhibited tumor growth over inhibition by single agent alone in a significant fashion (p < 0.004: lenalidomide and p < 0.0035: sorafenib). Furthermore, spontaneous lung metastasis development was completely inhibited in the combination treated animals. Sixty percent of vehicle treated animals developed lung metastases compared to 50% of lenalidomide treated animals, and 33% of sorafenib treated animals. CONCLUSION: Lenalidomide and sorafenib are effective at targeting endothelial cells, inhibiting growth of ocular melanoma cells and can inhibit growth of tumors in a xenograft model as well as inhibit development of metastases. Combining these agents works in an additive to synergistic way to inhibit the growth of tumors and development of metastases.

Real-time optoacoustic monitoring of vascular damage during photodynamic therapy treatment of tumor.

The optoacoustic technique is a noninvasive imaging method with high spatial resolution. It potentially can be used to monitor anatomical and physiological changes. Photodynamic therapy (PDT)-induced vascular damage is one of the important mechanisms of tumor destruction, and real-time monitoring of vascular changes can have therapeutic significance. A unique optoacoustic system is developed for neovascular imaging during tumor phototherapy. In this system, a single-pulse laser beam is used as the light source for both PDT and for concurrently generating ultrasound signals for optoacoustic imaging. To demonstrate its feasibility, this system is used to observe vascular changes during PDT treatment of chicken chorioallantoic membrane (CAM) tumors. The photosensitizer used in this study is protoporphyrin IX (PpIX) and the laser wavelength is 532 nm. Neovascularization in tumor angiogenesis is visualized by a series of optoacoustic images at different stages of tumor growth. Damage of the vascular structures by PDT is imaged before, during, and after treatment. Rapid, real-time determination of the size of targeted tumor blood vessels is achieved, using the time difference of positive and negative ultrasound peaks during the PDT treatment. The vascular effects of different PDT doses are also studied. The experimental results show that a pulsed laser can be conveniently used to hybridize PDT treatment and optoacoustic imaging and that this integrated system is capable of quantitatively monitoring the structural change of blood vessels during PDT. This method could be potentially used to guide PDT and other phototherapies using vascular changes during treatment to optimize treatment protocols, by choosing appropriate types and doses of photosensitizers and doses of light.

Sorafenib in advanced melanoma: a Phase II randomised discontinuation trial analysis.

The effects of sorafenib--an oral multikinase inhibitor targeting the tumour and tumour vasculature--were evaluated in patients with advanced melanoma enrolled in a large multidisease Phase II randomised discontinuation trial (RDT). Enrolled patients received a 12-week run-in of sorafenib 400 mg twice daily (b.i.d.). Patients with changes in bi-dimensional tumour measurements <25% from baseline were then randomised to sorafenib or placebo for a further 12 weeks (ie to week 24). Patients with > or =25% tumour shrinkage after the run-in continued on open-label sorafenib, whereas those with > or =25% tumour growth discontinued treatment. This analysis focussed on secondary RDT end points: changes in bi-dimensional tumour measurements from baseline after 12 weeks and overall tumour responses (WHO criteria) at week 24, progression-free survival (PFS), safety and biomarkers (BRAF, KRAS and NRAS mutational status). Of 37 melanoma patients treated during the run-in phase, 34 were evaluable for response: one had > or =25% tumour shrinkage and remained on open-label sorafenib; six (16%) had <25% tumour growth and were randomised (placebo, n=3; sorafenib, n=3); and 27 had > or =25% tumour growth and discontinued. All three randomised sorafenib patients progressed by week 24; one remained on sorafenib for symptomatic relief. All three placebo patients progressed by week-24 and were re-started on sorafenib; one experienced disease re-stabilisation. Overall, the confirmed best responses for each of the 37 melanoma patients who received sorafenib were 19% stable disease (SD) (ie n=1 open-label; n=6 randomised), 62% (n=23) progressive disease (PD) and 19% (n=7) unevaluable. The overall median PFS was 11 weeks. The six randomised patients with SD had overall PFS values ranging from 16 to 34 weeks. The most common drug-related adverse events were dermatological (eg rash/desquamation, 51%; hand-foot skin reaction, 35%). There was no relationship between V600E BRAF status and disease stability. DNA was extracted from the biopsies of 17/22 patients. Six had V600E-positive tumours (n=4 had PD; n=1 had SD; n=1 unevaluable for response), and 11 had tumours containing wild-type BRAF (n=9 PD; n=1 SD; n=1 unevaluable for response). In conclusion, sorafenib is well tolerated but has little or no antitumour activity in advanced melanoma patients as a single agent at the dose evaluated (400 mg b.i.d.). Ongoing trials in advanced melanoma are evaluating sorafenib combination therapies.

Echographic examination with new generation contrast agent of choroidal malignant melanomas.

PURPOSE: To determine the efficacy of echography with new generation contrast agents in visualizing vascularization of choroidal malignant melanomas. METHODS: An echographic contrast medium consisting of phospholipidic microbubbles filled with sulphur hexafluoride (Sonovue) was used to visualize microcirculation in 25 cases of choroidal lesions already diagnosed with standardized echography (21 choroidal malignant melanomas, four disciform lesions). RESULTS: In untreated malignant melanomas contrast agent echography revealed the presence of a dense microcirculation inside the mass. In one case vitreal seeding of the contrast agent was detectable before enucleation and histological examination revealed the presence of tumoral cells. In 12 cases treated with transpupillary thermotherapy, contrast agent echographic evaluation showed heavy regression of microcirculation after 1 week, confirmed in one case by histology, and a reduction of 70-80% in dimensions after 6 months (which appeared to have stabilized at subsequent examinations). In four cases treated with proton beam brachytherapy 2 years prior to our examination, contrast agent echography showed the absence of a microvascular network and the presence of large vessels and blood lakes. In four cases of disciform lesion, deep and superficial retina-associated vascularization was observed, with a weak spread of contrast agent inside the lesion. CONCLUSION: Live representation with good resolution of choroidal malignant melanoma microcirculation was obtained.

The impact of surgery and mild hyperthermia on tumor response and angioneogenesis of malignant melanoma in a rat perfusion model.

BACKGROUND: The aim of this experimental study was to determine the effect of mild hyperthermia on tumor response and angioneogenesis in an isolated limb perfusion model with a human melanoma xenograft. METHODS: A human melanoma xenograft was implanted into the hindlimbs of 30 athymic nude rats. The animals were randomized into five groups: group I: control, group II: sham group, group III: external hyperthermia with a tissue temperature of 41.5 degrees C for 30 minutes without ILP, group IV: normothermic ILP (tissue temperature 37 degrees C for 30 minutes, group V: hyperthermic ILP (tissue temperature 41.5 degrees C for 30 minutes). Tumor response was evaluated by tumor size determination and immunohistochemical analysis 6 weeks postoperatively. Tissue sections were investigated for expression of CD34 and basic fibroblast growth factor (bFGF). RESULTS: Average tumor volumes of the controls (I) increased from 105 mm3 to 1388 mm3. In the sham operated group (II) tumor volumes were significantly larger than in group I. Tumor volumes in group IV were significantly smaller than in group I and lowest in group V. There were no significant differences in size between group I and group III after six weeks. In group III and IV each, 5 animals showed tumor progression and one had a partial tumor response. In group V only 2 animals showed tumor progression. Immunhistochemical analysis of the tissue sections demonstrated that angioneogenesis was more pronounced in group II than in group I and less pronounced in group IV and V compared with group I. CONCLUSIONS: Our results suggest that even a surgical manipulation such as a skin incision promotes tumor growth, probably by induction of growth factors like bFGF. External hyperthermia of 41.5 degrees C tissue temperature for 30 minutes only has no impact on tumor growth and angioneogenesis in vivo.

In vivo and in vitro evaluation of erianin, a novel anti-angiogenic agent.

This study evaluated the anti-angiogenic activities of erianin in vivo and in vitro. Erianin, a natural product from Dendrobium chrysotoxum, caused moderate growth delay in xenografted human hepatoma Bel7402 and melanoma A375 and induced significant vascular shutdown within 4 h of administering 100 mg/kg of the drug. Erianin also displayed potent anti-angiogenic activities in vitro: it abrogated spontaneous or basic fibroblast growth factor-induced neovascularisation in chick embryo; it inhibited proliferation of human umbilical vein endothelial cells (EC(50) 34.1+/-12.7 nM), disrupted endothelial tube formation, and abolished migration across collagen and adhesion to fibronectin. Erianin also exerted selective inhibition toward endothelial cells, and quiescent endothelium showed more resistance than in proliferative and tumour conditions. In a cytoskeletal study, erianin depolymerised both F-actin and beta-tubulin, more significantly in proliferating endothelial cells than in confluent cells. In conclusion, erianin caused extensive tumour necrosis, growth delay and rapid vascular shutdown in hepatoma and melanoma models; it inhibited angiogenesis in vivo and in vitro and induced endothelial cytoskeletal disorganisation. These findings suggest that erianin has the therapeutic potential to inhibit angiogenesis in vivo and in vitro.

[Use of indocyanine green for photodynamic therapy of choroidal melanoma--preliminary report]. / Zastosowanie zieleni indocyjaninowej w leczeniu fotodynamicznym czerniaka naczyniówki--doniesienie wstepne.

PURPOSE: To evaluate the usefulness of indocyanine green (ICG) in the photodynamic therapy (iPDT) of choroidal melanoma. MATERIAL AND METHODS: Thirty eight patients with choroidal melanoma were treated with iPDT. Tumor thickness ranged from 2.6 to 4.1 mm in ultrasonography. Before iPDT Ruthenium-106 plaques were used in 18 cases, in 6 cases with additional transpupillary thermotherapy ("sandwich method"), and Iodine-125 plaques in 3 cases. In 11 eyes the iPDT was the only performed treatment. Therapy was performed with 810 nm diode laser after intravenous injection of 25 mg solution of indocyanine green. Six months after iPDT ophthalmological examination was performed with ultrasonography and ICGA images evaluation. RESULTS: The baseline ICG study showed pathological intrinsic vasculature in all examined cases. Six months after iPDT changes in microcirculation, as well as significant decrease of tumors thickness in ultrasonography (mean 38%), were detected in all cases. Complete regression of intrinsic vessels was demonstrated by ICGA in 26 cases, and partial regression of pathological vascularization was found in 12 patients. CONCLUSIONS: Our preliminary study suggests, that ICG mediated iPDT can be a new and promising way, to treat choroidal melanoma.

Primary transpupillary thermotherapy (TTT) for malignant choroidal melanoma.

PURPOSE: To evaluate the efficacy of transpupillary thermotherapy (TTT) as the only method of treatment for small choroidal melanoma. PATIENTS AND METHODS: In a prospective non-randomized analysis, 20 patients with primary choroidal melanoma (posterior to the equator with base < or = 2 and thickness < or = 4.5 mm) were treated with TTT as the only method of treatment (diode laser at 810 nm, beam diameter 3 mm, power setting 0.3-0.9 W, exposure time 20-37 min). During follow-up of at least 6 months, clinical aspects, ultrasonographic tumour thickness, fluorescein and indocyanine green angiographic patterns, visual acuity and ocular side-effects were recorded. RESULTS: In 17 eyes the tumour regressed significantly within 3 months after one treatment session (to a flat chorioretinal scar in 15 eyes). Despite a clinically flattened chorioretinal scar, fluorescein and indocyanine green angiography revealed that choriocapillary vessels in the heat-treated areas of 15 eyes remained perfused. Three amelanotic melanomas showed almost no response to TTT after repeated treatment at higher power settings. Visual acuity remained unchanged or improved in 12 eyes. Ocular side-effects included posterior synechia of the iris (1), macular oedema (2) and temporary retrobulbar pain (2). No patient showed tumour recurrence or metastases during follow-up. CONCLUSIONS: Preliminary results obtained by this study demonstrate good efficacy and visual outcome following TTT as the only method of treatment for small choroidal melanoma. However, indocyanine green angiographic findings suggest that tissue damage in the choroidal layer might be less effective, which perhaps may lead to a higher rate of tumour regrowth. Long-term follow-up is required to obtain data on late ocular side-effects, tumour recurrence and metastasis.

The response of human tumour blood flow to a fractionated course of thermoradiotherapy.

The response of human tumour blood flow to a fractionated course of thermoradiotherapy was documented in four superficial but bulky tumours (three adenocarcinomas, one melanoma). Blood flow was measured 15, 30, 45, and 60 min after the onset of heating. These measurements were made at the same intra-tumour point during each heat fraction by use of a modified thermal clearance technique in which a correction was made for the heat dissipated by thermal conduction. This point was at least 2 cm beneath the surface in the central portion of the tumour. Extracellular pH was measured within 1 cm of this point prior to the first heat fraction and 2-3 weeks later. Hyperthermia was administered for 60 min, twice a week for 4 weeks by use of a 16-channel 915 MHz microwave applicator. Each patient also received a radiation dose of 40 Gy fractionated at 2 Gy/fx, five times a week (adenocarcinomas) or 4 Gy/fx, twice a week (melanoma). Blood flow remained relatively constant during heating after steady state conditions were attained. However, an overall decrease in tumour blood flow was observed in each patient over the course of thermoradiotherapy. In each case, a relatively small decrease in blood flow occurred between most heat fractions which resulted in an overall decrease which ranged from 50-100%. However, there was a tendency for blood flow to increase following the initial heat fraction at points where the steady state temperature was approximately 41 degrees C or less. Extracellular pH increased in two of three patients and decreased in the other.

Expression of hyaluronidase by tumor cells induces angiogenesis in vivo.

Hyaluronic acid is a proteoglycan present in the extracellular matrix and is important for the maintenance of tissue architecture. Depolymerization of hyaluronic acid may facilitate tumor invasion. In addition, oligosaccharides of hyaluronic acid have been reported to induce angiogenesis. We report here that a hyaluronidase similar to the one on human sperm is expressed by metastatic human melanoma, colon carcinoma, and glioblastoma cell lines and by tumor biopsies from patients with colorectal carcinomas, but not by tissues from normal colon. Moreover, angiogenesis is induced by hyaluronidase+ tumor cells but not hyaluronidase- tumor cells and can be blocked by an inhibitor of hyaluronidase. Tumor cells thus use hyaluronidase as one of the "molecular saboteurs" to depolymerize hyaluronic acid to facilitate invasion. As a consequence, breakdown products of hyaluronic acid can further promote tumor establishment by inducing angiogenesis. Hyaluronidase on tumor cells may provide a target for anti-neoplastic drugs.

Local and systemic toxicity in 'borderline true' hyperthermic isolated perfusion for lower limb melanoma.

This paper reports clinical results of hyperthermal (41.5 degrees C) isolated perfusion of the lower limb for melanoma treatments in association with cytostatic drugs (L-PAM) in 10 consecutive patients. Attention is focussed on the toxicity effects in the search for a possible correlation between the treatment variables and toxicity. Careful heat administration techniques and thorough and accurate temperature monitoring have resulted in a uniform and closely controlled temperature distribution, allowing us to approach the limiting temperature value for possible damage both of the limb tissues and of the perfusate. Care was taken to avoid perfusate overheating (42 degrees C maximum). Local toxicity was observed between grades II and III. Systemic toxicity was of insignificant level. The clinical results show that high-temperature treatments with simultaneous administration of the cytostatic are feasible with acceptable toxicity. Further clinical investigations are recommended to ascertain the efficacy of the method.