Lack of lymphangiogenesis despite coexpression of VEGF-C and its receptor Flt-4 in uveal melanoma.

PURPOSE: Because lymphatic vessels are absent from the normal eye and because uveal melanomas are presumed to spread by a hematogenous route in the absence of tumor exposure to conjunctival lymphatics, this study was undertaken to investigate the presence of lymphatic vessels in primary uveal melanomas. METHODS: The presence of lymphatics in 2 control eyes and in 33 primary uveal, 10 primary cutaneous, and 3 metastatic cutaneous melanomas was evaluated by using a double-immunostaining protocol that differentially highlights blood and lymphatic vasculature. In addition, 14 uveal melanomas were immunostained for the lymphatic growth factor vascular endothelial growth factor (VEGF)-C (with anti-VEGF-C polyclonal antibodies [pAbs]), its receptors Flt-4 (with monoclonal antibody [mAb] 9D9) and KDR (with anti-KDR mAb [Clone KDR-2]), and the hemangiogenic factor VEGF-A (with anti-VEGF pAbs). RESULTS: Lymphatics were not detected in normal eyes or in uveal melanoma. As a consequence, signs of lymphangiogenesis were not present. There was coexpression of VEGF-C with Flt-4 and KDR in 6 (43%) of the 14 melanomas. Staining for VEGF-A was completely negative in 25 uveal melanomas analyzed. CONCLUSIONS: The strictly hematogenous metastasis of primary uveal melanomas is explained by the absence of lymphatics in and around the tumor. The current data suggest that, in the presence of endothelial Flt-4, VEGF-C expression is not sufficient to induce lymphangiogenesis from preexisting blood vessels in human cancer.

Complete response of melanoma-in-transit metastasis after isolated limb perfusion with tumor necrosis factor alpha and melphalan without massive tumor necrosis: a clinical and histopathological study of the delayed-type reaction pattern.

Treatment of stage IIIA/B melanoma patients by isolated limb perfusion (ILP) with a combination of tumor necrosis factor-alpha (TNF-alpha) and melphalan induces a complete response in 80-90% of the cases. The mechanism of tumor regression induced by the combination of TNF-alpha and melphalan is not precisely understood. Previous studies focused on the immediate (ie., within a few days) clinico-pathological changes after perfusion involving hemorrhagic necrosis. However, clinical data clearly indicate that complete tumor remission frequently requires a period of a few weeks to as much as months after ILP. Because the mechanism underlying this delayed-type reaction is completely unknown, we studied the clinico-pathological events in patients with such slowly regressing melanoma lesions. For this purpose, 94 biopsies of in-transit melanoma metastasis that were taken sequentially from 11 patients between 1 week and 9 months after ILP were analyzed by light and electron microscopy and immunohistochemistry. Clinical data included patient sex, age, anatomical localization and size of the tumor, and follow-up. All of the 11 patients ultimately responded to perfusion treatment (9 complete, 1 partial, 1 stable disease). Serial biopsies showed scattered individual tumor cell necrosis without hemorrhage. Most of the lesions with this delayed-type reaction pattern were less than 0.5 cm in diameter. They contained varying amounts of histologically viable-looking tumor cells and tumor-infiltrating melanophages. In addition, a marked but transient infiltrate of peritumoral eosinophils and moderate interstitial edema and dermal fibrosis were encountered. Only small numbers of lymphocytes were present. In comparison with the reaction pattern after treatment with melphalan alone, the delayed-type reaction pattern was similar but more intense. The scattered tumor cell necrosis in the latter type may be explained by a TNF-alpha-induced increase in permeability of the tumor vascular bed, which results in higher intratumoral concentrations of melphalan or in a prolongation of its effect. Subsequently, degenerated tumor cells are cleared by macrophages, and, finally, repair by fibrosis occurs. Because the immediate reaction type is evoked by hyperpermeability of the tumor vessels as well, quantitative differences seem to determine which reaction type ensues. We suggest that the extent of tumor vasculature that is sensitive to TNF-alpha determines the onset and histopathological pattern of tumor regression after ILP.

An improved procedure to quantify tumour vascularity using true colour image analysis. Comparison with the manual hot-spot procedure in a human melanoma xenograft model.

In a number of recent papers, the degree of tumour vascularization has been described as a promising new prognostic factor. Methods for the assessment of vascular density involve immunohistochemical staining of the vasculature, followed by counting the number of vessel profiles in the angiogenic hot spot. One of the problems of this procedure is the selection of the angiogenic hot spot, which has been described as being subject to inter-observer variation. In this study, the value of true colour image analysis in reducing inter-observer variation has been assessed. Highly (MV3) and poorly (M14) vascularized human melanoma xenografts were used to evaluate the image analysis procedure, and the image analysis results were compared with results from the conventional manual hot-spot procedure. Assessment by image analysis was performed on measurement fields covering the entire tumour tissue specimens rather than on a single hot-spot field. Also, by selecting the most densely vascularized area from all fields assessed by the semi-automatic procedure, it was possible to objectify the hot spot selection (automated hot-spot procedure). Manual assessment showed a good correlation between two independent observers for MV3 xenografts (r = 0.74, P = 0.014), but a poor correlation for M14 xenographs (r = 0.4, P > 0.05). Automated assessment by different operators showed good correlations for both MV3 xenografts (r = 0.99, P < 0.001) and M14 xenografts (r = 0.80, P = 0.006). It is concluded that although both manual vessel counting and semi-automated image analysis can differentiate between the level of vascularization in the two types of xenograft (P < 0.001 for both methods), the automated method is favourable in that it showed no significant inter-observer effects. In M14 xenografts, the manual hot-spot vessel densities did not correlate well with the automated hot-spot densities (r = 0.27, P > 0.05), indicating that selection of angiogenic hot spots in this tumour type is indeed subject to observer bias. The automated hot-spot vessel densities were a reliable indicator of overall tumour vessel density in both tumour types. Image analysis allows analysis of vessel subclasses based on morphological criteria such as vessel profile area or diameter. In the model system used, the discrimination between MV3 and M14 xenografts was further enhanced by selectively examining vessels with diameters between 6 and 9 microns (P < 0.0005). In conclusion, image analysis appears to offer an objective and more reproducible method to quantify tumour vascularity than manual counting of vessel profiles in the hot spot. Analysis of subclasses of vessels may further enhance the value of vessel density measurements in discriminating between tumour types differing in biological behaviour.

Tolerance of rat spinal cord to continuous interstitial irradiation.

PURPOSE: To study the kinetics of repair in rat spinal cord during continuous interstitial irradiation at different dose rates and to investigate the impact of a rapid dose fall off over the spinal cord thickness. MATERIAL AND METHODS: Two parallel catheters were inserted on each side of the vertebral bodies from the level of T10 to L4. These catheters were afterloaded with two 192Ir- wires of 4 cm length each (activity 1-10 mCi/cm) or connected to the HDR- microSelectron. Experiments have been carried out to obtain complete dose response curves at 7 different dose rates: 0.53, 0.90, 1.64, 2.56, 4.4, 9.9 and 120 Gy/h. Paralysis of the hindlegs after 5 - 6 months and histopathological examination of the spinal cord of each animal were used as experimental endpoints. RESULTS: The distribution of the histological damage was a good reflection of the rapid dose fall - off over the spinal cord, with white matter necrosis or demyelination predominantly seen in the dorsal tracts of the spinal cord or dorsal roots. With each reduction of the dose rate, spinal cord tolerance was significantly increased, with a maximum dose rate factor of 4.3 if the dose rate was reduced from 120 Gy/h to 0.53 Gy/h (ED50 of 17.3 Gy and 75.0 Gy, respectively). Estimates of the repair parameters using different types of analysis are presented. For the direct analysis the best fit of the data was obtained if a biexponential function for repair was used. For the 100% dose prescribed at the ventral side of the spinal cord the alpha/beta ratio is 1.8 Gy (0.8 - 2.8) and two components of repair are observed: a slow component of repair of 2.44 h (1.18 - infinity) and a fast component of 0.15 h (0.02 - infinity). The proportion of the damage repaired with the slow component is 0.59 (0.18 - 1). For the maximum of 150% of the prescribed dose at the dorsal side of the spinal cord the alpha/beta ratio is 2.7 Gy (1.5 - 4.4); the two components for the kinetics of repair remain the same. CONCLUSIONS: Spinal cord radiation tolerance is significantly increased by a reduction in dose rate. Depending on the dose prescription, the alpha/beta ratio is 1.8 or 2.7 Gy for the 100% and 150% of the reference dose (rate), respectively; for the kinetics of repair a biphasic pattern is observed, with a slow component of 2.44 hours and a fast component of 0.15 hours, which is independent of the dose prescription.

Vascular density in melanoma xenografts correlates with vascular permeability factor expression but not with metastatic potential.

We studied the relation between tumour vascular density and tumour growth rate, metastatic incidence and vascular permeability factor (VPF) mRNA levels in a human xenograft model described previously. Vascular density was determined by automated image analysis. Xenografts derived from cell lines BLM and MV3 showed the highest mean vascular density (MVD), the highest in vivo growth rate, high VPF mRNA levels and rapid development of lung metastases. Xenografts of cell lines M14, Mel57 and MV1 showed a significantly lower degree of vascularization, lower in vivo growth rates and lower levels of VPF mRNA, but formed lung metastases with a similar incidence as those of BLM and MV3. Xenografts from cell line 1F6 did not form lung metastases, whereas tumours derived from a spontaneous mutant of 1F6, designated 1F6m, gave rise to lung metastases to the same extent as Mel57, M14 and MV1 tumours. MVD values in 1F6 and 1F6m xenografts, VPF mRNA levels and in vivo growth rates of 1F6 and 1 F6m xenografts, however, were similar. In conclusion, in the melanoma xenograft model vascular density is correlated with in vivo growth rate and with in vitro VPF mRNA levels, but not with the ability to metastasize.

Synergistic effects of TNF-alpha and melphalan in an isolated limb perfusion model of rat sarcoma: a histopathological, immunohistochemical and electron microscopical study.

Isolated limb perfusion (ILP) with tumour necrosis factor alpha (TNF-alpha) and melphalan has shown impressive results in patients with irresectable soft tissue sarcomas and stage III melanoma of the extremities. The mechanisms of the reported in vivo synergistic anti-tumour effects of TNF-alpha and melphalan are not precisely understood. We have developed an ILP model in the rat using a non-immunogenic sarcoma in which similar in vivo synergy is observed. The aim of this present study was to analyse the morphological substrate for this synergistic response of TNF-alpha in combination with melphalan to shed more light on the pathomechanisms involved. Histology of the tumours from saline- (n = 14) and melphalan-treated (n = 11) rats revealed apparently vital tumour cells in over 80% of the cross-sections. Interstitial oedema and coagulation necrosis were observed in the remaining part of the tumour. Haemorrhage was virtually absent. TNF-alpha (n = 22) induced marked oedema, hyperaemia, vascular congestion, extravasation of erythrocytes and haemorrhagic necrosis (20-60% of the cross-sections). Oedema and haemorrhage suggested drastic alterations of permeability and integrity of the microvasculature. Using light and electron-microscopy, we observed that haemorrhage preceded generalised platelet aggregation. Therefore, we suggest that the observed platelet aggregation was the result of the microvascular damage rather than its initiator. Remarkably, these events hardly influenced tumour growth. However, perfusion with the combination of TNF-alpha and melphalan (n = 24) showed more extensive haemorrhagic necrosis (80-90% of the cross-sections) and revealed a prolonged remission (mean 11 days) in comparison with the other groups of rats. Electron microscopical analysis revealed similar findings as described after TNF-alpha alone, although the effects were more prominent at all time points after perfusion. In conclusion, our findings suggest that the enhanced anti-tumour effect after the combination of TNF-alpha with melphalan results from potentiation of the TNF-alpha-induced vascular changes accompanied by increased vascular permeability and platelet aggregation. This may result in additive cytotoxicity or inhibition of growth of residual tumour cells.

Transient induction of E-selectin expression following TNF alpha-based isolated limb perfusion in melanoma and sarcoma patients is not tumor specific.

Endothelial injury of the tumor microvasculature after isolated limb perfusion (ILP) with TNF-alpha and melphalan is considered to play an important role in the pathogenesis of tumor necrosis. It is thought to follow endothelial cell activation and subsequent attraction of polymorphonuclear cells (PMNs). The observed selectivity for the tumor could be due to preferential overexpression of cell-adhesion molecules by the tumor vasculature. We tested this proposition by analyzing sequential biopsies from both tumor and normal distant skin, taken from melanoma and sarcoma patients before ILP and at 30 min and 24 h after ILP. Histopathologically confirmed complete response was observed in six of seven melanoma patients, 1-8 months after ILP. By using immunohistochemistry on the light- and electron-microscopic level, the expression patterns of intercellular adhesion molecules-1 (ICAM-1), E-selectin (ELAM-1), VCAM-1, and PECAM-l were examined. In addition, the results were compared with the effects on HUVECs (human umbilical vein endothelial cells) in vitro of transient exposure of the agents used during ILP. ICAM-1 and PECAM-1 were constitutively expressed on vascular endothelial cells, both in normal tissues and in the tumor lesions. In biopsies taken 30 min after termination of the perfusion, a moderate induction of E-selectin expression on the vascular endothelium in the tumors and a marked expression on the vasculature in the perfused normal skin were observed. It decreased within 24 h after perfusion in both normal skin and in the tumor. The upregulation of E-selectin was accompanied neither by an influx of neutrophils nor by hemorrhagic necrosis. There were no drastic changes in the expression of VCAM-1, ICAM-1, or PECAM- 1. These findings imply that the upregulation of E-selectin after ILP is not restrfcted to the tumor microvasculature and that, therefore, these microvascular events seem not to be the decisive pathomechanism responsible for tumor regression.

VWF release and platelet aggregation in human melanoma after perfusion with TNF alpha.

Twenty-nine stage IIIA/B melanoma patients treated by isolated limb perfusion (ILP) with a high dose of recombinant human tumour necrosis factor alpha (rHuTNF alpha), interferon gamma (IFN gamma), and melphalan were histologically documented with emphasis on therapy-induced changes of the tumour vasculature. Sequential biopsies were taken at various intervals before and after the treatment to compare the morphological change. In order to visualize microvascular changes, immunostaining was performed for von Willebrand factor (VWF), type IV collagen, alpha-smooth muscle actin, endothelial antigen PAL-E, tissue factor, CD41 (thrombocyte marker), and fibrin. In biopsies prior to perfusion, necrosis, haemorrhage, and fibrin thrombi were not found. Within 3 h following triple combination therapy, a change in the distribution of VWF staining occurred, from a discrete endothelial pattern in the untreated lesions to a fuzzy perivascular and subepidermal pattern in the treated lesions. Within 24 h, this was accompanied by intravascular thrombocyte aggregation and erythrostasis, in the absence of tissue factor and fibrin deposits. These findings indicate that the thrombocyte aggregation observed is not caused by local procoagulant activity, but is rather the result of the therapy-associated vascular damage or haemostasis. Although it is difficult to derive the dynamics of this process from static images, we assume that TNF alpha induced endothelial cell damage, leading to VWF release. Release VWF may play a role in the adhesion between thrombocytes and the damaged endothelium or the denuded subendothelium. As a consequence, the blood flow is impaired, leading to congestion and oedema, compatible with an early stage of haemorrhagic infarction.

E-cadherin expression in head and neck squamous-cell carcinoma is associated with clinical outcome.

The cell-cell adhesion molecule E-cadherin has been shown to suppress invasive growth of epithelial cells in vitro, and loss of its expression is thought to be important in invasion and metastatic potential of epithelial tumors in vivo. We retrospectively studied the level of E-cadherin expression in 50 primary head and neck squamous-cell carcinomas (HNSCC) by immunohistochemical methods, on frozen sections, using anti-E-cadherin monoclonal antibody (MAb) 6F9. It concerned patients with different stages of carcinoma of larynx or oral cavity who had been treated with curative intention 30 months or more before. Percentages of membranous stained tumor cells were scored in 1 of 5 categories. Scores were generally low, as in 11/50 lesions < or = 5% cells were stained, and in 19/50 lesions only 6-25% cells showed membranous staining. In 9 lymph-node metastases evaluated, E-cadherin expression was in the same range as in the primary tumors. There was a significant correlation between the level of membranous E-cadherin expression in the primary tumor and the degree of differentiation. No relation was found with tumor size (pT) or regional lymph-node classification (pN). Nevertheless, 29 patients surviving > or = 30 months without evidence of disease had significantly higher levels of membranous E-cadherin expression in their primary tumors than 10 patients with unfavorable clinical course clearly related to recurrent and/or metastatic HNSCC. Moreover, this could only partially be explained by distinctions in differentiation grade between both groups. Our results suggest that membranous E-cadherin expression has prognostic importance in patients with HNSCC.

Human leukocyte antigen expression in renal cell carcinoma lesions does not predict the response to interferon therapy.

Human leukocyte antigen (HLA) classes I and II molecules are essential for antigen presentation to cytotoxic T cells and helper T cells, respectively. Consequently, they may play a role in anticancer immunotherapy as well. We studied whether the pretreatment HLA phenotype of the tumor is predictive for response to interferon immunotherapy in vivo. Therefore, renal cell carcinoma (RCC) primary tumor lesions from 31 patients treated with interferon-alpha and interferon-gamma (13 responders and 18 nonresponders) were analyzed retrospectively for HLA antigen expression with immunohistochemical methods. Furthermore, from eight patients, pretreatment metastatic lesions were examined. In the primary tumors HLA class I expression was high: in 26 of 30 lesions more than 50% cells were stained. HLA class II expression was mostly low: in 14 of 31 primary tumors less than 5% cells were stained. A significant correlation was found between HLA phenotype of primary tumors and corresponding metastases. There was no association between tumor HLA classes I and II antigen expression and clinical response to interferon therapy. In conclusion, pretreatment HLA phenotype of RCC has no predictive value for outcome of interferon immunotherapy. A role for treatment-induced changes in HLA expression in vivo, however, can not be excluded. These findings do not provide indications for the working mechanism of interferon immunotherapy in vivo.

HLA antigen expression in routinely processed head and neck squamous cell carcinoma primary lesions of different sites.

HLA class I and II antigen expression in routinely processed head and neck squamous cell carcinoma (HNSCC) primary lesions was evaluated. Paraffin embedded samples from 66 squamous cell carcinomas and 7 verrucous carcinomas were studied immunohistochemically using recently developed anti-HLA class I monoclonal antibodies (MAbs) HC10 and HCA2, and anti-HLA-DR rabbit serum. Percent stained tumor cells were scored in 1 of 5 categories. The scores of 40 tumors were compared to the staining results obtained on frozen sections of the corresponding lesions, including those of the anti-class I MAb W6/32. High percentage-matched scores for paraffin and frozen sections were obtained, with HC10 vs. HC10, HC10 vs. W6/32, and anti-HLA-DR vs. anti-HLA-DR showing the best correlations. In the squamous cell carcinomas HLA class I expression was high (i.e., in 49/66 lesions more than 50% cells were stained), and correlated with the degree of differentiation, and inversely with the modified Jakobsson score. HLA class II expression (more than 5% cells stained) was found in 21/66 tumors and correlated inversely with the degree of differentiation. All verrucous carcinomas exhibited very high HLA class I expression, whereas class II was locally expressed in 5/7 lesions. Comparison of 4 subsites of HNSCC showed that carcinomas of the oral cavity had the highest HLA class I expression. This suggests susceptibility to CD8+ T cells, and together with the well developed submucosal lymphoid tissue, makes the oral cavity carcinomas probably well suited for local immunotherapeutic approaches in HNSCC.