Diverging trends in incidence and mortality, and improved survival of non-Hodgkin's lymphoma, in the Netherlands, 1989-2007.

BACKGROUND: We studied progress in the fight against non-Hodgkin's lymphoma (NHL) in the Netherlands by describing the changes in incidence, treatment, relative survival, and mortality during 1989-2007. PATIENTS AND METHODS: We included all adult patients with NHL [i.e. all mature B-, T-, and natural killer (NK) cell neoplasms, with the exception of plasma cell neoplasms], newly diagnosed in the period 1989-2007 and recorded in the Netherlands Cancer Registry (n=55 069). Regular mortality data were derived from Statistics Netherlands. Follow-up was completed up to 1 January 2009. Annual percentages of change in incidence, mortality, and relative survival were calculated. RESULTS: The incidence of indolent B-cell and T- and NK-cell neoplasms rose significantly (estimated annual percentage change=1.2% and 1.3%, respectively); incidence of aggressive B-cell neoplasms remained stable. Mortality due to NHL remained stable between 1989 and 2003, and has decreased since 2003. Five-year relative survival rates rose from 67% to 75%, and from 43% to 52%, respectively, for indolent and aggressive mature B-cell neoplasms, but 5-year survival remained stable at 48% for T- and NK-cell neoplasms. CONCLUSIONS: In the Netherlands, incidence of indolent mature B-cell and mature T- and NK-cell neoplasms has increased since 1989 but remained stable for aggressive neoplasms. Survival increased for all mature B-cell neoplasms, preceding a declining mortality and increased prevalence of NHL (17 597 on 1 January 2008).

Chronic lymphocytic leukaemia in the Netherlands: trends in incidence, treatment and survival, 1989-2008.

We present trends in incidence, early treatment and survival of Chronic Lymphocytic Leukaemia (CLL) between 1989 and 2008, based on population-based data from the Netherlands Cancer Registry. Incidence rates were stable at 5.1 per 100,000 person-years for males, but increased from 2.3 to 2.5 for females, especially for females aged 50-64 years (from 3.6 to 4.3). Patients were less likely to receive chemotherapy within six months, i.e. from 29% to 24% among males and from 25% to 21% among females. Five-year relative survival increased from 61% in 1989-1993 to 70% 2004-2008 for males, and from 71% to 76% for females. The relative excess risk of dying decreased in time to 0.7 (males) and 0.9 (females) in 2004-2008, reference 1989-1993, and increased with age to 2.9 (males) and 1.8 (females) in patients aged 75-94 years, reference 30-64 years. The increasing incidence among females aged 50-64 coincided with the introduction of mass screening for breast cancer, which resulted in a large group of women under increased surveillance and possibly led to increased detection of CLL. The increase in survival might be underestimated due to possible decreased or delayed registration of indolent cases and the retroactive effect of the introduction of new therapies.

Hepatosplenic gammadelta T-cell lymphoma: A case report.

A 53-year-old man complained of weight loss, night sweats, and splenomegaly. The patient was diagnosed with stage IV hepatosplenic gammadelta T-cell non-Hodgkin's lymphoma, a highly aggressive and rare form of peripheral T-cell lymphoma. After completing CHOP chemotherapy, the patient relapsed. He did not respond to subsequent alemtuzumab therapy.

FOXP1, a gene highly expressed in a subset of diffuse large B-cell lymphoma, is recurrently targeted by genomic aberrations.

The transcription factor Forkhead box protein P1 (FOXP1) is highly expressed in a proportion of diffuse large B-cell lymphoma (DLBCL). In this report, we provide cytogenetic and fluorescence in situ hybridization (FISH) data showing that FOXP1 (3p13) is recurrently targeted by chromosome translocations. The genomic rearrangement of FOXP1 was identified by FISH in three cases with a t(3;14)(p13;q32) involving the immunoglobulin heavy chain (IGH) locus, and in one case with a variant t(2;3) affecting sequences at 2q36. These aberrations were associated with strong expression of FOXP1 protein in tumor cells, as demonstrated by immunohistochemistry (IHC). The cases with t(3p13) were diagnosed as DLBCL ( x 1), gastric MALT lymphoma ( x 1) and B-cell non-Hodgkin's lymphoma, not otherwise specified ( x 2). Further IHC and FISH studies performed on 98 cases of DLBCL and 93 cases of extranodal marginal zone lymphoma showed a high expression of FOXP1 in approximately 13 and 12% of cases, respectively. None of these cases showed, however, FOXP1 rearrangements by FISH. However, over-representation of the FOXP1 locus found in one additional case of DLBCL may represent another potential mechanism underlying an increased expression of this gene.

Massive reduction of tumour load and normalisation of hyperprolactinaemia after high dose cabergoline in metastasised prolactinoma causing thoracic syringomyelia.

In 1970 a 20 year old woman presented with a pituitary chromophobe adenoma for which she underwent transfrontal pituitary surgery. In 1978 she had to be reoperated on because of local tumour recurrence, resulting in hypopituitarism. Bromocriptine (5 mg/day) was given for 15 years, but the plasma prolactin levels remained elevated. In 2000 the patient presented with signs and symptoms suggestive of a spinal cord lesion at the mid-thoracic level. A magnetic resonance imaging (MRI) scan showed an extensive leptomeningeal mass extending from the brainstem to L5, with a thoracic syringomyelia at the T7-T8 level. The plasma prolactin level was very high (5114 microg/l). A biopsy showed the presence of a metastasised prolactinoma. On administration of high dose cabergoline, 0.5 mg twice a day orally, the plasma prolactin levels decreased within one month and then normalised within 26 months. Tumour load reduced considerably but unfortunately, her signs and symptoms did not improve. This case illustrates that a high dose dopamine agonist might be an important therapeutic option in patients with a metastasised prolactinoma.

Granulomatous angiopanniculitis of the breast. A case report.

BACKGROUND: Granulomatous angiopanniculitis (GAP) is a rare benign condition of the breast of unknown etiology. Clinically and by fine needle examination, GAP may simulate breast carcinoma. The cytologic characteristics have not been described before. CASE: A 63-year-old female exhibited a palpable mass in her left breast. The fine needle aspirate contained both epithelioid and stromal elements. The epithelioid component consisted of dissociated individual cells and small groups and clusters of atypical cells. The stromal component showed a uniform, not-atypical pattern. The lumpectomy specimen showed nonnecrotizing granulomatous panniculitis and lymphoid angiitis without involvement of ducts or lobules. CONCLUSION: Granulomatous lesions should be borne in mind in the differential diagnosis of breast cancer in fine needle aspiration cytology. GAP must be histopathologically distinguished from granulomatous inflammation in the breast of autoimmune or infectious origin as specific medical therapy may be available for these latter diseases.

Synergistic antitumour effect of TNF-SAM2 with melphalan and doxorubicin in isolated limb perfusion in rats.

An isolated limb perfusion model (ILP) using soft tissue sarcoma bearing rats (BN175) was used to study antitumour activity of a tumour necrosis factor alpha mutant (TNF-SAM2) in combination with melphalan and doxorubicin. Progressive disease was demonstrated after ILP without agents (sham) or with 50 micrograms TNF-SAM2. ILP with 40 micrograms melphalan or 400 micrograms doxorubicin resulted in no change of tumour volume or progressive disease five days after perfusion. Partial and complete response rates were demonstrated in 76% of rats when the combination of TNF-SAM2 and melphalan was used. TNF-SAM2 in combination with doxorubicin was synergistic as well with a 70% response rate. Histopathologically these responses consisted of hemorrhagic necrosis of the coagulative type. 2 In conclusion, TNF-SAM2 has similar antitumour activity in combination with melphalan or doxorubicin as rHuTNF in sarcoma-bearing rats and is eligible to be tested in clinical ILP or organ perfusion settings because of its potential decreased toxicity.

Low-dose tumor necrosis factor-alpha augments antitumor activity of stealth liposomal doxorubicin (DOXIL) in soft tissue sarcoma-bearing rats.

It has previously been demonstrated in the setting of an isolated limb perfusion that application of high-dose TNF-alpha in combination with chemotherapy (melphalan, doxorubicin) results in strong synergistic antitumor effects in both the clinical and preclinical settings. In this study, we demonstrate that systemic administration of low-dose TNF-alpha augments the antitumor activity of a liposomal formulation of doxorubicin (DOXIL(R)). Addition of TNF-alpha to a DOXIL(R) regimen, which by itself induced some tumor growth delay, resulted in massive necrosis and regression of tumors. Furthermore, we could demonstrate a significant increase of liposomal drug in the tumor tissue when TNF-alpha had been co-administered. Administration of TNF-alpha augmented DOXIL(R) accumulation only after repeated injections, whereas accumulation of free doxorubicin was not affected by TNF-alpha. Drug levels in the tumor interstitium appeared crucial as intracellular levels of free or liposome-associated doxorubicin were not increased by TNF-alpha. Therefore, we hypothesize that low-dose TNF-alpha augments leakage of liposomal drug into the tumor interstitium, explaining the observed improved antitumor effects. Regarding the effects of systemic administration of low doses of TNF-alpha, these findings may be important for enhanced tumor targeting of various liposomal drug formulations.

In vivo isolated kidney perfusion with tumour necrosis factor alpha (TNF-alpha) in tumour-bearing rats.

Isolated perfusion of the extremities with high-dose tumour necrosis factor alpha (TNF-alpha) plus melphalan leads to dramatic tumour response in patients with irresectable soft tissue sarcoma or multiple melanoma in transit metastases. We developed in vivo isolated organ perfusion models to determine whether similar tumour responses in solid organ tumours can be obtained with this regimen. Here, we describe the technique of isolated kidney perfusion. We studied the feasibility of a perfusion with TNF-alpha and assessed its anti-tumour effects in tumour models differing in tumour vasculature. The maximal tolerated dose (MTD) proved to be only 1 microg TNF-alpha. Higher doses appeared to induce renal failure and a secondary cytokine release with fatal respiratory and septic shock-like symptoms. In vitro, the combination of TNF-alpha and melphalan did not result in a synergistic growth-inhibiting effect on CC 531 colon adenocarcinoma cells, whereas an additive effect was observed on osteosarcoma ROS-1 cells. In vivo isolated kidney perfusion, with TNF-alpha alone or in combination with melphalan, did not result in a significant anti-tumour response in either tumour model in a subrenal capsule assay. We conclude that, because of the susceptibility of the kidney to perfusion with TNF-alpha, the minimal threshold concentration of TNF-alpha to exert its anti-tumour effects was not reached. The applicability of TNF-alpha in isolated kidney perfusion for human tumours seems, therefore, questionable.

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.

Assessment of the role of neutrophils on the antitumor effect of TNFalpha in an in vivo isolated limb perfusion model in sarcoma-bearing brown Norway rats.

INTRODUCTION: Isolated limb perfusion (ILP) with TNFalpha in combination with melphalan and IFNgamma has resulted in an immediate and dramatic tumor response in patients. Such an effect was also noted following ILP in a rat sarcoma model. This model enables us to investigate several factors responsible for the TNFalpha-induced tumor responses. We applied total body irradiation (TBI) to reduce white blood cell count, to investigate the contribution of leukocytes to the anti-tumor effect of TNFalpha. METHODS: Small fragments of the nonimmunogenic BN 175 sarcoma were implanted sc in the lower hind leg. A 5 Gy TBI was performed before ILP at a tumor diameter of approximately 15 mm. The hind limbs of 63 rats were perfused and were divided into 6 groups: group 1, sham perfusion, n = 9; group 2, TBI + sham perfusion, n = 6; group 3, TNFalpha 50 microgram, n = 9; group 4, melphalan 40 microgram, n = 9; group 5, TNFalpha 50 microgram + melphalan 40 microgram, n = 22; group 6, TBI + TNFalpha + melphalan ILP, n = 8. In addition, 10 rats were perfused for histological analysis at 24 h post-ILP. RESULTS: We observed in Group 1: 9/9 progressive disease (PD); Group 2: 6/6 PD; Group 3: 9/9 PD; Group 4: 9/9 no change (NC) of tumor diameter for at least 4 days; Group 5: 6/22 NC, 16/22 complete remission (CR), 12/16 of which showed skin necrosis at the tumor site; and Group 6: 7/8 NC and 1/8 CR (without skin necrosis). After TBI, WBC reduction of 80-95% was observed, while the number of platelets was not significantly reduced and platelet aggregation was maintained at 72 %. Histological analysis revealed decreased hemorrhagic necrosis associated with the absence of PMN infiltration at the tumor margins in the TBI rats. CONCLUSION: TBI and the associated reduction in WBC count decreased the tumor response by TNFalpha and melphalan significantly and abrogated the immediate response of skin necrosis at the tumor site, as found in rats treated with TNFalpha and melphalan without TBI. These data strongly suggest that leukocytes play an important role in the hemorrhagic effects of TNFalpha.

Tumor necrosis factor-alpha in isolated perfusion systems in the treatment of cancer: the Rotterdam preclinical-clinical program.

The clinical success of the application of tumor necrosis factor-alpha (TNFalpha) in isolated limb perfusions in patients with advanced sarcomas, melanomas and other tumors has renewed the interest in this agent as an anticancer drug. At the Rotterdam Cancer Center, we have developed an interactive preclinical-clinical TNFalpha program that explores new methods to use TNFalpha in various settings. Regional organ perfusion models were developed and the effectivity of targeting of TNFalpha to the tumor by means of systemic administration of liposomes are tested. Furthermore various drugs and mechanisms that may enhance the activity of TNFalpha are under investigation. A summary of this comprehensive program is presented here.

Endothelial P-selectin expression is reduced in advanced primary melanoma and melanoma metastasis.

Some malignant tumors induce a cellular immune response that results in the formation of an inflammatory infiltrate and subsequent tumor regression. The infiltrating leukocytes extravasate from the bloodstream after binding to adhesion receptors on the surface of the endothelium. One of these receptors is the P-selectin molecule (CD62P) that is constitutively present on normal capillaries. We observed that P-selectin expression is absent from the microvasculature in advanced primary melanoma and in melanoma metastasis in contrast to benign melanocytic lesions where P-selectin expression was identical to that in normal skin. We suggest that one of the mechanisms by which advanced melanoma lesions evade inflammatory regression operates via a decrease of endothelial P-selectin expression.

Lack of lymphangiogenesis in human primary cutaneous melanoma. Consequences for the mechanism of lymphatic dissemination.

Cutaneous melanoma has an initial preference for lymphatic spread. Remarkably, melanoma progression toward this metastasizing phenotype is accompanied by intense blood vessel angiogenesis (hemangiogenesis), but lymphangiogenesis, the formation of new lymph vessels in the tumor, has never been reported. To investigate how primary melanoma cells interact with the existing lymphatic microvasculature, and whether lymphangiogenesis occurs, an immunostaining was developed that differentially decorates blood and lymph vessels in frozen tissue sections. The density and distribution of both these vessel types in and around thin (< or = 1.5 mm) and thick (> or = 1.5 mm) primary melanoma lesions and in normal and uninvolved skin were determined. Although especially in thick melanoma lesions a significant increase in blood vessel density was observed, lymphatic density remained unaltered, showing that lymphangiogenesis did not occur. Morphological analysis indicated, however, that melanoma progression is accompanied by a sequence of events that involves hemangiogenesis supporting tumor expansion, especially in the vertical growth phase. Often, stromal sepia are formed around the blood capillaries in the tumor neovasculature protecting them from invasion. Lymph vessels inside the tumor were infrequently observed. However, subepidermal lymph vessels often seemed to be entrapped and penetrated by the expanding tumor mass. In this way, hemangiogenesis, as the driving force behind tumor expansion, might indirectly increase the chance of lymphatic invasion in the absence of lymphangiogenesis. This model explains the paradox that, although melanoma metastasis seems to require angiogenesis, a consistent relation of prognosis with blood capillary density in primary cutaneous melanoma is lacking.

Neuron-specific enolase, S-100 protein, myelin basic protein and lactate in CSF in dementia.

The pattern of injury of the specific cell structures of the central nervous system (CNS) is different in the various types of the dementia syndrome. We challenged the hypothesis that this could be reflected in specific patterns of brain-specific proteins in the cerebrospinal fluid (CSF). The neuron-specific enolase (NSE), S-100, myelin basic protein (MBP) and lactate levels were retrospectively analyzed in the CSF of 159 patients with various types of dementia. A previous study from our department demonstrated age-related reference values for the brain-specific proteins in the CSF. The present study affirmed the strikingly high NSE and S-100 values in the CSF of patients with autopsydiagnosed Creutzfeld-Jacob disease: NSE, S-100 and MBP levels in the CSF of patients with various other types of dementia, and controls, did not differ significantly. Therefore we concluded that a single determination of CSF concentrations of these brain-specific proteins were of little value in the differential diagnosis of the dementia syndrome. In the diagnosis of normal pressure hydrocephalus increased levels of CSF lactate may be helpful.

Immunohistochemical analysis of integrin alpha vbeta3 expression on tumor-associated vessels of human carcinomas.

Expression of the alpha vbeta3 integrin is upregulated on sprouting endothelia. Systemic application of antibody or peptidic inhibitors of alpha vbeta3 function disrupts tumor angiogenesis and reduces growth and invasiveness of human tumors in animal models. We systematically investigated alpha vbeta3 expression on tumor-associated vessels of 4 different human epithelial tumors and the corresponding normal tissues by means of immunohistochemistry on frozen sections using the alpha vbeta3-complex specific monoclonal antibody LM609. Variable levels of LM609 staining were found in all carcinoma lesions. A considerable number of tumor tissues (35/50) expressed alpha vbeta3 on more than 50% of their vessels. Inflammatory infiltrates and the possibly hypoxic conditions near necrotic areas of tumors were accompanied by an increased alpha vbeta3 expression. Remarkably, the vasculature in apparently normal tissue also stained for alpha vbeta3. However, the percentages of stained vessels and their staining intensity were lower than in neoplastic tissues. Besides the vascular alpha vbeta3 expression, several extravascular cell types stained positive, in both normal and tumor specimens. Taken together, our findings show a considerable number of colon, pancreas, lung and breast carcinoma lesions with many alpha vbeta3-expressing vessels that could be targets for anti-alpha vbeta3-therapy.

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.

Synergistic antitumour effect of recombinant human tumour necrosis factor alpha with melphalan in isolated limb perfusion in the rat.

The efficacy of isolated limb perfusion (ILP) for 'intransit' metastases from malignant melanoma and irresectable soft tissue sarcoma has been improved considerably by the addition of tumour necrosis factor (TNF) alpha. A rat sarcoma tumour model was, therefore, developed to evaluate the effects of TNF-alpha, melphalan and the combination of these drugs in the treatment of sarcoma. In BN rats bearing the non-immunogenic BN 175 sarcoma ILPs were performed with perfusate only, TNF-alpha, melphalan alone, or in combination when tumours had grown to approximately 1.5 cm in diameter. All rats treated with sham perfusion or perfusion with 50 micrograms TNF-alpha showed progressive disease. After perfusion with 40 micrograms melphalan no change in tumour diameter was observed in any rats at 4 days. After a combined perfusion with 40 micrograms melphalan and 50 micrograms TNF-alpha complete remission was noted in 12 of 16 rats. This synergistic effect in vivo between relatively ineffective doses of TNF-alpha and melphalan was not observed in vitro.

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.