A preliminary study on a new model system to evaluate tumour-detection and tumour-purging protocols in ovarian cortex tissue intended for fertility preservation.

STUDY QUESTION: Is it possible to create a model system that mimics ovarian metastatic disease in order to devise new strategies to detect cancer cells and prevent cancer cell transmission via ovarian tissue autotransplantation in cancer survivors? SUMMARY ANSWER: Injection of bovine or human ovarian cortex fragments with cells from different cancer types led to the formation of proliferating tumour masses and newly formed small metastatic lesions. WHAT IS KNOWN ALREADY: Autotransplantation of ovarian tissue comes with the major concern of cancer cells possibly being present in the tissue. A model system to develop strategies aimed at enhancing the safety of ovarian tissue autotransplantation is currently lacking. STUDY DESIGN, SIZE, DURATION: The ability of injected human leukaemia, lymphoma, Ewing's sarcoma or breast cancer cells to proliferate and form tumour-like structures in bovine and human ovarian cortex tissue in vitro was assessed. The injected cells were from human cancer cell lines. After 4 days of culture, some tissue fragments were harvested for standard histological staining and immunohistochemical staining of tumour cell specific antigens and the Ki67 proliferation marker, while the remaining fragments were incubated for an additional 6 days (bovine tissue) or 3 days (human tissue) before analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Experiments were performed with ovarian tissue from women after prophylactic salpingo-oophorectomy. Bovine ovarian tissue was obtained at an abattoir. Glucose uptake during in vitro culture was monitored to quantify the viability of tissue. Tumour formation was assessed at Day 4 and Day 10 in bovine ovarian tissue and at Day 4 and Day 7 in human ovarian tissue, using histology and immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: We found that bovine and human ovarian cortex tissue could be cultured for up to 10 and 7 days, respectively, without any loss of viability. Our preliminary results show that all cell lines tested were capable of forming proliferating tumours in ovarian cortex tissue in vitro. Lymphoma and breast cancer cells produced small metastases near the original lesions. LIMITATIONS, REASONS FOR CAUTION: The tumour model presented was based on the growth of human cancer cell lines in ovarian cortex tissue. It is unknown whether these cells behave differently from malignant cells derived from primary tumours. In addition, the human ovarian tissue was derived from women over 39 years of age, which is obviously considerably older than patients opting for ovarian tissue cryopreservation. WIDER IMPLICATIONS OF THE FINDINGS: Our model system will facilitate the development of procedures to detect cancer cells in, or purge cancer cells from, human ovarian tissue. STUDY FUNDING/COMPETING INTERESTS: Unconditional funding was received from the Radboud Institute for Health Sciences, KiKa Foundation and Merck Serono. There are no conflicts of interest to declare.

Efficacy of ovarian tissue cryopreservation in a major European center.

PURPOSE: To evaluate the effect of cryopreservation and thawing of ovarian tissue from oncological patients opting for fertility preservation on ovarian tissue viability. METHODS: In this prospective cohort study, the ovarian tissue viability before and after cryopreservation and thawing was measured for 25 newly diagnosed oncological patients who had their ovarian tissue cryopreserved. Outcome measures were follicle integrity (histology), follicle viability (Calcein viability assay), steroid hormone production (estradiol and progesterone production in vitro) and overall tissue viability (glucose uptake in vitro). This study was conducted at a Cryobank for storage of ovarian tissue in a university hospital. RESULTS: Cryopreserved/thawed ovarian tissue showed a decreased glucose uptake when compared to tissue that had not been cryopreserved. In addition, a diminished E2 and P4 production was observed after cryopreservation and thawing, despite the fact that numbers of viable follicles as determined by the Calcein viability assay were comparable. Histological examination revealed a higher percentage of degenerated follicles after cryopreservation and thawing. CONCLUSIONS: Ovarian tissue cryopreservation and thawing impairs the viability of ovarian tissue in oncological patients opting for fertility preservation.

Plasminogen activators are involved in angiostatin generation in vivo in benign and malignant ovarian tumor cyst fluids.

In many tumor types, angiogenesis is the net result of pro- and anti-angiogenic mediators and correlated with metabolic activity, growth, and degree of malignancy. One of the first discovered anti-angiogenic compounds is angiostatin, a proteolytic fragment of plasminogen. The requirements for in vivo angiostatin generation have not yet been determined. We investigated the levels of plasminogen and angiostatin by western blotting and of components of the plasminogen activator complex by ELISA in cyst fluid derived from benign and malignant ovarian tumors. Fluid samples from functional ovarian follicles, dermoid cysts and endometriotic lesions were evaluated separately. When no or minimal amounts of plasminogen were present in the cyst fluids, angiostatin was generally absent as well, irrespective of plasminogen activator concentrations. When plasminogen was present, the degree of conversion of plasminogen to angiostatin was significantly correlated with the level of uPA, and, to a lesser extent, to the tPA level. However, angiostatin was also found in a number of cyst fluid samples with minimal or no plasminogen activators, suggesting the involvement of other angiostatin generating proteases in these samples. Conversely, no angiostatin was observed in a number of cyst fluid samples containing both plasminogen and plasminogen activators. The presence of an inhibitor of the enzymatic activity of uPA and/or tPA, like PAI-1, may explain this finding. Our data show that plasminogen activators are clearly involved in in vivo angiostatin formation in ovarian cysts. Most likely, however, other proteases, as well as inhibitors of plasminogen activators, are involved as well.

Autotransplantation of cryopreserved ovarian tissue in cancer survivors and the risk of reintroducing malignancy: a systematic review.

BACKGROUND The risk of recurrent oncological disease due to the reintroduction of cancer cells via autotransplantation of cryopreserved ovarian tissue is unknown. METHODS A systematic review of literature derived from MEDLINE, EMBASE and the Cochrane Library was conducted. Studies on follow-up after autotransplantation; detection of cancer cells in ovarian tissue from oncological patients by histology, polymerase chain reaction or xenotransplantation; and epidemiological data on ovarian metastases were included. RESULTS A total of 289 studies were included. Metastases were repeatedly detected in ovarian tissue obtained for cryopreservation purposes from patients with leukaemia, as well as in one patient with Ewing sarcoma. No metastases were detected in ovarian tissue from lymphoma and breast cancer patients who had their ovarian tissue cryopreserved. Clinical studies indicated that one should be concerned about autotransplantation safety in patients with colorectal, gastric and endometrial cancer. For patients with low-stage cervical carcinoma, clinical data were relatively reassuring, but studies focused on the detection of metastases were scarce. Oncological recurrence has been described in one survivor of cervical cancer and one survivor of breast cancer who had their ovarian tissue autotransplanted, although these recurrences may not be related to the transplantation. CONCLUSIONS It is advisable to refrain from ovarian tissue autotransplantation in survivors of leukaemia. With survivors of all other malignancies, current knowledge regarding the safety of autotransplantation should be discussed. The most reassuring data regarding autotransplantation safety were found for lymphoma patients.

Glucose/lactate metabolism of cryopreserved intact bovine ovaries as a novel quantitative marker to assess tissue cryodamage.

For some patients, the autotransplantation of a cryopreserved-thawed intact ovary might be the best option to preserve their reproductive potential after fertility-threatening treatment. The best procedure to successfully cryopreserve a human ovary without inflicting a devastating level of cryodamage is to date unknown. To optimize this procedure, this study developed an assay to monitor the extent of cryodamage inflicted on bovine ovarian tissue by different cryopreservation protocols. The assay measures glucose and lactate metabolism of ovarian tissue fragments in vitro and determines the extent of cryodamage in cryopreserved ovaries. This study tested the cryoprotective effect of two different routes of administration of the cryoprotectant dimethylsulphoxide (DMSO). The cryoprotective effect was assessed in different tissue layers of the ovary, namely the cortex, the subcortex and the medulla. Submersion of intact ovaries in DMSO prior to freezing-thawing resulted in the complete protection of the glucose/lactate metabolism of the cortex, but not of the inner ovarian mass. Perfusion without simultaneous submersion, resulted in partial protection of cortex, subcortex and medulla, while the combination of submersion and perfusion conveyed the highest level of protection for all three ovarian tissue layers.

In vitro 17ß-oestradiol release as a marker for follicular survival in cryopreserved intact bovine ovaries.

Transplantation of cryopreserved intact ovaries from cancer patients is a technically challenging option for restoring fertility after sterilizing cancer therapy. In this paper we describe an assay based on 17ß-oestradiol (oestradiol) production, to monitor the functional damage sustained by the ovarian tissue during the freeze/thawing procedure. To this end, fresh bovine ovarian cortical biopsies were cultured in vitro for 7 days. As a control, the oestradiol release of biopsies that had sustained maximal cryodamage was analyzed. In addition the oestradiol release by cortical biopsies from two ME2SO perfused and cryopreserved intact ovaries was analyzed. Oestradiol production could be measured in culture supernatants, while oestradiol release of maximal cryo-damaged biopsies was at background levels. In vitro oestradiol release by cortical biopsies can be used as a functional marker for cryo-damage and indicates that our assay is suitable to optimize the cryopreservation procedure of intact ovaries.

Toxicity testing of human assisted reproduction devices using the mouse embryo assay.

Systems to assess the toxicity of materials used in human assisted reproduction currently lack efficiency and/or sufficient discriminatory power. The development of 1-cell CBA/B6 F1 hybrid mouse embryos to blastocysts, expressed as blastocyst rate (BR), is used to measure toxicity. The embryos were divided into control and test groups, and were exposed to either control medium or to a potentially toxic test medium. Inferences on toxicity were based on differences in BR between the two groups. The mouse embryo assay followed a stratified (mouse), randomized (embryo), and balanced (equal number of embryos per group and per mouse) design. The number of embryos needed was calculated using power analysis. The basal BR of the hybrid strain was determined in a historical population. Sixty-nine mouse embryos per group were required to detect toxic materials with sufficient sensitivity and to account for the considerable inter-mouse variation in blastocyst development. Fifty-two samples, divided over batches of seven different products were tested before use in the study IVF centre and five of these were found to be toxic. This test system, presented as the Nijmegen mouse embryo assay (NMEA), can be used to detect embryo-toxic materials in daily IVF practice, and this report may provide a starting point for standardization.

Optimal perfusion of an intact ovary as a prerequisite for successful ovarian cryopreservation.

BACKGROUND: Cryopreservation and subsequent reimplantation of intact ovaries from cancer patients, offers potentially the best prognosis for restoring fertility after sterilizing cancer treatment. We used bovine ovaries as a model system to explore the perfusion procedure that is required for cryopreservation of intact ovaries. METHODS: The arteria ovarica was cannuled, and ovaries were flushed with Indian ink for 5 min. RESULTS: Successful perfusion of blood vessels was immediately visible macroscopically by a grey to black discoloration of the ovary and was confirmed microscopically, by examining tissue sections. There was no correlation between the time interval from removal of the ovary to the start of the perfusion, and success of perfusion. We determined the percentage of Indian ink-perfused vessels and scored blood vessels in four different size classes. The percentage of perfused vessels increased with an increase in vessel size. In a limited set of preliminary experiments with human ovaries, comparable results were obtained. CONCLUSIONS: Our results show that bovine ovaries are a suitable and adequate model system for optimizing the cryopreservation of human ovaries. As bovine are at least of comparable size to human ovaries, we expect that our results can be extrapolated to the human situation.

Globozoospermia revisited.

Globozoospermia is a rare (incidence <0.1%) but severe disorder in male infertility. Total globozoospermia is diagnosed by the presence of 100% round-headed spermatozoa lacking an acrosome. It is still unclear whether patients whose ejaculate contains both normal and globozoospermic cells (partial globozoospermia) suffer from a variation of the same syndrome. Apart from the fact that affected males suffer from reduced fertility or even infertility, no other physical characteristics can be associated with the syndrome. ICSI is a treatment option for these patients, although low fertilization rates after ICSI show a reduced ability to activate the oocyte. In globozoospermic cells, the use of acrosome markers has demonstrated an absent or severely malformed acrosome. Chromatin compaction appears to be disturbed but is not consistently over- or undercondensed. In some cases, an increased number of cells with DNA fragmentation have been observed. The analysis of the cytogenetic composition revealed an increased aneuploidy rate in some cases. Nonetheless, no increased number of spontaneous abortions or congenital defects has been reported in pregnancies conceived after ICSI. The pathogenesis of globozoospermia most probably originates in spermiogenesis, more specifically in acrosome formation and sperm head elongation. In several knockout mouse models, a phenotype similar to that in humans was found. Together with the occurrence of affected siblings, these findings indicate a genetic origin, which makes globozoospermia a good candidate for genetic analysis. More research is needed to elucidate the pathogenesis of human globozoospermia to further understand globozoospermia as well as (abnormalities in) spermiogenesis and spermatogenesis in general.

Sample size calculations for a split-cluster, beta-binomial design in the assessment of toxicity.

Mouse embryo assays are recommended to test materials used for in vitro fertilization for toxicity. In such assays, a number of embryos is divided in a control group, which is exposed to a neutral medium, and a test group, which is exposed to a potentially toxic medium. Inferences on toxicity are based on observed differences in successful embryo development between the two groups. However, mouse embryo assays tend to lack power due to small group sizes. This paper focuses on the sample size calculations for one such assay, the Nijmegen mouse embryo assay (NMEA), in order to obtain an efficient and statistically validated design. The NMEA follows a stratified (mouse), randomized (embryo), balanced design (also known as a split-cluster design). We adopted a beta-binomial approach and obtained a closed sample size formula based on an estimator for the within-cluster variance. Our approach assumes that the average success rate of the mice and the variance thereof, which are breed characteristics that can be easily estimated from historical data, are known. To evaluate the performance of the sample size formula, a simulation study was undertaken which suggested that the predicted sample size was quite accurate. We confirmed that incorporating the a priori knowledge and exploiting the intra-cluster correlations enable a smaller sample size. Also, we explored some departures from the beta-binomial assumption. First, departures from the compound beta-binomial distribution to an arbitrary compound binomial distribution lead to the same formulas, as long as some general assumptions hold. Second, our sample size formula compares to the one derived from a linear mixed model for continuous outcomes in case the compound (beta-)binomial estimator is used for the within-cluster variance.

Unintended consequences of a quality improvement program designed to improve treatment of alcohol withdrawal in hospitalized patients.

BACKGROUND: New guidelines, accompanied by an educational campaign, introduced standardized monitoring of withdrawal severity while emphasizing prophylactic fixed-schedule benzodiazepine (BDZ) treatment of at-risk patients. EVALUATION: Preliminary analysis showed more deaths during the year after introduction of the guidelines. Investigation revealed some evidence of guideline adherence and a decrease in the number of patients requiring transfer to a higher level of care. However, an 18% increase in the median length of stay was also found, as was an increase in the total dose of benzodiazepines administered to patients with cirrhosis and severe concurrent illness, and the risk of in-hospital death persisted even after adjustment for patient mix. RESPONSE: This feedback led to guideline revision and redoubled educational efforts focused on safe benzodiazepine prescribing. Ongoing monitoring of patient outcomes showed no further deterioration and some evidence of improved quality of care. CONCLUSION: Evaluation of such quality improvement efforts should include measurement of both treatment patterns and patient outcomes.

[Cryopreservation of semen of adolescents and young adult men with cancer]. / Invriezen van sperma bij pubers en jonge volwassen mannen met kanker.

The importance of cryopreserving semen for young male cancer patients is illustrated in three case descriptions. A 28-year-old man with chronic myeloid leukaemia that resulted in azoospermia, later fathered a child with his semen that had been stored prior to chemotherapy. In an 18-year-old adolescent with non-Hodgkin lymphoma the possibility to store cryopreserved semen was only raised after chemotherapy had been started and had caused azoospermia. This caused the patient serious regret. A 14-year-old boy with acute lymphatic leukaemia had his semen stored despite initial hesitations due to his young age. The cancer hardly ever affects the semen quality to the extent that cryopreservation of the semen becomes impossible. The aim should be to obtain several ejaculates prior to the cancer therapy and to store multiple portions, so that later a number of fertilisation attempts are possible. The primary attending physician is initially responsible for raising the possibility of semen cryopreservation. Ideally, however, all health professionals involved should be aware of this aspect. There is a need for multidisciplinary protocols for oncology centres and sperm banks, so that the timely informing of patients is guaranteed, responsibilities are recorded--with appropriate procedures to prevent unnecessary delay--and procedures that concur with legal requirements and financial constraints are established.

Elements regulating angiogenesis and correlative microvessel density in benign hyperplastic and malignant prostate tissue.

PURPOSE: To quantify the ex vivo production of proangiogenic proteins (vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (tPA)) and angiogenesis inhibitors (plasminogen activator inhibitor type-1 (PAI-1) and angiostatin) from epithelial and stromal components of primary prostate cancer (CaP) and benign prostatic hyperplasia (BPH) cultures. To perform microvessel density (MVD) counts on sections of BPH and CaP from the same prostatectomy specimens. SCOPE: Angiogenic cytokine expression was measured by immunoassays and in vitro angiostatin generating capacities assessed using immunoblotting. CaP and BPH tissue was immunostained using factor VIII antibody to determine MVD. CONCLUSIONS: Elements regulating angiogenesis are present in both primary cultures of CaP and BPH, suggesting that angiogenic ability is well established in the absence of carcinoma.

The pattern of metastasis of human melanoma to the central nervous system is not influenced by integrin alpha(v)beta(3) expression.

We investigated the effect of integrin alpha(v)beta(3) expression on the metastatic pattern of human melanoma cells in the central nervous system (CNS). For this purpose, we developed a hematogenous CNS melanoma metastasis model in nude mice using a modified internal carotid artery infusion technique. This protocol revealed 2 different patterns of CNS metastasis. The integrin alpha(v)beta(3)-expressing melanoma lines Mel57 and Zkr nearly exclusively produced metastases in the brain parenchyma, whereas cells of the BLM and MV3 lines, devoid of integrin alpha(v)beta(3) expression, preferentially metastasized to dura mater and leptomeninges. Treatment with hyaluronidase to obtain single BLM cell suspensions did not influence the metastatic pattern, indicating that this was not simply the result of entrapment of tumor cell aggregates in large-sized leptomeningeal vessels. The role of integrin alpha(v)beta(3) expression in the process of metastasis was tested by transfection of BLM, but did not lead to an altered pattern of metastasis. We did observe, however, slower growth of the transfected tumors, although the in vitro growth rate was unaltered, indicating a reduction in tumorigenicity. We conclude from our findings that CNS metastasis of melanoma cells in the mouse xenograft model occurs in at least 2 different but very reproducible patterns. Although it is predicted that adhesion of tumor cells to endothelial cells plays a role in this phenomenon, tumor cell integrin alpha(v)beta(3) expression per se does not explain the difference in metastatic behavior in the CNS. We assume that other, as yet unknown factors, must be involved.

Coexpression of integrin alpha(v)beta3 and matrix metalloproteinase-2 (MMP-2) coincides with MMP-2 activation: correlation with melanoma progression.

Tumor cell invasion and metastasis formation depend on both adhesive and proteolytic mechanisms. Previous studies have shown that expression of matrix metalloproteinase-2 and integrin alphavbeta3 correlate with melanoma progression. Recently, direct binding of matrix metalloproteinase-2 to alpha(v)beta3 was implicated in presenting activated matrix metalloproteinase-2 on the cell surface of invasive cells. In this study we investigated this, using the highly metastatic, alpha(v)beta3-negative melanoma cell lines MV3 and BLM, their beta3-transfected alpha(v)beta3 expressing counterparts, xenografts derived from these cell lines, and fresh human cutaneous melanoma lesions comprising all stages of melanoma progression. Expression and activation status of matrix metalloproteinase-2 were studied by reverse transcription-polymerase chain reaction, immunohistochemistry, western blotting, and zymographic analysis, respectively. Matrix metalloproteinase-2 protein expression in vitro was similar in both alpha(v)beta3-negative and alpha(v)beta3-positive cell lines Remarkable differences, however, exist in the localization of inactive and active matrix metalloproteinase-2. Soluble active matrix metalloproteinase-2 was detectable only in the conditioned medium of alpha(v)beta3-negative cell lines and undetectable in the alpha(v)beta3-positive cell lines. Conversely, active matrix metalloproteinase-2 was present exclusively on the cell surface of the alpha(v)beta3 expressing transfectants. Western blot analysis of other components that are involved in matrix metalloproteinase-2 activation showed that processing of proMT1-matrix metalloproteinase to the activated form was enhanced in beta3 transfectants, whereas secretion of tissue inhibitor of metalloproteinase-2 was decreased. In vivo, the presence of functionally active matrix metalloproteinase-2 was significantly higher in xenografts derived from the alpha(v)beta3 expressing MV3 and BLM cell lines. In human cutaneous melanoma lesions, neither matrix metalloproteinase-2 nor integrin alpha(v)beta3 is detectable in melanoma in situ as determined by immunohistochemistry. In contrast, the number of matrix metalloproteinase-2-positive and alphavbeta3-positive tumor cells was clearly increased in primary melanomas, and melanoma metastases. Double staining experiments and confocal laser microscopy demonstrated that the percentage of cells coexpressing matrix metalloproteinase-2 and alpha(v)beta3 increased in advanced primary melanomas and melanoma metastases. In addition, zymography showed that functionally active matrix metalloproteinase-2 was frequently present in melanoma metastases. In these lesions a high proportion of matrix metalloproteinase-2- and alphavbeta3-double-positive melanoma cells were detectable. Our study demonstrates that the presence of activated matrix metalloproteinase-2 correlates with expression of alpha(v)beta3 in human melanoma cells both in vitro and in vivo, and also in fresh human melanoma lesions. These findings strongly suggest that co-ordinated expression of both factors may be required for melanoma cell invasion and metastasis formation.

Gambling in the south: implications for physicians.

Three historical cycles of legalized gambling have occurred in the South. Currently, every southern state has legalized some form of gaming. Adult past-year prevalence rates of problem gambling in southern states are within the national range. Higher prevalence rates occur in states with casinos and multiple forms of legalized gambling. States with lotteries have higher prevalence rates of adolescent problem gambling. Problem gambling can cause stress-induced physical diseases and psychiatric symptoms in gamblers and their families. Physicians can reduce personal, family, and social costs of problem gambling through increased awareness, strategic screening, and early intervention. Treatment approaches include inpatient treatment centers, self-help fellowship groups, and cognitive-behavioral and addiction-based psychotherapies. Although no standard pharmacologic treatments for gambling disorders exist, use of selective serotonin re-uptake inhibitors is under investigation. Referral resources are available to physicians in states with state-funded treatment programs for problem gamblers and/or state councils for problem gambling.

A statewide survey of ECT policies and procedures.

The 1990 American Psychiatric Association (APA) Electroconvulsive Therapy (ECT) Task Force Recommendations include facility policy and procedure guidelines. The objectives of this study were to determine and to improve the adherence to the 1990 APA ECT Task Force Recommendations on policies and procedures among the providers of ECT in Louisiana. Completed surveys on ECT policy and procedures were obtained from the seven major Louisiana ECT providers from the last quarter of 1996. Project coordinators distributed copies of the survey results and a comprehensive set of ECT policies and procedures at a statewide meeting of participating hospitals during the spring of 1997. Most facilities had policies for electrical safety of ECT equipment, testing of new ECT equipment, pre-ECT work-up, ECT informed consent, patient instruction sheets, outpatient ECT, documentation of ECT procedures, clinical privileging, and ECT quality assurance monitoring. Subsequent telephone follow-up found that all participants changed their policies and procedures as a result of the project. Louisiana ECT providers showed general compliance with the facility policy and procedure aspects of the 1990 APA ECT Task Force Recommendations. The awareness model of guideline compliance was applicable to improving facility policies and procedures.

Matrix metalloproteinases in human melanoma.

Cutaneous melanoma is a highly invasive and metastatic tumor. Degradation of basement membranes and extracellular matrix is an essential step in melanoma cell migration, invasion, and metastasis formation. Matrix metalloproteinases and their tissue inhibitors play a crucial role in these complex multistep processes. Melanoma cells may express a number of matrix metalloproteinase family members (MMP-1, MMP-2, MMP-9, MMP-13, and MT1-MMP) as well as their tissue inhibitors (TIMP-1, TIMP-2, and TIMP-3). Numerous studies have examined matrix metalloproteinases, their tissue inhibitors, and the molecules that regulate their expression and/or activation in melanoma cell lines in vitro and in vivo, and in human melanocytic lesions. Recent results have indicated that adhesion molecules such as CD44 and integrin alphavbeta3 are involved in positioning activated matrix metalloproteinase molecules on the cell surface of invasive tumor cells. In this review we evaluate these novel aspects of the role of matrix metalloproteinases and their tissue inhibitors in melanoma progression. We conclude that the balance between levels of activated matrix metalloproteinases and expression levels of their tissue inhibitors, and the coexpression of activated matrix metalloproteinases and adhesion molecules are important factors in determining melanoma cell invasion, tumor growth, and metastasis formation.

EORTC Receptor and Biomarker Study Group Report: a sandwich enzyme-linked immunosorbent assay for vascular endothelial growth factor in blood and tumor tissue extracts.

A four-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for vascular endothelial growth factor (VEGF) for application in blood (serum and plasma) and tumor tissue extracts was set up within the framework of the EORTC Receptor and Biomarker Study Group (RBSG). Polyclonal antibodies against VEGF165 were raised in chickens and rabbits, and used in a previously described assay format. The assay was validated and characterized for use in serum, plasma and tumor tissue extracts. The resulting VEGF ELISA was found to be specific for VEGF165 and VEGF121, the main isoforms of VEGF. The assay showed good precision and parallelism in serial dilutions of samples. The assay was not susceptible to interference by heterophilic antibodies because avian antibodies (duck anti-chicken and chicken anti-VEGF) were used in the pre-analyte stage and mammalian antibodies (rabbit anti-VEGF and goat anti-rabbit) in the post-analyte stage. In conclusion, a sensitive, robust and specific VEGF ELISA has been developed. Research into the prognostic value of VEGF employing this assay is currently underway.