Prognostic factors among patients with brain metastases from cancer of unknown primary site.

PURPOSE: Cancers of an unknown primary site (CUPs) have a dismal prognosis, and the situation is even worse for CUPs patients with brain metastases (BM-CUPs). This study aims to give better insight into the occurrence and survival of BM-CUPs patients. METHODS: Cases were selected from the Netherlands Cancer Registry (1,430 BM-CUPs/17,140 CUPs). Baseline characteristics between CUPs patients with and without BM were tested using chi-square tests and Mann-Whitney U tests. Patients' overall survival (OS) times were estimated by the Kaplan-Meier method and prognostic factors on OS was assessed using Cox proportional hazards regression analyses. RESULTS: The proportion of BM-CUPs patients among CUPs increased from 8% in 2009-2010 to 10% in 2017-2018 (p < 0.001). Most patients presented with multiple brain lesions (53%). Survival of BM-CUPs improved over time: one-year OS increased from 10% for patients diagnosed in 2009-2010 to 17% (2017- 2018) (p < 0.01), and median survival times increased from 1.8 months to 2.2 months. Independent predictors of poor survival were multiple (HR 1.25; p < 0.01) or unknown (HR 1.48; p < 0.01) locations of BM, unknown/poorly/undifferentiated carcinoma histology (HR 1.53; p < 0.01), or clinical symptoms of BM (HR 1.74; p < 0.01), accompanying liver metastasis (HR 1.43; p < 0.01) and more than one metastatic site outside the brain compared to none (HR 1.52; p < 0.01). CONCLUSION: The incidence of patients with BM-CUPs is steadily increasing over time and overall prognosis remains dismal. Our results, however, show distinct patient subgroups that exhibit comparatively better outcomes, and more predictors may likely still be identified.

The development of locomotor kinetics in the foal and the effect of osteochondrosis.

REASON FOR PERFORMING STUDY: Foals stand and walk immediately after birth, but insight into the subsequent longitudinal development of their gait kinetics in the early juvenile phase and the possible influence of osteochondrosis thereon is lacking. OBJECTIVES: To quantify gait kinetics in foals during the first half year of life, taking into account their osteochondrosis status. STUDY DESIGN: Prospective, cohort study performed at a single stud farm. METHODS: Pressure plate measurements at walk and trot from 11 Dutch Warmblood foals during the first 24 weeks of life were used to determine body mass normalised peak vertical force, normalised vertical impulse and stance duration. Coefficients of variation of peak vertical force and stance duration were used as measures for gait maturity. Radiographs of tarsocrural and femoropatellar joints were taken at age 4-6 weeks and after 6 months to check for osteochondrosis. A linear mixed model was used to determine the effects of age, limb, presence of osteochondrosis and speed on gait parameters. RESULTS: Mean walking and trotting velocity increased over time as did stance duration and normalised vertical impulse, normalised peak vertical force values however remained relatively constant. During the first weeks of their life only the coefficient of variation of stance duration decreased significantly, while the coefficient of variation of peak vertical force did not. None of the foals was visibly lame, but the presence of osteochondrosis resulted in a temporarily but significantly reduced normalised peak vertical force. MAIN LIMITATIONS: This study is a relatively small sample size of one breed from a single stud farm. A stand-alone pressure plate was used and body mass was estimated rather than measured. CONCLUSIONS: Despite being precocious, foals need time to mature their gait. During growth, velocity at walk and trot increases, but normalised peak vertical force remains relatively constant. Although not visibly lame, a temporary reduction in normalised peak vertical force was detected in osteochondrosis positive foals using a pressure plate.

An impressive response to pazopanib in a patient with metastatic endometrial carcinoma.

The incidence of endometrial carcinoma is rising and the patients with distant metastases have a poor prognosis, especially when progression of disease occurs after systemic treatment with hormonal therapy or chemotherapy. Pazopanib, a multi-targeted inhibitor of several oncogenic receptor tyrosine kinases, has been investigated in patients with chemotherapy-resistant endometrial carcinoma or patients for whom chemotherapy is contraindicated. In this report we will describe a spectacular response to pazopanib in a patient with recurrent metastatic endometrial carcinoma.

Characterisation of tumour vasculature in mouse brain by USPIO contrast-enhanced MRI.

To enhance the success rate of antiangiogenic therapies in the clinic, it is crucial to identify parameters for tumour angiogenesis that can predict response to these therapies. In brain tumours, one such parameter is vascular leakage, which is a response to tumour-derived vascular endothelial growth factor-A and can be measured by Gadolinium-DTPA (Gd-DTPA)-enhanced magnetic resonance imaging (MRI). However, as vascular permeability and angiogenesis are not strictly coupled, tumour blood volume may be another potentially important parameter. In this study, contrast-enhanced MR imaging was performed in three orthotopic mouse models for human brain tumours (angiogenic melanoma metastases and E34 and U87 human glioma xenografts) using both Gd-DTPA to detect vascular leakage and ultrasmall iron oxide particles (USPIO) to measure blood volume. Pixel-by-pixel maps of the enhancement in the transverse relaxation rates (Delta R(2) and Delta R(2)(*)) after injection of USPIO provided an index proportional to the blood volume of the microvasculature and macrovasculature, respectively, for each tumour. The melanoma metastases were characterised by a blood volume and vessel leakage higher than both glioma xenografts. The U87 glioblastoma xenografts displayed higher permeability and blood volume in the rim than in the core. The E34 glioma xenografts were characterised by a relatively high blood volume, accompanied by only a moderate blood-brain barrier disruption. Delineation of the tumour was best assessed on post-USPIO gradient-echo images. These findings suggest that contrast-enhanced MR imaging using USPIOs and, in particular, Delta R(2) and Delta R(2)(*) quantitation, provides important additional information about tumour vasculature.

The endogenous anti-angiogenic VEGF isoform, VEGF165b inhibits human tumour growth in mice.

Vascular endothelial growth factor-A is widely regarded as the principal stimulator of angiogenesis required for tumour growth. VEGF is generated as multiple isoforms of two families, the pro-angiogenic family generated by proximal splice site selection in the terminal exon, termed VEGFxxx, and the anti-angiogenic family formed by distal splice site selection in the terminal exon, termed VEGFxxxb, where xxx is the amino acid number. The most studied isoforms, VEGF165 and VEGF165b have been shown to be present in tumour and normal tissues respectively. VEGF165b has been shown to inhibit VEGF- and hypoxia-induced angiogenesis, and VEGF-induced cell migration and proliferation in vitro. Here we show that overexpression of VEGF165b by tumour cells inhibits the growth of prostate carcinoma, Ewing's sarcoma and renal cell carcinoma in xenografted mouse tumour models. Moreover, VEGF165b overexpression inhibited tumour cell-mediated migration and proliferation of endothelial cells. These data show that overexpression of VEGF165b can inhibit growth of multiple tumour types in vivo indicating that VEGF165b has potential as an anti-angiogenic, anti-tumour strategy in a number of different tumour types, either by control of VEGF165b expression by regulation of splicing, overexpression of VEGF165b, or therapeutic delivery of VEGF165b to tumours.

Micronodular transformation as a novel mechanism of VEGF-A-induced metastasis.

How and why tumors metastasize is still a matter of debate. The assumption is that mutations render tumor cells with a metastatic phenotype, enabling entrance in and transport through lymph or blood vessels. Distant outgrowth is thought to occur only in a suitable microenvironment (the seed and soil hypothesis). However, the anatomical location of most metastases in cancer patients suggests entrapment of tumor cells in the first microcapillary bed that is encountered. We here investigated how vascular endothelial growth factor-A (VEGF-A) attributes to the metastatic process. We describe here that VEGF-A enhances spontaneous metastasis by inducing intravasation of heterogeneous tumor cell clusters, surrounded by vessel wall elements, via an invasion-independent mechanism. These tumor clusters generate metastatic tissue embolisms in pulmonary arteries. Treatment of tumor-bearing mice with the antiangiogenic compound ZD6474 prevented the development of this metastatic phenotype. This work shows that tumors with high constitutive VEGF-A expression metastasize via the formation of tumor emboli and provides an alternative rationale for anti-VEGF-A therapy, namely to inhibit metastasis formation.

Development of luciferase tagged brain tumour models in mice for chemotherapy intervention studies.

The blood-brain barrier (BBB) is considered one of the major causes for the low efficacy of cytotoxic compounds against primary brain tumours. The aim of this study was to develop intracranial tumour models in mice featuring intact or locally disrupted BBB properties, which can be used in testing chemotherapy against brain tumours. These tumours were established by intracranial injection of suspensions of different tumour cell lines. All cell lines had been transfected with luciferase to allow non-invasive imaging of tumour development using a super-cooled CCD-camera. Following their implantation, tumours developed which displayed the infiltrative, invasive or expansive growth patterns that are also found in primary brain cancer or brain metastases. Contrast-enhanced magnetic resonance imaging showed that the Mel57, K1735Br2 and RG-2 lesions grow without disruption of the BBB, whereas the BBB was leaky in the U87MG and VEGF-A-transfected Mel57 lesions. This was confirmed by immunohistochemistry. Bioluminescence measurements allowed the visualisation of tumour burden already within 4 days after injection of the tumour cells. The applicability of our models for performing efficacy studies was demonstrated in an experiment using temozolomide as study drug. In conclusion, we have developed experimental brain tumour models with partly disrupted, or completely intact BBB properties. In vivo imaging by luciferase allows convenient follow-up of tumour growth and these models will be useful for chemotherapeutic intervention studies.

In vivo activities of mutants of vascular endothelial growth factor (VEGF) with differential in vitro activities.

VEGF mutants in which Cys51 or Cys60 are converted into a serine are poor inducers of proliferation in human umbilical vein endothelial cells, but they have wild-type activity in the Miles vascular permeability assay. To assess the contribution of proliferation vs. other VEGF activities such as vascular permeability, to tumor angiogenesis and growth, C127I cells, transfected with BPV-based expression plasmids carrying wild-type or mutated VEGF cDNAs, were injected subcutaneously in BALB/c nu/nu mice. From C127I cells expressing wtVEGF(165), intensely vascularized and invasive tumors developed within 2 to 3 weeks. From cells expressing VEGF-Cys51Ser or VEGF-Cys60Ser, tumors developed only after 2 to 3 months, comparable to the time of development of control tumors, i.e., tumors from cells transfected with empty vector. Despite the late take, the VEGF-Cys51Ser and VEGF-Cys60Ser tumors developed an extensive vascular bed with an architecture comparable to that of recombinant wtVEGF-producing tumors whereas control tumors had a considerably lower vascular density. No metastases were detected in mice carrying either wtVEGF or mutant VEGF expressing tumors. Thus, because proliferation-defective VEGF-mutants cannot induce angiogenesis, we conclude that the proliferation-inducing effect of VEGF is crucial for tumor angiogenesis and growth. The hypervasculature in the tumors expressing these VEGF-mutants suggests, however, that other VEGF-activities, such as the induction of vascular permeability, strongly affects vascular density and vascular structure. Furthermore, neither overexpression of VEGF or a high vascular density or hyperpermeability of tumor vasculature is necessarily followed by metastasis.

Targetting VEGF in anti-angiogenic and anti-tumour therapy: where are we now?

Since the recognition of the importance of the vascular bed for growth and metastasis of solid tumours, many researchers have investigated the approach of attacking the tumour vascular bed instead of the tumour cells themselves in anti-cancer therapy. Such approaches have become possible with the increasing knowledge of the angiogenic process and the factors that regulate it. Especially the potent angiogenic factor VEGF has been the subject of extensive study in this regard. A number of studies showed that inactivation of this factor or its receptors led to a profound negative effect on the development of experimental tumours. However, despite the encouraging results obtained in animal studies, it remains to be established whether human tumours, which might be in a state of relative quiescence, are as sensitive to anti-VEGF treatment as the fast-growing tumours that are generally used in animal studies. If so, anti-VEGF treatment might certainly represent a powerful tool in anti-cancer therapy, either or not in combination with other blockers of angiogenesis.

Synergism between temporally distinct growth factors: bFGF, insulin and lens cell differentiation.

Fibroblast growth factors (FGFs) are the only known factors that can induce differentiation of the mammalian lens epithelial cell, while insulin acts only as a mitogen, not as a morphogen. We show here that insulin enhances expression of the alphaA-crystallin gene in lens epithelial cells and induces the synthesis of lens fibre cell specific betaB2- and gamma-crystallins in early differentiated fibre cells. Different signal transduction pathways are required for bFGF or insulin maintained fibre cell differentiation. A 15 min preincubation with bFGF was sufficient for the lens epithelial cells to become competent to undergo insulin maintained differentiation. The phorbol ester TPA could replace bFGF. The bFGF instructed competence to differentiate decays with a half-life of about 30 h. Hence, bFGF and insulin can act in concert to produce a differentiated phenotype even when they are not present simultaneously.

Mutants of basic fibroblast growth factor identify different cellular response programs.

Mutations, expected to affect the intracellular routing, i.e. additional nuclear localization sequences (NLS; the natural 23 kDa isoform and a 17D27R mutant) and/or a deletion of amino acids 26-29 (23 delta 26-29 and 17 delta 26-29), were introduced in basic fibroblast growth factor (bFGF). The mutants were assayed for their mitotic activity and their capacity to induce a tissue-specific response in human umbilical vein endothelial cells [HUVECs; induction of urokinase plasminogen activator receptor (u-PAR)], or in rat lens epithelial cells (fibre cell differentiation). In HUVECs, the 17D27R mutant had wild type activity, the 23 kDa and the delta 26-29 proteins were impaired in the induction of both mitosis and u-PAR. The delta 26-29 proteins, but not the 23 kDa protein or 17D27R mutant, were also impaired in receptor binding in that they bound only to a subset of receptors. The concentration of 17 kDa bFGF required for half maximal u-PAR response was 30 fold higher than for the half maximal 3H-thymidine incorporation. Addition of an NLS to bFGF strongly inhibited the induction of fibre cell differentiation, though it had little effect on the stimulation of DNA synthesis. The 17 delta 26-29 kDa mutant had wild type differentiation activity but was a poor mitogen for lens epithelial cells.

Species specificity for HBsAg binding protein endonexin II.

BACKGROUND/AIMS: Hepatitis B virus displays a distinct species and tissue tropism. Previously we have demonstrated that a human liver plasma membrane protein with a molecular weight of approximately 34 kiloDalton specifically binds to HBsAg. This protein was identified as endonexin II, a Ca2+ dependent phospholipid binding protein. METHODS: Using a mouse monoclonal antibody, directed against the HBsAg binding epitope on human endonexin II, liver tissue from various non-human species, human liver tissue and some extra-hepatic human tissues were screened for the presence of endonexin II. RESULTS: Endonexin II was detectable in human, chimpanzee and rhesus monkey liver and in all tested extra-hepatic human tissues, using western blot and immunohistochemical techniques. In rat, mouse, cow and pig liver tissues endonexin II could not be detected with the antibody. CONCLUSIONS: The species specific distribution of the HBsAg binding protein endonexin II apparently correlates with the species tropism of hepatitis B virus. Furthermore, the detection of HBV-DNA, RNA transcripts and antigens in a variety of tissues in chronic infected patients, is in agreement with the wide distribution of the HBsAg binding endonexin II in various tissues.

Hepatitis B virus: specific binding and internalization of small HBsAg by human hepatocytes.

Previously, we identified human liver endonexin II (EII) present on human hepatocyte plasma membrane as a specific hepatitis B surface antigen (HBsAg) binding protein. We also showed the spontaneous development of anti-idiotypic (anti-HBsAg) antibodies in rabbits immunized with EII and in chicken immunized with the F(ab')2 fragment of rabbit anti-EII IgG. These findings suggest the existence of a receptor-ligand relationship between EII and HBsAg. In the present study, we demonstrate that small HBsAg conjugated to 10 nm colloidal gold also binds specifically to human hepatocytes. Invagination of the coated pit region at the HBsAg binding sites on the human hepatocyte plasma membrane results in the internalization of the HBsAg-gold particles. The binding and consequently the internalization of HBsAg is inhibited by anti-EII or anti-idiotypic (anti-HBsAg) antibodies. These findings indicate that EII is directly involved in the binding and uptake of hepatitis B envelope proteins.

Cloning and production of functional active recombinant hepatitis B virus surface antigen binding protein.

Endonexin II present on the surface of human hepatocytes has recently been identified as a hepatitis B virus surface antigen (HBsAg) binding protein. A full-length cDNA clone encoding human endonexin II was isolated from a human liver cDNA library and was placed under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Infection of Spodoptera frugiperda cells with recombinant virus resulted in the production of high amounts of recombinant protein. This protein has the same molecular weight and iso-electric point as native human endonexin II. It can be easily purified by methods analogous to those described for the native protein. Moreover, the recombinant product binds very efficiently to hepatitis B surface proteins (HBsAg) in a similar fashion as native human endonexin II.

Spontaneous development of anti-hepatitis B virus envelope (anti-idiotypic) antibodies in animals immunized with human liver endonexin II or with the F(ab')2 fragment of anti-human liver endonexin II immunoglobulin G: evidence for a receptor-ligand-like relationship between small hepatitis B surface antigen and endonexin II.

In a previous study, we have identified endonexin II (E-II) on human liver plasma membranes as a specific, Ca(2+)-dependent, small hepatitis B surface antigen (HBsAg)-binding protein. In this article, we describe the spontaneous development of anti-HBs antibodies in rabbits immunized with native or recombinant human liver E-II and in chickens immunized with the F(ab')2 fragment of rabbit anti-human liver E-II immunoglobulin G. Anti-HBs activity was not observed in rabbits immunized with rat liver E-II. Cross-reactivity of anti-E-II antibodies to HBsAg epitopes was excluded, since anti-HBs and anti-E-II activities can be separated by E-II affinity chromatography. The existence of an anti-idiotypic antibody is further demonstrated by competitive binding of human liver E-II and this antibody (Ab2) to small HBsAg, suggesting that Ab2 mimics a specific E-II epitope that interacts with small HBsAg. In addition, it was demonstrated that anti-HBs antibodies developed in rabbits after immunization with intact human liver E-II or in chickens after immunization with F(ab')2 fragments of rabbit anti-human liver E-II immunoglobulin G recognize the same epitopes on small HBsAg. These findings strongly indicate that human liver E-II is a very specific small HBsAg-binding protein and support the assumption that human liver E-II is the hepatitis B virus receptor protein.

In vitro binding properties of the hepatitis delta antigens to the hepatitis B virus envelope proteins: potential significance for the formation of delta particles.

To investigate the possible existence of (a) reactive binding site(s) on the hepatitis B surface antigen (HBsAg) for the hepatitis delta antigen (delta Ag) in the hepatitis delta virus (HDV), we performed binding studies using recombinant (rec)Small, recMiddle, recLarge HBsAg and recombinant small (S) and large (L) hepatitis delta antigen (recS delta Ag, recL delta Ag). Rec delta Ag was immobilized onto microtiter plates and incubated with recSmall, recMiddle and recLarge HBsAg. Of the three HBsAg proteins only the recMiddle HBsAg was found to bind to recS delta Ag. This binding was inhibited by the addition of synthetic PreS2 peptide but not by small HBsAg, indicating that the S delta Ag exhibits a PreS2 binding site. RecL delta Ag bound to all three forms of HBsAg. The binding of the HBsAg to recL delta Ag was saturable and could be blocked with an excess of HBsAg, but not with BSA. The region of the additional 19 amino acids of the L delta Ag is therefore responsible for the creation of the small HBsAg binding site on the L delta Ag. We therefore suggest that all HBsAg proteins but particularly the small HBsAg in the HDV coat seem to be involved in the interaction with the HDV core particle and that the PreS2 region of the middle HBsAg plays a crucial role in binding to small delta Ag during HDV particle formation, probably to increase the stability of the HDV particle.

Hepatitis delta virus attaches to human hepatocytes via human liver endonexin II, a specific HBsAg binding protein.

Recently we identified human liver endonexin II (EII) as a specific hepatitis B surface antigen (HBsAg) binding protein. To investigate whether EII is also able to interact with the HBsAg envelope of the hepatitis delta virus (H delta V), immunoprecipitation experiments were performed. H delta V particles could be co-precipitated by polyclonal rabbit anti-EII, but not by rabbit anti-glutathiontransferase (GST pi) antibodies from an H delta V-enriched fraction containing EII or GST pi. These findings suggest that H delta V particles were co-precipitated by anti-EII as a consequence of the binding between HBsAg present in the H delta V envelope and EII. Furthermore, binding of H delta V particles to human hepatocytes could be inhibited by incubation of the liver cells with rabbit anti-EII IgG or the H delta V particles with anti-idiotypic (anti-HBsAg) antibodies, developed spontaneously in rabbits immunized with EII. These findings support the assumption that small HBsAg present in the H delta V envelope is important for the attachment to the hepatocytes and that EII plays an important role in this process.

Endonexin II, present on human liver plasma membranes, is a specific binding protein of small hepatitis B virus (HBV) envelope protein.

Binding of viral envelope proteins to specific receptors on human hepatocytes is considered to be an important step in HBV infection. In this study, we demonstrate that a 34-kDa human liver plasma membrane protein specifically binds to small HBsAg in a Ca(2+)-dependent manner. By partial amino acid sequence analysis of preparatively isolated 34-kDa protein comigrating with HBsAg-binding protein obtained from binding assay on IEF/SDS-PAGE, we have identified this HBsAg-binding protein as Endonexin II (E-II). Native human liver E-II inhibits binding of HBsAg to intact human hepatocytes and shows specific binding to small HBsAg. This binding can be inhibited by human liver plasma membrane proteins, recombinant E-II, or anti-E-II antibodies. Despite 90% sequence homology, rat liver E-II does not bind to small HBsAg and does not inhibit significantly (less than 20%) binding of HBsAg to intact hepatocytes. Cross-linking of small HBsAg and radiolabeled human liver E-II resulted in a specific additional protein complex on PAGE with an apparent molecular weight of 90 kDa, corresponding to a complex of E-II and small HBsAg with a ratio of 2 to 1 or 1 to 2. These findings indicate that E-II, found in human liver, is a specific HBsAg-binding protein and might play an important role in the initiation of HBV infection.

Binding of the major and large HBsAg to human hepatocytes and liver plasma membranes: putative external and internal receptors for infection and secretion of hepatitis B virus.

A likely mechanism of the strong hepatotrophism of the hepatitis B virus is the presence of specific receptors for the surface antigen of hepatitis B virus on hepatocyte membranes. To examine this hypothesis, we have performed binding studies using recombinant large (preS1 + preS2 + S) and major (S) proteins with adult human hepatocytes, rat hepatocytes, human fibroblasts, human peripheral blood mononuclear cells and plasma membranes derived from these cell types. We found that major HBsAg was able to bind specifically to human hepatocytes, human fibroblasts and human blood mononuclear cells. This binding could be inhibited by recombinant middle (preS2 + S) protein but not by the recombinant large protein. No binding could be demonstrated between large HBsAg and human hepatocytes. However, large protein bound specifically to plasma membranes derived from human liver tissue, human fibroblasts and HepG2A16 cells. This binding could be partially inhibited by the major protein and by a synthetic preS1 peptide but not by a synthetic preS2 peptide. These results support the assumption that hepatitis B virus absorption and penetration into human hepatocytes is mediated by specific receptors recognizing an amino acid sequence in the S-region. This recognition site is not displayed by the recombinant large protein. However, the large protein is recognized by its preS1 region and by a second binding site in the S-region by a receptor molecule, located on the inner surface of the plasma membranes or intracellular membranes of human hepatocytes and of some other cell types derived from human tissue.