Targeting the endothelin axis with atrasentan, in combination with IFN-alpha, in metastatic renal cell carcinoma.

BACKGROUND: The endothelin system is involved in tumour growth. Atrasentan, a selective endothelin-A-receptor antagonist, blocks endothelin signalling. This phase I trial studied combining treatment of interferon-alpha (IFN-α) with atrasentan in renal cell carcinoma (RCC). PATIENTS AND METHODS: This study evaluated the safety and tolerance of IFN-α (9MU subcutaneously (s.c.) three times a week) in combination with atrasentan (2.5, 5 and 10 mg orally once daily) in untreated metastatic RCC. Cohort 10 mg was extended to obtain insights in efficacy and pharmacodynamics. RESULTS: Observed toxicities mainly consisted of known IFN-like toxicities (anorexia, chills, fever, fatigue and nausea), and of nasal congestion (associated to atrasentan). None of these toxicities were considered dose limiting. Cohort 10 mg was extended up to 32 patients; in a subset of patients treated according to the protocol (n=27), median overall survival (OS) was 17.3 months. One patient (3.1%) showed a partial response lasting 14.3 months. In an exploratory analysis, we observed that in the subset of patients with declining vascular endothelial growth factor (VEGF) levels (in combination with rising Endothelin-1 levels), median OS was 22.2 months compared with 2.2 months in patients with increasing VEGF levels. CONCLUSION: Combination treatment of IFN-α 9MU-α s.c. three times a week and atrasentan 10 mg once daily is tolerated. Clinical activity, especially OS, and biomarkers in our view warrant further studies targeting the endothelin axis.

Supernatants of human leukocytes contain mediator, different from interferon gamma, which induces expression of MHC class II antigens.

In this report, data are presented on the regulation of MHC class II antigen expression by a mediator present in supernatants of human mixed leukocyte cultures (MLC-SN), and which is different from IFN-gamma. The capacity of supernatants to induce antigen expression did not correspond to titers of IFN-gamma. Removal of IFN-gamma using either dialysis against pH 2 or neutralizing mAb against human IFN-gamma did not abrogate the MHC class II antigen expression-inducing capacity of MLC-SN when tested on adenocarcinoma cell lines, kidney epithelial cells, and fibroblasts in vitro in an indirect immunofluorescence assay. Therefore, supernatants of human leukocytes contain a mediator, different from IFN-gamma, which induces expression of MHC class II antigens. Dose-response studies revealed that the mediator is produced after allogeneic and lectin stimulation of human leukocytes, and by unstimulated leukocytes. Activation of leukocytes resulted in increased titers of the mediator. The mediator markedly enhances expression of both HLA-DR and HLA-DQ antigens, whereas IFN-gamma had a similar effect on HLA-DR antigens, and only a minor effect on HLA-DQ antigens. Interaction of the mediator and IFN-gamma resulted in a potentiating effect of these two factors on MHC class II antigen expression. Biochemical analysis revealed a mediator, distinguishable by FPLC from IL-1, IL-2, and human IFN-gamma, and which has a molecular mass of 32 kD.

Two-protein signature of novel serological markers apolipoprotein-A2 and serum amyloid alpha predicts prognosis in patients with metastatic renal cell cancer and improves the currently used prognostic survival models.

BACKGROUND: In metastatic renal cell cancer (mRCC), the Memorial Sloan-Kettering Cancer Center (MSKCC) risk model is widely used for clinical trial design and patient management. To improve prognostication, we applied proteomics to identify novel serological proteins associated with overall survival (OS). PATIENTS AND METHODS: Sera from 114 mRCC patients were screened by surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS). Identified proteins were related to OS. Three proteins were subsequently validated with enzyme-linked immunosorbent assays and immunoturbidimetry. Prognostic models were statistically bootstrapped to correct for overestimation. RESULTS: SELDI-TOF MS detected 10 proteins associated with OS. Of these, apolipoprotein A2 (ApoA2), serum amyloid alpha (SAA) and transthyretin were validated for their association with OS (P = 5.5 x 10(-9), P = 1.1 x 10(-7) and P = 0.0004, respectively). Combining ApoA2 and SAA yielded a prognostic two-protein signature [Akaike's Information Criteria (AIC) = 732, P = 5.2 x 10(-7)]. Including previously identified prognostic factors, multivariable Cox regression analysis revealed ApoA2, SAA, lactate dehydrogenase, performance status and number of metastasis sites as independent factors for survival. Using these five factors, categorization of patients into three risk groups generated a novel protein-based model predicting patient prognosis (AIC = 713, P = 4.3 x 10(-11)) more robustly than the MSKCC model (AIC = 729, P = 1.3 x 10(-7)). Applying this protein-based model instead of the MSKCC model would have changed the risk group in 38% of the patients. CONCLUSIONS: Proteomics and subsequent validation yielded two novel prognostic markers and survival models which improved prediction of OS in mRCC patients over commonly used risk models. Implementation of these models has the potential to improve current risk stratification, although prospective validation will still be necessary.

Impact of integration of clinical and outpatient units on cancer patient satisfaction.

OBJECTIVE: There is an ongoing drive to measure and improve quality of care. Donabedians' quality framework with structure, process and outcome domains provides a useful hold to examine quality of care. The aim of this study was to address the effect of an intervention in hospital structure (integration of three units into one) with the purpose of improving processes (increase meeting, cooperation and communication between professionals and patients) and its effect on the outcome (cancer patient satisfaction). DESIGN: Pre-test-post-test. SETTING: University Medical Center Utrecht, The Netherlands, Department of Medical Oncology. PARTICIPANTS: Cancer patients (n = 174, n = 97). INTERVENTIONS: Physical integration by bringing separately located units (outpatient clinic, day-care clinic, clinical ward) together in one wing of the hospital and adjustments in communication and coordination structures. MAIN OUTCOME MEASURE: Patient satisfaction questionnaire. RESULTS: Satisfaction with care improved for six scales (27%) after integration. Effect sizes (ESs) ranged from 0.36 to 0.80, indicating a small to moderate effect. The most important improvement was found at the day-care clinic on aspects like 'the degree in which the nurses were informed about a patients situation', 'privacy', 'interior design', 'quality of hospital equipment', 'sanitary supplies' and 'waiting periods'. With regard to continuity and coordination of care, satisfaction increased for five items (28% of items concerning continuity and coordination of care). ESs ranged from 0.42 to 0.75. CONCLUSIONS: Integration of three oncology units into one unit had a positive impact on care delivery processes and resulted in improved patient satisfaction concerning care and treatment.

[Angiogenesis inhibitors for the systemic treatment of metastatic renal cell carcinoma: sunitinib, sorafenib, bevacizumab and temsirolimus]. / Angiogeneseremmers voor de systemische behandeling van gemetastaseerd niercelcarcinoom: sunitinib, sorafenib, bevacizumab en temsirolimus.

Treatment of patients with metastatic renal cell carcinoma is evolving rapidly due to the advent of novel targeted therapies. Improved knowledge of the underlying pathogenesis has led to the development of drugs that modulate the dominant signal transduction pathways for this disease, which results in inhibition of angiogenesis. Recent evidence indicates that the receptor tyrosine kinase inhibitor sunitinib prolongs progression-free survival compared with interferon-alpha, especially in patients with intermediate risk. Immunotherapy with interferon-alpha or high-dose interleukin-2 should still be considered for low-risk patients, particularly those with clear-cell tumours and metastases of the lung only. In patients who fail treatment with interferon-alpha, sorafenib has been shown to improve progression-free survival. High-risk patients may benefit from treatment with temsirolimus, which inhibits mammalian target of rapamycin (mTOR) kinase activity and has shown to improve overall survival. These angiogenesis inhibitors did not receive mention in the recently published guideline 'Renal cell carcinoma'.

Endothelial intercellular adhesion molecule-1 expression is suppressed in human malignancies: the role of angiogenic factors.

Intercellular adhesion molecule 1 (ICAM-1) is involved in the recirculation of blood leukocytes and, presumably, in the infiltration of cytolytic effector leukocytes into tumors. The present report describes a down-regulated expression of vascular ICAM-1 on tumor-infiltrating endothelial cells (EC) in renal cell carcinoma. This finding was obtained by flow cytometric analysis of tumor EC compared to EC obtained from healthy tissue. Since growth of solid tumors is dependent on the formation of new blood vessels (angiogenesis), we hypothesized that angiogenic factors are responsible for the down-regulation of ICAM-1. This hypothesis was investigated in vitro using human umbilical vein- and dermis-derived EC. Using flow cytometry, we found a biphasic regulation of ICAM-1 during stimulation of cultured EC with the angiogenic agent basic fibroblast growth factor (bFGF). Although 16-24 h after activation a marked up-regulation of ICAM-1 was observed, expression was significantly decreased after 48h. The longevity of this down-regulation was at least 7 days. Northern blot analysis revealed down-regulation of the steady-state mRNA level of the gene. ICAM-2 showed similar results of intial up- and later down-regulation. Functional relevance for the changes in ICAM-1 expression was demonstrated by a corresponding biphasic regulation of EC-leukocyte adhesion after EC activation by bFGF. The described effects are specific for bFGF since other angiogenic factors (such as vascular endothelial growth factor, transforming growth factor beta, and interleukin 8) did not affect adhesion molecule expression. Subsequent experiments showed that angiogenic factors decrease the sensitivity of EC to activation with tumor necrosis factor-alpha in regard to adhesion molecule expression. The present results reveal a tumor-derived escape mechanism from cytolytic effector leukocytes by down-regulation of vascular adhesion molecules in vivo and in vitro and decreased responsiveness to proinflammatory cytokines.

The angiogenic factor bFGF impairs leukocyte adhesion and rolling under flow conditions.

Recirculation of leukocytes is mediated by the intricately regulated expression of adhesion molecules on both the vessel wall and leukocyte membranes. In the present paper it is demonstrated that tumor angiogenesis factors impair leukocyte rolling and adhesion under flow conditions. Three lines of evidence presented in this paper support this finding; (i) treatment of cultured endothelial cells (EC) with the angiogenic factor basic fibroblast growth factor (bFGF) results in decreased ICAM-1 expression and decreased numbers of adhering leukocytes under flow conditions. (ii) flow induced upregulation of endothelial ICAM-1 in the presence of bFGF does not yield ICAM-1 levels higher than on resting EC. (iii) bFGF decreases the TNFalpha mediated induction of E-selectin and ICAM-1 expression, resulting in decreased rolling and firm adhesion of leukocytes on the endothelial surface. For ICAM-1 it is demonstrated that bFGF inhibits TNFalpha induced levels of mRNA, and that this effects is transcriptionally regulated. These findings support our earlier described hypothesis that angiogenic factors are involved in the tumor derived escape mechanism from immune surveillance, since we demonstrate here that these mechanisms are operative under physiologic flow conditions.

Limiting dilution assays. Experimental design and statistical analysis.

Two issues in limiting dilution analysis are considered. The first concerns the experimental design: a mathematical algorithm has been developed which calculates the number of replicate culture groups, and the (mean) number of cells per well to be used on the basis of the experimenter's a priori information about the unknown frequency. The procedure guarantees useful data if the a priori interval estimate of the frequency to be determined is correct and the cells are willing to grow. The second issue concerns the statistical method to be used for estimation of the unknown frequency. Several methods (minimum chi-square, maximum likelihood and the jackknife version of the maximum likelihood method) have been evaluated with artificial data from extensive Monte Carlo experiments. All three methods were useful in the statistical analysis of data. As a result of these experiments and theoretical considerations the jackknife version of the maximum likelihood estimation procedure is proposed as the statistical procedure of choice. The next best method is the maximum likelihood procedure.

Alloantigen presentation by canine endothelial cells. Presentation of autologous alloantigens in vitro.

Presentation of autologous alloantigen by certain cells in an allograft can result in allograft rejection. The precise type of cell in a graft responsible for this aspect of allograft rejection remains to be established. Here we report the capacity of canine venous endothelial cells to activate, in vitro, allogeneic lymphocytes for proliferation and differentiation by presentation of alloantigen. Antigen-presenting cell (APC)-depleted lymphocyte populations, prepared in a multistep procedure and tested for absence of APC, were cocultured with allogeneic venous endothelial cells. Proliferation and differentiation into cytotoxic T lymphocytes were measured. While mixed lymphocyte culture of APC-depleted lymphocytes did not result in proliferation and differentiation, coculture of allogeneic APC-depleted lymphocytes with venous endothelial cells resulted in proliferation and generation of cell-mediated cytotoxicity in these cultures. It is concluded that canine venous endothelial cells in vitro have the capacity to present alloantigen. The data suggest an essential role for endothelium in the initial phase of allograft rejection.

Effect of cyclosporine on MHC class II antigen expression on arterial and venous endothelium in vitro.

MHC class II antigens play a crucial role in immunological responses. The expression of MHC class II antigens on monocytes and endothelial cells is reported to be variable and able to be induced by gamma-interferon. In this study we report on MHC class II antigen expression in vitro by arterial and venous canine endothelial cells, as detected with FACS analysis and indirect immunofluorescence with a monoclonal antibody against canine MHC class II antigens. It appears that cultured endothelial cells do not express MHC class II antigens. Their expression could be induced during a three-day incubation period in lymphokine-containing supernatant produced in mixed leukocyte culture (MLC). Cyclosporine (CsA) added to allogeneically stimulated or unstimulated canine lymphocytes in MLC inhibited the induction of expression by the MLC supernatant. The addition of CsA to MLC supernatant did not have an inhibitory effect. It is concluded that CsA inhibits the production of an MHC class-II-antigen-inducing lymphokine produced by lymphocytes in mixed cultures; allogeneic stimulation is not necessary for production of the lymphokine. It is postulated that a possible mode of action of CsA in prolongation of allograft survival is based on prevention of the induction of MHC class II antigen expression by endothelial cells.

Cell-mediated cytotoxicity patterns of cloned cytotoxic T lymphocytes. Cytotoxicity directed against canine lymphoblasts, monocytes, and endothelial cells.

Knowledge of the antigens recognized during allograft rejection is still incomplete. Cloned cytotoxic T lymphocytes were used to study the distribution of target determinants in the dog. CTL clones were obtained with a limiting dilution technique from effector cells generated in mixed lymphocyte culture. The clones have been tested for cell-mediated cytotoxicity against PHA-stimulated lymphoblasts, monocytes, and arterial and venous endothelial cells. A limited number of patterns of lysis of one, or more than one, of the four different target cells was observed. The nature of the possible target determinants recognized by these CTL-clones is discussed.

Limiting dilution analysis of canine alloantigen-reactive T lymphocytes.

A sensitive limiting dilution assay for the determination of canine cytotoxic T lymphocyte precursors (CTL-P) has been developed. Murine second MLC supernatant as a source of Interleukin 2 (IL 2) was used to expand stimulated CTL-P to numbers that were easily detectable with the classic 51Cr lysis assay. The frequencies of CTL-P that reacted to allogeneic stimulator cells in canine peripheral blood ranged from 1:1000 to 1:2000 lymphocytes. During in vitro stimulation in a mixed leukocyte culture a rapid increase in frequency was noted, and at day 7 a frequency as high as 1:5.4 was found. The presence of irradiated stimulator cells during limiting dilution culture restricted proliferation of the CTL-P; only those which recognized the stimulator cells proliferated. The determination of cytotoxicity in large numbers of individual microcultures with CTL-P seeded at clonal conditions toward 2 allogeneic target cells permitted quantitation of the frequency of CTL-P with specificity for different alloantigens. These techniques greatly facilitate monitoring of cellular immune reactions after renal allografting because both determination of the influence of cytostatic drug treatment on CTL-P frequency, and analysis of the specificity and function of allograft infiltrating cells are now possible.

In vitro stimulation of lymphocytes by vascular endothelial cells. A study with canine arterial and venous endothelial cells.

Data are presented on the ability of arterial and venous endothelial cells to stimulate allogeneic leukocytes. Mixed cultures of allogeneic endothelium and lymphocytes result in proliferation of lymphocytes and generation of cell-mediated cytotoxicity, which do not occur in cultures of syngeneic combinations of endothelium and lymphocytes. Studies of kinetics showed a peak in proliferation at days 6-7. The optimal responder-stimulator ratio appeared to be 15:1. Lymphocytes stimulated with venous endothelial cells were cytotoxic both for arterial and for venous endothelial cells and PHA blasts of the stimulator dog, whereas lymphocytes stimulated with arterial endothelial cells lysed only arterial endothelial cells and PHA blasts of the stimulator. Lysis of syngeneic or third-party allogeneic control targets was virtually absent. Optimal conditions for long-term culture of effector cells from mixed leukocyte endothelial cell cultures were analyzed. Addition of Il 2 every 3 days and the original stimulating antigen every 6 days permitted continuous proliferation of these cytotoxic lymphocytes with preservation of the cytotoxicity pattern.

Influence of a skin allograft on frequencies of CTL precursor in peripheral blood of the dog.

Alloantigen-specific cytotoxic T lymphocytes (CTL) and their precursors (CTL-P) have been determined in the peripheral blood of skin allografted dogs. CTL-P frequencies increased rapidly after transplantation and reached maximal values after complete rejection of the skin allograft. Differences in the time response kinetics of CTL-P frequencies between recipients were not correlated with the length of graft survival. The CTL-P frequencies declined after days 13-20 and appeared still to be elevated 30 days after rejection of the graft.

Endothelial CD34 is suppressed in human malignancies: role of angiogenic factors.

We report the suppressed vascular CD34 expression in renal cell carcinoma. This was found by quantitatively analyzing CD34 expression on normal and tumor derived EC by flow cytometry. In vitro studies revealed that culture of umbilical cord or dermis derived microvascular EC with angiogenic factors such as basic fibroblast growth factor (bFGF) and vascular endothelial cell growth factor induced downregulation of CD34. This angiogenesis-induced downregulated expression of CD34 adhesion molecule may contribute to the tumor mediated escape mechanism from immune surveillance. It is concluded that there are quantitative differences in expression of endothelial CD34 in different compartments of the vasculature, that angiogenic factors affect this expression and that subpopulations of EC exist with differences in EAM expression.

Analysis of buoyant density of canine peripheral blood leukocytes with PVP-Silica (Percoll) density gradients.

Isolation of canine peripheral blood mononuclear cells with a one step centrifugal separation procedure has not been very successful sofar. Significant contamination with polymorphonuclear cells has been reported. An analysis of the buoyant density of canine peripheral blood leukocytes on a self-generating Percoll gradient showed that the buoyant densities of polymorphonuclear cells and lymphocytes are so near that separation with high purity and yield is not possible with the use of a density gradient. Transient changes in buoyant density of polymorphonuclear cells have been observed. In such situations differences in buoyant density between cell types have been observed which permit separation of mononuclear cells from polymorphonuclear cells at a reasonable yield.

Canine IL-2: characterization and optimal conditions for production.

The culture requirements for the production of canine I1-2 are reported. Several parameters have been tested, such as concentration of lectin, length of culture period and presence of the additives serum, polyethylene glycol (PEG) and phorbol myristic acetate (PMA). Optimal results have been obtained by stimulation of peripheral blood leukocytes with the lectin PHA (8 micrograms/ml) for 48 h. Techniques for the production of I1-2 containing supernatant free of PHA have been evaluated. Gel filtration chromatography of culture supernatant revealed that canine I1-2 has a molecular weight (m.w.) of approximately 30,000 daltons, similar to the m.w. of I1-2 produced by murine T cells.

Effect of culture conditions on endothelial cell growth and responsiveness.

The in vitro culture of endothelial cells (EC) is dependent on the presence of a coated surface and the availability of growth factors in the medium. The aim of the present research is to investigate whether in vitro EC culture conditions, such as serum source and surface coating, determine the growth characteristics of EC. The phenotype of EC was studied at the level of adhesion molecule expression and down-regulation by angiogenic factors. We found that human umbilical vein EC adhere well to and stretch well with plastic coated with fibronectin, collagen, gelatin and hyaluronan in contrast to non-coated plastic. While low in hyaluronan-coated wells, the spontaneous proliferation of EC was enhanced in fibronectin-collagen and gelatin-coated wells as compared to non-coated wells. Basic fibroblast growth factor bFGF-induced proliferation, however, was best on hyaluronan-coated plastic. A markedly up-regulated proliferation was measured on fibronectin and collagen while EC on gelatin-coated plastic only showed moderate bFGF-induced proliferation. On non-coated plastic EC were not inducible with bFGF. The induction of apoptosis by serum deprivation on these different matrices was most efficient when no coat was available or when wells were coated with hyaluronan, and bFGF inhibited apoptosis induction under all conditions. The use of different culture media demonstrated that human and bovine serum both can be used for human EC assays. The synthetic medium Utroser G prevented both spontaneous and growth factor-induced proliferation. We found that apart from some magnitude differences, the down-regulation of intercellular adhesion molecule-1 (ICAM-1) by angiogenic factors such as bFGF is not dependent on specific culture conditions.

[Interferon for adjuvant therapy in melanoma; although approved, not indicated]. / Interferon als adjuvante therapie bij het melanoom: wel geregistreerd, niet geïndiceerd.

The Dutch melanoma group reconsidered their 1997 consensus statement on treatment of melanoma because new studies on adjuvant treatment with interferon(IFN)-alpha have been published. These have resulted in its registration for stage IIa; for stage IIb/III IFN-alpha was already registered. Overall survival should be the main endpoint of adjuvant clinical studies, especially when treatment is associated with toxicity. Since a benefit has not been unequivocally demonstrated in melanoma with Breslow thickness > 1.5 mm and/or regional lymph node metastases, there is no need to change the Dutch consensus statement. Drug registration authorities and medical professionals should cooperate more closely.