Follow-up after focal therapy in renal masses: an international multidisciplinary Delphi consensus project.

PURPOSE: To establish consensus on follow-up (FU) after focal therapy (FT) in renal masses. To formulate recommendations to aid in clinical practice and research. METHODS: Key topics and questions for consensus were identified from a systematic literature research. A Web-based questionnaire was distributed among participants selected based on their contribution to the literature and/or known expertise. Three rounds according to the Delphi method were performed online. Final discussion was conducted during the "8th International Symposium on Focal Therapy and Imaging in Prostate and Kidney Cancer" among an international multidisciplinary expert panel. RESULTS: Sixty-two participants completed all three rounds of the online questionnaire. The panel recommended a minimum follow-up of 5 years, preferably extended to 10 years. The first FU was recommended at 3 months, with at least two imaging studies in the first year. Imaging was recommended biannually during the second year and annually thereafter. The panel recommended FU by means of CT scan with slice thickness ≤3 mm (at least three phases with excretory phase if suspicion of collecting system involvement) or mpMRI. Annual checkup for pulmonary metastasis by CT thorax was advised. Outside study protocols, biopsy during follow-up should only be performed in case of suspicion of residual/persistent disease or radiological recurrence. CONCLUSIONS: The consensus led to clear FU recommendations after FT of renal masses supported by a multidisciplinary expert panel. In spite of the low level of evidence, these recommendations can guide clinicians and create uniformity in the follow-up practice and for clinical research purposes.

Inhibition of the alpha-nu integrins with a cyclic RGD peptide impairs angiogenesis, growth and metastasis of solid tumours in vivo.

Anti-angiogenetic cancer therapy is a potential new form for treatment of solid tumours. The alpha(v)-integrins (alpha(v)beta3, alpha(v)beta5) mediate the contact of activated endothelial cells to proteins of the extracellular matrix during tumour angiogenesis as a prerequisite for survival of endothelial cells. The aim of this study was to investigate the effects of application of a methylated cyclic RGD-peptide as an alpha(v)-integrin antagonist on angiogenesis, microcirculation, growth and metastasis formation of a solid tumour in vivo. Experiments were performed in the dorsal skinfold preparation of Syrian Golden hamsters bearing the amelanotic hamster melanoma A-Mel-3. Animals were injected intraperitoneally with a methylated cyclic RGD-peptide every 12 h, the control group received an inactive peptide. Microcirculatory parameters of tumour angiogenesis including functional vessel density, red blood cell velocity, vessel diameter and leucocyte-endothelium interaction were analysed using intravital microscopy. In an additional study the effects on growth and metastasis of subcutaneous A-Mel-3 were quantified. Functional vessel density was markedly reduced on day 3 in treated animals compared to controls (37.2 +/- 12.1 vs 105.2 +/- 11.2 cm(-1); mean +/- s.e.m.; P<0.05) and increased subsequently in both groups. Red blood cell velocity at day 3 was below values of controls (0.026 +/- 0.01 vs 0.12 +/- 0.03 mm x s(-1); P<0.05). No differences were observed in vessel diameters and leucocyte-endothelium interaction was almost absent in both groups. Furthermore, growth and metastasis of subcutaneous tumours after administration of the cyclic RGD-peptide was significantly delayed in comparison to controls (P<0.05). Inhibition of alpha(v)-integrins by a cyclic RGD-peptide resulted in significant reduction of functional vessel density, retardation of tumour growth and metastasis in vivo. Taken together, these results implicate RGD-peptides as agents which have anti-tumour and anti-metastatic activity in vivo.

High density culturing of porcine hepatocytes immobilized on nonwoven polyurethane-based biomatrices.

OBJECTIVE: Hepatocytes are increasingly used as functional units in bioartificial liver devices. The objective of the present study was to investigate the feasibility of culturing porcine hepatocytes in high density on a novel polyurethane-based nonwoven three-dimensional matrix. We investigated (1) the optimal cell density within this culture configuration, (2) the maintenance of liver-specific morphology and cell functions over long-term periods and (3) the necessity to apply an additional extracellular matrix component (collagen gel). METHODS: Nonwoven polyurethane matrices were manufactured by a specially developed fiber extrusion technology. Pig hepatocytes were cultured at various cell densities of 0.1, 0.25, 0.5, 0.75, 1 and 2 x 10(6) cells/cm(2) on three-dimensional networks of nonwoven polyurethane matrices and cell adhesion as well as functional parameters (DNA of nonattached/attached cells, lactate dehydrogenase release and cytochrome P450 activity) were determined. To assess the performance of cells within this configuration albumin and urea excretion was measured over 8 days. The potentially beneficial effect of an additional extracellular matrix configuration was evaluated by comparing the average albumin synthesis in groups of identical cell numbers. RESULTS: The optimal cell density in this three-dimensional culture configuration was 1 x 10(6) cells/cm(2). The functional capacity of hepatocytes was stable for 8 days at an average level of 53.7 +/- 5.6 ng/h/microg DNA and of 1.8 +/- 0.14 microg/h/microg DNA for albumin and urea excretion, respectively. The supplementation of an extracellular matrix configuration did not improve functional activity of cells. Average albumin synthesis was 35.6 ng/h/microg DNA (28.7, 42.8) and 32.7 ng/h/microg DNA (23.4, 49.2) for collagen-immobilized and control cultures, respectively. CONCLUSION: The results of the study indicate that nonwoven polyurethane sheets supply a biocompatible support structure for functionally active high density cultures. Thus, nonwoven polyurethane matrices should be further investigated on with respect to their role in the development, optimization and design of bioartificial liver systems.

Validation of MR thermometry technology: a small animal model for hyperthermic treatment of tumours.

BACKGROUND: Local hyperthermia has been shown to be an effective adjuvant therapy for cancer. However, progress in this treatment modality requires the non-invasive assessment of temperature distribution in the entire tumour to enable administration of an efficient thermal dose to all tumour areas. Magnetic resonance (MR) imaging offers a promising tool to quantify, non-invasively and three-dimensionally, temperature distribution within tumours. An animal model taking into account the complex interrelationship between pathophysiological changes within a tumour during hyperthermia and temperature-sensitive MR parameters is warranted for the development and validation of new MR thermometry technology. METHODS: An experimental set-up was implemented to allow simultaneous measurements of temperature, tumour blood flow and temperature-sensitive MR parameters under standardised conditions in vivo. Local hyperthermia was induced at 44 degrees C for 20 min under inhalation anaesthesia on seven Syrian Golden hamsters bearing an amelanotic melanoma. Fibreoptic probes were used for reference temperature measurements. Laser Doppler flowmetry served for on-line tumour blood flow determination, and MR thermometry was performed using longitudinal T1 relaxation time measurements. RESULTS: The experimental design enables multifunctional MR thermometry. T1 relaxation times of tumours were 1.44 s (1.36, 1.46) and 1.53 s (1. 48, 1.75) at 37 degrees C and during hyperthermia at 44 degrees C, respectively (median, 25% and 75% quartiles, respectively; P<0.05). At the end of 20 min of hyperthermic treatment at 44 degrees C, relative tumour blood flow was reduced to 40.5% (20.7, 43.3) compared to values before treatment (median, 25% and 75% quartiles, respectively; P<0.05). Imaging of T1 relaxation times revealed a heterogeneous distribution in temperature during hyperthermic treatment. CONCLUSION: This novel in vivo model allows standardised investigations for the development and validation of MR thermography methods.

Pharmacokinetics and selectivity of aminolevulinic acid-induced porphyrin synthesis in patients with cervical intra-epithelial neoplasia.

Photodynamic therapy (PDT), due to its tumor selectivity, represents an alternative approach to diagnose and treat cervical intra-epithelial neoplasia (CIN) without altering normal surrounding tissue. Our aim was to investigate the pharmacokinetics and the selectivity of 5-aminolevulinic acid (5-ALA)-induced porphyrin fluorescence after topical administration, to obtain basic clinical data for future diagnostic fluorescence imaging and PDT protocols for CIN. Twenty-eight non-pregnant women with a cytological diagnosis of low-grade or high-grade squamous intra-epithelial lesions were included. An aqueous solution containing 3% 5-ALA was topically applied 1 to 6 hrs prior to conization using a cervical cap. After excision, porphyrin-induced fluorescence was quantified in dysplastic (n = 14) and normal epithelium (n = 28) by means of quantitative fluorescence microscopy. High values of porphyrin fluorescence were found in squamous epithelium between 150 and 450 min, with a maximum at 300 min following administration of 5-ALA. Ratios of porphyrin fluorescence of dysplastic vs. surrounding normal epithelium were 1.3 and 1.21 for CIN 1 (n = 3) and CIN 2 (n = 3), respectively. In CIN 3 patients (n = 8), this ratio was 2.35; the best selectivity of 5-ALA-induced porphyrin fluorescence in CIN 3 lesions (ratio 3) was observed with a topical administration time of between 150 and 250 min. Our results demonstrate that patients with CIN 3 show higher 5-ALA-induced fluorescence compared with normal epithelium. The optimal administration time of topically applied 5-ALA was between 3 and 4 hr. Our data suggest that topical ALA-PDT and photodynamic diagnosis might be suitable for detecting CIN.

Distribution and pharmacokinetics of Photofrin in human bile duct cancer.

Prognosis of patients with bile duct tumors is mostly poor due to late diagnosis and a lack of adequate curative and palliative treatment modalities. To evaluate the potential of photodynamic therapy (PDT) as a novel and alternative treatment approach, we have investigated the uptake and tumor-specific localization of the photosensitizer Photofrin in human biliary tract neoplasms. We have quantified the distribution and the pharmacokinetics of Photofrin in normal and tumor tissue biopsies of the human bile duct by quantitative fluorescence microscopy and digital image analysis of cryosections. Fluorescence intensities (expressed as a percentage of a standard) are 19.0 +/- 11.4% and 25.2 +/- 12.7% for tumors and 10.9 +/- 2.9% and 13.2 +/- 9.1% (mean +/- SD) for normal bile duct tissue at 24 h (n = 5) and 48 h (n = 8) after Photofrin administration (2 mg kg-1 i.v.), respectively, and decrease afterwards in normal bile duct tissue over the period of investigation (4-35 days). The ratios of fluorescence in tumor versus normal tissue are found to be 1.7 +/- 0.7 and 2.3 +/- 1.2 (mean +/- SD) at days one and two after Photofrin administration, respectively. Thus, Photofrin preferentially accumulates in bile duct neoplasms, reaching peak values during the first two days. These data suggest that laser irradiation should be performed within this period after Photofrin injection to achieve tumor selectivity of PDT for effective treatment of bile duct carcinoma.

Characterization of the insulin-like growth factor axis in a human hepatoma cell line (PLC).

The insulin-like growth factors (IGF-I and -II) are structurally related peptides participating in the regulation of metabolism, growth and cellular differentiation. In the present study, the human hepatoma cell line PLC was studied for the expression of individual components of the IGF axis. Northern blot analysis using IGF-I and -II coding cDNAs failed to detect IGF-I- or -II-specific transcripts in total RNA from PLC cells. Biosynthesis of type I and II IGF receptors was demonstrated by northern blotting and binding studies as well as cross-linking of the respective radiolabeled ligand. Both IGF-I and -II stimulated [3H]-thymidine incorporation dose-dependently. The mitogenic activity of exogenously added IGFs was reduced by the presence of IGF-binding proteins of 24, 30, 34, 41 and 45 kDa in supernatants of PLC cells identified as IGFBP-4, -1, -2 and -3, respectively, by [125I]IGF-I ligand-, immuno- and northern blotting. Biosynthesis of IGFBP-3 was stimulated dose-dependently by IGF-I and -II, while IGFBP-1, -2 and -4 were not affected. The increase of IGFBP-3 in response to IGF-I and -II was due to a stimulation of IGFBP-3 specific mRNA as well as to an inhibition of IGFBP-3 endocytosis. Proteolytic activity for rhIGFBP-3 was detected in media from PLC cells at acidic pH that was inhibited by the aspartyl protease inhibitor pepstatin A as well as after immunodepletion of cathepsin D from media of PLC cells. Thus, a role of cathepsin D for the regulation of IGFBP-3 bioavailability via endocytosis in acidic prelysosomal compartments was suggested. The susceptibility of PLC for IGF-I and -II was restricted by their ability to increase the abundance of inhibitory IGFBPs and to decrease the level of IGF-I receptor expression. The present data point to the IGF axis as a complex regulatory system that self limits the mitogenic activity of exogenous IGFs.

Porcine hepatocytes for biohybrid artificial liver devices: a comparison of hypothermic storage techniques.

Two hypothermic preservation techniques were investigated to assess their possible role in on-demand cell supply for bioartificial liver support devices. Porcine hepatocytes from slaughterhouse organs were isolated and either cold stored in a modified University of Wisconsin solution for up to 72 h or directly cultured in a sandwich configuration, frozen at Day 3 of culture, and stored for up to 30 days with subsequent long-term culture (14 days) in both groups. Cold storage for 72 h resulted in a decreased viability of cells (58.7 +/- 7.9%) with well preserved ultrastructures in the remainder of cells. In subsequent culture, albumin secretion was slightly increased, and cytochrome P450 IA1 dependent 7-ethoxy-coumarine deethylation activity was reduced to about 40% of control values. After cryopreservation, hepatocyte cultures revealed no severe damage to ultrastructures of cells, and functional parameters (albumin, 7-ethoxycoumarine deethylation) were comparable with controls after an initial drop in activity directly after thawing. Length of storage time did not influence results. Both hypothermic preservation protocols might eventually play an important role for bioartificial liver processing and on-demand cell supply, dependent on the individual reactor design.

Hypothernic storage of pig hepatocytes: influence of different storage solutions and cell density.

For clinical use of bioartifical liver devices a constant supply of primary liver cells has to be provided. Hypothermic storage of isolated pig hepatocytes could support large-scale stocking of cells. Freshly isolated pig hepatocytes from slaughterhouse livers were stored at 4 degrees C for 24, 48, and 72 h three different solutions: Leibovitz L-15 + 5% polyethylene glycol (PEG), University of Wisconsin (UW) solution, and a simplified UW solution. After storage, cells were cultured for 2 weeks in the collagen sandwich configuration. Viability of hepatocytes was 65, 85, and 83% after 24 h storage, 21, 74, and 70% after 48 h, and 5, 65, and 59% after 72 h in Leibovitz L-15 medium, UW, and the simplified UW, respectively. After storage in L-15 medium, cells attached poorly to collagen matrices and exhibited ultrastructural lesions. Functional performance in this group, as judged by albumin secretion and cytochrome P450-dependent activity in subsequent culture, decreased rapidly as a function of storage time, with zero values after 48 h storage. In contrast, hypothermia of hepatocytes in both UW solutions resulted in well-preserved cells with respect to ultrastructural appearance, attachment rates, and functional performance during culture. No significant differences were observed between the original and the simplified UW solution. Higher cell concentrations up to 5 x 10(7) cells/ml improved viability of hepatocytes on warmup. In terms of cell supply for hybrid artificial liver support, hypothermic storage of hepatocytes at 4 degrees C could mean an alternative to the cryopreservation of cells, which usually results in a substantial loss of cells and vital function of cells. Thus, pig hepatocytes could be stored at 4 degrees C for several days and meet the logistical need of bioartificial liver devices while avoiding the hazards of cell freezing.

Synthesis of insulin-like growth factor binding proteins and of the acid-labile subunit of the insulin-like growth factor ternary binding protein complex in primary cultures of human hepatocytes.

BACKGROUND/AIMS: The liver is the main source of circulating insulin-like growth factor binding proteins. In man, the cellular origin of insulin-like growth factor binding proteins has remained obscure. METHODS: Human hepatocytes isolated from surgical specimens were purified and cultured using a collagen gel immobilization technique. Gene expression of individual insulin-like growth factor binding proteins and of the acid-labile subunit of the insulin-like growth factor binding proteins by Western ligand blotting and immunoblot analysis. Neutral size chromatography of medium samples was used to detect insulin-like growth factors binding protein complexes. RESULTS: In cultured hepatocytes transcripts for insulin-like growth factor binding protein-1, -2, -3, -4 and for acid labile subunit could be demonstrated. Ligand blotting revealed the secretion of insulin-like growth factor binding proteins of molecular weights of 24 kD, 30 kD, 34 kD, 43 kD and 46 kD, respectively. Using polyclonal antisera, these proteins were identified as insulin-like growth factor binding protein-1, -2 and the insulin-like growth factor binding protein-3 doublet. Neural size chromatography of culture supernatants showed the presence of an insulin-like growth factor binding protein complex of approximately 40 kD, but absence of the high molecular weight ternary complex of 150 kD. CONCLUSIONS: It is concluded that in man parenchymal liver cells have to be regarded as a source of acid-labile subunit and of circulating insulin-like growth factor binding proteins including insulin-like growth factor binding protein-3.

Metabolism of pimobendan in long-term human hepatocyte culture: in vivo-in vitro comparison.

The aim of this study was to investigate further the potential of a new hepatocyte culture based on the hypothesis that liver cells in an appropriate in vitro environment (immobilizing gel technique) maintain high metabolic activity comparable with that in vivo. Pimobendan (UD-CG 115), a pyridazinone derivative, is a cardiotonic vasodilator that increases myocardial contractility through calcium sensitization and relaxation of vascular smooth muscle, probably due to phosphodiesterase inhibition. In man, pimobendan is O-demethylated to UD-CG 212. This latter is metabolized to O- and N-glucuronides. Pimobendan itself is also glucuronidated to a N-glucuronide. Human hepatocytes immobilized in collagen gel were incubated with pimobendan to investigate their metabolic activity in the long-term and to compare the results to the data from clinical trials. 14C-labelled pimobendan was incubated at two concentrations (10 and 100 microM) at day 3, 11 and 22 of culture, and samples were analysed after 4, 24 and 48-h incubation. Metabolic patterns were evaluated by hplc with radioactivity-, diode array-, and mass spectral-detection. In vitro, pimobendan was O-demethylated and subsequently O-glucuronidated. The rate of metabolism of pimobendan could be maintained in this culture system for > 3 weeks. However, the relative amount of a putative N-glucuronide under in vitro conditions was lower than in vivo.

Porcine hepatocytes from slaughterhouse organs. An unlimited resource for bioartificial liver devices.

Most recent strategies for the development of hybrid artificial liver devices focus on the use of parenchymal liver cells (hepatocytes). For clinical application of these devices, a sufficient cell supply is mandatory. Because human liver tissue is rarely available, isolated porcine hepatocytes from laboratory animals have been suggested for use in bioartificial livers. The authors introduce a modified isolation protocol to yield large scale numbers of viable porcine hepatocytes from slaughterhouse organs. Perfusion and enzymatic digestion of the left medial liver lobe (n = 74) resulted in 1.0 +/- 0.3 x 10(7) viable hepatocytes per gram of tissue, and an overall yield of 1.92 +/- 0.5 x 10(9) viable cells per isolation (viability: 93 +/- 2%). Collagen gel immobilization maintained morphologic integrity and functional activity of hepatocyte cultures over long-term periods. Cell morphology, as assessed by light microscopic evaluation, was maintained for 2 weeks. Stable DNA content (51 +/- 5 micrograms) and low values of alanine aminotransferase release (8 microU/hr/micrograms DNA) indicated structural stability of cultures after a short period of post isolational adaptation. Albumin secretion (4.5 micrograms/hr/micrograms DNA) and persistent Cytochrome P450 IA1 dependent deethylation of 7-ethoxycoumarin (4.5 nmol/hr/micrograms DNA) indicated long-term metabolic activity of cultured hepatocytes. Hepatocytes from livers of slaughtered pigs represent an unlimited resource of viable material for cell culture, and their usefulness as functional units of bioartificial liver support devices should be tested.

[An artificial liver on its way to clinical reality?]. / Die künstliche Leber auf dem Weg in die klinische Realität?

Temporary extracorporeal liver assist devices based on liver cell cultures represent a promising approach for the treatment of liver failure as well as for perioperative care in liver surgery and transplantation. Primary hepatocytes from donor animals as well as human cell lines have been discussed as cell sources. A viable cell mass of 110-220 g (= 2.5-5 x 10(10) liver cells) has to be functionally active in bioreactors over a time period of several days. This cell number correlates to 10-20% of liver cells in an adult human liver. Neither in animal experiments, nor in clinical trials a complete liver replacement could yet be shown for cell-based bioreactors, however, beneficial effects have been demonstrated: partial detoxification and specific synthetic functions have been reported in animal experiments. Two authors have shown first preliminary clinical data on different cell-based liver assist devices with influences on blood parameters and the neurological status of treated patients. Nevertheless, clinical improvements also have been achieved by using non-biological support, like charcoal-hemoperfusion and modified hemodialysis. In order to assess the influence of liver support devices on better results in therapy of liver failure, randomized clinical trials have to be performed. Prior to this, artificial liver support systems have to demonstrate their biocompatibility and biosafety for clinical use as well as their efficacy and availability.