Gerald H. Mickisch

Chemosensitivity testing of primary human renal cell carcinoma by a tetrazolium based microculture assay (MTT)

SummaryMTT staining procedures have been used in chemosensitivity testing of established cell lines of human and other sources as well as of human leukaemias, but only limited information on its application in primary solid human tumors is presently available. We have evaluated MTT staining in primary human Renal Cell Carcinomas (RCCs), studied various factors interfering with the optimal use, and finally applied it in subsequent chemosensitivity testing. The method depends on the conversion of a water-soluble tetrazolium salt (MTT) to a purple colored formazan precipitate, a reaction effected by enzymes active only in living cells. Single cell suspensions of RCCs were obtained either by enzymatic dispersion or by mechanical dissagregation, filtered through gauze, and purified by Ficoll density centrifugation. Tests were carried out in 96-well microculture plates. 104 viable tumor cells per well at 4 h incubation time with 20 μg MTT/100 μl total medium volume yielded best results. Formazan crystals were dissolved with DMSO, and the plates were immediately measured on a microculture plate reader at 540 nm. Under these criteria, linearity of the system could be demonstrated. For chemosensitivity testing, cells were continuously exposed to a number of drugs prior to the MTT staining procedure. Reproducibility of results was assessed and confirmed by culturing RCCs in flasks additionally, resubmitting them after 1, 2, and 4 weeks to the MTT assay. We conclude that the semiautomated MTT assay offers a valid, rapid, reliable and simple method to determine the degree of chemoresistance in primary human RCCs.

Mechanisms and Modulation of Multidrug Resistance in Primary Human Renal Cell Carcinoma

Human renal cell carcinomas show a high degree of intrinsic multidrug resistance. In experimental cell lines, the membrane bound P-170 glycoprotein and the glutathione redox cycle seem to contribute to this phenomenon. P-170 may be inactivated by calcium antagonists; the glutathione redox cycle by buthionine sulfoximine. We studied the resistance patterns of 35 human renal cell carcinomas against vinblastine, doxorubicin and carboplatinum in a tetrazolium-based microculture assay. Concomitantly, P-170 expression was traced immunohistochemically using moab C219 and the glutathione content was determined enzymatically. Reversal of multidrug resistance was examined by applying the R-stereoisomer of verapamil and/or by addition of buthionine sulfoximine. A high degree of chemoresistance was seen in 27 tumors against vinblastine, in 30 tumors against doxorubicin and in 31 tumors against carboplatinum. Chemoresponse was found in eight, five or four cases respectively. P-170 was detected in 70% of highly vinblastine resistant and in 63% of highly doxorubicin resistant tumors, but in none of the less resistant cases. Resistance against carboplatinum and doxorubicin was significantly associated with elevated glutathione levels as compared to less resistant renal cell carcinomas. R-verapamil lead to a strong reversal of vinblastine resistance and to a distinct circumvention of doxorubicin resistance, but revealed no effect in carboplatinum resistance. Buthionine sulfoximine overcame carboplatinum resistance and modified doxorubicin resistance, but had no influence on vinblastine resistance. The combined application of R-verapamil and buthionine sulfoximine reversed doxorubicin resistance but did not act synergistically in vinblastine or carboplatinum resistance. Both mechanisms, P-170 and glutathione, occurred independently of each other and may well explain multidrug resistance of human renal cell carcinomas.

P-170 Glycoprotein, Glutathione and Associated Enzymes in Relation to Chemoresistance of Primary Human Renal Cell Carcinomas

High intrinsic chemoresistance contributes to the dismal outcome of patients with disseminated renal cell carcinoma (RCC). In experimental cell lines, two defined defence mechanisms, P-170 glycoprotein and glutathione metabolism, have been established in multidrug resistance, a cross-resistance to cytotoxic compounds without functional or structural similarities. In 21 primary human RCCs, P-170 expression was examined, glutathione content and activities of related enzymes determined and the results were correlated to the degree of in vitro chemoresistance. P-170 was found in 10 of 17 resistant tumors but in none of the sensitive cases. The glutathione content was significantly higher and the related enzyme distinctively enhanced in resistant RCCs. Both mechanisms occurred independently and may well explain the multidrug resistance of RCC. Therefore, reversal of one or both systems may have a clinical impact on the chemotherapy of RCCs.

In vivo models of P-glycoprotein-mediated multidrug resistance

This chapter is intended as a critical discussion of the various approaches that can be taken to study reversal of multidrug resistance in tumors growing in animals. For this discussion, multidrug resistance will be taken to mean cross-resistance to a variety of amphipathic, hydrophobic drugs that are substrates for the energy-dependent multidrug transporter, P-glycoprotein (recently reviewed in [1,2]). Although there are certainly other forms of multidrug resistance that occur clinically, most of these have not yet been defined clearly at a biochemical or molecular level, and hence development of precise in vivo model systems to study these other mechanisms of multidrug resistance have yet to be developed. The systems that will be discussed in this chapter will be evaluated with respect to their ability to detect agents capable of reversing P-glycoprotein-mediated resistance in resistant cells, with little or no sensitizing effect in control cells that lack P-glycoprotein.

Cross-resistance patterns related to glutathione metabolism in primary human renal cell carcinoma

SummaryIn 59 cases of primary human renal cell carcinoma (RCC), cross-resistance and collateral susceptibility patterns were determined in an MTT microculture assay. Concomitantly, the glutathione (GSH) content and the enzymatic activity of γ-glutamyl transpeptidase (GGT) were measured as distinct resistance characteristics. Resistance or chemoresponse towards Vinca alkaloids and anthracyclines were found to be highly coincident, suggesting that the classical multidrug resistance mechanism is active in human RCC. Strong resistance to platinum complexes combined with relative sensitivity to bleomycin was significantly associated with elevated glutathione levels, providing evidence for another pathway instigating chemoresistance. In contrast, despite substantial enzymatic activity, GGT effects revealed no correlation to the chemoresistance pattern. This result implies that it is the GSH-linked binding and reduction potential rather than the GGT-associated transportation capacity that has an impact on the expression of chemoresistance in human RCC.

The European Medicines Agency Approval of Axitinib (Inlyta) for the Treatment of Advanced Renal Cell Carcinoma After Failure of Prior Treatment With Sunitinib or a Cytokine: Summary of the Scientific Assessment of the Committee for Medicinal Products for Human Use

Axitinib is a tyrosine kinase inhibitor of vascular endothelial growth factor receptor 1 (VEGFR-1), VEGFR-2, and VEGFR-3. Based on the positive opinion from the European Medicines Agency (EMA), a marketing authorization valid throughout the European Union (EU) was issued for the treatment of advanced renal cell carcinoma (RCC) after failure of prior treatment with sunitinib or a cytokine. The demonstration of clinical benefit for axitinib was based on a phase III, randomized, open-label, multicenter study of axitinib compared with sorafenib in patients with advanced RCC after failure of a prior systemic first-line regimen containing one or more of the following agents: sunitinib, bevacizumab plus interferon-α, temsirolimus, or cytokines. In the primary analysis, a 2-month increase in median progression-free survival (PFS) was observed for axitinib compared with sorafenib (hazard ratio [HR]: 0.665; 95% confidence interval [CI]: 0.544-0.812; p < .0001). In the subgroup of patients with a prior cytokine-containing regimen, the increase in median PFS associated with axitinib was 5.4 months (updated analysis, HR: 0.519; 95% CI: 0.375-0.720; p < .0001). In the subgroup of patients with prior sunitinib treatment, the increase in median PFS was 1.4 months (updated analysis, HR: 0.736; 95% CI: 0.578-0.937; p = .0063). The analysis of overall survival showed no statistically significant survival benefit of axitinib over sorafenib in patients previously treated with cytokine-containing regimens (HR: 0.813; 95% CI: 0.556-1.191) or sunitinib (HR: 0.997; 95% CI: 0.782-1.270). The most common treatment-related adverse events associated with axitinib included diarrhea, hypertension, fatigue, nausea, decreased appetite, dysphonia, and palmar-plantar erythrodysesthesia. Most of these events were mild or moderate in severity. This paper summarizes the scientific review of the application leading to approval in the EU. The detailed scientific assessment report and product information, including the summary of product characteristics, are available on the EMA website (http://www.ema.europa.eu).

Circumvention of multidrug resistance mediated by P-170 glycoprotein using calcium antagonists in primary human renal cell carcinoma

In experimental cell lines and in some human tumors, calcium antagonists reversed multidrug resistance mediated by P-170 glycoprotein in vitro. So far, clinical trials have not been very rewarding as intrinsic cardiovascular activities of these compounds impeded sufficient dosage. Renal cell carcinomas are considered to be good models for the evaluation of this new therapeutic concept. In 35 primary human renal cell carcinomas, the potency of 7 different calcium antagonists in combination with vinblastine monotherapy was examined in a tetrazolium-based microculture assay (MTT test) in order to circumvent chemoresistance. Concomitantly, P-170 glycoprotein expression was traced immunohistochemically using moab C 219. Substances derived from piperazine (flunarizine) showed only minor effects in this respect. The calcium antagonists of the papaverine type such as verapamil etc. revealed the strongest reversal of chemoresistance. Derivatives of benzothiazepine (diltiazem) or of dihydropyridine (nifedipine etc.) acted similarly and reached about 70% of the verapamil activity. All calcium antagonists lead to a significant enhancement of vinblastine cytotoxicity. An obvious link of P-170 glycoprotein to vinblastine chemoresistance was demonstrated. This particular resistance characteristic was detected in 19 of 27 resistant cases, but in none of the tumors displaying a chemoresponse. In particular, the new stereoisomer R-verapamil, which showed strong reversal of chemoresistance but which exerts 10 times lower cardiovascular side effects than racemic verapamil, seems to be suitable for further evaluation with regard to the clinical application.

Prognostic parameters for the management of advanced testis tumours.

The need for prognostic parameters in testicular germ cell tumours is sometimes questioned based on an overall cure rate of more than 80% of the patients regardless of tumour stage. However, the trend for an earlier and more accurate diagnosis amenable to curative treatment as well as the high effectiveness of standard Cisplatinum containing chemotherapy has masked the continuing need for intensifying therapy in patients with adverse risk factors. This intense treatment is often associated with worrysome morbidity and the assessment of prognostic factors, stage by stage, is warranted on which patient at risk can be identified and treated accordingly. Traditional prognostic factors, on which most classification systems are based, include large tumour volume, the presence of liver, bone or brain metastasis, grossly elevated tumour markers and an extragonadal primary site, particularly in the mediastinum. Novel prognostic factors are either (1) independent from the patient and his disease, (2) inherent on the patients characteristics or (3) based on tumour biology. Clearly, the infrastructure and the experience of the treating uro-oncology unit (see 1) is decisive for treatment outcomes, and - at least - ‘difficult to treat’ patients should be referred to properly resourced cancer centres. Patients with higher socio-economic status, willing to travel and well educated enough to be worried about their diseases status apparently gain access to expert centres more easily (see 2), translating into an upgrade on prognosis. Finally, biologic factors (see 3) such as β-human chorionic gonadotrophin or MAGE epitopes in seminoma or the percentage of embryonal carcinoma components or vascular invasion may or may not inversely influence the prognosis and need further assessment in prospective trials. However, the search for even better (molecular) biologic factors is speeding up because more complex treatment decisions such as in advanced testicular cancers rely on a more precise determination of prognosis, enabling a more tailored selection of individualized therapeutic options.

Chemoresistance of renal cell carcinoma

The past decade has seen the successful application of genetic techniques to the dissection of the most important phenotypes of cancer cells. In the case of drug resistance, the elucidation of the genes involved in resistance to anti-cancer drugs has led to new and unexpected information about tumor physiology and may well open therapeutic options by virtue of reversing clinical drug resistance. For the time being, data on renal cell carcinoma (RCC) are mainly preclinical, but quite compelling, and warrant careful evaluation in ongoing clinical studies.

Verapamil induzierte Durchbrechung der „multidrug-resistance“ des Nierenzellkarzinoms im in-vitro Kurzzeittest

30–40% der Patienten mit Nierenzellkarzinom weisen bei Diagnosestellung bereits Metastasen auf; ein nahezu ebenso hoher Prozentsatz entwickelt diese im Verlauf der Nachsorge. Das Problem einer adjuvanten Therapie stellt sich damit haufig. Zur schlechten Prognose fortgeschrittener Hypernephro-me tragt die primare (intrinsische) Chemoresistenz (bis 80% der Falle) masgeblich bei [4].