Loss of ORP3 induces aneuploidy and promotes bladder cancer cell invasion through deregulated microtubule and actin dynamics.

We have recently shown that loss of ORP3 leads to aneuploidy induction and promotes tumor formation. However, the specific mechanisms by which ORP3 contributes to ploidy-control and cancer initiation and progression is still unknown. Here, we report that ORP3 is highly expressed in ureter and bladder epithelium while its expression is downregulated in invasive bladder cancer cell lines and during tumor progression, both in human and in mouse bladder cancer. Moreover, we observed an increase in the incidence of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced invasive bladder carcinoma in the tissue-specific Orp3 knockout mice. Experimental data demonstrate that ORP3 protein interacts with γ-tubulin at the centrosomes and with components of actin cytoskeleton. Altering the expression of ORP3 induces aneuploidy and genomic instability in telomerase-immortalized urothelial cells with a stable karyotype and influences the migration and invasive capacity of bladder cancer cell lines. These findings demonstrate a crucial role of ORP3 in ploidy-control and indicate that ORP3 is a bona fide tumor suppressor protein. Of note, the presented data indicate that ORP3 affects both cell invasion and migration as well as genome stability through interactions with cytoskeletal components, providing a molecular link between aneuploidy and cell invasion and migration, two crucial characteristics of metastatic cells.

The Role of TKS5 in Chromosome Stability and Bladder Cancer Progression.

TKS5 promotes invasion and migration through the formation of invadopodia in some tumour cells, and it also has an important physiological function in cell migration through podosome formation in various nontumour cells. To date, the role of TKS5 in urothelial cells, and its potential role in BC initiation and progression, has not yet been addressed. Moreover, the contribution of TKS5 to ploidy control and chromosome stability has not been reported in previous studies. Therefore, in the present study, we wished to address the following questions: (i) Is TKS5 involved in the ploidy control of urothelial cells? (ii) What is the mechanism that leads to aneuploidy in response to TKS5 knockdown? (iii) Is TKS5 an oncogene or tumour-suppressor gene in the context of BC? (iv) Does TKS5 affect the proliferation, migration and invasion of BC cells? We assessed the gene and protein expressions via qPCR and Western blot analyses in a set of nontumour cell strains (Y235T, HBLAK and UROtsa) and a set of BC cell lines (RT4, T24, UMUC3 and J82). Following the shRNA knockdown in the TKS5-proficient cells and the ectopic TKS5 expression in the cell lines with low/absent TKS5 expression, we performed functional experiments, such as metaphase, invadopodia and gelatine degradation assays. Moreover, we determined the invasion and migration abilities of these genetically modified cells by using the Boyden chamber and wound-healing assays. The TKS5 expression was lower in the bladder cancer cell lines with higher invasive capacities (T24, UMUC3 and J82) compared to the nontumour cell lines from human ureter (Y235T, HBLAK and UROtsa) and the noninvasive BC cell line RT4. The reduced TKS5 expression in the Y235T cells resulted in augmented aneuploidy and impaired cell division. According to the Boyden chamber and wound-healing assays, TKS5 promotes the invasion and migration of bladder cancer cells. According to the present study, TKS5 regulates the migration and invasion processes of bladder cancer (BC) cell lines and plays an important role in genome stability.

Improving the Quality of Human Upper Urinary Tract Specimens by Cryobiopsy.

OBJECTIVE: The quality of histopathological specimens obtained from the upper urinary tract with conventional flexible ureterorenoscopic biopsy needs to be improved. We investigated the feasibility and biopsy quality of specimens obtained by cryobiopsy, compared with standard ureterorenoscopic biopsy techniques in a human ex vivo model. MATERIALS AND METHODS: Human ureters obtained from nephrectomy specimens (N=12) were dissected and canulated with an ureteral access sheath. Ureterorenoscopic biopsies were randomly obtained from different sites of the renal pelvic caliceal system using different types of instruments. The performance of two newly developed flexible cryoprobes with outer diameters of 1.1 mm (CB11) and 0.9 mm (CB09) was compared with that of the biopsy forceps(FB) and Bigopsy®(BiG) and two different Dormia baskets N-Gage (NG) and Zero-Tip (ZT). We assessed the feasibility of the various biopsy techniques based on the number of biopsy attempts needed to obtain macroscopically discernible biopsies. The specimens were examined histopathologically for size, biopsy quality, presence of various artifact types, and representativeness. RESULTS: Biopsies taken with the cryoprobes showed a higher biopsy quality than biopsies taken with the comparative instruments. The CB11 provided significantly larger biopsies than forceps biopsies and also than biopsies with ZT. The CB09 was able to collect larger samples when compared with the FB and BiG biopsy forceps. There were no significant differences in artifact area, except for the CB11 cryoprobe compared with the NG. To clarify the results a subdivision of larger or smaller than 20% artifact area was performed. A significant difference was found between CB11 and the forceps biopsies, as well as between CB11 and NG and ZT in favor of the cryoprobe. The representation of the histopathological sample was also determined. Biopsies taken with CB11 were more representative compared with forceps biopsies BiG and FB and basket biopsies NG and ZT. CONCLUSIONS: In a standardized comparative ex vivo setting, larger biopsies were obtained by using the cryobiopsy technique with the CB11 probe. Qualitatively, cryobiopsy specimens were overlaid by fewer artifacts and a higher biopsy quality was achieved in histopathologic examination compared with standard instrumentation. Further stepwise development will transfer the promising cryobiopsy technique into the clinical setting.

Shorter Leukocyte Telomere Length Is Associated with Worse Survival of Patients with Bladder Cancer and Renal Cell Carcinoma.

BACKGROUND: Telomeres are protein-DNA complexes at the tips of linear chromosomes. They protect the DNA from end-to-end fusion and exonucleolytic degradation. Shortening of telomeric DNA during aging can generate dysfunctional telomeres, promoting tumorigenesis. More recent data indicate that both short and long telomeres of peripheral blood leukocyte (PBL) cells can serve as prognostic biomarkers for cancer risk and may be associated with survival of patients with solid cancers. Telomere length in PBL cells could also be a potential prognostic biomarker for survival in bladder cancer (BC) or renal cell carcinoma (RCC). METHODS: The relative telomere length (RTL) of PBL cells was assessed in patients with BC (n = 144) and RCC (n = 144) by using qPCR. A control population of patients without malignant disease (NC, n = 73) was included for comparison. The correlation and association of RTL with histopathological parameters and overall survival (OS) were evaluated. RESULTS: Patients with BC and RCC had significantly shorter telomeres compared to patients without malignant disease. Within the cancer cohorts, multivariate analysis revealed that short RTL is an independent predictor of worse survival in BC (p = 0.039) and RCC (p = 0.041). CONCLUSION: Patients with BC and RCC had significantly shorter telomeres compared to the normal population. Shorter RTL in BC and RCC was an independent predictor of reduced survival.

The Role of Interleukin-1-Receptor-Antagonist in Bladder Cancer Cell Migration and Invasion.

BACKGROUND: The interleukin-1-receptor antagonist IL1RA (encoded by the IL1RN gene) is a potent competitive antagonist to interleukin-1 (IL1) and thereby is mainly involved in the regulation of inflammation. Previous data indicated a role of IL1RA in muscle-invasive urothelial carcinoma of the bladder (UCB) as well as an IL1-dependent decrease in tissue barrier function, potentially contributing to cancer cell invasion. OBJECTIVE: Based on these observations, here we investigated the potential roles of IL1RA, IL1A, and IL1B in bladder cancer cell invasion in vitro. METHODS: Cell culture, real-time impedance sensing, invasion assays (Boyden chamber, pig bladder model), qPCR, Western blot, ELISA, gene overexpression. RESULTS: We observed a loss of IL1RA expression in invasive, high-grade bladder cancer cell lines T24, UMUC-3, and HT1197 while IL1RA expression was readily detectable in the immortalized UROtsa cells, the non-invasive bladder cancer cell line RT4, and in benign patient urothelium. Thus, we modified the invasive human bladder cancer cell line T24 to ectopically express IL1RA, and measured changes in cell migration/invasion using the xCELLigence Real-Time-Cell-Analysis (RTCA) system and the Boyden chamber assay. The real-time observation data showed a significant decrease of cell migration and invasion in T24 cells overexpressing IL1RA (T24-IL1RA), compared to cells harboring an empty vector (T24-EV). Concurrently, tumor cytokines, e.g., IL1B, attenuated the vascular endothelial barrier, which resulted in a reduction of the Cell Index (CI), an impedance-based dimensionless unit. This reduction could be reverted by the simultaneous incubation with IL1RA. Moreover, we used an ex vivo porcine organ culture system to evaluate cell invasion capacity and showed that T24-IL1RA cells showed significantly less invasive capacity compared to parental T24 cells or T24-EV. CONCLUSIONS: Taken together, our results indicate an inverse correlation between IL1RA expression and tumor cell invasive capacity and migration, suggesting that IL1RA plays a role in bladder carcinogenesis, while the exact mechanisms by which IL1RA influences tumor cells migration/invasion remain to be clarified in future studies. Furthermore, we confirmed that real-time impedance sensing and the porcine ex vivo organ culture methods are powerful tools to discover differences in cancer cell migration and invasion.

Grainyhead-Like 3 Influences Migration and Invasion of Urothelial Carcinoma Cells.

Invasive urothelial carcinomas of the bladder (UCB) characteristically show a loss of differentiation markers. The transcription factor Grainyhead-like 3 (GRHL3) plays an important role in the development and differentiation of normal urothelium. The contribution to UCB progression is still elusive. Differential expression of GRHL3 was assessed in normal human urothelium and in non-invasive and invasive bladder cancer cell lines. The contribution of GRHL3 to cell proliferation, viability and invasion in UCB cell lines was determined by gain- and loss-of-function assays in vitro and in an organ culture model using de-epithelialized porcine bladders. GRHL3 expression was detectable in normal human urothelial cells and showed significantly higher mRNA and protein levels in well-differentiated, non-invasive RT4 urothelial carcinoma cells compared to moderately differentiated RT112 cells. GRHL3 expression was absent in anaplastic and invasive T24 cells. Ectopic de novo expression of GRHL3 in T24 cells significantly impaired their migration and invasion properties in vitro and in organ culture. Its downregulation improved the invasive capacity of RT4 cells. The results indicate that GRHL3 may play a role in progression and metastasis in UCB. In addition, this work demonstrates that de-epithelialized porcine bladder organ culture can be a useful, standardized tool to assess the invasive capacity of cancer cells.

Urothelial Carcinoma of the Bladder Induces Endothelial Cell Activation and Hypercoagulation.

Cancer-related venous thromboembolisms (VTE) are associated with metastasis and reduced survival in patients with urothelial cancer of the bladder. Although previous reports suggest the contribution of tissue factor and podoplanin, the mechanistic linkage between VTE and bladder cancer cell-derived molecules is unknown. Therefore, we compared distinct procoagulant pathways in four different cell lines. In vitro findings were further confirmed by microfluidic experiments mimicking the pathophysiology of tumor blood vessels and in tissue samples of patients with bladder cancer by transcriptome analysis and immunohistology. In vitro and microfluidic experiments identified bladder cancer-derived VEGF-A as highly procoagulant because it promoted the release of von Willebrand factor (VWF) from endothelial cells and thus platelet aggregation. In tissue sections from patients with bladder cancer, we found that VWF-mediated blood vessel occlusions were associated with a poor outcome. Transcriptome data further indicate that elevated expression levels of enzymes modulating VEGF-A availability were significantly connected to a decreased survival in patients with bladder cancer. In comparison with previously postulated molecular players, we identified tumor cell-derived VEGF-A and endothelial VWF as procoagulant mediators in bladder cancer. Therapeutic strategies that prevent the VEGF-A-mediated release of VWF may reduce tumor-associated hypercoagulation and metastasis in patients with bladder cancer. IMPLICATIONS: We identified the VEGF-A-mediated release of VWF from endothelial cells to be associated with bladder cancer progression.

Differences of the tumour cell glycocalyx affect binding of capsaicin-loaded chitosan nanocapsules.

The glycocalyx regulates the interaction of mammalian cells with extracellular molecules, such as cytokines. However, it is unknown to which extend the glycocalyx of distinct cancer cells control the binding and uptake of nanoparticles. In the present study, exome sequencing data of cancer patients and analysis of distinct melanoma and bladder cancer cell lines suggested differences in cancer cell-exposed glycocalyx components such as heparan sulphate. Our data indicate that glycocalyx differences affected the binding of cationic chitosan nanocapsules (Chi-NCs). The pronounced glycocalyx of bladder cancer cells enhanced the internalisation of nanoencapsulated capsaicin. Consequently, capsaicin induced apoptosis in the cancer cells, but not in the less glycosylated benign urothelial cells. Moreover, we measured counterion condensation on highly negatively charged heparan sulphate chains. Counterion condensation triggered a cooperative binding of Chi-NCs, characterised by a weak binding rate at low Chi-NC doses and a strongly increased binding rate at high Chi-NC concentrations. Our results indicate that the glycocalyx of tumour cells controls the binding and biological activity of nanoparticles. This has to be considered for the design of tumour cell directed nanocarriers to improve the delivery of cytotoxic drugs. Differential nanoparticle binding may also be useful to discriminate tumour cells from healthy cells.

The endothelial glycocalyx anchors von Willebrand factor fibers to the vascular endothelium.

The dynamic change from a globular conformation to an elongated fiber determines the ability of von Willebrand factor (VWF) to trap platelets. Fiber formation is favored by the anchorage of VWF to the endothelial cell surface, and VWF-platelet aggregates on the endothelium contribute to inflammation, infection, and tumor progression. Although P-selectin and ανß3-integrins may bind VWF, their precise role is unclear, and additional binding partners have been proposed. In the present study, we evaluated whether the endothelial glycocalyx anchors VWF fibers to the endothelium. Using microfluidic experiments, we showed that stabilization of the endothelial glycocalyx by chitosan oligosaccharides or overexpression of syndecan-1 (SDC-1) significantly supports the binding of VWF fibers to endothelial cells. Heparinase-mediated degradation or impaired synthesis of heparan sulfate (HS), a major component of the endothelial glycocalyx, reduces VWF fiber-dependent platelet recruitment. Molecular interaction studies using flow cytometry and live-cell fluorescence microscopy provided further evidence that VWF binds to HS linked to SDC-1. In a murine melanoma model, we found that protection of the endothelial glycocalyx through the silencing of heparanase increases the number of VWF fibers attached to the wall of tumor blood vessels. In conclusion, we identified HS chains as a relevant binding factor for VWF fibers at the endothelial cell surface in vitro and in vivo.

Economy of Standards: European Association of Urology Guideline Changes Influence Treatment Costs in Stage I Testicular Cancer Patients.

OBJECTIVE: The study aimed to calculate direct medical costs (DMC) during the first year of diagnosis and to evaluate the impact of guideline changes on treatment costs in clinical stage (CS) I testicular germ cell tumor (TGCT) patients in a German healthcare system. MATERIALS AND METHODS: Healthcare expenditures as DMC during the first year of diagnosis for 307 TGCT patients in CS I treated at our institution from 1987 to 2013 were calculated from the statutory health insurance perspective using patient level data. Three periods were defined referring to the first European Association of Urology (EAU) guideline in 2001 as well as to subsequent major guideline changes in 2005 and 2010. Data source for cost calculations were the German Diagnosis Related Groups system for inpatient stays (version 2014) and the German system for reimbursement of outpatient care (EBM - Einheitlicher Bewertungsmaßstab, edition 2014). RESULTS: During our 25 years of study period, mean DMC in the first year after diagnosis for the entire cohort of TGCT patients in CS I almost halved from EUR 13.000 to EUR 6.900 (p < 0.001). From 1987 to 2001, DMC for CS I seminomatous germ cell tumor (SGCT) patients were EUR 13.790 ± 4.700. From 2002 to 2010, mean costs were EUR 10.900 ± 5.990, and from 2011 to 2013, mean costs were EUR 5.190 ± 3.700. For CS I non-seminomatous germ cell tumor (NSGCT) patients, from 1987 to 2001, mean DMC were EUR 11.650 ± 5.690. From 2002 to 2010, mean costs were EUR 11.230 ± 5.990, and from 2011 to 2013, mean costs were EUR 11.170 ± 7.390. Follow-up examinations became less frequent over time, which caused a significant cost reduction for NSGCT (p = 0.042) while costs remained stable for SGCT. When adding costs of relapse treatment, active surveillance (AS) was the most cost-effective adjuvant treatment option in CS I NSGCT whereas one course carboplatin or AS caused similar expenditures in SGCT patients. CONCLUSION: The introduction of the EAU guidelines in 2001 caused a decrease in DMC in CS I seminoma patients. This cost reduction mainly took place due to the declining importance of radiation therapy. No substantial changes were seen in patients with CS I NSGCT. Costs for follow-up care also diminished, but to a lesser degree. Even when considering expenditures for relapse treatment, AS remained cost-effective in CS I TCGT patients. Our data show that evidence-based medicine in TGCT can reduce DMC in the first year after diagnosis.

Decreased Invasion of Urothelial Carcinoma of the Bladder by Inhibition of Matrix-Metalloproteinase 7.

OBJECTIVES: To measure and to modulate the invasive potential of urothelial carcinoma of the bladder (UCB) cells in a standardized preclinical setting using broad-spectrum matrix-metalloproteinase (MMPs) inhibitors and specific targeting of MMP7. MATERIALS AND METHODS: MMP expression levels in UCB cells were determined by quantitative real-time PCR (qRT-PCR) and gel zymographies of cell supernatants (MMP9, MMP2 and MMP1) and cell lysates (MMP7). The invasiveness of human UCB cells (HT1197 and T24/83) and human benign urothelial cells (UROtsa) was modulated by a broad-spectrum MMP inhibitor (4-Aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamic acid; AHA) and by MMP7 specific siRNAs. MMP7 knockdown efficiency was assessed by qRT-PCR and western blot. Invasive potential of UCB cells was measured by a standardized trans-epithelial electrical resistance (TEER) assay. RESULTS: Different MMP secretion profiles were measured in UCB cells. The active form of MMP7 was exclusively detected in HT1197 cells. Characteristic TEER breakdown patterns were observed in UCB cells when compared to benign cells. Invasive potentials were significantly higher in HT1197 cells than in T24/83 and in UROtsa cells [14.8±5.75 vs. 1.5±0.56 and 1.2±0.15, respectively; p < 0.01]. AHA treatment reduced the invasive potential of HT1197 cells. Also the specific downregulation of MMP7 by siRNA lowered the HT1197 cell invasiveness [20±1.0 vs. 16±2.8; p < 0.05]. Neither AHA nor MMP-7 siRNA transfection altered the invasive potential of T24/83 cells. CONCLUSIONS: Invasion of UCB is partially dependent on MMPs. Specific targeting of MMP7 by siRNA reduces the invasive potential in a subgroup of UCB cells. Therefore, MMP7 represents a potential therapeutic target which warrants further investigation.

Role of the Coagulation System in Genitourinary Cancers: Review.

Tumor progression is associated with aberrant hemostasis, and patients with malignant diseases have an elevated risk of developing thrombosis. A crosstalk among the vascular endothelium, components of the coagulation cascade, and cancer cells transforms the intravascular milieu to a prothrombotic, proinflammatory, and cell-adhesive state. We review the existing evidence on activation of the coagulation system and its implication in genitourinary malignancies and discuss the potential therapeutic benefit of antithrombotic agents. A literature review was performed searching the Medline database and the Cochrane Library for original articles and reviews. A second search identified studies reporting on oncological benefit of anticoagulants in genitourinary cancer. An elevated expression of procoagulatory tissue factor on tumor cells and tumor-derived microparticles seems to stimulate cancer development and progression. Several components of the hemostatic system, including D-dimers, von Willebrand Factor, thrombin, fibrin-/ogen, soluble P-selectin, and prothrombin fragments 1 + 2 were either overexpressed or overactive in genitourinary cancers. Hypercoagulation was in general associated with a poorer prognosis. Experimental models and small trials in humans showed reduced cancer progression after treatment with anticoagulants. Main limitations of these studies were heterogeneous experimental methodology, small patient numbers, and a lack of prospective validation. In conclusion, experimental and clinical evidence suggests procoagulatory activity of genitourinary neoplasms, particularly in prostate, bladder and kidney cancer. This may promote the risk of vascular thrombosis but also metastatic progression. Clinical studies linked elevated biomarkers of hemostasis with poor prognosis in patients with genitourinary cancers. Thus, anticoagulation may have a therapeutic role beyond prevention of thromboembolism.

Drain placement can safely be omitted for open partial nephrectomy: Results from a prospective randomized trial.

OBJECTIVES: To examine the benefit of drain placement during open partial nephrectomy. METHODS: Overall, 106 patients treated with open partial nephrectomy were enrolled in a prospective randomized trial. Based on the randomization, a drain was placed or omitted. Complications were assessed according to the Clavien classification. Pain level and requirement for analgesics was evaluated according to a customized pattern. RESULTS: There was no significant difference in the two groups regarding age, body mass index, American Society of Anesthesiologists score, tumor size and nephrometry (preoperative aspects and dimensions used for an anatomical classification). In terms of overall and drain-related complications, no advantage of placing a drain could be proven (P = 0.249). Patients with a drain suffered from a significantly higher pain level (P = 0.01) and showed prolonged mobilization (P < 0.001). There was no difference in bowel movements and requirement of additional analgesics (P = 0.347 and 0.11). CONCLUSIONS: The results of the study suggest that drain placement during open partial nephrectomy can safely be omitted, even in cases with violation of the collecting system.

Cisplatin-Based Chemotherapy for Testicular Germ Cell Tumors: Complication Rates of Peripheral versus Central Venous Administration.

OBJECTIVE: Despite the low local toxicity of the used agents, Cisplatin-based chemotherapy (CBP) for patients with testicular germ cell tumors (TGCT) is mostly delivered via a central venous access (CVA). Since 2008, CBP is given peripherally in our hospital. METHODS: Medical reports of TGCT patients who received CBP between September 1991 and August 2014 were evaluated. Complications regarding the way of administration (CVA vs. peripheral venous catheter [PVC]) were classified according to the Common Terminology Criteria of Adverse Events. The complication rates were compared using chi square test and propensity score matching. RESULTS: During 288 cycles in 109 patients, 85 complications (29.5%) were observed with similar rates for overall (PVC 31.3%, CVA 29.9%; p = 0.820) and grade I complications (21.3%, 25.4%; p = 0.470). More grade II complications were observed in the PVC group (10.0 vs. 1.5%; p < 0.001). Grade III complications requiring invasive treatment were found only in the CVA group (3.0%; p = 0.120). Using propensity score matching, no differences in overall (p = 0.950), grade I (p = 0.540) and grades II/III (p = 0.590) complications were seen. CONCLUSION: The peripheral and central administration of CBP has similar overall complication rates. Despite more grade II complications, the peripheral administration of CBP is a safe alternative for TGCT patients. Additionally, no severe grade III complications occurred.

Preference-based Health status in a German outpatient cohort with multiple sclerosis.

BACKGROUND: To prospectively determine health status and health utility and its predictors in patients with multiple sclerosis (MS). METHODS: A total of 144 MS patients (mean age: 41.0 ± 11.3 y) with different subtypes (patterns of progression) and severities of MS were recruited in an outpatient university clinic in Germany. Patients completed a questionnaire at baseline (n = 144), 6 months (n = 65) and 12 months (n = 55). Health utilities were assessed using the EuroQol instrument (EQ-5D, EQ VAS). Health status was assessed by several scales (Expanded Disability Severity Scale (EDSS), Modified Fatigue Impact Scale (M-FIS), Functional Assessment of MS (FAMS), Beck Depression Inventory (BDI-II) and Multiple Sclerosis Functional Composite (MSFC)). Additionally, demographic and socioeconomic parameters were assessed. Multivariate linear and logistic regressions were applied to reveal independent predictors of health status. RESULTS: Health status is substantially diminished in MS patients and the EQ VAS was considerably lower than that of the general German population. No significant change in health-status parameters was observed over a 12-months period. Multivariate analyses revealed M-FIS, BDI-II, MSFC, and EDSS to be significant predictors of reduced health status. Socioeconomic and socio-demographic parameters such as working status, family status, number of household inhabitants, age, and gender did not prove significant in multivariate analyses. CONCLUSION: MS considerably impairs patients' health status. Guidelines aiming to improve self-reported health status should include treatment options for depression and fatigue. Physicians should be aware of depression and fatigue as co-morbidities. Future studies should consider the minimal clinical difference when health status is a primary outcome.

Economic burden in a German cohort of patients with multiple sclerosis.

AIMS: To estimate costs of multiple sclerosis (MS) in a German cohort according to severity of the disease and clinical symptoms. METHODS: 144 patients were recruited from an MS outpatient clinic. Costs were calculated according to current German health-economic guidelines from the perspective of the social health insurance system. Patients were either interviewed or completed a questionnaire. Cost assessment covered a 3-month period. Health outcomes were: Expanded Disability Status Scale, MS Functional Composite, Functional Assessment of MS, fatigue, depression (Beck Depression Inventory II) and patients' socioeconomic status. Multivariate linear regression identified independent cost predictors. RESULTS: Total quarterly costs per patient were EUR 10,329 (95% CI 9,357-11,390). Direct costs were EUR 5,344 for the social health insurance system and EUR 140 for the patient. Drugs represented the major share of direct costs (and 35% of total costs); indirect costs accounted for 47% of total costs. Univariate and multivariate analyses identified age, disability, fatigue and depression as independent predictors for total, indirect or drug costs. CONCLUSION: MS represents a high economic burden, with direct costs exceeding indirect costs. To reduce costs, research should focus on prevention that slows down progression of MS. Rehabilitation and symptomatic treatment may have merits in decreasing indirect costs.