Thomas Höfner

Identification of a population of blood circulating tumor cells from breast cancer patients that initiates metastasis in a xenograft assay.

It has been hypothesized that carcinoma metastasis is initiated by a subpopulation of circulating tumor cells (CTCs) found in the blood of patients. However, although the presence of CTCs is an indicator of poor prognosis in several carcinoma entities, the existence and phenotype of metastasis-initiating cells (MICs) among CTCs has not been experimentally demonstrated. Here we developed a xenograft assay and used it to show that primary human luminal breast cancer CTCs contain MICs that give rise to bone, lung and liver metastases in mice. These MIC-containing CTC populations expressed EPCAM, CD44, CD47 and MET. In a small cohort of patients with metastases, the number of EPCAM+CD44+CD47+MET+ CTCs, but not of bulk EPCAM+ CTCs, correlated with lower overall survival and increased number of metastasic sites. These data describe functional circulating MICs and associated markers, which may aid the design of better tools to diagnose and treat metastatic breast cancer.

Expression and prognostic significance of cancer stem cell markers CD24 and CD44 in urothelial bladder cancer xenografts and patients undergoing radical cystectomy.

OBJECTIVES To evaluate CD24/CD44/CD47 cancer stem cell marker expressions in bladder cancer (BCa) and provide data on their prognostic significance for clinical outcome in patients undergoing radical cystectomy (RC). MATERIAL AND METHODS Primary BCa tissue was used for xenograft studies. A tissue microarray was prepared using specimens from a cohort of 132 patients. All patients underwent RC for urothelial BCa between 2001 and 2010. Expression of CD24, CD44, and CD47 was examined in primary samples and xenografts by fluorescence-activated cell sorting. Populations of CD24(low)- and CD24(high)-expressing cells were sorted and evaluated for tumorigenicity in vivo. Tissue microarray was analyzed for CD24/CD44 staining intensity and tumor-specific vs. stromal cell staining. Associations with BCa survival, BCa stage, and lymph node status were evaluated by univariate and multivariate analyses. RESULTS CD24 and CD44/CD47 expressions mark distinct cell populations within the normal urothelium as well as in BCa. CD24(high/low) expression was not sufficient to characterize CD24 as a BCa-initiating marker in in vivo primary xenotransplants. CD24 and CD44 expressions correlated with lower cancer-specific survival in patients. However, multivariate analyses of CD24 or CD44 did not demonstrate significantly increased hazards for cancer-specific death if analyzed together with stage, grade, and nodal status of patients. CONCLUSIONS Cancer stem cell markers CD24/CD44/CD47 are differentially expressed in cells of urothelial BCa in patients undergoing RC and influence cancer-specific survival of patients. Further evaluation of CD24/CD44/CD47 protein expression could be of high therapeutic value in BCa. However, both CD24 and CD44 expressions cannot be regarded as independent prognostic parameters for patients undergoing RC.

Defined Conditions for the Isolation and Expansion of Basal Prostate Progenitor Cells of Mouse and Human Origin

Summary Methods to isolate and culture primary prostate epithelial stem/progenitor cells (PESCs) have proven difficult and ineffective. Here, we present a method to grow and expand both murine and human basal PESCs long term in serum- and feeder-free conditions. The method enriches for adherent mouse basal PESCs with a Lin−SCA-1+CD49f+TROP2high phenotype. Progesterone and sodium selenite are additionally required for the growth of human Lin−CD49f+TROP2high PESCs. The gene-expression profiles of expanded basal PESCs show similarities to ESCs, and NF-kB function is critical for epithelial differentiation of sphere-cultured PESCs. When transplanted in combination with urogenital sinus mesenchyme, expanded mouse and human PESCs generate ectopic prostatic tubules, demonstrating their stem cell activity in vivo. This novel method will facilitate the molecular, genomic, and functional characterization of normal and pathologic prostate glands of mouse and human origin.

Efficacy of Targeted Treatment Beyond Third-Line Therapy in Metastatic Kidney Cancer: Retrospective Analysis From a Large-Volume Cancer Center

INTRODUCTION/BACKGROUND Currently, 7 agents are approved for the first- and second-line therapy for metastatic renal cell carcinoma. In contrast, data supporting their use beyond second line are limited. Here we summarize our experience in patients treated with more than 4 lines of therapy. METHODS We retrospectively assessed the outcome of 24 patients treated at our institution with at least 4 lines of therapy. Progression-free survival (PFS) and overall survival (OS) were calculated using Kaplan-Meier estimates. RESULTS Median OS from the initiation of first-line therapy for the whole cohort is 64.7 months. Up to 96% of the patients received a tyrosine kinase inhibitor (TKI) and mammalian target of rapamycin (mTOR) inhibitor (mTOR-I) within the first 3 lines of treatment. In the fourth or following lines, patients were treated with TKI, mTOR-I, bevacizumab/interferon, or experimental drugs. Seven patients continued treatment with a sixth-line agent; one has been treated up to the ninth line. Sixteen percent of the patients receiving fourth-line therapy and 13% receiving fifth-line therapy experienced a partial remission, which was independent from response to previous therapies. Median OS from fourth and fifth line was 30.8 and 26.2 months, respectively. Median PFS for fourth-line therapy was 5.8 months. No significant difference in PFS was observed for patients with disease that responded or did not respond to first-line therapy. CONCLUSION Despite the limitations of a retrospective analysis, our study suggests that selected patients benefit from multiple lines of treatment, independent of response to first-line therapy. However, the optimal sequence of treatment with regard to later lines remains to be determined.

Protein profile of basal prostate epithelial progenitor cells—stage-specific embryonal antigen 4 expressing cells have enhanced regenerative potential in vivo

The long‐term propagation of basal prostate progenitor cells ex vivo has been very difficult in the past. The development of novel methods to expand prostate progenitor cells in vitro allows determining their cell surface phenotype in greater detail. Mouse (Lin−Sca‐1+ CD49f+ Trop2high‐phenotype) and human (Lin− CD49f+ TROP2high) basal prostate progenitor cells were expanded in vitro. Human and mouse cells were screened using 242 anti‐human or 176 antimouse monoclonal antibodies recognizing the cell surface protein profile. Quantitative expression was evaluated at the single‐cell level using flow cytometry. Differentially expressed cell surface proteins were evaluated in conjunction with the known CD49f+/TROP2high phenotype of basal prostate progenitor cells and characterized by in vivo sandwich‐transplantation experiments using nude mice. The phenotype of basal prostate progenitor cells was determined as CD9+/CD24+/CD29+/CD44+/CD47+/CD49f+/CD104+/CD147+/CD326+/Trop2high of mouse as well as human origin. Our analysis revealed several proteins, such as CD13, Syndecan‐1 and stage‐specific embryonal antigens (SSEAs), as being differentially expressed on murine and human CD49f+ TROP2+ basal prostate progenitor cells. Transplantation experiments suggest that CD49f+ TROP2high SSEA‐4high human prostate basal progenitor cells to be more potent to regenerate prostate tubules in vivo as compared with CD49f+ TROP2high or CD49f+ TROP2high SSEA‐4low cells. Determination of the cell surface protein profile of functionally defined murine and human basal prostate progenitor cells reveals differentially expressed proteins that may change the potency and regenerative function of epithelial progenitor cells within the prostate. SSEA‐4 is a candidate cell surface marker that putatively enables a more accurate identification of the basal PESC lineage.

Advantages and Disadvantages of Bone Protective Agents in Metastatic Prostate Cancer: Lessons Learned

Nine out of ten metastatic prostate cancer (PCa) patients will develop osseous metastases. Of these, every second will suffer from skeletal-related events (SRE). SRE are associated with an increased risk for death, which is markedly increased in the presence of pathological fracture. Moreover, health insurance costs nearly double in the presence of SRE. Zoledronic acid and denosumab are both approved drugs for the prevention or delay of SRE in castration-resistant prostate cancer (CRPC) patients with osseous metastases. However, long-term treatment with one of these two drugs is associated with the development of medication-related osteonecrosis of the jaw (MRONJ). Routine inspections of the oral cavity before and during treatment are mandatory in these patients. Regarding imaging techniques, bone scintigraphy seems to be a promising tool to detect early stage MRONJ. Zoledronic acid does not reduce the incidence of SRE in hormone-sensitive PCa. First data shows 3-monthly application of zoledronic acid to be equi-effective to monthly application.

Nationwide analysis on the impact of socioeconomic land use factors and incidence of urothelial carcinoma

BACKGROUND Incidence rates for urothelial carcinoma (UC) have been reported to differ between countries within the European Union (EU). Besides occupational exposure to chemicals, other substances such as tobacco and nitrite in groundwater have been identified as risk factors for UC. We investigated if regional differences in UC incidence rates are associated with agricultural, industrial and residential land use. METHODS Newly diagnosed cases of UC between 2003 and 2010 were included. Information within 364 administrative districts of Germany from 2004 for land use factors were obtained and calculated as a proportion of the total area of the respective administrative district and as a smoothed proportion. Furthermore, information on smoking habits was included in our analysis. Kulldorff spatial clustering was used to detect different clusters. A negative binomial model was used to test the spatial association between UC incidence as a ratio of observed versus expected incidence rates, land use and smoking habits. RESULTS We identified 437,847,834 person years with 171,086 cases of UC. Cluster analysis revealed areas with higher incidence of UC than others (p=0.0002). Multivariate analysis including significant pairwise interactions showed that the environmental factors were independently associated with UC (p<0.001). The RR was 1.066 (95% CI 1.052-1.080), 1.066 (95% CI 1.042-1.089) and 1.067 (95% CI 1.045-1.093) for agricultural, industrial and residential areas, respectively, and 0.996 (95% CI 0.869-0.999) for the proportion of never smokers. CONCLUSION This study displays regional differences in incidence of UC in Germany. Additionally, results suggest that socioeconomic factors based on agricultural, industrial and residential land use may be associated with UC incidence rates.

The influence of prostatic anatomy and neurotrophins on basal prostate epithelial progenitor cells

Based on findings of surface marker, protein screens as well as the postulated near‐urethral location of the prostate stem cell niche, we were interested whether androgen ablation, distinct anatomic regions within the prostate or neurotrophins have an influence on basal prostate epithelial progenitor cells (PESCs).

530 EXTERNAL VALIDATION OF DISEASE-FREE SURVIVAL AT 2 OR 3 YEARS AS A SURROGATE AND NEW PRIMARY ENDPOINT FOR PATIENTS UNDERGOING RADICAL CYSTECTOMY FOR MUSCLE INVASIVE UROTHELIAL CANCER OF THE BLADDER

Patrick J. Bastian*, Munchen, Germany; Matthias May, Straubing, Germany; Hans-Martin Fritsche, Regensburg, Germany; Alexander Buchner, Munchen, Germany; Sabine Brookman-May, Regensburg, Germany; Christian Bolenz, Mannheim, Germany; Edwin Hermann, Munster, Germany; Maximillian Burger, Regensburg, Germany; Thomas Hofner, Heidelberg, Germany; Jorg Elliger, Bonn, Germany; Derya Tilki, Munchen, Germany; Lutz Trojan, Mannheim, Germany; Christian Wulfing, Munster, Germany; Axel Haferkamp, Frankfurt, Germany; Christian Gilfrich, Straubing, Germany; Markus Hohenfellner, Heidelberg, Germany; Wolf F. Wieland, Regensburg, Germany; Stefan C. Muller, Bonn, Germany; Philipp Nuhn, Munchen, Germany

487 LYMPH NODE DENSITY AFFECTS CANCER-SPECIFIC SURVIVAL IN PATIENTS WITH LYMPH NODE-POSITIVE UROTHELIAL BLADDER CANCER FOLLOWING RADICAL CYSTECTOMY

Background: The prognosis for patients with lymph node (LN)–positive bladder cancer (BCa) is likely affected by the extent of lymphadenectomy in radical cystectomy (RC) cases. Specifically, the prognostic significance of the LN density (ratio of positive LNs to the total number removed) has been demonstrated. Objective: To evaluate the prognostic signature of lymphadenectomy variables, including the LN density, for a large, multicentre cohort of RC patients with LNpositive BCa. Design, setting, and participants: The clinical and histopathologic data from 477 patients with LN-positive urothelial BCa (pN1–2) were analysed. The median follow-up period for all living patients was 28 mo. Measurements: Multivariable Cox regression analysis was used to test the effect of various pelvic lymph node dissection (PLND) variables on cancer-specific survival (CSS) based on colinearity in various models. 1