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Background Extensive DNA sequencing has led to an unprecedented view of the diversity of individual genomes and their evolution among patients with clear cell renal cell carcinoma (ccRCC). Objective To understand subclonal architecture and dynamics of patient-derived two-dimensional (2D) and three-dimensional (3D) ccRCC models in vitro, in order to determine whether they mirror ccRCC inter- and intratumor heterogeneity. Design, setting, and participants We have established a comprehensive platform of living renal cancer cell models from ccRCC surgical specimens. Outcome measurements and statistical analysis We confirmed the concordance of 2D and 3D patient-derived cell (PDC) models with the original tumor tissue in terms of histology, biomarker expression, cancer driver mutations, and copy number alterations. We addressed inter- and intrapatient heterogeneity by analyzing clonal dynamics during serial passaging. Results and limitations In-depth genetic characterization verified the presence of heterogeneous cell populations, and revealed a high degree of similarity between subclonal compositions of monolayer and organoid cell cultures and the corresponding parental ccRCCs. Clonal dynamics were evident during serial passaging of cells in vitro, suggesting that PDC cultures can offer insights into evolutionary potential and treatment susceptibility of ccRCC subclones in vivo. Proof-of-concept drug profiling using selected ccRCC-targeted therapy agents highlighted patient-specific vulnerabilities in PDC models that could not be anticipated by interrogating commercially available cell lines. Conclusions We demonstrate that PDC models mirror inter- and intratumor heterogeneity of ccRCC in vitro. Based on our findings, we envision that the use of these models will advance our understanding of the trajectories that cause genetic diversity and their consequences for treatment on an individual level. Patient summary In this study, we developed two- and three-dimensional patient-derived models from clear cell renal cell carcinoma (ccRCC) as “mini-tumors in a dish.” We show that these cell models retain important features of the human ccRCCs such as the profound tumor heterogeneity, thus highlighting their importance for cancer research and precision medicine., European Urology Focus, 7 (1), ISSN:2405-4569

Aims Epithelial ovarian cancer (EOC) can be classified into four major types (serous, endometrioid, clear cell, mucinous). The prevalence of driver gene mutations in the different subtypes is controversial. High-grade serous carcinomas show frequent TP53 mutations, whereas KRAS and BRAF mutations are less common. In non-serous EOC, the relevance of these gene mutations remains to be elucidated. Methods We investigated 142 formalin-fixed, paraffin-embedded EOC, including serous (n = 63), endometrioid (n = 29), clear cell (n = 25), mucinous (n = 14), and others (n = 11) for mutations in TP53 exons 5–8, KRAS exons 2 and 3, and BRAF exon 15 by pyro-sequencing using the GS Junior 454 platform. The mutational status was correlated with clinicopathological features and patient overall survival. Results We identified mutations in the coding region of TP53 in 51.4% (73/142), and of KRAS in 9.9% (14/142) but not of BRAF . TP53 mutations occurred frequently not only in high-grade serous carcinomas (58.7%), but also in mucinous (57%) and clear cell EOC (52%). TP53 mutations were associated with high-grade carcinomas (p = 0.014), advanced FIGO stage (p = 0.001), intraoperative residual disease > 1 cm (p = 0.004), as well as poor overall survival (p = 0.002). KRAS mutations were mainly identified in mucinous EOC (57%) and were concomitantly with TP53 mutations in five mucinous carcinomas (36%). Conclusions TP53 gene driver mutations are a common feature of all advanced ovarian cancer subtypes, whereas BRAF mutations seem to be a rare event in EOC. KRAS mutations with synchronous TP53 mutations occur predominantly in low-grade mucinous carcinomas, suggesting a specific molecular background of this ovarian cancer type.

The combinations of genetic alterations that cooperate with von Hippel–Lindau (VHL) mutation to cause clear cell renal cell carcinoma (ccRCC) remain poorly understood. We show that the TP53 tumour suppressor gene is mutated in approximately 9% of human ccRCCs. Combined deletion of Vhl and Trp53 in primary mouse embryo fibroblasts causes proliferative dysregulation and high rates of aneuploidy. Deletion of these genes in the epithelium of the kidney induces the formation of simple cysts, atypical cysts and neoplasms, and deletion in the epithelia of the genital urinary tract leads to dysplasia and tumour formation. Kidney cysts display a reduced frequency of primary cilia and atypical cysts and neoplasms exhibit a pro‐proliferative signature including activation of mTORC1 and high expression of Myc, mimicking several cellular and molecular alterations seen in human ccRCC and its precursor lesions. As the majority of ccRCC is associated with functional inactivation of VHL, our findings suggest that for a subset of ccRCC, loss of p53 function represents a critical event in tumour development. ISSN:1757-4676 ISSN:1757-4684

AIM OF THE STUDY To evaluate the frequency of MRE11/RAD50/NBS1 (MRN)-complex loss of protein expression in endometrial cancers (EC) and to determine whether loss of MRE11 renders the cancer cells sensitive to Poly(ADP-ribose) polymerase (PARP)-inhibitory treatment. METHODS MRN expression was examined in 521 samples of endometrial carcinomas and in 10 cancer cell lines. A putative mutation hotspot in the form of an intronic poly(T) allele in MRE11 was sequenced in selected cases (n = 26). Sensitivity to the PARP-inhibitor, BMN673 was tested in colony formation assays before and after MRE11 silencing using siRNA. Homologous recombination (HR) DNA repair was evaluated by RAD51-foci formation assay upon irradiation and drug treatment. RESULTS Loss of MRE11 protein was found in 30.7% of EC tumours and significantly associated with loss of RAD50, NBS1 and mismatch repair protein expression. One endometrial cell line showed a markedly reduced MRE11 expression due to a homozygous poly(T) mutation of MRE11, thereby exhibiting an increased sensitivity to BMN673. MRE11 depletion sensitizes MRE11 expressing EC cell lines to the treatment with BMN673. The increased sensitivity to PARP-inhibition correlates with reduced RAD51 foci formation upon ionizing radiation in MRE11-depleted cells. CONCLUSION Loss of the MRE11 protein predicts sensitivity to PARP-inhibitor sensitivity in vitro, defining it as an additional synthetic lethal gene with PARP. The high incidence of MRE11 loss in ECs can be potentially exploited for PARP-inhibitor therapy. Furthermore, MRE11 protein expression using immunohistochemistry could be investigated as a predictive biomarker for PARP-inhibitor treatment.

The complement system has been shown to facilitate peripheral prion pathogenesis. Mice lacking complement receptors CD21/35 partially resist terminal prion disease when infected i.p. with mouse-adapted scrapie prions. Chronic wasting disease (CWD) is an emerging prion disease of captive and free-ranging cervid populations that, similar to scrapie, has been shown to involve the immune system, which probably contributes to their relatively facile horizontal and environmental transmission. In this study, we show that mice overexpressing the cervid prion protein and susceptible to CWD (Tg(cerPrP)5037 mice) but lack CD21/35 expression completely resist clinical CWD upon peripheral infection. CD21/35-deficient Tg5037 mice exhibit greatly impaired splenic prion accumulation and replication throughout disease, similar to CD21/35-deficient murine prion protein mice infected with mouse scrapie. TgA5037;CD21/35−/− mice exhibited little or no neuropathology and deposition of misfolded, protease-resistant prion protein associated with CWD. CD21/35 translocate to lipid rafts and mediates a strong germinal center response to prion infection that we propose provides the optimal environment for prion accumulation and replication. We further propose a potential role for CD21/35 in selecting prion quasi-species present in prion strains that may exhibit differential zoonotic potential compared with the parental strains.

The O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation status is a predictive parameter for the response of malignant gliomas to alkylating agents such as temozolomide. First clinical trials with temozolomide plus bevacizumab therapy in metastatic melanoma patients are ongoing, although the predictive value of the MGMT promoter methylation status in this setting remains unclear. We assessed MGMT promoter methylation in formalin-fixed, primary tumor tissue of metastatic melanoma patients treated with first-line temozolomide and bevacizumab from the trial SAKK 50/07 by methylation-specific polymerase chain reaction. In addition, the MGMT expression levels were also analyzed by MGMT immunohistochemistry. Eleven of 42 primary melanomas (26%) revealed a methylated MGMT promoter. Promoter methylation was significantly associated with response rates CR + PR versus SD + PD according to RECIST (response evaluation criteria in solid tumors) (p0.05) with a trend to prolonged median progression-free survival (8.1 versus 3.4 months, p0.05). Immunohistochemically different protein expression patterns with heterogeneous and homogeneous nuclear MGMT expression were identified. Negative MGMT expression levels were associated with overall disease stabilization CR+PR+SD versus PD (p=0.05). There was only a poor correlation between MGMT methylation and lack of MGMT expression. A significant proportion of melanomas have a methylated MGMT promoter. The MGMT promoter methylation status may be a promising predictive marker for temozolomide therapy in metastatic melanoma patients. Larger sample sizes may help to validate significant differences in survival type endpoints.

The identification of cell surface accessible biomarkers enabling diagnosis, disease monitoring, and treatment of renalcell carcinoma (RCC) is as challenging as the biology and progression of RCC is unpredictable. A hallmark of most RCCis the loss-of-function of the von Hippel–Lindau (pVHL) protein by mutation of its gene (VHL). Using the cell surfacecapturing (CSC) technology, we screened and identified cell surface N-glycoproteins in pVHL-negative and positive786-O cells. One hundred six cell surface N-glycoproteins were identified. Stable isotope labeling with amino acidsin cell culture–based quantification of the CSC screen revealed 23 N-glycoproteins whose abundance seemed tochange in a pVHL-dependent manner. Targeted validation experiments using transcriptional profiling of primary RCCsamples revealed that nine glycoproteins, including CD10 and AXL, could be directly linked to pVHL-mediated transcrip-tional regulation. Subsequent human tumor tissue analysis of these cell surface candidate markers showed a correla-tion between epithelial AXL expression and aggressive tumor phenotype, indicating that pVHL-dependent regulation ofglycoproteins may influence the biologic behavior of RCC. Functional characterization of the metalloprotease CD10 incell invasion assays demonstrated a diminished penetrating behavior of pVHL-negative 786-O cells on treatment withthe CD10-specific inhibitor thiorphan. Our proteomic surfaceome screening approach in combination with transcrip-tional profiling and functional validation suggests pVHL-dependent cell surface glycoproteins as potential diagnosticmarkers for therapeutic targeting and RCC patient monitoring., Neoplasia, 14 (6), ISSN:1522-8002, ISSN:1476-5586

The extracellular matrix N-glycoprotein periostin (OSF-2, POSTN) is a major constituent of the desmoplastic stroma around solid tumors. It promotes tumor invasion and metastasis via epithelial-mesenchymal transition (EMT). In this study we investigated periostin expression at both RNA and protein level as well as the expression pattern of its splice isoforms in non-small cell lung cancer (NSCLC).Thirty fresh frozen and corresponding formalin-fixed NSCLC tissues (adeno- and squamous cell carcinoma subtype, each n=15) and their matched non-neoplastic tissues were investigated. Periostin mRNA levels were analyzed by quantitative RT-PCR. The EMT-markers periostin and vimentin were analyzed by immunohistochemistry. Laser capture microdissection allowed for analysis of periostin expression in tumor epithelia and stroma, separately. Isoform patterns were investigated by isoform-specific PCR following sequencing in NSCLC, fetal and adult normal lung tissue.The qRT-PCR analysis showed periostin mRNA up-regulation in NSCLC tissue in relation to normal lung, with significantly higher levels in the adeno-compared to the squamous cell subtype (p0.05). However, protein levels in both tumor epithelia and stroma correlated with squamous cell carcinoma (p0.001) and larger tumor size (p0.05). Further, periostin tumor epithelia expression, correlated with higher tumor grade (p0.05). Sequence analysis detected eight periostin isoforms in fetal lung, but only five in both NSCLC and matched normal lung tissue. Among the eight isoforms, four are new and were labelled 5, 7, 8 and 9. The exclusive presence of isoforms 1 and 9 in fetal tissue suggests splice-specific regulation during lung embryogenesis. Finally, laser capture microdissection demonstrated that both tumor epithelia and stromal cells can be a source of periostin production in NSCLC.This study represents the first analysis of periostin isoform expression patterns in NSCLC and a characterization of periostin expression in cancer versus stromal cells at both RNA and protein level.

Multilocular cystic renal cell carcinoma is a rare renal cell carcinoma with an excellent prognosis. To clarify the relationship with typical clear cell renal cell carcinoma, we evaluated 15 cases of multilocular cystic renal cell carcinomas diagnosed according to the 2004 WHO classification. Von Hippel Lindau (VHL) gene mutations were determined by whole genome amplification and direct sequencing. Carbonic anhydrase 9 (CAIX), a hypoxia-inducible factor (HIF) target, paired box gene 2 (PAX2), cyclin-dependent kinase inhibitor p27 and glycogen synthase kinase 3-β (GSK3β) were immunohistochemically evaluated as members of the VHL protein (pVHL)- and phosphatase and tensin homolog (PTEN)-controlled pathways. VHL mutations were identified in 3 of 12 (25%) tumors. Inactivated GSK3β, decreased PTEN expression and PAX2 positivity were observed in the vast majority of the multilocular cystic renal cell carcinomas. Strong nuclear staining of p27 was seen in 14 of 15 cases. Compared with multilocular cystic renal cell carcinomas, expression frequencies of PAX2, p-GSK3β, PTEN and CAIX were similar in a set of low-grade, early-stage clear cell renal cell carcinomas, whereas only 30% had strong p27 positivity. These results are consistent with the hypothesis that multilocular cystic renal cell carcinomas are related at the molecular level with clear cell renal cell carcinomas. Maintenance of a strong subcellular p27 expression in all multilocular cystic renal cell carcinomas analyzed may in part explain the excellent prognosis of these tumor patients.

Inheritance of a mutant allele of the von Hippel-Lindau tumor suppressor gene predisposes affected individuals to develop renal cysts and clear cell renal cell carcinoma. Von Hippel-Lindau gene inactivation in single renal tubular cells has indirectly been showed by immunohistochemical staining for the hypoxia-inducible factor alpha target gene product carbonic anhydrase IX. In this study we were able to show von Hippel-Lindau gene deletion in carbonic anhydrase IX positive nonneoplastic renal tubular cells, in epithelial cells lining renal cysts and in a clear cell renal cell carcinoma of a von Hippel-Lindau patient. This was carried out by means of laser confocal microscopy and immunohistochemistry in combination with fluorescence in situ hybridization. Carbonic anhydrase IX negative normal renal tubular cells carried no von Hippel-Lindau gene deletion. Furthermore, recent studies have indicated that the von Hippel-Lindau gene product is necessary for the maintenance of primary cilia stability in renal epithelial cells and that disruption of the cilia structure by von Hippel-Lindau gene inactivation induces renal cyst formation. In our study, we show a significant shortening of primary cilia in epithelial cells lining renal cysts, whereas, single tubular cells with a von Hippel-Lindau gene deletion display to a far lesser extent signs of cilia shortening. Our in vivo results support a model in which renal cysts represent precursor lesions for clear cell renal cell carcinoma and arise from single renal tubular epithelial cells owing to von Hippel-Lindau gene deletion.

We have studied the role of CD21/35, which bind derivatives of complement factors C3 and C4, in extraneural prion replication and neuroinvasion. Upon administration of small prion inocula, CD21/35−/− mice experienced lower attack rates and delayed disease over both wild-type (WT) mice and mice with combined C3 and C4 deficiencies. Early after inoculation, CD21/35−/− spleens were devoid of infectivity. Reciprocal adoptive bone marrow transfers between WT and CD21/35−/− mice revealed that protection from prion infection resulted from ablation of stromal, but not hemopoietic, CD21/35. Further adoptive transfer experiments between WT mice and mice devoid of both the cellular prion protein PrPC and CD21/35 showed that splenic retention of inoculum depended on stromal CD21/35 expression. Because both PrPC and CD21/35 are highly expressed on follicular dendritic cells, CD21/35 appears to be involved in targeting prions to follicular dendritic cells and expediting neuroinvasion following peripheral exposure to prions.