Purpose
Non-invasive high-grade (HG) bladder cancer is a heterogeneous disease that is characterized insufficiently. First-line Bacillus Calmette-Guérin instillation fails in a substantial amount of ...cases and alternative bladder-preserving treatments are limited, underlining the need to promote a further molecular understanding of non-invasive HG lesions. Here, we characterized pure HG papillary urothelial bladder cancer (pure pTa HG), a potential subgroup of non-invasive HG bladder carcinomas, with regard to molecular subtype affiliation and potential for targeted therapy.
Methods
An immunohistochemistry panel comprising luminal (KRT20, ERBB2, ESR2, GATA3) and basal (KRT5/6, KRT14) markers as well as p53 and FGFR3 was used to analyze molecular subtype affiliations of 78 pure pTa HG/papillary pT1(a) HG samples. In 66 of these,
ERBB2
fluorescence
in situ
hybridization was performed. Additionally, targeted sequencing (31 genes) of 19 pTa HG cases was conducted, focusing on known therapeutic targets or those described to predict response to targeted therapies noted in registered clinical trials or that are already approved.
Results
We found that pure pTa HG/papillary pT1(a) HG lesions were characterized by a luminal-like phenotype associated with frequent (58% of samples) moderate to high ERBB2 protein expression, rare FGFR3 alterations on genomic and protein levels, and a high frequency (89% of samples) of chromatin-modifying gene alterations. Of note, 95% of pTa HG/papillary pT1 HG cases harbored at least one potential druggable genomic alteration.
Conclusions
Our data should help guiding the selection of targeted therapies for investigation in future clinical trials and, additionally, may provide a basis for prospective mechanistic studies of pTa HG pathogenesis.
Oncogenic drivers such as mutated
are the preferred targets in modern drug development. However, restoring the lost function of tumor suppressor proteins could also be a valid approach to combatting ...cancer. ITIH5 has been revealed as a potent metastasis suppressor in both breast and pancreatic cancer. Here, we show that ITIH5 overexpression in MDA-MB-231 breast cancer cells can also locally suppress tumor growth by 85%, when transplanted into the mammary fat pad of nude mice. For a potential drug development approach, we further aimed to define downsized ITIH5 polypeptides that still are capable of mediating growth inhibitory effects. By cloning truncated and His-tagged ITIH5 fragments, we synthesized two recombinant
-terminal polypeptides (ITIH5
and ITIH5
), both covering the ITI heavy chain specific "vault protein inter-alpha-trypsin" (VIT) domain. Truncated ITIH5 variants caused dose-dependent cell growth inhibition by up to 50% when applied to various cancer cell lines (e.g., MDA-MB-231, SCaBER, A549) reflecting breast, bladder and lung cancer in vitro. Thus, our data suggest the substantial role of the ITIH5-specific VIT domain in ITIH5-mediated suppression of tumor cell proliferation. As extracellularly administered ITIH5 peptides mimic the growth-inhibitory effects of the full-length ITIH5 tumor suppressor protein, they may constitute the basis for developing anticancer drugs in the future.
Secreted frizzled related protein 3 (SFRP3) contains a cysteine-rich domain (CRD) that shares homology with Frizzled CRD and regulates WNT signaling. Independent studies showed epigenetic silencing ...of SFRP3 in melanoma and hepatocellular carcinoma. Moreover, a tumor suppressive function of SFRP3 was shown in androgen-independent prostate and gastric cancer cells. The current study is the first to investigate SFRP3 expression and its potential clinical impact on non-small cell lung carcinoma (NSCLC). WNT signaling components present on NSCLC subtypes were preliminary elucidated by expression data of The Cancer Genome Atlas (TCGA). We identified a distinct expression signature of relevant WNT signaling components that differ between adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Of interest, canonical WNT signaling is predominant in LUAD samples and non-canonical WNT signaling is predominant in LUSC. In line, high SFRP3 expression resulted in beneficial clinical outcome for LUAD but not for LUSC patients. Furthermore, SFRP3 mRNA expression was significantly decreased in NSCLC tissue compared to normal lung samples. TCGA data verified the reduction of SFRP3 in LUAD and LUSC patients. Moreover, DNA hypermethylation of SFRP3 was evaluated in the TCGA methylation dataset resulting in epigenetic inactivation of SFRP3 expression in LUAD, but not in LUSC, and was validated by pyrosequencing of our NSCLC tissue cohort and in vitro demethylation experiments. Immunohistochemistry confirmed SFRP3 protein downregulation in primary NSCLC and indicated abundant expression in normal lung tissue. Two adenocarcinoma gain-of-function models were used to analyze the functional impact of SFRP3 on cell proliferation and regulation of CyclinD1 expression in vitro. Our results indicate that SFRP3 acts as a novel putative tumor suppressor gene in adenocarcinoma of the lung possibly regulating canonical WNT signaling.
We determined if improved tumor detection using hexaminolevulinate (HAL) fluorescence cystoscopy could lead to improved treatment in patients with bladder cancer.
A total of 146 patients with known ...or suspected bladder cancer were assessed in this open, comparative, within patient, controlled phase III study. Patients received intravesical HAL for 1 hour and were assessed with standard white light cystoscopy and blue light fluorescence cystoscopy. All lesions were mapped onto a bladder chart and biopsies were taken from suspicious areas for assessment by an independent pathologist. An independent urologist blinded to the detection method used recommended treatment plans based on biopsy results and medical history according to European Association of Urology bladder cancer guidelines. Any differences in recommended treatment plans arising from the 2 cystoscopy methods were recorded.
HAL imaging improved overall tumor detection. Of all tumors 96% were detected with HAL imaging compared with 77% using standard cystoscopy. This difference was particularly noticeable for dysplasia (93% vs 48%), carcinoma in situ (95% vs 68%) and superficial papillary tumors (96% vs 85%). As a result of improved detection, additional postoperative procedures were recommended in 15 patients (10%) and more extensive treatment was done intraoperatively in a further 10. Overall 17% of patients received more appropriate treatment at the time of the study following blue light fluorescence cystoscopy, that is 22% or 1 of 5 if patients without tumors were excluded.
HAL imaging is more effective than standard white light cystoscopy for detecting bladder tumors and lesions. This leads to improved treatment in a significant number of patients (p <0.0001).
Dysfunction of the SWI/SNF complex has been observed in various cancers including urothelial carcinomas. However, the clinical impact of the SWI/SNF complex in squamous-differentiated bladder cancers ...(sq-BLCA) remains unclear. Therefore, we aimed to analyze potential expression loss and genetic alterations of (putative) key components of the SWI/SNF complex considering the co-occurrence of genetic driver mutations and PD-L1 expression as indicators for therapeutic implications. Assessment of ARID1A, SMARCA2, SMARCA4, SMARCB1/INI1, SMARCC1, SMARCC2 and PBRM1 mutations in a TCGA data set of sq-BLCA (n = 45) revealed that ARID1A was the most frequently altered SWI/SNF gene (15%) while being associated with protein downregulation. Genetic alterations and loss of ARID1A were confirmed by Targeted Next Generation Sequencing (NGS) (3/6) and immunohistochemistry (6/116). Correlation with further mutational data and PD-L1 expression revealed co-occurrence of ARID1A loss and TP53 mutations, while positive correlations with other driver mutations such as PIK3CA were not observed. Finally, a rare number of sq-BLCA samples were characterized by both ARID1A protein loss and strong PD-L1 expression suggesting a putative benefit upon immune checkpoint inhibitor therapy. Hence, for the first time, our data revealed expression loss of SWI/SNF subunits in sq-BLCA, highlighting ARID1A as a putative target of a small subgroup of patients eligible for novel therapeutic strategies.
In the 2016 WHO classification of genitourinary tumors Muellerian tumors of the urinary tract (MTUT) comprise clear cell adenocarcinomas and endometrioid carcinomas. Since these rare tumors remained ...understudied, we aimed to characterize their molecular background by performing DNA- and RNA-based targeted panel sequencing. All tumors (n = 11) presented single nucleotide alterations (SNVs), with ARID1A mutations being the most prevalent (5/11, 45%). Besides frequent ARID1A mutations, loss of ARID1A protein is not a suitable marker since protein expression is (partly) preserved also in mutated cases. Copy number alterations (CNVs) were found in 64% of cases (7/11), exclusively gene amplifications. Interestingly, a functionally relevant RSPO2 gene fusion/microdeletion was discovered in the endometrioid adenocarcinoma case. Comparing our findings with mutational profiles of other tumor entities, absence of TERT promoter mutations argues for a non-urothelial origin. No similarities were also found between MTUT and kidney cancers while parallels were observed for specific SNVs with endometrial carcinomas. In conclusion, immunohistochemical PAX8-positivity and lack of TERT promoter mutations could serve as key diagnostic features in difficult cases. Thus, understanding the molecular background of these tumors helps to refine treatment options and offers the possibility of targeted therapies in cases where needed.
This study aims at characterizing the role of the putative tumor suppressor ITIH5 in basal-type bladder cancers (BLCA). By sub-classifying TCGA BLCA data, we revealed predominant loss of ITIH5 ...expression in the basal/squamous-like (BASQ) subtype. ITIH5 expression inversely correlated with basal-type makers such as KRT6A and CD44. Interestingly, Kaplan-Meier analyses showed longer recurrence-free survival in combination with strong CD44 expression, which is thought to mediate ITIH-hyaluronan (HA) binding functions. In vitro, stable ITIH5 overexpression in two basal-type BLCA cell lines showing differential CD44 expression levels, i.e., with (SCaBER) and without squamous features (HT1376), demonstrated clear inhibition of cell and colony growth of BASQ-type SCaBER cells. ITIH5 further enhanced HA-associated cell-matrix attachment, indicated by altered size and number of focal adhesion sites resulting in reduced cell migration capacities. Transcriptomic analyses revealed enrichment of pathways and processes involved in ECM organization, differentiation and cell signaling. Finally, we provide evidence that ITIH5 increase sensitivity of SCaBER cells to chemotherapeutical agents (cisplatin and gemcitabine), whereas responsiveness of HT1376 cells was not affected by ITIH5 expression. Thus, we gain further insights into the putative role of ITIH5 as tumor suppressor highlighting an impact on drug response potentially via the HA-CD44 axis in BASQ-type BLCA.
Background: Perihilar cholangiocarcinoma (pCCA) is a rare primary liver malignancy. Even in patients amenable to surgery, outcomes are often dismal. Here, we aimed to identify prognostic markers for ...patient outcomes by analyzing functionally relevant single-nucleotide polymorphisms (SNPs) in genes with a role in tumor inflammation and angiogenesis. We analyzed 11 polymorphisms in the inflammation-angiogenesis axis (VEGF, EGF, EGFR, IL-1b, IL-6, CXCL8 (IL-8), IL-10, CXCR1, HIF1A, and COX2 genes) for their prediction of tumor recurrence and survival in pCCA patients undergoing surgery in a curative intent. Methods: Samples were obtained from 111 patients with pCCA undergoing liver resection in curative intent. DNA was extracted and analyzed using polymerase chain reaction-restriction fragment length polymorphism protocols and correlated with patients’ outcomes. Results: Out of the assessed variants, only the CXCR1 (also: interleukin-8-receptor alpha – IL-8RA) +860C>G heterozygous polymorphism (rs2234671) was associated with decreased disease-free survival (DFS), cancer-specific survival (CSS), and overall survival (OS) (18/111 (16.2%), median DFS 14 months, log-rank p = 0.007; median CSS 31 months, log-rank p = 0.007; and median OS 6 months, log-rank p = 0.002), compared to the GG genotype (92/111 (82.9%), median DFS 55 months, median CSS 63 months, and median OS 33 months). In the multivariate analysis, +860C>G remained an independent prognostic factor for DFS (adjusted p = 0.008), CSS (adjusted p = 0.001), and OS (adjusted p = 0.001). Conclusion: Genetic variant of CXCR1 +860C>G may serve as a molecular marker for DFS, CSS, and OS in patients undergoing curative-intent surgery for pCCA, indicating that the analysis of SNPs in genes involved in immune-mediated angiogenesis may help to identify patient subgroups at high risk for dismal oncological and overall outcome.
OBJECTIVE—Medial artery calcification in patients with chronic kidney disease proceeds through intramembranous ossification resulting from osteoblast-induced calcification of the collagen ...extracellular matrix. The current study is based on the hypothesis that mesenchymal stem cells (MSC) constitute critical cells for procalcific extracellular matrix remodeling in patients with chronic kidney disease.
METHODS AND RESULTS—Human MSC were cultured in media supplemented with pooled sera from either healthy or uremic patients (20%). Exposure to uremic serum enhanced the proliferation of MSC (cell counting, BrdU incorporation) whereas apoptosis and necrosis were not affected (annexin V and 7-amino-actinomycin staining). Uremic serum–exposed MSC recapitulated osteogenesis by matrix calcification and expression of bone-related genes (bone morphogenetic protein BMP-2 receptor, alkaline phosphatase, osteopontin, and Runx2) in 35 days. The uremic serum–induced osteogenesis was completely blocked by a BMP-2/4 neutralizing antibody or the natural antagonist NOGGIN. Calcification and matrix remodeling were further analyzed in a collagen-embedded osteogenesis model recapitulating the vascular collagen I/III environment. The uremic serum–induced calcification was shown to occur along collagen fibers as shown by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and von Kossa staining and was accompanied by extensive matrix remodeling.
CONCLUSION—Uremic serum induced in a BMP-2/4-dependent manner an osteoblast-like phenotype in MSC accompanied by matrix remodeling and calcification.
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