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FGFR1 expression in cancer cell lines.

Detailed description of STS Cohort 1 including the clinical data and FGFR1 status.

Soft-tissue sarcoma cohorts.

Detailed description of STS Cohort 2 including the clinical data and FGFR1 status.

Radiographic assessment of target lesions following BGJ398 treatment.

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PDF file - 57K, Significant decrease of p-(Tyr416)-SRC levels upon siRNA-mediated knockdown of IGF-1R in CME-1 synovial sarcoma cells.

PDF file - 70K, As an indirect proof of specific SRC-related action of dasatinib in synovial sarcoma cells, CME-1 did not display any significant effects upon dasatinib treatement after siRNA-mediated SRC-knockdown.

PDF file - 57K, Densitometric analysis of the top five phosphorylated kinases as found in phospho kinase arrays in 1273/99 and HS-SY-II synovial sarcoma cells. Flow cytometric analysis of three synovial sarcoma cell lines treated with different doses of dasatinib for 48 hours.

Increasingly extensive genomic diagnostics in cancer precision medicine require uniform evaluation criteria for the classification of variants with regard to their functional and therapeutic implications. In this review we present the most important guidelines and classification systems currently used in daily clinical practice, explain their advantages and disadvantages as well as differences and similarities, and present the step-by-step, systematic process that enables successful variant interpretation.

Background Cystic fibrosis (CF) is the most common genetic disorder in the Caucasian population. Despite remarkable improvements in morbidity and mortality during the last decades, the disease still limits survival and reduces quality of life of affected patients. Moreover, CF still represents substantial economic burden for healthcare systems. Inflammation and infection already start in early life and play important roles in pulmonary impairment. The aim of this study is to analyze the potential role of DMBT1, a protein with functions in inflammation, angiogenesis, and epithelial differentiation, in CF. Results Immunohistochemically DMBT1 protein expression was upregulated in lung tissues of CF patients compared to healthy controls. Additionally, pulmonary expression of Dmbt1 was approximately 6-fold increased in an established transgenic mouse model of CF-like lung disease (ENaC tg) compared to wild-type mice as detected by qRT-PCR. Since acetylcysteine (ACC) has been shown to reduce inflammatory markers in the airways, its potential influence on DMBT1 expression was analyzed. A549 cells stably transfected with an expression plasmid encoding the largest (8kb) DMBT1 variant (DMBT1+ cells) or an empty vector control (DMBT1- cells) and incubated with ACC both showed significantly reduced DMBT1 concentrations in the culture medium (p = 0.0001). To further elucidate the function of DMBT1 in pulmonary airways, respiratory epithelial cells were examined by phase contrast microscopy. Addition of human recombinant DMBT1 resulted in altered cilia motility and irregular beat waves (p < 0.0001) suggesting a potential effect of DMBT1 on airway clearance. Conclusions DMBT1 is part of inflammatory processes in CF and may be used as a potential biomarker for CF lung disease and a potential tool to monitor CF progression. Furthermore, DMBT1 has a negative effect on ciliary motility thereby possibly compromising airway clearance. Application of ACC, leading to reduced DMBT1 concentrations, could be a potential therapeutic option for CF patients.

Background Hypoxia and asphyxia are known to induce surfactant inactivation in newborns. Deleted in Malignant Brain Tumors 1 (DMBT1) is an innate immunity protein with functions in epithelial differentiation and angiogenesis. It was detected in hyaline membranes of infants with respiratory distress syndrome. Human recombinant DMBT1 is able to increase the surface tension of exogenous surfactant preparations in a dose-dependent manner. Methods Immunohistochemistry was performed on lung sections of infants who died due to pre-, peri- or postnatal hypoxia. The lung epithelial cell line A549 was stably transfected with a DMBT1 (DMBT1+ cells) expression plasmid or with an empty plasmid (DMBT1- cells). The cells were cultured in normoxic or hypoxic conditions, and then DMBT1 as well as HIF-1α RNA expression were analyzed by using real-time-polymerase chain reaction. Human recombinant DMBT1 was added to the modified porcine natural surfactant Curosurf to examine the effect of DMBT1 on surfactant ultrastructure with electron microscopy. Results DMBT1 expression was upregulated in human lung tissue after fetal/peri-/postnatal hypoxia. In addition, in vitro experiments showed increased DMBT1 RNA expression in A549 cells after hypoxia. HIF-1α was upregulated in both DMBT1+ and DMBT1- cells in response to hypoxia. The addition of human recombinant DMBT1 to Curosurf caused an impaired surfactant ultrastructure. Conclusions DMBT1 is upregulated in response to hypoxia and there seems to be a link between hypoxia and surfactant inactivation.

The multi-receptor tyrosine kinase inhibitor pazopanib is approved for the treatment of advanced soft-tissue sarcoma and has also shown activity in other sarcoma subtypes. However, its clinical efficacy is highly variable, and no reliable predictors exist to select patients who are likely to benefit from this drug.We analysed the molecular profiles and clinical outcomes of patients with pazopanib-treated sarcoma enrolled in a prospective observational study by the German Cancer Consortium, DKTK MASTER, that employs whole-genome/exome sequencing and transcriptome sequencing to inform the care of young adults with advanced cancer across histology and patients with rare cancers.Among 109 patients with available whole-genome/exome sequencing data, there was no correlation between clinical parameters, specific genetic alterations or mutational signatures and clinical outcome. In contrast, the analysis of a subcohort of 62 patients who underwent molecular analysis before pazopanib treatment and had transcriptome sequencing data available showed that mRNA levels of NTRK3 (hazard ratio [HR] = 0.53, p = 0.021), IGF1R (HR = 1.82, p = 0.027) and KDR (HR = 0.50, p = 0.011) were independently associated with progression-free survival (PFS). Based on the expression of these receptor tyrosine kinase genes, i.e. the features NTRK3-high, IGF1R-low and KDR-high, we developed a pazopanib efficacy predictor that stratified patients into three groups with significantly different PFS (p lt; 0.0001). Application of the pazopanib efficacy predictor to an independent cohort of patients with pazopanib-treated sarcoma from DKTK MASTER (n = 43) confirmed its potential to separate patient groups with significantly different PFS (p = 0.02), whereas no such association was observed in patients with sarcoma from DKTK MASTER (n = 97) or The Cancer Genome Atlas sarcoma cohort (n = 256) who were not treated with pazopanib.A score based on the combined expression of NTRK3, IGF1R and KDR allows the identification of patients with sarcoma and with good, intermediate and poor outcome following pazopanib therapy and warrants prospective investigation as a predictive tool to optimise the use of this drug in the clinic.

Background Deleted in malignant brain tumors 1 (DMBT1) is involved in innate immunity and epithelial differentiation. It has been proven to play a role in various states of inflammation or hypoxia of fetal gastrointestinal and pulmonary diseases. Discrimination of pathogenesis in necrotizing enterocolitis (NEC) based on cardiac status improves the understanding of NEC in different patient subgroups. We aimed at examining DMBT1 expressions regarding their association with cardiac status leading to impaired intestinal perfusion, intraoperative bacteria proof, and a fulminant course of NEC. Methods Twenty-eight patients with NEC were treated surgically between 2010 and 2019 at our institution. DMBT1 expression was examined in intestinal sections using immunohistochemistry to detect DMBT1 protein. Associations of clinical parameters and DMBT1 expression were analyzed. Results We examined DMBT1 levels in 10 patients without cardiac defects and 18 patients with persisting ductus arteriosus (PDA) and congenital heart defects (CHD). Compared to patients without cardiac malformations, DMBT1 levels tended to score higher in patients with PDA/CHD (p = 0.2113) and were negatively correlated with C-reactive protein in these infants (p = 0.0172; r = − 0.5533). The number of DMBT1-expressing macrophages was elevated in the PDA/CHD-subgroup (p = 0.0399). Ratios of neutrophils and monocytes to lymphocytes were significantly higher in infants with PDA/CHD (p = 0.0319 and 0.0493). DMBT1 expression was significantly associated with positive bacterial culture of intraoperative swabs (p = 0.0252) and DMBT1 expression of the serosa was associated with a fulminant course of NEC (p = 0.0239). Conclusions This study demonstrates that DMBT1 expression may be influenced by cardiac anomalies with an impaired intestinal perfusion in the neonatal intestine. NEC in PDA/CHD infants is associated with more DMBT1-positive macrophages and a significantly elevated neutrophil-to-lymphocyte ratio.

Background Approvals of cancer therapeutics are primarily disease entity specific. Current molecular diagnostic approaches frequently identify actionable alterations in rare cancers or rare subtypes of common cancers for which the corresponding treatments are not approved and unavailable within clinical trials due to entity-related eligibility criteria. Access may be negotiated with health insurances. However, approval rates vary, and critical information required for a scientific evaluation of treatment-associated risks and benefits is not systematically collected. Thus clinical trials with optimized patient selection and comprehensive molecular characterization are essential for translating experimental treatments into standard care. Patients and methods Continuous ReAssessment with Flexible ExTension in Rare Malignancies (CRAFT) is an open-label phase II trial for adults with pretreated, locally advanced, or metastatic solid tumors. Based on the evaluation by a molecular tumor board, patients are assigned to combinations of six molecularly targeted agents and a programmed death-ligand 1 (PD-L1) antagonist within seven study arms focusing on (i) BRAF V600 mutations; (ii) ERBB2 amplification and/or overexpression, activating ERBB2 mutations; (iii) ALK rearrangements, activating ALK mutations; (iv and v) activating PIK3CA and AKT mutations, other aberrations predicting increased PI3K–AKT pathway activity; (vi) aberrations predicting increased RAF–MEK–ERK pathway activity; (vii) high tumor mutational burden and other alterations predicting sensitivity to PD-L1 inhibition. The primary endpoint is the disease control rate (DCR) at week 16; secondary and exploratory endpoints include the progression-free survival ratio, overall survival, and patient-reported outcomes. Using Simon’s optimal two-stage design, 14 patients are accrued for each study arm. If three or fewer patients achieve disease control, the study arm is stopped. Otherwise, 11 additional patients are accrued. If the DCR exceeds 7 of 25 patients, the null hypothesis is rejected for the respective study arm. Conclusions CRAFT was activated in October 2021 and will recruit at 10 centers in Germany. Trial registration numbers EudraCT: 2019-003192-18; ClinicalTrials.gov: NCT04551521., Highlights • Actionable genetic alterations are detected in many cancers with unknown efficacy of the corresponding targeted therapies. • CRAFT, an open-label multicenter phase II trial, uses six molecularly targeted agents and a PD-L1 antagonist in seven treatment arms. • Patient allocation to trial arms is based on evaluation of molecular tumor characteristics by the German Cancer Consortium. • The in-depth molecular characterization helps to understand mechanisms underlying primary and acquired therapy resistance. • The CRAFT trial has been active in Germany since October 2021 and will open at 10 sites during 2022.

Amniotic fluid is a mixture containing many different proteins as immunomodulatory peptides and growth factors. The glycoprotein Deleted in Malignant Brain Tumors 1 (DMBT1) is participated in innate immunity, angiogenesis and epithelial differentiation. We analyzed the DMBT1 concentration in amniotic fluid during gestation.DMBT1 concentration was quantified by ELISA. Amniotic fluid samples were collected from preterm and term neonates. Effects of maternal or neonatal parameters were analyzed. To evaluate the source of DMBT1 we examined RNA of fetal tissue in relation to DMBT1 expression.The median DMBT1 concentration in amniotic fluid was 54.4 ng/ml. Amniotic fluid obtained28 weeks of gestation revealed significantly lower DMBT1 concentrations compared to ≥28 weeks. We found a positive correlation between DMBT1 concentration and gestational age (The results showed that DMBT1 concentrations in amniotic fluid correlate with the gestational age during gestation and that the fetal gastrointestinal tract is a potential source of DMBT1.Amniotic fluid contains not only nutrients, but also many immunomodulatory peptides and growth factors. Deleted in Malignant Brain Tumors 1 (DMBT1) is an innate immunity protein with functions in epithelial differentiation and angiogenesis. The aim of this research was to study the DMBT1 content and the factors affecting its concentration in amniotic fluid during gestation. In summary, the results obtained in this study showed that DMBT1 is a component of amniotic fluid and that DMBT1 concentrations in amniotic fluid correlate with gestational age. In addition to this, the fetal gastrointestinal tract is a potential source of DMBT1 detected in amniotic fluid.

Lipid droplets, a morphologic feature of adipocytic tumors, are strongly regulated by associated proteins of the perilipin/PAT (perilipin, adipophilin, and tail-interacting protein of 47 kD) family. So far, the use of perilipins as markers for differential diagnosis of soft tissue tumors has only been studied in a few cases. The aim of this study was to investigate the expression of perilipins in 478 human soft tissue tumors and 60 respective normal tissues. Perilipin 1 was immunohistochemically positive in all studied cases of well-differentiated liposarcomas, >90% of myxoid round cell liposarcomas, and >70% of pleomorphic liposarcomas, whereas only the differentiated components of dedifferentiated liposarcomas were immunohistochemically positive for perilipin 1. All other types of soft tissue sarcomas were negative for perilipin 1. Perilipin 2 was more prominent in dedifferentiated and pleomorphic liposarcomas and nearly all other high-grade sarcomas. In well-differentiated liposarcomas, lipomas, or normal adipose tissue, perilipin 2 was virtually absent. In addition, long-term stimulation of adipogenesis in the liposarcoma cell line LiSa-2 restored perilipin 1 expression, as exhibited in the source tumor. Furthermore, knockdown of perilipin 2 or perilipin 3 in LiSa-2 cells influenced lipid droplet number and size as well as cell vitality. In summary, perilipin 1 is a promising marker for the differential diagnosis of liposarcomas from other soft tissue sarcomas, whereas perilipin 2 correlates negatively with tumor grade and may be therapeutically useful.

Carboxylesterase 2 (CES2) is instrumental for conversion of ester-containing prodrugs in cancer treatment. Novel treatment strategies are exceedingly needed for cholangiocarcinoma (CCA) patients. Here, we assessed CES2 expression by immunohistochemistry in a CCA cohort comprising 171 non-liver fluke associated, intrahepatic (n = 72) and extrahepatic (perihilar: n = 56; distal: n = 43) CCAs. Additionally, 80 samples of high-grade biliary intraepithelial neoplastic tissues and 158 corresponding samples of histological normal, non-neoplastic biliary tract tissues were included. CES2 expression was highest in non-neoplastic biliary tissue and significantly decreased in CCA. Patients showing any CES2 expression in tumor cells had a significantly better overall survival compared to negative cases (p = 0.008). This survival benefit was also maintained after stratification of CES2-positive cases, by comparing low, medium and high CES2 expression levels (p-trend = 0.0006). Evaluation of CCA subtypes showed the survival difference to be restricted to extrahepatic tumors. Correlation of CES2 expression with data of tumor-infiltrating immune cells showed that particularly CD8+ T cells were more frequently detected in CES2-positive CCAs. Furthermore, treatment of CCA cell lines with the prodrug Irinotecan reduced cell viability, increased cytotoxicity and modulated inflammatory gene expression. In conclusion, reduced CES2 expression is associated with poor outcome and low CD8+ T cell infiltration in CCA patients. Further clinical studies could show, whether CES2 expression may serve as a predictive marker in patients treated with prodrugs converted by CES2.

Modern concepts in precision cancer medicine are based on increasingly complex genomic analyses and require standardized criteria for the functional evaluation and reporting of detected genomic alterations in order to assess their clinical relevance. In this article, we propose and address the necessary steps in systematic variant evaluation consisting of bioinformatic analysis, functional annotation and clinical interpretation, focusing on the latter two aspects. We discuss the role and clinical application of current variant classification systems and point out their scope and limitations. Finally, we highlight the significance of the molecular tumor board as a platform for clinical decision-making based on genomic analyses.

Increasingly extensive genomic diagnostics in cancer precision medicine require uniform evaluation criteria for the classification of variants with regard to their functional and therapeutic implications. In this review we present the most important guidelines and classification systems currently used in daily clinical practice, explain their advantages and disadvantages as well as differences and similarities, and present the step-by-step, systematic process that enables successful variant interpretation.Die immer umfangreichere genomische Diagnostik im Rahmen der Präzisionsonkologie erfordert einheitliche Bewertungskriterien für die Klassifizierung von Varianten im Hinblick auf ihre funktionelle Relevanz und therapeutischen Implikationen. In dieser Übersichtsarbeit stellen wir die wichtigsten derzeit gebräuchlichen Leitlinien und Klassifikationssysteme vor, erläutern Vor- und Nachteile sowie Unterschiede und Gemeinsamkeiten und beschreiben den schrittweisen, systematischen Prozess, der eine erfolgreiche Variantenbewertung ermöglicht.

Background Colorectal familial adenomatous polyposis (FAP) adenomas exhibit a uniform pathogenetic basis caused by a germline mutation in the adenomatous polyposis gene (APC), but the molecular changes leading to their development are incompletely understood. However, dysregulated apoptosis is known to substantially affect the development of colonic adenomas. One of the key regulatory proteins involved in apoptosis is apoptosis repressor with caspase recruitment domain (ARC). Methods The expression of nuclear and cytoplasmic ARC in 212 adenomas from 80 patients was analyzed by immunohistochemistry. We also compared expression levels of ARC with the expression levels of p53, Bcl-2, COX-2, and MMR proteins. Statistical analyses were performed by Spearman’s rank correlation and linear regression test. Results ARC was overexpressed in the nuclei and cytoplasm of most FAP adenomas investigated. Cytoplasmic ARC staining was moderately stronger (score 2) in 49.1% (n = 104/212) and substantially stronger (score 3) in 32.5% (n = 69/212) of adenomas compared to non-tumorous colorectal mucosa. In 18.4% (n = 39/212) of adenomas, cytoplasmic ARC staining was equivalent to that in non-tumorous mucosa. Nuclear expression of ARC in over 75% of cells was present in 30.7% (n = 65/212) of investigated adenomas, and nuclear expression in 10–75% of cells was detected in 62.7% (n = 133/212). ARC expression in under 10% of nuclei was found in 6.6% (n = 14/212) of adenomas. The correlation between nuclear ARC expression and cytoplasmic ARC expression was highly significant (p = 0.001). Moreover, nuclear ARC expression correlated positively with overexpression of Bcl-2, COX-2 p53 and β-catenin. Cytoplasmic ARC also correlated with overexpression of Bcl-2. Sporadic MMR deficiency was detected in very few FAP adenomas and showed no correlation with nuclear or cytoplasmic ARC. Conclusions Our results demonstrated that both cytoplasmic and nuclear ARC are overexpressed in FAP adenomas, thus in a homogenous collective. The highly significant correlation between nuclear ARC and nuclear β-catenin suggested that ARC might be regulated by β-catenin in FAP adenomas. Because of its further correlations with p53, Bcl-2, and COX-2, nuclear ARC might play a substantial role not only in carcinomas but also in precursor lesions.

Background Cholangiocarcinoma is a rapidly fatal cancer entity with a median survival of less than one year. In contrast to many other malignancies, no substantial therapeutic breakthrough has been made in the past few decades, thereby limiting the treatment to cytotoxic chemotherapy with little beneficial effect for most patients. Targeted therapy tailored to the individual has shown substantial success in the recent past as a promising avenue for cancer therapy. Methods In this study, we determined the frequency of amplification of the HER2 gene in a comprehensive and well-characterized European cholangiocarcinoma cohort encompassing 436 patients including intrahepatic (n = 155), proximal (n = 155) and distal (n = 126) cholangiocarcinoma by strict application of a combined immunohistochemical and in situ hybridization algorithm following the current guidelines for HER2 assessment in gastric cancer. Results We identified a proportion of 1.4% (n = 6) patients that demonstrated HER2 gene amplification, with the highest rate among the distal cholangiocarcinoma patients (2.4%). None of the patients with equivocal (2+) immunohistochemical staining results exhibited gene amplification molecularly. In four of the five patients with HER2 positivity, gene amplification was already present in concomitantly tested high-grade biliary intraepithelial neoplasia (80%). HER2 gene amplification was not significantly associated with other clinical parameters, including survival. Conclusions This study identifies HER2 gene amplification as a rare event in cholangiocarcinoma of the Western population, occurring already in high-grade BilIN in a subset of patients. Furthermore, we provide a robust testing algorithm that may be used prior to therapy administration in future clinical trials evaluating the role of HER2 as a predictive marker in cholangiocarcinoma.

Liver metastasis in colorectal cancer is common and the primary treatment is chemotherapy. To date, there is no routinely used test in clinical practice to predict the effectiveness of conventional chemotherapy. Therefore, biomarkers with predictive value for conventional chemotherapy would be of considerable benefit in treatment planning. We analysed three proteins [excision repair cross-complementing 1 (ERCC1), ribonucleoside-diphosphate reductase 1 (RRM1) and class III β-tubulin (TUBB3)] in colorectal cancer liver metastasis. We used tissue microarray slides with 101 liver metastasis samples, stained for ERCC1, RRM1 and TUBB3 and established scoring systems (fitted for tissue microarray) for each protein. In statistical analysis, we compared the expression of ERCC1, RRM1 and TUBB3 to mismatch proteins (MLH1, MSH2, MSH6 and PMS2), p53 and to apoptosis repressor protein (ARC). Statistically significant correlations were found between ERCC1, TUBB3 and MLH1, MSH2 and RRM1 and MSH2, MSH6. Noteworthy, our analysis revealed a strong significant correlation between cytoplasmic ARC expression and RRM1, TUBB3 (p=0.000 and p=0.001, respectively), implying an additional role of TUBB3 and RRM1 not only in therapy resistance, but also in the apoptotic machinery. Our data strengthens the importance of ERCC1, TUBB3 and RRM1 in the prediction of chemotherapy effectiveness and suggest new functional connections in DNA repair, microtubule network and apoptotic signaling (i.e. ARC protein). In conclusion, we showed the importance and need of predictive biomarkers in metastasized colorectal cancer and pointed out the relevance not only of single predictive markers but also of their interactions with other known and newly explored relations between different signaling pathways.

The Scavenger Receptor Cysteine-Rich (SRCR) proteins are an archaic group of proteins characterized by the presence of multiple SRCR domains. They are membrane-bound or secreted proteins, which are generally related to host defense systems in animals. Deleted in Malignant Brain Tumors 1 (DMBT1) is a SRCR protein which is secreted in mucosal fluids and involved in host defense by pathogen binding by its SRCR domains. Genetic polymorphism within DMBT1 leads to DMBT1-alleles giving rise to polypeptides with interindividually different numbers of SRCR domains, ranging from 8 SRCR domains (encoded by 6 kb DMBT1 variant) to 13 SRCR domains (encoded by the 8 kb DMBT1 variant). In the present study, we have investigated whether reduction from 13 to 8 amino-terminal SRCR domains leads to reduction of bacterial binding. The 6 kb variant bound ~20–45% less bacteria compared to the 8 kb variant. These results support the hypothesis that genetic variation in DMBT1 may influence microbial defense.

Purpose: Altered FGFR1 signaling has emerged as a therapeutic target in epithelial malignancies. In contrast, the role of FGFR1 in soft-tissue sarcoma (STS) has not been established. Prompted by the detection and subsequent therapeutic inhibition of amplified FGFR1 in a patient with metastatic leiomyosarcoma, we investigated the oncogenic properties of FGFR1 and its potential as a drug target in patients with STS. Experimental Design: The frequency of FGFR1 amplification and overexpression, as assessed by FISH, microarray-based comparative genomic hybridization and mRNA expression profiling, SNP array profiling, and RNA sequencing, was determined in three patient cohorts. The sensitivity of STS cell lines with or without FGFR1 alterations to genetic and pharmacologic FGFR1 inhibition and the signaling pathways engaged by FGFR1 were investigated using viability assays, colony formation assays, and biochemical analysis. Results: Increased FGFR1 copy number was detected in 74 of 190 (38.9%; cohort 1), 13 of 79 (16.5%; cohort 2), and 80 of 254 (31.5%; cohort 3) patients. FGFR1 overexpression occurred in 16 of 79 (20.2%, cohort 2) and 39 of 254 (15.4%; cohort 3) patients. Targeting of FGFR1 by RNA interference and small-molecule inhibitors (PD173074, AZD4547, BGJ398) revealed that the requirement for FGFR1 signaling in STS cells is dictated by FGFR1 expression levels, and identified the MAPK–ERK1/2 axis as critical FGFR1 effector pathway. Conclusions: These data identify FGFR1 as a driver gene in multiple STS subtypes and support FGFR1 inhibition, guided by patient selection according to the FGFR1 expression and monitoring of MAPK–ERK1/2 signaling, as a therapeutic option in this challenging group of diseases. Clin Cancer Res; 23(4); 962–73. ©2016 AACR.

Tumors of soft tissue and bone represent a heterogeneous group of neoplasias characterized by a wide variety of genetic aberrations. Albeit knowledge on tumorigenesis in mesenchymal tumors is continuously increasing, specific insights on altered signaling pathways as a basis for molecularly targeted therapeutic strategies are still sparse. The aim of this study was to determine the involvement of YAP1/TAZ-mediated signals in tumors of soft tissue and bone. Expression levels of YAP1 and TAZ were analyzed by immunohistochemistry in a large cohort of 486 tumor specimens, comprising angiosarcomas (AS), Ewing sarcomas, leiomyosarcomas, malignant peripheral nerve sheath tumors (MPNST), solitary fibrous tumors, synovial sarcomas (SySa), well-differentiated/dedifferentiated/pleomorphic and myxoid liposarcomas (MLS). Moderate to strong nuclear staining of YAP1 and TAZ was detected in 53% and 33%, respectively. YAP1 nuclear expression was most prevalent in MPNST, SySa and MLS, whereas nuclear TAZ was predominately detected in AS, MLS and MPNST. In a set of sarcoma cell lines, immunoblotting confirmed nuclear localization of YAP1 and TAZ, corresponding to their transcriptionally active pool. Suppression of YAP1/TAZ-TEAD mediated transcriptional activity significantly impaired sarcoma cell viability in vitro and in vivo. Our findings identify nuclear YAP1 and TAZ positivity as a common feature in subsets of sarcomas of soft tissue and bone and provide evidence of YAP1/TAZ-TEAD signaling as a specific liability to be considered as a new target for therapeutic intervention. Nuclear YAP1/TAZ expression may represent a biomarker suited to identify patients that could benefit from YAP1/TAZ-TEAD directed therapeutic approaches within future clinical trials.

Gallbladder carcinoma (GBC) is a biliary tract cancer with few treatment options and poor prognosis. Radical surgery is the only potentially curative treatment option but most patients diagnosed with GBC are unresectable. Thus, there is a great need for the development of new treatment options including targeted therapy. Here, we aimed at identifying deregulated miRNAs and affected pathways involved in GBC development and progression. We performed global miRNA profiling of 40 GBC and 8 normal gallbladder tissues and identified large differences with 30% of miRNAs being differentially expressed (false discovery rate: FDR

Aim Nuclear α-thalassemia/mental retardation X-linked (ATRX) loss and alternative lengthening of telomeres (ALT) are linked in distinct malignancies. We therefore aimed to determine the nuclear ATRX expression correlated with ALT in a comprehensive series of sarcomas. Methods and results A total of 573 formalin-fixed paraffin-embedded sarcomas comprising 28 entities were investigated for nuclear ATRX expression by immunohistochemistry. Telomere-specific fluorescence in-situ hybridization (FISH) was used to determine the ALT phenotype in 50 sarcomas with complete or heterogeneous ATRX loss. Complete nuclear ATRX loss was detected in 58 of 573 sarcomas, all high-grade, with the highest prevalence in undifferentiated pleomorphic sarcomas (38%) and pleomorphic liposarcomas (38%), followed by dedifferentiated liposarcomas (24%), osteosarcomas (21%), leiomyosarcomas (17%), myxofibrosarcomas (11%) and malignant peripheral nerve sheath tumours (4%). Interestingly, a further 20 sarcomas, all belonging to the aforementioned entities with complete ATRX loss, presented with a heterogeneous ATRX expression pattern. ALT was observed in 41 of 42 sarcomas with complete ATRX loss, but only in two of eight sarcomas with heterogeneous expression. Conclusion Nuclear ATRX loss, either complete or heterogeneous, is encountered in a considerable number of high-grade sarcomas with non-specific genetic alterations. A causal relationship with ALT might be indicated at least in cases with a complete nuclear ATRX loss.

Background: Biliary tract cancers (BTC) are rare malignant tumours with a poor prognosis. Previously, we have presented a detailed characterisation of the inflammatory infiltrate in BTC. Here, we analysed the impact of the expression of major histocompatibility complex class I (MHC I) on patient survival and the quantity, as well as the quality of tumour-infiltrating immune cell types in BTC. Methods: MHC I expression was assessed semi-quantitatively in 334 BTC, including extrahepatic (n=129) and intrahepatic cholangiocarcinomas (n=146), as well as adenocarcinomas of the gallbladder (n=59). In addition, 71 high-grade biliary intraepithelial lesions (BilIN 3) were included. Results were correlated with data on antitumour inflammation and investigated with respect to their association with clinicopathological variables and patient survival. Results: BTC showed a wide spectrum of different MHC I expression patterns ranging from complete negativity in some tumours to strong homogenous expression in others. In BilIN 3, significantly higher MHC I expression levels were seen compared to invasive tumours (P=0.004). Patients with strong tumoural MHC I expression had a significantly higher overall survival probability (median survival benefit: 8 months; P=0.006). MHC I expression strongly correlated with the number of tumour-infiltrating T-lymphocytes (CD4+ and CD8+) and macrophages. Conclusions: Differences of MHC I expression predict patient outcome and show correlations with specific components of the inflammatory infiltrate in BTC. These findings contribute to a better understanding of immune response and immune escape phenomena in cholangiocarcinogenesis.

Liposarcomas (LS) are the most common malignant mesenchymal tumors, with an overall long-term mortality rate of 60%. LS comprise three major subtypes, i.e., well-differentiated/dedifferentiated liposarcoma (WDLS/DDLS), myxoid/round cell liposarcoma (MLS) and pleomorphic liposarcoma (PLS). Aiming at the preclinical identification of novel therapeutic options, we here investigate the functional significance of SRC in primary human LS and in LS-derived cell lines. Immunohistochemical and Western blot analyses reveal relevant levels of activated p-(Tyr416)-SRC in LS of the different subtypes with particular activation in MLS and PLS. Dysregulation of the SRC modifiers CSK and PTP1B was excluded as major reason for the activation of the kinase. Consistent siRNA-mediated knockdown of SRC or inhibition by the SRC inhibitor Dasatinib led to decreased proliferation of LS cell lines of the different subtypes, with MLS cells reacting particularly sensitive in MTT assays. Flow cytometric analyses revealed that this effect was due to a significant decrease in mitotic activity and an induction of apoptosis. SRC inhibition by Dasatinib resulted in dephosphorylation of SRC itself, its interacting partners FAK and IGF-IR as well as its downstream target AKT. Consistent with a particular role of SRC in cell motility, Dasatinib reduced the migratory and invasive potential of MLS cells in Boyden chamber and Matrigel chamber assays. In summary, we provide evidence that SRC activation plays an important role in LS biology and therefore represents a potential therapeutic target, particularly in MLS and PLS.

Leiomyosarcoma (LMS) is an aggressive mesenchymal malignancy with few therapeutic options. The mechanisms underlying LMS development, including clinically actionable genetic vulnerabilities, are largely unknown. Here we show, using whole-exome and transcriptome sequencing, that LMS tumors are characterized by substantial mutational heterogeneity, near-universal inactivation of TP53 and RB1, widespread DNA copy number alterations including chromothripsis, and frequent whole-genome duplication. Furthermore, we detect alternative telomere lengthening in 78% of cases and identify recurrent alterations in telomere maintenance genes such as ATRX, RBL2, and SP100, providing insight into the genetic basis of this mechanism. Finally, most tumors display hallmarks of “BRCAness”, including alterations in homologous recombination DNA repair genes, multiple structural rearrangements, and enrichment of specific mutational signatures, and cultured LMS cells are sensitive towards olaparib and cisplatin. This comprehensive study of LMS genomics has uncovered key biological features that may inform future experimental research and enable the design of novel therapies., The molecular genetic landscape of leiomyosarcoma (LMS) is largely unknown. Here, the authors identify frequent DNA copy number alterations, whole-genome duplication, TP53 and RB1 inactivation, alternative telomere lengthening, and genomic imprints of defective DNA repair via homologous recombination as a potential therapeutic target in LMS patients.

AIMS: Deleted in malignant brain tumours 1 (DMBT1) exerts functions in the regulation of epithelial differentiation and inflammation and has been proposed as a tumour suppressor. Because chronic inflammation is a hallmark of cholangiocarcinogenesis, the aim of this study was to investigate the expression of DMBT1 in biliary tract cancer (BTC) and to correlate this expression with clinicopathological data.METHODS AND RESULTS: The expression of DMBT1 protein was examined immunohistochemically in 157 BTC patients [41 intrahepatic (ICC), 60 extrahepatic cholangiocarcinomas (ECC) and 56 adenocarcinomas of the gallbladder (GBAC)]. Additionally, 56 samples of high-grade biliary intraepithelial neoplasia (BilIN 3) and 92 corresponding samples of histological non-neoplastic biliary tract tissues were included. DMBT1 expression was increased significantly in BilIN 3 compared to normal tissue (P < 0.0001) and BTC (P < 0.0001). BTC showed no significant difference in DMBT1 expression compared to non-neoplastic biliary tissue (P = 0.315). Absent DMBT1 expression in non-neoplastic biliary tissue of BTC patients was associated with poorer survival (P = 0.027). DMBT1 expression was correlated significantly with patients' age (P < 0.001).CONCLUSION: DMBT1 is expressed differently in cholangiocarcinogenesis and poorer patients' survival rates are associated with absent DMBT1 expression in non-neoplastic biliary tissue, suggesting a tumour-suppressive role of DMBT1 in early cholangiocarcinogenesis.

The far upstream element binding protein (FBP) and the FBP-interacting repressor (FIR) represent molecular tools for transcriptional fine tuning of target genes. Strong overexpression of FBP in human hepatocellular carcinoma (HCC) supports tumor growth and correlates with poor patient prognosis. However, the role of the transcriptional repressor FIR in hepatocarcinogenesis remains poorly delineated. We show that overexpression of FIR correlates with tumor dedifferentiation and tumor cell proliferation in about 60% of primary HCCs. Elevated FIR levels are associated with genomic gains of the FIR gene locus at chromosome 8q24.3 in human HCC specimens. In vitro, nuclear enrichment of FIR supports HCC cell proliferation and migration. Expression profiling of HCC cells after small interfering RNA (siRNA)-mediated silencing of FIR identified the transcription factor DP-1 (TFDP1) as a transcriptional target of FIR. Surprisingly, FIR stimulates the expression of FBP in a TFDP1/E2F1-dependent manner. FIR splice variants lacking or containing exon 2 and/or exon 5 are expressed in the majority of HCCs but not in normal hepatocytes. Specific inhibition of FIR isoforms with and without exon 2 revealed that both groups of FIR splice variants facilitate tumor-supporting effects. This finding was confirmed in xenograft transplantation experiments with lentiviral-infected short hairpin RNA (shRNA) targeting all FIR variants as well as FIR with and without exon 2. Conclusion: High-level nuclear FIR does not facilitate repressor properties but supports tumor growth in HCC cells. Thus, the pharmacological inhibition of FIR might represent a promising therapeutic strategy for HCC patients with elevated FIR expression. (Hepatology 2014;60:1241–1250)

Introduction: Soft tissue and bone tumors represent a heterogeneous group of neoplasias characterized by a wide variety of genetic aberrations. Albeit knowledge on tumorigenesis in mesenchymal tumors is continuously increasing, specific findings on altered signal transduction pathways as a basis for molecularly targeted therapeutic strategies is still sparse. Given recent studies on aberrant activation of the Hippo pathway transcriptional coactivators YAP and TAZ in different cancer types, the aim of this study was to determine the involvement of YAP/TAZ-mediated signal transduction in soft tissue and bone tumors. Experimental procedures: The expression levels of nuclear YAP and TAZ were analyzed by immunohistochemistry in a large cohort of 486 soft tissue and bone tumors. The comprehensive set of tissue specimens comprised 10 diagnostic categories: Angiosarcomas (AS; n=29), Ewing’s sarcomas (ES; n=20), leiomyosarcomas (LMS; n=68), malignant peripheral nerve sheath tumors (MPNST; n=45), solitary fibrous tumors (SFT; n=36), synovial sarcomas (SySa; n=65), well-differentiated liposarcomas (WDLS; n=55), dedifferentiated liposarcomas (DDLS; n=74), myxoid liposarcomas (MLS; n=85), and pleomorphic liposarcomas (PLS; n=9). The biological effects of the small molecule YAP/TAZ-TEAD inhibitor verteporfin on sarcoma cell proliferation (MPNST, SySa and MLS) were monitored by immunoblotting and cell viability assays in vitro. Results: Moderate to strong nuclear staining of YAP and TAZ was detected in 53% and 33% of soft tissue and bone tumor specimens, respectively. YAP nuclear expression was most prevalent in MPNST (58%), SySa (78%) and MLS (91%), whereas nuclear TAZ was predominately found in AS (55%), MLS (55%) and MPNST (71%). Immunoblotting confirmed the nuclear localization of YAP and TAZ in MPNST, SySa and MLS cell lines. Inhibition of the transcriptionally active YAP/TAZ-TEAD interaction employing the small molecular inhibitor verteporfin resulted in a significant suppression of sarcoma cell viability. Conclusions: This study identifies elevated transcriptional activity of nuclear YAP/TAZ as specific liability of subgroups of soft tissue and bone tumors. We provide preclinical evidence that YAP/TAZ-mediated signal transduction represents a rational target for therapeutic intervention in sarcoma cell lines that warrants further investigation. Citation Format: Ilka Isfort, Sandra Elges, Magdalene Cyra, Danielle Brandes, Ruth Berthold, Marcus Renner, Gunhild Mechtersheimer, Olle Larsson, Sebastian Huss, Eva Wardelmann, Wolfgang Hartmann, Marcel Trautmann. Hippo pathway transcriptional coactivators YAP/TAZ in soft tissue and bone tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2139.

The molecular mechanisms underlying the genesis of cholangiocarcinomas (CCs) are poorly understood. Epigenetic changes such as aberrant hypermethylation and subsequent atypical gene expression are characteristic features of most human cancers. In CC, data regarding global methylation changes are lacking so far. We performed a genome-wide analysis for aberrant promoter methylation in human CCs. We profiled 10 intrahepatic and 8 extrahepatic CCs in comparison to non-neoplastic biliary tissue specimens, using methyl-CpG immunoprecipitation (MCIp) combined with whole-genome CpG island arrays. DNA methylation was confirmed by quantitative mass spectrometric analysis and functional relevance of promoter hypermethylation was shown in demethylation experiments of two CC cell lines using 5-aza-2′deoxycytidine (DAC) treatment. Immunohistochemical staining of tissue microarrays (TMAs) from 223 biliary tract cancers (BTCs) was used to analyze candidate gene expression at the protein level. Differentially methylated, promoter-associated regions were nonrandomly distributed and enriched for genes involved in cancer-related pathways including Wnt, transforming growth factor beta (TGF-β), and PI3K signaling pathways. In CC cell lines, silencing of genes involved in Wnt signaling, such as SOX17, WNT3A, DKK2, SFRP1, SFRP2, and SFRP4 was reversed after DAC administration. Candidate protein SFRP2 was substantially down-regulated in neoplastic tissues of all BTC subtypes as compared to normal tissues. A significant inverse correlation of SFRP2 protein expression and pT status was found in BTC patients. Conclusion: We provide a comprehensive analysis to define the genome-wide methylation landscape of human CC. Several candidate genes of cancer-relevant signaling pathways were identified, and closer analysis of selected Wnt pathway genes confirmed the relevance of this pathway in CC. The presented global methylation data are the basis for future studies on epigenetic changes in cholangiocarcinogenesis. (Hepatology 2014;59:544–554)

Synovial sarcoma is a high-grade soft tissue malignancy characterized by a specific reciprocal translocation t(X;18), which leads to the fusion of the SS18 (SYT) gene to one of three SSX genes (SSX1, SSX2 or SSX4). The resulting chimeric SS18-SSX protein is suggested to act as an oncogenic transcriptional regulator. Despite multimodal therapeutic approaches, metastatic disease is often lethal and the development of novel targeted therapeutic strategies is required. Several expression-profiling studies identified distinct gene expression signatures, implying a consistent role of Wnt/β-catenin signaling in synovial sarcoma tumorigenesis. Here we investigate the functional and therapeutic relevance of Wnt/β-catenin pathway activation in vitro and in vivo. Immunohistochemical analyses of nuclear β-catenin and Wnt downstream targets revealed activation of canonical Wnt signaling in a significant subset of 30 primary synovial sarcoma specimens. Functional aspects of Wnt signaling including dependence of Tcf/β-catenin complex activity on the SS18-SSX fusion proteins were analyzed. Efficient SS18-SSX-dependent activation of the Tcf/β-catenin transcriptional complex was confirmed by TOPflash reporter luciferase assays and immunoblotting. In five human synovial sarcoma cell lines, inhibition of the Tcf/β-catenin protein-protein interaction significantly blocked the canonical Wnt/β-catenin signaling cascade, accompanied by the effective downregulation of Wnt targets (AXIN2, CDC25A, c-MYC, DKK1, CyclinD1 and Survivin) and the specific suppression of cell viability associated with the induction of apoptosis. In SYO-1 synovial sarcoma xenografts, administration of small molecule Tcf/β-catenin complex inhibitors significantly reduced tumor growth, associated with diminished AXIN2 protein levels. In summary, SS18-SSX-induced Wnt/β-catenin signaling appears to be of crucial biological importance in synovial sarcoma tumorigenesis and progression, representing a potential molecular target for the development of novel therapeutic strategies.

Biliary tract cancers (BTC) are a diverse group of tumours that arise from the biliary tract epithelium. Biliary tract cancers can be divided into three major clinical phenotypes: cholangiocarcinomas (CC) of intrahepatic (ICC) and extrahepatic (ECC) origin and adenocarcinomas of the gallbladder (GBAC). In the United States and Europe, risk factors for BTC include chronic biliary tract diseases, such as primary sclerosing cholangitis, hepatolithiasis, choledochal cysts, and other anatomical malformations of the bile ducts (Blechacz et al, 2011). Non-biliary diseases such as heavy alcohol abuse, obesity, non-alcoholic fatty liver disease, chronic hepatitis C, and cirrhosis are also more prevalent in BTC patients compared with the general population. For BTC patients with locally advanced or metastatic disease, the prognosis is poor with median survival less than 1 year (Hezel et al, 2010). Common therapeutic options include surgery and chemotherapy but as BTC patients often present in an unresectable state, there is an urgent need of novel chemotherapeutic options (Patel, 2011). As some BTC patients are in a general condition insufficient to undergo aggressive systemic treatment, finding better and reliable prognostic markers for BTC is of importance for further stratification of patients for therapeutic trials. Tumour cells are often surrounded by infiltrating inflammatory cells, particularly lymphocytes and macrophages (Smyth et al, 2006). Tumour antigens drive the development of tumour-specific adaptive immune responses (Boon et al, 1994). CD4+ and CD8+ T lymphocytes are crucial components of tumour-specific cellular adaptive immunity. CD8+ T lymphocytes attack tumour cells presenting tumour-associated antigen peptide with major histocompatibility complex class I (MHC I) on their surface by producing interferon-γ. Interferon-γ-dependent mechanisms of tumour cell cytostasis and killing comprise cell cycle inhibition, apoptosis, angiostasis, and induction of antitumourigenic activity of macrophages (Dunn et al, 2004). Immunohistochemical studies have found that tumour-infiltrating CD8+ T lymphocytes have a favourable effect on patient survival in several malignant tumours including colorectal (Galon et al, 2006), ovarian (Zhang et al, 2003), breast (Mahmoud et al, 2011), and pancreatic (Fukunaga et al, 2004) cancer. In colorectal cancer (CRC), density and location of CD8+ T lymphocytes have a prognostic value superior to and independent of the International Union Against Cancer (UICC) – TNM classification (Mlecnik et al, 2011). Further, density of immune cells at the invasive margin of metastatic CRC is predictive for response to chemotherapy (Halama et al, 2011). In BTC, sub-populations of immune cells have so far only been studied in parts, using comparably small patient cohorts and without specifying anatomical/histological subtypes (Oshikiri et al, 2003; Takagi et al, 2004). Therefore, our aim was to analyse the influence of density and distribution of tumour-infiltrating immune cells on patient prognosis in a large, well-characterized series of BTC patients with long-time follow-up.

Aims In adult humans, the follicle-stimulating hormone receptor (FSHR) is expressed only in the granulosa cells of the ovary and the Sertoli cells of the testis. Recently, it has been shown that FSHR is expressed selectively on the surface of blood vessels in a wide range of tumours. So far, the expression of FSHR in mesenchymal tumours has not been studied. Methods and results We performed a semiquantitative evaluation of FSHR protein expression in a large cohort of soft tissue sarcomas (STS; n = 335), including 11 subtypes. FSHR-positive vessels were detected in all sarcoma subtypes analysed. Among liposarcomas, significantly more cases of dedifferentiated liposarcomas (28 of 44) showed FSHR expression compared to well-differentiated liposarcomas (WDLS; four of 21; P