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Purpose: The human SIM2 gene is located within the Down's syndrome critical region of chromosome 21 and encodes transcription factors involved in brain development and neuronal differentiation. SIM2 has been assigned a possible role in the pathogenesis of solid tumors, and the SIM2-short isoform (SIM2-s) was recently proposed as a molecular target for cancer therapy. We previously reported SIM2 among the highly up-regulated genes in 29 prostate cancers, and the purpose of our present study was to examine the expression status of SIM2 at the transcriptional and protein level as related to outcome in prostate cancer.Experimental Design: By quantitative PCR, mRNA in situ hybridization, and immunohistochemistry, we evaluated the expression and significance of SIM2 isoforms in 39 patients with clinically localized prostate cancer and validated the expression of SIM2-s protein in an independent cohort of 103 radical prostatectomies from patients with long and complete follow-up.Results: The SIM2 isoforms (SIM2-s and SIM2-l) were significantly coexpressed and increased in prostate cancer. Tumor cell expression of SIM2-s protein was associated with adverse clinicopathologic factors like increased preoperative serum prostate-specific antigen, high histologic grade, invasive tumor growth with extra-prostatic extension, and increased tumor cell proliferation by Ki-67 expression. SIM2-s protein expression was significantly associated with reduced cancer-specific survival in multivariate analyses.Conclusions: These novel findings indicate for the first time that SIM2 expression might be important for clinical progress of human cancer and support the recent proposal of SIM2-s as a candidate for targeted therapy in prostate cancer.

Supplementary Figure 2 from Increased Expression of SIM2-s Protein Is a Novel Marker of Aggressive Prostate Cancer

Supplementary Figure 1 from Increased Expression of SIM2-s Protein Is a Novel Marker of Aggressive Prostate Cancer

Supplementary Figure 2 Legend from Increased Expression of SIM2-s Protein Is a Novel Marker of Aggressive Prostate Cancer

Metastatic castration-resistant prostate cancer (mCRPC) is an immunologically cold disease with dismal outcomes. Cryoablation destroys cancer tissue, releases tumor-associated antigens and creates a pro-inflammatory microenvironment, while dendritic cells (DCs) activate immune responses through processing of antigens. Immunotherapy combinations could enhance the anti-tumor efficacy. This open-label, single-arm, single-center phase I trial determined the safety and tolerability of combining cryoablation and autologous immature DC, without and with checkpoint inhibitors. Immune responses and clinical outcomes were evaluated. Patients with mCRPC, confirmed metastases and intact prostate gland were included. The first participants underwent prostate cryoablation with intratumoral injection of autologous DCs in a 3 + 3 design. In the second part, patients received cryoablation, the highest acceptable DC dose, and checkpoint inhibition with either ipilimumab or pembrolizumab. Sequentially collected information on adverse events, quality of life, blood values and images were analyzed by standard descriptive statistics. Neither dose-limiting toxicities nor adverse events > grade 3 were observed in the 18 participants. Results indicate antitumor activity through altered T cell receptor repertoires, and 33% durable (> 46 weeks) clinical benefit with median 40.7 months overall survival. Post-treatment pain and fatigue were associated with circulating tumor cell (CTC) presence at inclusion, while CTC responses correlated with clinical outcomes. This trial demonstrates that cryoimmunotherapy in mCRPC is safe and well tolerated, also for the highest DC dose (2.0 × 108) combined with checkpoint inhibitors. Further studies focusing on the biologic indications of antitumor activity and immune system activation could be considered through a phase II trial focusing on treatment responses and immunologic biomarkers.

Background The STAT3 pathway may drive prostate cancer (PCa) progression to metastatic castration-resistant prostate cancer (mCRPC). STAT3 may serve as a good target for the treatment of prostate cancer. The function of STAT3 relies significantly on its SH2 domain, which promotes STAT3 homo- or hetero-dimerization, protein-protein interaction and nuclear translocation of the STAT3 dimers needed for transcription. Due to this important role, the STAT3 SH2 domain becomes an attractive therapeutic target. In a drug discovery and development program we have found two small molecular compounds named 323-1 and 323-2 that were identified as novel STAT3 SH2 domain inhibitors in a series of experiments. Material and methods In silico computational modeling, Drug Affinity Responsive Target Stability (DARTS) and Fluorescence Polarization (FP) assays were performed to examine whether compounds 323-1 and 323-2 could bind to the STAT3 protein. The antitumor activity of compounds 323-1 and 323-2 was firstly determined by clonogenic assays in different prostate tumor cells, then validated in the humanised NOD/SCID mouse CRPC model. Effects of compound 323-1 and 323-2 on STAT3 transcriptional activity were determined by the luciferase assay and protein levels by western blotting. Results In silico computational modeling, DARTS and FP assays altogether determined 323-1 and 323-2 as direct STAT3 inhibitors targeting the STAT3 SH2 domain and inhibiting both phosphorylated and non-phosphorylated STAT3 dimerization. Computational docking predicted that both 323 compounds bind to three subpockets of the STAT3 SH2 domain with full inhibition of STAT3. FP assay further confirmed that 323s target the STAT3 SH2 domain by competitively abrogating the interaction between STAT3 and the SH2-binding peptide GpYLPQTV. Compared with S3I-201, the 323 compounds exhibited stronger inhibition of STAT3 and reduced the level of IL-6-stimulated phosphorylation of STAT3 (Tyr705) in LNCaP cells over the phosphorylation of STAT1 (Tyr701) induced by IFN-ɣ in PC3 cells or the phosphorylation of STAT1 (Ser727) in DU145 cells. Both compounds down-regulated STAT3 target genes MCL1 and cyclin D1. The in-vivo data supported that 323s significantly inhibited LNCaP cell generated CRPC tumor growth. Thus, the two compounds are promising leading compounds for the treatment of cancers with hyper-activated STAT3. Conclusions Here we report that these compounds modulate the IL-6/STAT3 pathway by 1) inhibition of STAT3 phosphorylation on Tyr705 and 2) disruption of STAT3 dimerization by directly targeting its SH2 domain; 3) inhibition of STAT3 transcriptional activity. Thus, the compounds 323-1 and 323-2 are promising new leading compounds for therapeutic STAT3 inhibition. Citation Format: Yaping Hua, Waqas Azeem, Anne Margrete Øyan, Karl-Henning Kalland. Novel STAT3 inhibitors targeting the STAT3 dimerization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1404.

Background: Dendritic cells (DCs) are fundamental to adaptive immunity by detecting, engulfing and presenting foreign or changed antigenic peptides to lymphocytes that thereafter initiate antigen-specific immune attacks. DC-based immunotherapy against cancer has been promising for a long time, but has not yet met expectations. Cancer cell heterogeneity and numerous immune evasion mechanisms represent challenges. According to textbook renderings, DCs may induce either tolerance or immune activation depending upon the nature of the encountered antigen and the status of the microenvironment. Recently, we have discovered a more complicated scenario according to which monocyte-derived DCs (moDCs) exhibit dual pro- and anti-inflammatory features, suggesting that selected conditions that enhance pro-inflammatory features and counteract tolerogenic features could generate more robust and potent DCs for cancer therapy. Materials and Methods: Healthy blood donor buffy coats were used for the isolation of monocytes and conventional CD141+ and CD1c+ natural circulating DCs. IL-4 and GM-CSF were used to differentiate monocytes into immature moDCs. Toll-like receptor agonists were used to induce DC maturation, and specific agonists and inhibitors were tested in different DC variants. Flow cytometry, ELISA determination of cytokine secretion, HPLC-based quantitation of kynurenine secretion, genome-wide gene expression analyses and functional assays, such as antigen uptake, migration and the mixed leukocyte reaction were used for evaluation of treated DCs. Results: Conventional DCs were more susceptible to apoptosis, and dependent upon culture conditions, than moDCs. Inhibition of β-catenin signaling stimulated several pro-inflammatory features, e.g. increased IL-12 and decreased IL-10 secretion, with reciprocal effects caused by β-catenin stimulation. Maturation of moDCs was accompanied by very strong induction of indoleamine 2, 3-dioxygenase 1 (IDO1) transcription and paralleled by increased kynurenine secretion, but this was partially counteracted by β-catenin stimulation. Conclusions: Preparation of more potent DCs for cancer therapy needs to take into account DC longevity and conflicting activation patterns of pro- and anti-inflammatory abilities. Citation Format: Waqas Azeem, Seyed Mohammad Lellahi, Yaping Hua, Anne Margrete Øyan, Benjamin Gabriel, Karl-Henning Kalland. Robust and potent dendritic cells for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3568.

Modulation of β-catenin signaling has attractive therapeutic potential in cancer immunotherapy. Several studies have found that β-catenin can mediate immune evasion in cancer and promote anti-inflammatory features of antigen-presenting dendritic cells. Many small molecular compounds that inhibit Wnt/β-catenin signaling are currently in clinical development, but none have entered routine clinical use. New inhibitors of β-catenin signaling are consequently desirable. Here, we have tested, in monocyte-derived dendritic cells, the effects of two small molecular compounds, axitinib and nitazoxanide, that previously have been discovered to inhibit β-catenin signaling in colon cancer cells. Immature and lipopolysaccharide-matured dendritic cells prepared from healthy blood donor buffy coats were stimulated with 6-bromoindirubin-3′-oxime (6-BIO) to boost basal β-catenin activity, and the effects of axitinib and nitazoxanide were compared with the commercial β-catenin inhibitor ICG-001. Assays, including genome-wide RNA-sequencing, indicated that neither axitinib nor nitazoxanide demonstrated considerable β-catenin inhibition. Both compounds were found to be less toxic to monocyte-derived dendritic cells than either 6-BIO or ICG-001. Axitinib stimulated several aspects of dendritic cell function, such as IL12-p70 secretion, and counteracted IL-10 secretion, according to the present study. However, neither axitinib nor nitazoxanide were found to be efficient β-catenin inhibitors in monocyte-derived dendritic cells.

Background: Despite successful implementation of drugs targeting the human epidermal growth factor receptor 2 (HER2) receptor in breast and gastric cancers, the potential of HER2 as a therapeutic target in other cancers has been less studied, including endometrial cancer. We investigated expression levels of HER2 (ERBB2) in a large cohort of endometrial cancer lesions, also including complex atypical hyperplasia and metastatic lesions. Methods: 67 precursor lesions, 790 primary endometrial cancers and 383 metastatic lesions were investigated for HER2 expression in relation to clinicopathologic features and outcome. Protein levels were assessed by immunohistochemistry (using the HercepTest and staining index (SI) criteria), mRNA levels by microarrays and amplification status by chromogenic in situ hybridisation. Results: High HER2 protein levels were significantly associated with features of aggressive disease and increased mRNA ERBB2 levels. HER2 expression defined by the SI proved to be a better predictor of survival compared with the HercepTest. A discordant HER2 expression pattern between paired primary and metastatic lesions was detected, revealing substantial reduction in HER2 expression from primary to metastatic disease. Conclusions: Loss of HER2 expression is common in metastatic endometrial cancer lesions and assessment of HER2 levels in the metastatic lesions may be important to define the potential benefit of anti-HER2 treatments in endometrial cancer patients. publishedVersion

// Karen Klepsland Mauland 1, 2 , Zhenlin Ju 3 , Ingvild Loberg Tangen 1, 2 , Anna Berg 1, 2 , Karl-Henning Kalland 1, 4 , Anne Margrete Oyan 1, 4 , Line Bjorge 1, 2 , Shannon N. Westin 5 , Camilla Krakstad 1, 6 , Jone Trovik 1, 2 , Gordon B. Mills 7 , Erling A. Hoivik 1, 2 and Henrica Maria Johanna Werner 1, 2 1 Centre for Cancer Biomarkers CCBIO, Department of Clinical Science (K2), University of Bergen, Bergen, Norway 2 Department of Obstetrics and Gynecology, Haukeland University Hospital, Bergen, Norway 3 Department of Bioinformatics and Computational Biology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA 4 Department of Microbiology, Haukeland University Hospital, Bergen, Norway 5 Department of Gynecologic Oncology and Reproductive Medicine, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA 6 Centre for Cancer Biomarkers CCBIO, Department of Biomedicine, University of Bergen, Bergen, Norway 7 Department of Systems Biology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA Correspondence to: Karen Klepsland Mauland, email: Karen.Mauland@uib.no Henrica Maria Johanna Werner, email: Henrica.Werner@uib.no Keywords: endometrial cancer; obesity; biomarkers; hormone receptors; MAPK signaling Received: April 27, 2017 Accepted: September 20, 2017 Published: October 31, 2017 ABSTRACT Endometrial cancer development is strongly linked to obesity, but knowledge regarding the influence of excess weight on endometrial tumor signaling pathways remains scarce. We therefore analyzed reverse phase protein array (RPPA) data for obesity-related protein expression patterns, using one training (n=272) and two test cohorts (n=68; n=178) of well-annotated samples from women treated for endometrioid endometrial cancer. Gene expression profiling and immunohistochemistry were used for cross-platform validation. Body mass index (BMI) was significantly correlated with progesterone receptor (PR) expression and a hormone receptor protein signature, across all cohorts. In two of the cohorts, BMI was negatively correlated with RTK- and MAPK-pathway activation, particularly phosphorylated MAPK T202 Y204 (p-MAPK) level. Using stepwise selection modelling, a BMI-associated protein signature, including phosphorylated estrogen receptor α S118 (p-ERα) and p-MAPK, was identified. In the subset of FIGO stage 1, grade 1-2 tumors, obese patients (BMI≥30) had better survival compared to non-obese patients in the two cohorts with longest follow-up time (p=0.042, p=0.058). Non-obese patients had higher p-MAPK levels, whereas obese patients had higher p-ERα levels and enrichment of gene signatures related to estrogen signaling, inflammation, immune signaling and hypoxia. In subgroup analysis of non-obese patients with FIGO stage 1 tumors, low PI3K-activation was associated with reduced survival (p=0.002, training cohort). In conclusion, increasing BMI is associated with increased PR and p-ERα levels and reduced MAPK signaling, both in all patients and in subsets with predicted excellent prognosis. The MAPK-pathway represents a potential therapeutic target in non-obese patients with low stage and low grade tumors.

// Karen Klepsland Mauland 1, 2 , Elisabeth Wik 3, 4, * , Erling A. Hoivik 1, 2, * , Kanthida Kusonmano 2, 5, 6 , Mari Kylleso Halle 1, 2 , Anna Berg 1, 2 , Hans Kristian Haugland 4 , Anne Margrete Oyan 1, 7 , Karl-Henning Kalland 1, 7 , Ingunn Marie Stefansson 4 , Lars A. Akslen 3, 4 , Camilla Krakstad 1, 8 , Jone Trovik 1, 2 , Henrica Maria Johanna Werner 1, 2 and Helga Birgitte Salvesen 1, 2, ** 1 Center for Cancer Biomarkers CCBIO, Department of Clinical Science (K2), University of Bergen, Bergen, Norway 2 Department of Obstetrics and Gynecology, Haukeland University Hospital, Bergen, Norway 3 Center for Cancer Biomarkers CCBIO, Department of Clinical Medicine (K1), Section for Pathology, University of Bergen, Bergen, Norway 4 Department of Pathology, Haukeland University Hospital, Bergen, Norway 5 Computational Biology Unit, University of Bergen, Bergen, Norway 6 Bioinformatics and Systems Biology Program, School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, Bangkhuntien, Bangkok, Thailand 7 Department of Microbiology, Haukeland University Hospital, Bergen, Norway 8 Center for Cancer Biomarkers CCBIO, Department of Biomedicine, University of Bergen, Bergen, Norway * These authors have contributed equally to this work ** Deceased Correspondence to: Karen Klepsland Mauland, email: Karen.Mauland@uib.no Keywords: endometrial cancer, DNA ploidy, prognosis, transcriptional alterations, chromosome 15q Received: October 06, 2016 Accepted: November 23, 2016 Published: December 25, 2016 ABSTRACT Aneuploidy is a widely studied prognostic marker in endometrial cancer (EC), however, not implemented in clinical decision-making. It lacks validation in large prospective patient cohorts adjusted for currently standard applied prognostic markers, including estrogen/progesterone receptor status (ER/PR). Also, little is known about aneuploidy-related transcriptional alterations, relevant for understanding its role in EC biology, and as therapeutic target. We included 825 EC patients with available ploidy status and comprehensive clinicopathologic characterization to analyze ploidy as a prognostic marker. For 144 patients, gene expression data were available to explore aneuploidy-related transcriptional alterations. Aneuploidy was associated with high age, FIGO stage and grade, non-endometrioid histology, ER/PR negativity, and poor survival (p-values

The transcription factor β-catenin is able to induce tolerogenic/anti-inflammatory features in different types of dendritic cells (DCs). Monocyte-derived dendritic cells (moDCs) have been widely used in dendritic cell-based cancer therapy, but so far with limited clinical efficacy. We wanted to investigate the hypothesis that aberrant differentiation or induction of dual pro- and anti-inflammatory features may be β-catenin dependent in moDCs. β-catenin was detectable in both immature and lipopolysaccharide (LPS)-stimulated DCs. The β-catenin inhibitor ICG-001 dose-dependently increased the pro-inflammatory signature cytokine IL-12p70 and decreased the anti-inflammatory signature molecule IL-10. The β-catenin activator 6-bromoindirubin-3'-oxime (6-BIO) dose-dependently increased total and nuclear β-catenin, and this was associated with decreased IL-12p70, increased IL-10, and reduced surface expression of activation markers, such as CD80 and CD86, and increased expression of inhibitory markers, such as PD-L1. 6-BIO and ICG-001 competed dose-dependently regarding these features. Genome-wide mRNA expression analyses further underscored the dual development of pro- and anti-inflammatory features of LPS-matured moDCs and suggest a role for β-catenin inhibition in production of more potent therapeutic moDCs.

3029 Background: Dendritic cell (DC)-based cryoimmunotherapy (CryoIT) was used to treat metastatic castration-resistant prostate cancer in a Phase I clinical trial. Primary objective was safety of treatment. Secondarily, clinical, radiological and immunological treatment responses were investigated. Methods: In 18 patients cryoablation by a freeze-thaw process under general anesthesia was performed, followed by intratumoral autologous immature DC injection. In the last 9 patients checkpoint inhibition of either CTLA-4 or PD-1 was added. Subjects had minimum 46 weeks follow-up. Adverse events (AEs) and blood analyses were registered at all visits. Disease progression was determined by three imaging modalities according to (i)RECISTv1.1 and progression-free survival (PFS) by Kaplan-Meier method. Circulating tumor cells (CTC/7.5 mL, CellSearch) and ultradeep T-cell receptor (TCR) b-chain sequences (TCRSafe) were enumerated. Patients were separated by CTC into none (n=10), 1-4 (n=4) and ≥ 5 (n=4). Health related quality of life (HRQoL) measured by EORTC-QLQ C30 questionnaire were answered at inclusion, and 10, 22 and 46 weeks post CryoIT. Scores were calculated according to the EORTC manual. Results: Subjects progressing within 22 weeks had higher PSA (p=0.03). AE profile of the total cohort (n=18) was comparable with interim reports (n=13); of 20 possible DC-related AEs one was severe (urinary retention) and 19 mild-to-moderate, and spread independent of treatment regime. Maximum tolerated dose of DC was not reached. By 46 weeks, imaging showed 6 patients partial response or stable disease. Median PFS was 150 days in total cohort. Pretreatment CTC counts ≥5 indicated higher progression rates and recurring CTC. Ultradeep TCR-sequencing showed more prevalent and higher expressed (>5-fold) new TCR clonotypes at 2-6 weeks in men without progression. Participants reported high and stable HRQoL scores throughout the study. However, presence of CTC was associated with worse HRQoL scores at week 10 (p=0.031) and 22 (p=0.005). Conclusions: DC treatment seems safe and well tolerated, also combined with checkpoint inhibitors. Effect is indicated in subjects with moderate pre-treatment PSA levels. Immune responses are suggested by higher number of novel TCR clonotypes in men with non-progressive disease. Clinical trial information: NCT02423928 .

Prostate cancer (PCa) often recurs as incurable castration‐resistant prostate cancer (CRPC) after the failure of androgen deprivation therapy. CRPC development relies on androgen receptor (AR) signaling. The IL6/STAT3 pathway is also a key driver of CRPC. The crosstalk between IL6/STAT3 and the AR pathways provides opportunities to explore next‐generation agents to treat PCa. Through screening of around 600 natural compounds in our newly established prostate tumorigenesis model, potential STAT3 signaling inhibitors were found and additionally examined for effects on AR signaling. The small molecular compound 154 exhibited dual effects on IL6/STAT3 and AR pathways. We show here that compound 154 inhibits AR and STAT3 transcriptional activity, reduces the expression of phosphorylation of STAT3 (Y705) and downregulates the mRNA levels of AR target genes. Compound 154 also inhibits protein expression of AR and AR splice variants (ARv567es and AR‐V7) without altering AR mRNA levels. Compound 154 binds to AR directly, but not to STAT3 and is identified as an antagonist of the AR amino‐terminal domain (NTD) by disrupting protein‐protein interactions between STAT3 and the AR NTD. Moreover, compound 154 does not reduce AR nuclear translocation. Compound 154 possesses the potential to become a leading compound in novel therapies against CRPC.

We have identified nine highly connected and differentially expressed gene subnetworks between aggressive primary tumors and metastatic lesions in endometrial carcinomas. We implemented a novel pipeline combining gene set and network approaches, which here allows integration of protein-protein interactions and gene expression data. The resulting subnetworks are significantly associated with disease progression across tumor stages from complex atypical hyperplasia, primary tumors to metastatic lesions. The nine subnetworks include genes related to metastasizing features such as epithelial-mesenchymal transition (EMT), hypoxia and cell proliferation. TCF4 and TWIST2 were found as central genes in the subnetwork related to EMT. Two of the identified subnetworks display statistically significant association to patient survival, which were further supported by an independent validation in the data from The Cancer Genome Atlas data collection. The first subnetwork contains genes related to cell proliferation and cell cycle, while the second contains genes involved in hypoxia such as HIF1A and EGLN3. Our findings provide a promising context to elucidate the biological mechanisms of metastasis, suggest potential prognostic markers and further identify therapeutic targets. The pipeline R source code is freely available, including permutation tests to assess statistical significance of the identified subnetworks.

// Geir Bredholt 1, * , Monica Mannelqvist 1, * , Ingunn M. Stefansson 1, 2 , Even Birkeland 1 , Trond Hellem Bo 1, 3 , Anne M. Oyan 4, 5 , Jone Trovik 5, 6 , Karl-Henning Kalland 4, 5 , Inge Jonassen 3 , Helga B. Salvesen 5, 6 , Elisabeth Wik 1, 2 , Lars A. Akslen 1, 2 1 Centre for Cancer Biomarkers CCBIO, Department of Clinical Medicine, Section for Pathology, University of Bergen, Bergen, Norway 2 Department of Pathology, Haukeland University Hospital, Bergen, Norway 3 CCBIO, Department of Informatics, University of Bergen, Bergen, Norway 4 Department of Microbiology, Haukeland University Hospital, Bergen, Norway 5 Center for Cancer Biomarkers CCBIO, Department of Clinical Science, University of Bergen, Norway 6 Department of Gynecology and Obstetrics, Haukeland University Hospital, Bergen, Norway * These authors have contributed equally to this work Correspondence to: Lars A. Akslen, e-mail: lars.akslen@uib.no Keywords: necrosis, hypoxia, angiogenesis, inflammation, gene signatures Received: June 11, 2015 Accepted: August 26, 2015 Published: October 14, 2015 ABSTRACT Aims: Tumor necrosis is associated with aggressive features of endometrial cancer and poor prognosis. Here, we investigated gene expression patterns and potential treatment targets related to presence of tumor necrosis in primary endometrial cancer lesions. Methods and Results: By DNA microarray analysis, expression of genes related to tumor necrosis reflected multiple tumor-microenvironment interactions like tissue hypoxia, angiogenesis and inflammation pathways. A tumor necrosis signature of 38 genes and a related patient cluster (Cluster I, 67% of the cases) were associated with features of aggressive tumors such as type II cancers, estrogen receptor negative tumors and vascular invasion. Further, the tumor necrosis signature was increased in tumor cells grown in hypoxic conditions in vitro . Multiple genes with increased expression are known to be activated by HIF1A and NF-kB. Conclusions: Our findings indicate that the presence of tumor necrosis within primary tumors is associated with hypoxia, angiogenesis and inflammation responses. HIF1A, NF-kB and PI3K/mTOR might be potential treatment targets in aggressive endometrial cancers with presence of tumor necrosis.

// Ingunn M. Stefansson 1,2,* , Maria Raeder 1,3,* , Elisabeth Wik 1,2 , Monica Mannelqvist 1,2 , Kanthida Kusonmano 1,3 , Goril Knutsvik 1,2 , Ingfrid Haldorsen 4,5 , Jone Trovik 1,3 , Anne M. Oyan 1,6 , Karl-H. Kalland 1,6 , Anne Cathrine Staff 7 , Helga B. Salvesen 1,3 and Lars A. Akslen 1,2 1 Centre for Cancer Biomarkers CCBIO, Department of Clinical Medicine, Section for Pathology, University of Bergen, Bergen, Norway 2 Department of Pathology, Haukeland University Hospital, Bergen, Norway 3 Department of Obstetrics and Gynaecology, Haukeland University Hospital, Bergen, Norway 4 Department of Radiology, Haukeland University Hospital, Postbox, Bergen, Norway 5 Section for Radiology, University of Bergen, Bergen, Norway 6 Department of Microbiology, Haukeland University Hospital, Bergen, Norway 7 Department of Obstetrics and Gynaecology, Women and Children’s Division, Oslo University Hospital, University of Oslo, Norway * These authors contributed equally to this work Correspondence to: Lars A. Akslen, email: // Keywords : angiogenesis, endometrial cancer, gene expression signature, prognosis Received : January 19, 2015 Accepted : February 17, 2015 Published : March 10, 2015 Abstract Background: Angiogenesis is a hallmark of cancer. The aim of this study was to explore whether microvessel proliferation is associated with gene expression profiles or copy number alterations in endometrial cancer. Methods: A prospective series of endometrial carcinomas was studied for angiogenesis markers, gene expression profiles, and gene copy number data. For validation, an independent series of endometrial carcinomas as well as an external cohort of endometrial cancer patients were examined by gene expression microarrays. Results: Increased microvessel proliferation (MVP) was associated with aggressive tumor features and reduced survival, and a 32-gene expression signature was found to separate tumors with high versus low MVP. An increased 32-gene signature score was confirmed to associate with high-grade tumor features and reduced survival by independent cohorts. Copy number studies revealed that amplification of the 6p21 region was significantly associated with MVP, a high 32-gene score, as well as reduced survival. Conclusion: Increased MVP was significantly associated with aggressive endometrial cancer and reduced survival. Integrated analyses demonstrated significant associations between increased vascular proliferation, amplification of the 6p21 region, VEGF-A mRNA expression, and the 32-gene angiogenesis signature. Our findings indicate amplification of 6p21 as a possible driver of tumor vascular proliferation in endometrial cancer.

Mutations of the phosphoinositide-3-kinase (PI3K) catalytic subunit alpha gene (PIK3CA) are frequent in endometrial cancer. We sequenced exon9 and exon20 of PIK3CA in 280 primary endometrial cancers to assess the relationship with clinicopathologic variables, patient survival and associations with PIK3CA mRNA and phospho-AKT1 by gene expression and protein data, respectively. While PIK3CA mutations generally had no impact on survival, and were not associated with clinicopathological variables, patients with exon9 charge-changing mutations, providing a positive charge at the substituted amino acid residue, were associated with poor survival (p = 0.018). Furthermore, we characterized PIK3CA mutations in the metastatic setting, including 32 patients with matched primary tumors and metastases, and found a high level of concordance (85.7%; 6 out of 7 patients), suggesting limited heterogeneity. PIK3CA mRNA levels were increased in metastases compared to the primary tumors (p = 0.031), independent of PIK3CA mutation status, which rather associated with reduced PIK3CA mRNA expression. PIK3CA mutated tumors expressed higher p-AKT/AKT protein levels, both within primary (p PIK3CA mutations, an aspect that should be considered when designing PIK3 pathway targeting strategies in endometrial cancer.

Background Little is known about the role of glial host cells in brain tumours. However, supporting stromal cells have been shown to foster tumour growth in other cancers. Methods We isolated stromal cells from patient-derived glioblastoma (GBM) xenografts established in GFP-NOD/scid mice. With simultaneous removal of CD11b+ immune and CD31+ endothelial cells by fluorescence activated cell sorting (FACS), we obtained a population of tumour-associated glial cells, TAGs, expressing markers of terminally differentiaed glial cell types or glial progenitors. This cell population was subsequently characterised using gene expression analyses and immunocytochemistry. Furthermore, sphere formation was assessed in vitro and their glioma growth-promoting ability was examined in vivo. Finally, the expression of TAG related markers was validated in human GBMs. Results TAGs were highly enriched for the expression of glial cell proteins including GFAP and myelin basic protein (MBP), and immature markers such as Nestin and O4. A fraction of TAGs displayed sphere formation in stem cell medium. Moreover, TAGs promoted brain tumour growth in vivo when co-implanted with glioma cells, compared to implanting only glioma cells, or glioma cells and unconditioned glial cells from mice without tumours. Genome-wide microarray analysis of TAGs showed an expression profile distinct from glial cells from healthy mice brains. Notably, TAGs upregulated genes associated with immature cell types and self-renewal, including Pou3f2 and Sox2. In addition, TAGs from highly angiogenic tumours showed upregulation of angiogenic factors, including Vegf and Angiopoietin 2. Immunohistochemistry of three GBMs, two patient biopsies and one GBM xenograft, confirmed that the expression of these genes was mainly confined to TAGs in the tumour bed. Furthermore, their expression profiles displayed a significant overlap with gene clusters defining prognostic subclasses of human GBMs. Conclusions Our data demonstrate that glial host cells in brain tumours are functionally distinct from glial cells of healthy mice brains. Furthermore, TAGs display a gene expression profile with enrichment for genes related to stem cells, immature cell types and developmental processes. Future studies are needed to delineate the biological mechanisms regulating the brain tumour-host interplay. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3109-8) contains supplementary material, which is available to authorized users.

Human prostate cancer is initiated in a benign prostate epithelial cell which gains the potential to progress to metastatic disease. The exact cell of origin of prostate cancer has been debated in recent years based upon different models. Primary prostate epithelial cells have restricted life-spans in culture, but can be immortalized. Prostate cancer cell lines have been difficult to establish and new ones are desirable. Attempts to transform benign prostate epithelial cells in vitro have proved difficult without the use of strong carcinogens or oncogenes in processes not likely to mimic closely carcinogenesis in the aging human prostate. Models of epithelial-to-mesenchymal transition (EMT) and cancer stem cells in prostate carcinogenesis have become available, and advances in three-dimensional organoid culture technology represent a breakthrough in prostate cancer research. Organoids may recapitulate multiple features of prostate cancer and have the potential to replace costly and laborious animal experiments. Still, animal models are needed to investigate and validate molecular mechanisms and to develop therapeutic principles in the pipeline between in vitro experiments and clinical applications. Although mice represent the most common experimental animal in prostate cancer research, species like rat, dog, and zebrafish may have advantages depending upon the hypothesis or question. Animal models can generally be categorized into spontaneous or induced development of cancer, immunodeficient animals with xenografts, and genetically engineered animals. In prostate cancer, neuroendocrine differentiation and bone metastases are prevalent in the final stages of cancer progression and animal models that recapitulate these processes are available.

Obesity is linked to increased incidence of endometrioid endometrial cancer (EEC) and complex atypical hyperplasia (CAH). We here explore pattern and sequence of molecular alterations characterizing endometrial carcinogenesis in general and related to body mass index (BMI), to improve diagnostic stratification and treatment strategies. We performed molecular characterization of 729 prospectively collected EEC and CAH. Candidate biomarkers were identified in frozen samples by whole-exome and Sanger sequencing, oligonucleotide gene expression and Reverse Phase Protein Arrays (investigation cohort) and further explored in formalin fixed tissues by immunohistochemistry and Fluorescent in Situ Hybridization (validation cohort). We here demonstrate that PIK3CA mutations, PTEN loss, PI3K and KRAS activation are early events in endometrial carcinogenesis. Molecular changes related to KRAS activation and inflammation are more common in obese CAH patients, suggesting different prevention and systemic treatment strategies in obese and non-obese patients. We also found that oncoprotein Stathmin might improve preoperative diagnostic distinction between premalignant and malignant endometrial lesions.

Background: Recent identification of a specific role of HSF1 in cancer progression has led to new relevance of HSF1 as both a prognostic and a predictive marker. The role of HSF1 in endometrial cancer has so far been unexplored. Methods: A total of 823 lesions from endometrial carcinoma precursors, primary tumours and metastases were prospectively collected and explored for HSF1 protein expression in relation to established markers for aggressive disease and survival. Transcriptional alterations related to HSF1 protein level were investigated by microarray analysis for 224 freshly frozen samples in parallel. Results: High expression of HSF1 protein in endometrial carcinoma is significantly associated with aggressive disease and poor survival (all P-values ⩽0.02), also among ERα-positive patients presumed to have good prognosis. The HSF1-related gene signatures increase during disease progression and were also found to have prognostic value. Gene expression analyses identified HSP90 inhibition as a potential novel therapeutic approach for cases with high protein expression of HSF1. Conclusions: We demonstrate for the first time in endometrial cancer that high expression of HSF1 and measures for transcriptional activation of HSF1 associate with poor outcome and disease progression. The HSP90 inhibitors are suggested as new targeted therapeutics for patients with high HSF1 levels in tumour in particular.

The angiogenic switch, a rate-limiting step in tumor progression, has already occurred by the time most human tumors are detectable. However, despite significant study of the mechanisms controlling this switch, the kinetics and reversibility of the process have not been explored. The stability of the angiogenic phenotype was examined using an established human liposarcoma xenograft model. Nonangiogenic cells inoculated into immunocompromised mice formed microscopic tumors that remained dormant for approximately 125 days (vs. 95%) initiated angiogenic growth with second-order kinetics. These original, clonally derived angiogenic tumor cells were passaged through four in vivo cycles. At each cycle, a new set of single-cell clones was established from the most angiogenic clone and characterized for in vivo for tumorigenic activity. A total of 132 single-cell clones were tested in the second, third, and fourth in vivo passage. Strikingly, at each passage, a portion of the single-cell clones formed microscopic, dormant tumors. Following dormancy, like the original cell line, these revertant tumors spontaneously switched to the angiogenic phenotype. Finally, revertant clones were transcriptionally profiled and their angiogenic output determined. Collectively, these data demonstrate that the angiogenic phenotype in tumors is malleable and can spontaneously revert to the nonangiogenic phenotype in a population of human tumor cells. Implications: Leveraging the rate of reversion to the nonangiogenic phenotype and tumor dormancy may be a novel anticancer strategy. Mol Cancer Res; 12(5); 754–64. ©2014 AACR.

Cancers arise through accumulating genetic and epigenetic alterations, considered relevant for phenotype and approaches to targeting new therapies. We investigated a unique collection of endometrial cancer precursor samples and clinically annotated primary and metastatic lesions for two evolutionary and functionally related transcription factors, CCCTC-binding factor (zinc finger protein) (CTCF) and its paralogue CTCF-like factor, also denoted Brother of the Regulator of Imprinted Sites (CTCFL/BORIS). CTCF, a chromatin modeling- and transcription factor, is normally expressed in a ubiquitous fashion, while CTCFL/BORIS is restricted to the testis. In cancer, CTCF is thought to be a tumor suppressor, while CTCFL/BORIS has been suggested as an oncogene. CTCF mutations were identified in 13 %, with CTCF hotspot frameshift mutations at p.T204, all observed solely in the endometrioid subtype, but with no association with outcome. Interestingly, CTCFL/BORIS was amongst the top ranked genes differentially expressed between endometrioid and non-endometrioid tumors, and increasing mRNA level of CTCFL/BORIS was highly significantly associated with poor survival. As aberrant CTCFL/BORIS expression might relate to loss of methylation, we explored methylation status in clinical samples from complex atypical hyperplasia, through primary tumors to metastatic lesions, demonstrating a pattern of DNA methylation loss during disease development and progression in line with the increase in CTCFL/BORIS mRNA expression observed. Thus, CTCF and CTCFL/BORIS are found to diverge in the different subtypes of endometrial cancer, with CTCFL/BORIS activation through demethylation from precursors to metastatic lesions. We thus propose, CTCFL/BORIS as an Epi-driver gene in endometrial cancer, suggesting a potential for future vaccine development.

Objectives Proliferative capacity of acute myelogenous leukaemia (AML) blasts is important for leukaemogenesis, and we have investigated whether proliferative capacity of primary human AML cells could be used for subclassification of patients. Materials and methods In vitro proliferative capacity of AML cells derived from two independent groups was investigated. Cells were cultured under highly standardized conditions and proliferation assayed by 3H-thymidine incorporation after seven days culture. Patients were subclassified by clustering models, and gene expression profile was examined by microarray analyses. Results Based on proliferative capacity of the AML cells, three different patient clusters were identified: (i) autocrine proliferation that was increased by exogenous cytokines; (ii) detectable proliferation only in presence of exogenous cytokines; and (iii) low or undetectable proliferation even in presence of exogenous cytokines. Patients with highest proliferative capacity cells had no favourable prognostic impact by NPM-1 mutation. Analysis of gene expression profiles showed that the most proliferative cells generally had altered expression of genes involved in regulation of transcription/RNA functions, whereas patients with high proliferative capacity and internal tandem duplications (ITDs) in the FLT3 cytokine receptor gene had altered expression of several molecules involved in cytoplasmic signal transduction. Conclusions In vitro proliferative capacity of primary human AML cells was considerably variable between patients and could be used to identify biologically distinct patient subsets.

MicroRNAs play critical roles in tumorigenesis and metastasis. Here, we report the dual functions of miR-182 and miR-203 in our previously described prostate cell model. MiR-182 and miR-203 were completely repressed during epithelial to mesenchymal transition (EMT) from prostate epithelial EP156T cells to the progeny mesenchymal nontransformed EPT1 cells. Re-expression of miR-182 or miR-203 in EPT1 cells and prostate cancer PC3 cells induced mesenchymal to epithelial transition (MET) features. Simultaneously, miR-182 and miR-203 provided EPT1 cells with the ability to self-sufficiency of growth signals, a well-recognized oncogenic feature. Gene expression profiling showed high overlap of the genes affected by miR-182 and miR-203. SNAI2 was identified as a common target of miR-182 and miR-203. Knock-down of SNAI2 in EPT1 cells phenocopied re-expression of either miR-182 or miR-203 regarding both MET and self-sufficiency of growth signals. Strikingly, considerable overlaps of changed genes were found between the re-expression of miR-182/203 and knock-down of SNAI2. Finally, P-cadherin was identified as a direct target of SNAI2. We conclude that miR-182 and miR-203 induce MET features and growth factor independent growth via repressing SNAI2 in prostate cells. Our findings shed new light on the roles of miR-182/203 in cancer related processes.

Oncogenic mutations of the Wnt (wingless)/β-catenin pathway are frequently observed in major cancer types. Thus far, however, no therapeutic agent targeting Wnt/β-catenin signaling is available for clinical use. Here we demonstrate that axitinib, a clinically approved drug, strikingly blocks Wnt/β-catenin signaling in cancer cells, zebrafish, and Apc(min/+) mice. Notably, axitinib dramatically induces Wnt asymmetry and nonrandom DNA segregation in cancer cells by promoting nuclear β-catenin degradation independent of the GSK3β (glycogen synthase kinase3β)/APC (adenomatous polyposis coli) complex. Using a DARTS (drug affinity-responsive target stability) assay coupled to 2D-DIGE (2D difference in gel electrophoresis) and mass spectrometry, we have identified the E3 ubiquitin ligase SHPRH (SNF2, histone-linker, PHD and RING finger domain-containing helicase) as the direct target of axitinib in blocking Wnt/β-catenin signaling. Treatment with axitinib stabilizes SHPRH and thereby increases the ubiquitination and degradation of β-catenin. Our findings suggest a previously unreported mechanism of nuclear β-catenin regulation and indicate that axitinib, a clinically approved drug, would provide therapeutic benefits for cancer patients with aberrant nuclear β-catenin activation.

Wnt (wingless)/β-catenin signaling is critical for tumor progression and is frequently activated in colorectal cancer as a result of the mutation of adenomatous polyposis coli (APC); however, therapeutic agents targeting this pathway for clinical use are lacking. Here we report that nitazoxanide (NTZ), a clinically approved antiparasitic drug, efficiently inhibits Wnt signaling independent of APC. Using chemoproteomic approaches, we have identified peptidyl arginine deiminase 2 (PAD2) as the functional target of NTZ in Wnt inhibition. By targeting PAD2, NTZ increased the deamination (citrullination) and turnover of β-catenin in colon cancer cells. Replacement of arginine residues disrupted the transcriptional activity, and NTZ induced degradation of β-catenin. In Wnt-activated colon cancer cells, knockout of either PAD2 or β-catenin substantially increased resistance to NTZ treatment. Our data highlight the potential of NTZ as a modulator of β-catenin citrullination for the treatment of cancer patients with Wnt pathway mutations.

Background Expression of the androgen receptor (AR) is associated with androgen-dependent proliferation arrest and terminal differentiation of normal prostate epithelial cells. Additionally, activation of the AR is required for survival of benign luminal epithelial cells and primary cancer cells, thus androgen deprivation therapy (ADT) leads to apoptosis in both benign and cancerous tissue. Escape from ADT is known as castration-resistant prostate cancer (CRPC). In the course of CRPC development the AR typically switches from being a cell-intrinsic inhibitor of normal prostate epithelial cell proliferation to becoming an oncogene that is critical for prostate cancer cell proliferation. A clearer understanding of the context dependent activation of the AR and its target genes is therefore desirable. Methods Immortalized human prostate basal epithelial EP156T cells and progeny cells that underwent epithelial to mesenchymal transition (EMT), primary prostate epithelial cells (PrECs) and prostate cancer cell lines LNCaP, VCaP and 22Rv1 were used to examine context dependent restriction and activation of the AR and classical target genes, such as KLK3. Genome-wide gene expression analyses and single cell protein analyses were applied to study the effect of different contexts. Results A variety of growth conditions were tested and found unable to activate AR expression and transcription of classical androgen-dependent AR target genes, such as KLK3, in prostate epithelial cells with basal cell features or in mesenchymal type prostate cells. The restriction of androgen- and AR-dependent transcription of classical target genes in prostate basal epithelial cells was at the level of AR expression. Exogenous AR expression was sufficient for androgen-dependent transcription of AR target genes in prostate basal epithelial cells, but did not exert a positive feedback on endogenous AR expression. Treatment of basal prostate epithelial cells with inhibitors of epigenetic gene silencing was not efficient in inducing androgen-dependent transcription of AR target genes, suggesting the importance of missing cofactor(s). Conclusions Regulatory mechanisms of AR and androgen-dependent AR target gene transcription are insufficiently understood and may be critical for prostate cancer initiation, progression and escape from standard therapy. The present model is useful for the study of context dependent activation of the AR and its transcriptome. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2453-4) contains supplementary material, which is available to authorized users.

// Ingvild Loberg Tangen 1, 2 , Therese Bredholt Onyango 1, 2 , Reidun Kopperud 1, 2 , Anna Berg 1, 2 , Mari K. Halle 1, 2 , Anne M. Oyan 3, 4 , Henrica M.J. Werner 1, 2 , Jone Trovik 1, 2 , Karl Henning Kalland 3, 4 , Helga B. Salvesen 1, 2, * , Camilla Krakstad 2, 5, * 1 Centre for Cancer Biomarkers, Department of Clinical Science, University of Bergen, Norway 2 Department of Gynecology and Obstetrics, Haukeland University Hospital, Norway 3 Centre for Cancer Biomarkers, Department of Clinical Medicine, University of Bergen, Norway 4 Department of Microbiology, Haukeland University Hospital, Norway 5 Centre for Cancer Biomarkers, Department of Biomedicine, University of Bergen, Norway * These authors contributed equally to this work Correspondence to: Camilla Krakstad, email: camilla.krakstad@uib.no Keywords: androgen receptor, endometrial cancer, biomarker, survival Received: April 06, 2016 Accepted: June 13, 2016 Published: June 30, 2016 ABSTRACT Purpose: The expression and involvement of estrogen (ER) and progesterone receptor (PR) is extensively studied in endometrial cancer. Androgen receptor (AR) is a hormone receptor less studied in female cancers, and we here aim to investigate the expression level of AR in endometrial cancer precursor lesions, primary tumors and metastases, and its potential as therapeutic target. Results: Expression of AR was observed in 93% of hyperplasias, but only in 41% of non-endometrioid tumors. Compared to estrogen and progesterone receptor AR is more commonly expressed in metastatic lesions, and AR status is discordant in primary and metastatic lesions in a large proportion of cases. AR protein level was significantly associated with survival ( P < 0.001), and a calculated AR to ERα ratio identified a subgroup of patients with particular poor outcome. The anti-androgen enzalutamide may have a growth inhibitory effect in endometrial cancer cells based on experiments with primary endometrial tumor cells. Materials and Methods: 718 primary endometrial cancers and 298 metastatic lesions (from 142 patients) were investigated for expression of AR in relation to survival, clinical and histopathological data. Protein levels were investigated by immunohistochemistry and reverse phase protein array; mRNA levels by DNA oligonucleotide microarray. The effect of androgen stimulation and inhibition was tested on primary endometrial tumor cells. Conclusions: A large proportion of metastatic endometrial cancer lesions express AR, which may be a potential target in these patients. Treatment targeting AR may be of particular benefit in patients with high AR levels compared to ERα levels.

Background: Three quarter of endometrial carcinomas are treated at early stage. Still, 15 to 20% of these patients experience recurrence, with little effect from systemic therapies. Homo sapiens v-Ki-ras2 Kirsten rat sarcoma viral oncogenes homologue (KRAS) mutations have been reported to have an important role in tumorigenesis for human cancers, but there is limited knowledge regarding clinical relevance of KRAS status in endometrial carcinomas. Methods: We have performed a comprehensive and integrated characterisation of genome-wide expression related to KRAS mutations and copy-number alterations in primary- and metastatic endometrial carcinoma lesions in relation to clinical and histopathological data. A primary investigation set and clinical validation set was applied, consisting of 414 primary tumours and 61 metastatic lesions totally. Results: Amplification and gain of KRAS present in 3% of the primary lesions and 18% of metastatic lesions correlated significantly with poor outcome, high International Federation of Gynaecology and Obstetrics stage, non-endometrioid subtype, high grade, aneuploidy, receptor loss and high KRAS mRNA levels, also found to be associated with aggressive phenotype. In contrast, KRAS mutations were present in 14.7% of primary lesions with no increase in metastatic lesions, and did not influence outcome, but was significantly associated with endometrioid subtype, low grade and obesity. Conclusion: These results support that KRAS amplification and KRAS mRNA expression, both increasing from primary to metastatic lesions, are relevant for endometrial carcinoma disease progression.

Background: Endometrial cancer incidence is increasing in industrialised countries. High body mass index (BMI, kg m−2) is associated with higher risk for disease. We wanted to investigate if BMI is related to clinico-pathological characteristics, hormone receptor status in primary tumour, and disease outcome in endometrial cancer. Patients and methods: In total, 1129 women primarily treated for endometrial carcinoma at Haukeland University Hospital during 1981–2009 were studied. Body mass index was available for 949 patients and related to comprehensive clinical and histopathological data, hormone receptor status in tumour, treatment, and follow-up. Results: High BMI was significantly associated with low International Federation of Gynaecology and Obstetrics (FIGO) stage, endometrioid histology, low/intermediate grade, and high level of progesterone receptor (PR) mRNA by qPCR (n=150; P=0.02) and protein expression by immunohistochemistry (n=433; P=0.003). In contrast, oestrogen receptor (ERα) status was not associated with BMI. Overweight/obese women had significantly better disease-specific survival (DSS) than normal/underweight women in univariate analysis (P=0.035). In multivariate analysis of DSS adjusting for age, FIGO stage, histological subtype, and grade, BMI showed no independent prognostic impact. Conclusion: High BMI was significantly associated with markers of non-aggressive disease and positive PR status in a large population-based study of endometrial carcinoma. Women with high BMI had significantly better prognosis in univariate analysis of DSS, an effect that disappeared in multivariate analysis adjusting for established prognostic markers. The role of PR in endometrial carcinogenesis needs to be further studied. publishedVersion

The presence of tumor cells entering vascular channels is a prognostic marker for many cancers, including endometrial carcinoma. Vascular invasion is considered to be an early step in the metastatic process and important for the progress of malignant tumors. Here, we investigated the gene expression patterns related to vascular involvement in 57 primary endometrial cancers, using DNA microarray and quantitative PCR techniques. A vascular invasion signature of 18 genes was significantly associated with patient survival and clinicopathological phenotype. Vascular involvement was also related to gene sets for epithelial-mesenchymal transition, wound response, endothelial cells, and vascular endothelial growth factor (VEGF) activity. With immunohistochemical validation, both collagen 8 and matrix metalloproteinase 3 (MMP3) were associated with vascular invasion, whereas ANGPTL4 and IL-8 were associated with patient survival. Our findings indicate that vascular involvement within primary tumors is associated with gene expression profiles related to angiogenesis and epithelial-mesenchymal transition. These data could contribute to an improved understanding of potential targets for metastatic spread and may provide clinically important information for better management of endometrial cancer.

Epithelial to mesenchymal transition (EMT) is pivotal in tumor metastasis. Our previous work reported an EMT model based on primary prostate epithelial cells (EP156T) which gave rise to cells with mesenchymal phenotype (EPT1) without malignant transformation. To promote prostate cell transformation, cells were maintained in saturation density cultures to select for cells overriding quiescence. Foci formed repeatedly following around 8 weeks in confluent EPT1 monolayers. Only later passage EPT1, but not EP156T cells of any passage, could form foci. Cells isolated from the foci were named EPT2 and formed robust colonies in soft agar, a malignant feature present neither in EP156T nor in EPT1 cells. EPT2 cells showed additional malignant traits in vitro, including higher ability to proliferate following confluence, higher resistance to apoptosis and lower dependence on exogenous growth factors than EP156T and EPT1 cells. Microarray profiling identified gene sets, many of which belong to cell junction modules, that changed expression from EP156T to EPT1 cells and continued to change from EPT1 to EPT2 cells. Our findings provide a novel stepwise cell culture model in which EMT emerges independently of transformation and is associated with subsequent accumulation of malignant features in prostate cells. Reprogramming of cell junction modules is involved in both steps.

Interim analysis data are presented for 13 patients with metastatic castration resistant prostate cancer treated with dendritic cell based cryoimmunotherapy (CryoIT) and at least three months follow-up (FU). In CryoIT autologous immature dendritic cells (iDCs) mature in cryoablated tumor tissue and engulf tumor associated antigens and thereafter migrate to lymph nodes to instruct systemic immune attacks on heterogenous cancer cells. Primary endpoint of the trial is to investigate safety of CryoIT while secondary aims include clinical, radiological and immunological responses to treatment. Adverse events (AEs) and blood analyses were registered at all visits and imaging performed before and three months post-treatment. Norwegian regulatory authorities have approved the study. CryoIT of the prostate was performed as a freeze-thaw process under anesthesia and autologous iDCs injected after last thawing cycle. Radiologic responses according to RECISTv1.1 could categorize participants into either Group 1; stable disease (n=5) or Group 2; progressive cancer (n=4). Three patients showed mixed response and one had no follow-up imaging. Hematologic and immunologic values were evaluated according to response group. To identify peripheral blood leukocyte subsets and circulating tumor cells (CTCs), ultradeep T-cell receptor (TCR) β-chain sequences of complementarity-determining region 3, we utilized flow / mass cytometry, Cellsearch System, and TCRSafe Technology, respectively. At inclusion median age was 69 (62-73) years, BMI 25.9 (25-35), ECOG score 0 (0-1) and median PSA 8 (5-11). Group 2 demonstrated higher PSA (p=0.03), median 3.9 (IQR 2.7-6.8) and 74.6 (IQR 47.4-99.0) for Group 1 and Group 2, respectively. In total, 13 possible iDC-related AEs were reported by 10 individuals, whereof one severe and 12 mild-to-moderate, with urinary retention in seven subjects. All but one resolved by use of indwelling urinary catheter for median 8 (1-40) days. Participants with high CTC numbers (>5) before therapy had higher rates of progression, moderate pre-treatment CTC counts (3-5) decreased and no patient acquired CTCs after therapy. Ultradeep TCR-sequencing showed more prevalent and higher expression (>5-fold) of new TCR clonotypes 2-6 weeks after treatment in Group 1. The treatment seems safe and well tolerated. Higher PSA indicates more advanced disease at baseline for Group 2. The data indicate immune responses to treatment with higher increases in TCR clonotypes in radiologic stable disease. Clinical trial ID: NCT02423928. Citation Format: Liv Cecilie V. Thomsen, Alfred Honorè, Bjarte Almås, Lars A. Reisæter, Jannicke Frugård, Einar K. Kristoffersen, Guro Melve, Torjan Haslerud, Jarle Rørvik, Martin Biermann, Svein Inge Helle, Gunnar Kvalheim, Waqas Azeem, Jan Roger Olsen, Ole Johan Halvorsen, Lars Akslen, Duke K. Bahn, Klaus Pantel, Sabine Riethdorf, Haakon Ragde, Bjørn Tore Gjertsen, Anne M. Øyan, Karl-Henning Kalland, Christian Beisland. Dendritic cell based cryoimmunotherapy associates with clinical variables and changes in T-cell receptor expression in a prospective phase I trial of metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT066.

e17014Background: Interim analysis data are presented for 13 pts with mCRPC treated with DC based CryoIT and at least 3 months follow-up (FU). CryoIT exposes tumor antigens in cryoablated tissue to...

Bone marrow angiogenesis is suggested to play a role in the pathogenesis of acute myeloid leukaemia (AML) and endothelial cells may mediate chemosensitivity. This study investigated in vitro endothelial effects of coculture of microvascular endothelial cells (MVEC) with AML cells derived from 33 consecutive AML patients. A proliferation assay showed that (i) AML cells from the majority of patients examined increased endothelial cell proliferation, while cytokine neutralizing experiments had divergent effects on proliferation and (ii) the angiopoietin/Tie2 system was important for growth of AML cells, and angiopoietin-1 induced phosphorylation of signal transducers and activators of transcription (STAT) proteins in AML cells. Finally, gene expression profiling of MVEC cocultured with AML cells was conducted in non-contact cultures. Microarray analysis revealed that the majority of significantly expressed genes could be categorized into gene ontology terms involved in transcription, cellular organization and intracellular signalling. Our study indicates a role for the leukaemic-endothelium crosstalk in leukaemogenesis with enhancement of endothelial cell growth and increased AML cell proliferation possibly mediated by angiopoietin-1 and the STAT signalling pathway.

The balance between proangiogenic Angiopoietin-1 (Ang-1) and the antagonistic Ang-2 is important both for leukemogenesis and chemosensitivity in human acute myelogenous leukemia (AML). We examined the release of Ang-1 and Ang-2 by AML cells cultured alone and in cocultures with stromal cells. Detectable Ang-1 release from AML cells was observed for most patients (62/91), whereas Ang-2 release was detected only for a minority (23/91). Coculture of AML and stromal cells led to increased Ang-1 levels. Furthermore, the role of the angiopoietin system was investigated by characterizing whether the differences in angiopoietin expression in AML patients can be related to nucleophosmin (NPM1) mutations. We compared the gene expression profiles of AML cells derived from 19 patients with FLT3 mutations and normal cytogenetics with and without NPM1 mutations and observed increased expression of Ang-1 in patients with NPM1 mutations. Finally, we found significantly higher Ang-2 levels in serum of AML patients compared with healthy controls. Our results suggest that AML cells are a major source of Ang-1 in leukemic bone marrow, especially in patients with NPM1 mutations, but the local levels are also influenced by stromal cells. Local Ang-2 release from AML cells is less common, but high systemic levels of Ang-2 may affect bone marrow angioregulation.

This study describes the biological effects of hyperoxic treatment on BT4C rat glioma xenografts in vivo with special reference to tumor growth, angiogenesis, apoptosis, general morphology and gene expression parameters. One group of tumor bearing animals was exposed to normobaric hyperoxia (1 bar, pO(2) = 1.0) and another group was exposed to hyperbaric hyperoxia (2 bar, pO(2) = 2.0), whereas animals housed under normal atmosphere (1 bar, pO(2) = 0.2) served as controls. All treatments were performed at day 1, 4 and 7 for 90 min. Treatment effects were determined by assessment of tumor growth, vascular morphology (immunostaining for von Willebrand factor), apoptosis by TUNEL staining and cell proliferation by Ki67 staining. Moreover, gene expression profiles were obtained and verified by real time quantitative PCR. Hyperoxic treatment caused a approximately 60% reduction in tumor growth compared to the control group after 9 days (p < 0.01). Light microscopy showed that the tumors exposed to hyperoxia contained large "empty spaces" within the tumor mass. Moreover, hyperoxia induced a significant increase in the fraction of apoptotic cells ( approximately 21%), with no significant change in cell proliferation. After 2 bar treatment, the mean vascular density was reduced in the central parts of the tumors compared to the control and 1 bar group. The vessel diameters were significantly reduced (11-24%) in both parts of the tumor tissue. Evidence of induced cell death and reduced angiogenesis was reflected by gene expression analyses.Increased pO(2)-levels in experimental gliomas, using normobaric and moderate hyperbaric oxygen therapy, caused a significant reduction in tumor growth. This process is characterized by enhanced cell death, reduced vascular density and changes in gene expression corresponding to these effects.

The use of gene expression arrays in the evaluation and classification of tumors is becoming increasingly important in a number of malignancies. This is a powerful technique able to disclose interpatient variance in gene expression. Such variation in gene expression may be the cause of different disease outcome and may reflect disease phenotypes or chemoresistance. Acute myeloid leukemia is a malignant disease of the bone marrow where overall long-term disease-free survival is less than 50%. The need for better disease classification and evaluation is consequently evident. Gene expression profiling in acute myeloid leukemia has, in recent years, proven able to distinguish acute myeloid leukemia subclasses and predict clinical outcome and is, as such, a promising technique for improved disease evaluation. The early detection of gene expression in response to chemotherapy may be a novel way of monitoring disease management. The immediate gene response may be an indication of whether the drug of choice is efficient in leukemic cell eradication and may early indicate the need for other therapeutic measures. Furthermore, these early alterations in gene expression could facilitate identification of new treatment targets, thereby enabling better patient care and follow-up in the future.