TempEasy 3D Hydrogel Coculture System Provides Mechanistic Insights into Prostate Cancer Bone Metastasis.

Patients diagnosed with advanced prostate cancer (PCa) often experience incurable bone metastases; however, a lack of relevant experimental models has hampered the study of disease mechanisms and the development of therapeutic strategies. In this study, we employed the recently established Temperature-based Easy-separable (TempEasy) 3D cell coculture system to investigate PCa bone metastasis. Through coculturing PCa and bone cells for 7 days, our results showed a reduction in PCa cell proliferation, an increase in neovascularization, and an enhanced metastasis potential when cocultured with bone cells. Additionally, we observed increased cell proliferation, higher stemness, and decreased bone matrix protein expression in bone cells when cocultured with PCa cells. Furthermore, we demonstrated that the stiffness of the extracellular matrix had a negligible impact on molecular responses in both primary (PCa cells) and distant malignant (bone cells) sites. The TempEasy 3D hydrogel coculture system is an easy-to-use and versatile coculture system that provides valuable insights into the mechanisms of cell-cell communication and interaction in cancer metastasis.

TTLL12 has a potential oncogenic activity, suppression of ligation of nitrotyrosine to the C-terminus of detyrosinated α-tubulin, that can be overcome by molecules identified by screening a compound library.

Tubulin tyrosine ligase 12 (TTLL12) is a promising target for therapeutic intervention since it has been implicated in tumour progression, the innate immune response to viral infection, ciliogenesis and abnormal cell division. It is the most mysterious of a fourteen-member TTL/TTLL family, since, although it is the topmost conserved in evolution, it does not have predicted enzymatic activities. TTLL12 seems to act as a pseudo-enzyme that modulates various processes indirectly. Given the need to target its functions, we initially set out to identify a property of TTLL12 that could be used to develop a reliable high-throughput screening assay. We discovered that TTLL12 suppresses the cell toxicity of nitrotyrosine (3-nitrotyrosine) and its ligation to the C-terminus of detyrosinated α-tubulin (abbreviated to ligated-nitrotyrosine). Nitrotyrosine is produced by oxidative stress and is associated with cancer progression. Ligation of nitrotyrosine has been postulated to be a check-point induced by excessive cell stress. We found that the cytotoxicities of nitrotyrosine and tubulin poisons are independent of one another, suggesting that drugs that increase nitrotyrosination could be complementary to current tubulin-directed therapeutics. TTLL12 suppression of nitrotyrosination of α-tubulin was used to develop a robust cell-based ELISA assay that detects increased nitrotyrosination in cells that overexpress TTLL12 We adapted it to a high throughput format and used it to screen a 10,000 molecule World Biological Diversity SETTM collection of low-molecular weight molecules. Two molecules were identified that robustly activate nitrotyrosine ligation at 1 µM concentration. This is the pioneer screen for molecules that modulate nitrotyrosination of α-tubulin. The molecules from the screen will be useful for the study of TTLL12, as well as leads for the development of drugs to treat cancer and other pathologies that involve nitrotyrosination.

A potential osteogenic role for microRNA-181a-5p during palatogenesis.

BACKGROUND: In a previous study, we found that the highly conserved hsa-miR-181a-5p is downregulated in palatal fibroblasts of non-syndromic cleft palate-only infants. OBJECTIVES: To analyze the spatiotemporal expression pattern of mmu-miR-181a-5p during palatogenesis and identify possible mRNA targets and their involved molecular pathways. MATERIAL AND METHODS: The expression of mmu-miR-181a-5p was analyzed in the developing palates of mouse embryos from E11 to E18 using qPCR and ISH. Mouse embryonic palatal mesenchyme cells from E13 were used to analyze mmu-miR-181a-5p expression during osteogenic differentiation. Differential mRNA expression and target identification were analyzed using whole transcriptome RNA sequencing after transfection with a mmu-miR-181a-5p mimic. Differentially expressed genes were linked with underlying pathways using gene set enrichment analysis. RESULTS: The expression of mmm-miR-181a-5p in the palatal shelves increased from E15 and overlapped with palatal osteogenesis. During early osteogenic differentiation, mmu-miR-181a-5p was upregulated. Transient overexpression resulted in 49 upregulated mRNAs and 108 downregulated mRNAs (adjusted P-value < 0.05 and fold change > ± 1.2). Ossification (Stc1, Mmp13) and cell-cycle-related GO terms were significantly enriched for upregulated mRNAs. Analysis of possible mRNA targets indicated significant enrichment of Hippo signaling (Ywhag, Amot, Frmd6 and Serpine1) and GO terms related to cell migration and angiogenesis. LIMITATIONS: Transient overexpression of mmu-miR-181a-5p in mouse embryonic palatal mesenchyme cells limited its analysis to early osteogenesis. CONCLUSION: Mmu-miR-181-5p expression is increased in the developing palatal shelves in areas of bone formation and targets regulators of the Hippo signaling pathway.

Transcriptome Profiling of Circulating Tumor Cells to Predict Clinical Outcomes in Metastatic Castration-Resistant Prostate Cancer.

The clinical utility of circulating tumor cells (CTC) as a non-invasive multipurpose biomarker is broadly recognized. The earliest methods for enriching CTCs from whole blood rely on antibody-based positive selection. The prognostic utility of CTC enumeration using positive selection with the FDA-approved CellSearchTM system has been demonstrated in numerous studies. The capture of cells with specific protein phenotypes does not fully represent cancer heterogeneity and therefore does not realize the prognostic potential of CTC liquid biopsies. To avoid this selection bias, CTC enrichment based on size and deformability may provide better fidelity, i.e., facilitate the characterization of CTCs with any phenotype. In this study, the recently FDA-approved Parsortix® technology was used to enrich CTCs from prostate cancer (PCa) patients for transcriptome analysis using HyCEADTM technology. A tailored PCa gene panel allowed us to stratify metastatic castration-resistant prostate cancer (mCRPC) patients with clinical outcomes. In addition, our findings suggest that targeted CTC transcriptome profiling may be predictive of therapy response.

Precision oncology using organoids of a secretory carcinoma of the salivary gland treated with TRK-inhibitors.

The use of anticancer drugs targeting specific molecular tumor characteristics is rapidly increasing in clinical practice, but selecting patients to benefit from these remains a challenge. It has been suggested that organoid cultures would be ideally suited to test drug responses in vitro. Here we describe and characterize in depth a case of ETV6-NTRK3 gene fusion-positive secretory carcinoma of the salivary glands and corresponding organoid cultures that responded and subsequently acquired resistance to TRK targeting therapy with larotrectinib. This case-culture-characterization illustrates the advances made in precision oncology, but also exposes important caveats in using organoids to predict treatment response.

The Androgen Regulated lncRNA NAALADL2-AS2 Promotes Tumor Cell Survival in Prostate Cancer.

Castration resistance is the leading cause of death in men with prostate cancer. Recent studies indicate long noncoding RNAs (lncRNAs) to be important drivers of therapy resistance. The aim of this study was to identify differentially expressed lncRNAs in castration-resistant prostate cancer (CRPC) and to functionally characterize them in vitro. Tumor-derived RNA-sequencing data were used to quantify and compare the expression of 11,469 lncRNAs in benign, primary prostate cancer, and CRPC samples. CRPC-associated lncRNAs were selected for semi-quantitative PCR validation on 68 surgical tumor specimens. In vitro functional studies were performed by antisense-oligonucleotide-mediated lncRNA knockdown in hormone-sensitive prostate cancer (HSPC) and CRPC cell line models. Subsequently, cell proliferation, apoptosis, cell cycle, transcriptome and pathway analyses were performed using the appropriate assays. Transcriptome analysis of a prostate cancer tumor specimens unveiled NAALADL2-AS2 as a novel CRPC-upregulated lncRNA. The expression of NAALADL2-AS2 was found to be particularly high in HSPC in vitro models and to increase under androgen deprived conditions. NAALADL2-AS2 knockdown decreased cell viability and increased caspase activity and apoptotic cells. Cellular fractionization and RNA fluorescent in situ hybridization identified NAALADL2-AS2 as a nuclear transcript. Transcriptome and pathway analyses revealed that NAALADL2-AS2 modulates the expression of genes involved with cell cycle control and glycogen metabolism. We hypothesize that the nuclear lncRNA, NAALADL2-AS2, functions as a pro-survival signal in prostate cancer cells under pressure of targeted hormone therapy.

Development and characterization of patient-derived salivary gland cancer organoid cultures.

OBJECTIVE: Three-dimensional organoid cell cultures have been established for a variety of human cancers. For most rare cancers, including salivary gland cancer (SGC), these models are lacking, despite the great unmet need to study cancer biology in these diseases. Therefore, we aimed to develop patient-derived organoid (PDO) models for different subtypes of SGC. METHODS: Tumor samples of SGC patients were processed and embedded in Matrigel. Successful PDOs (expandable > 1*106 cells) were phenotypically characterized using immunohistochemistry (IHC) and genotypically by gene fusion analysis and by targeted and whole-exome sequencing. Successfully established PDOs were subjected to small-scale drug screening. RESULTS: Out of 37 attempts, 7 viable short-term PDOs were established (19 % success rate; 3 salivary duct carcinoma, 3 adenoid cystic carcinoma and 1 mucoepidermoid carcinoma). Each PDO showed close phenotypical mimicry to parental tissue. Genotypic characterization revealed that in each PDO > 97.6 % of all COSMIC annotated variants and all MYB, MYBL1 and NFIB gene rearrangements were retained. Drug screening was proven feasible in all PDOs. CONCLUSION: We present the first comprehensively characterized short-term SGC PDO models for three subtypes of SGC with close phenotypic and genotypic resemblance to parental tissue, which can be used for drug screening applications.

Identification of Fusion Genes and Targets for Genetically Matched Therapies in a Large Cohort of Salivary Gland Cancer Patients.

Introduction: Salivary gland cancer (SGC) is a rare cancer for which systemic treatment options are limited. Therefore, it is important to characterize its genetic landscape in search for actionable aberrations, such as NTRK gene fusions. This research aimed to identify these actionable aberrations by combining NGS-based analysis of RNA (gene fusions) and DNA (single and multiple nucleotide variants, copy number variants, microsatellite instability and tumor mutational burden) in a large cohort of SGC patients. Methods: RNA and DNA were extracted from archival tissue of 121 patients with various SGC subtypes. Gene fusion analysis was performed using a customized RNA-based targeted NGS panel. DNA was sequenced using a targeted NGS panel encompassing 523 cancer-related genes. Cross-validation of NGS-based NTRK fusion detection and pan-TRK immunohistochemistry (IHC) was performed. Results: Fusion transcripts were detected in 50% of the cases and included both known (MYB-NFIB, MYBL1-NFIB, CRTC1-MAML2) and previously unknown fusions (including transcripts involving RET, BRAF or RAD51B). Only one NTRK fusion transcript was detected, in a secretory carcinoma case. Pan-TRK IHC (clone EPR17341) was false positive in 74% of cases. The proportion of patients with targets for genetically matched therapies differed among subtypes (salivary duct carcinoma: 82%, adenoid cystic carcinoma 28%, mucoepidermoid carcinoma 50%, acinic cell carcinoma 33%). Actionable aberrations were most often located in PIK3CA (n = 18, 15%), ERBB2 (n = 15, 12%), HRAS and NOTCH1 (both n = 9, 7%). Conclusions: Actionable genetic aberrations were seen in 53.7% of all SGC cases on the RNA and DNA level, with varying percentages between subtypes.

Delivery of antisense oligonucleotides for splice-correction of androgen receptor pre-mRNA in castration-resistant prostate cancer models using cell-penetrating peptides.

BACKGROUND: Cell-penetrating peptides (CPPs) are a promising approach for delivering antisense oligonucleotides (AONs) as they form nanosized complexes through noncovalent interactions that show efficient cellular uptake. Previously, we have designed an AON system to correct splicing of the androgen receptor (AR) pre-mRNA, thereby preventing the generation of the splice variant AR-V7 mRNA. AON-mediated knockdown of AR-V7 resulted in inhibition of androgen-independent cell proliferation. In this study, we evaluated the CPP-mediated delivery of this AON into castration-resistant prostate cancer cell line models 22Rv1, DuCaP (dura mater cancer of the prostate), and VCaP (vertebral cancer of the prostate). METHODS: Nanoparticles (polyplexes) of AONs and CPPs were formed through rapid mixing. The impact of the peptide carrier, the formulation parameters, and cell incubation conditions on cellular uptake of fluorescently labeled AONs were assessed through flow cytometry. The cytotoxic activity of these formulations was measured using the CellTiter-Glo cell viability assay. The effectivity of CPP-mediated delivery of the splice-correcting AON-intronic splicing enhancer (ISE) targeting the ISE in the castration-resistant prostate cancer (CRPC)-derived 22Rv1, DuCaP, and VCaP cells was determined by measuring levels of AR-V7 mRNA normalized to those of the human heterochromatin protein 1 binding protein 3 (HP1BP3). Western blot analysis was used to confirm AR-V7 downregulation at a protein level. The cellular distribution of fluorescently labeled AON delivered by a CPP or a transfection reagent was determined through confocal laser scanning microscopy. RESULTS: The amphipathic and stearylated CPP PepFect 14 (PF14) showed higher uptake efficiency than arginine-rich CPPs. Through adjustment of formulation parameters, concentration and incubation time, an optimal balance between carrier-associated toxicity and delivery efficiency was found with a formulation consisting of an amino/phosphate ratio of 3, 0.35 µM AON concentration and 30 min incubation time of the cells with polyplexes. Cellular delivery of AON-ISE directed against AR pre-mRNA achieved significant downregulation of AR-V7 by 50%, 37%, and 59% for 22Rv1, DuCaP, and VCaP cells, respectively, and reduced androgen-independent cell proliferation of DuCaP and VCaP cells. CONCLUSIONS: This proof-of-principle study constitutes the basis for further development of CPP-mediated delivery of AONs for targeted therapy in prostate cancer.

RNA Biomarkers as a Response Measure for Survival in Patients with Metastatic Castration-Resistant Prostate Cancer.

Treatment evaluation in metastatic castration-resistant prostate cancer is challenging. There is an urgent need for biomarkers to discriminate short-term survivors from long-term survivors, shortly after treatment initiation. Thereto, the added value of early RNA biomarkers on predicting progression-free survival (PFS) and overall survival (OS) were explored. The RNA biomarkers: KLK3 mRNA, miR-375, miR-3687, and NAALADL2-AS2 were measured in 93 patients with mCRPC, before and 1 month after start of first-line abiraterone acetate or enzalutamide treatment, in two prospective clinical trials. The added value of the biomarkers to standard clinical parameters in predicting PFS and OS was tested by Harell's C-index. To test whether the biomarkers were independent markers of PFS and OS, multivariate Cox regression was used. The best prediction model for PFS and OS was formed by adding miR-375 and KLK3 (at baseline and 1 month) to standard clinical parameters. Baseline miR-375 and detectable KLK3 after 1 month of therapy were independently related to shorter PFS, which was not observed for OS. In conclusion, the addition of KLK3 and miR-375 (at baseline and 1 month) to standard clinical parameters resulted in the best prediction model for survival assessment.

Molecular Phenotyping of AR Signaling for Predicting Targeted Therapy in Castration Resistant Prostate Cancer.

Castration-resistant prostate cancer (CRPC) is defined by resistance of the tumor to androgen deprivation therapy (ADT). Several molecular changes, particularly in the AR signaling cascade, have been described that may explain ADT resistance. The variety of changes may also explain why the response to novel therapies varies between patients. Testing the specific molecular changes may be a major step towards personalized treatment of CRPC patients. The aim of our study was to evaluate the molecular changes in the AR signaling cascade in CRPC patients. We have developed and validated several methods which are easy to use, and require little tissue material, for exploring AR signaling pathway changes simultaneously. We found that the AR signaling pathway is still active in the majority of our CRPC patients, due to molecular changes in AR signaling components. There was heterogeneity in the molecular changes observed, but we could classify the patients into 4 major subgroups which are: AR mutation, AR amplification, active intratumoral steroidogenesis, and combination of AR amplification and active intratumoral steroidogenesis. We suggest characterizing the AR signaling pathway in CRPC patients before beginning any new treatment, and a recent fresh tissue sample from the prostate or a metastatic site should be obtained for the purpose of this characterization.

Predictive and Prognostic Biomarker Identification in a Large Cohort of Androgen Receptor-Positive Salivary Duct Carcinoma Patients Scheduled for Combined Androgen Blockade.

Patients suffering from recurrent or metastatic (R/M) salivary duct carcinoma (SDC) are often treated with combined androgen blockade (CAB). However, CAB frequently fails, resulting in a worse prognosis. Therefore, biomarkers that can predict treatment failure are urgently needed. mRNA from 76 R/M androgen receptor (AR)-positive SDC patients treated with leuprorelin acetate combined with bicalutamide was extracted from pre-treatment tumor specimens. AR, Notch, MAPK, TGFß, estrogen receptor (ER), Hedgehog (HH), and PI3K signaling pathway activity scores (PAS) were determined based on the expression levels of target genes. Additionally, 5-alpha reductase type 1 (SRD5A1) expression was determined. These markers were related to clinical benefit (complete/partial response or stable disease ≥6 months) and progression-free and overall survival (PFS/OS). SRD5A1 expression had the highest general predictive value for clinical benefit and positive predictive value (PPV: 85.7%). AR PAS had the highest negative predictive value (NPV: 93.3%). The fitting of a multivariable model led to the identification of SRD5A1, TGFß, and Notch PAS as the most predictive combination. High AR, high Notch, high ER, low HH PAS, and high SRD5A1 expression were also of prognostic importance regarding PFS and SRD5A1 expression levels for OS. AR, Notch PAS, and SRD5A1 expression have the potential to predict the clinical benefit of CAB treatment in SDC patients. SRD5A1 expression can identify patients that will and AR PAS patients that will not experience clinical benefit (85.7% and 93.3% for PPV and NPV, respectively). The predictive potential of SRD5A1 expression forms a rational basis for including SRD5A1-inhibitors in SDC patients' treatment.

Androgen receptor signalling confers clonogenic and migratory advantages in urothelial cell carcinoma of the bladder.

Bladder urothelial cell carcinoma (UCC) incidence is about three times higher in men compared with women. There are several indications for the involvement of hormonal factors in the aetiology of UCC. Here, we provide evidence of androgen signalling in UCC progression. Microarray and qPCR analysis revealed that the androgen receptor (AR) mRNA level is upregulated in a subset of UCC cases. In an AR-positive UCC-derived cell line model, UM-UC-3-AR, androgen treatment increased clonogenic capacity inducing the formation of big stem cell-like holoclones, while AR knockdown or treatment with the AR antagonist enzalutamide abrogated this clonogenic advantage. Additionally, blockage of AR signalling reduced the cell migration potential of androgen-stimulated UM-UC-3-AR cells. These phenotypic changes were accompanied by a rewiring of the transcriptome with almost 300 genes being differentially regulated by androgens, some of which correlated with AR expression in UCC patients in two independent data sets. Our results demonstrate that AR signals in UCC favouring the development of an aggressive phenotype and highlights its potential as a therapeutic target for bladder cancer.

Partial Adrenalectomy Carries a Considerable Risk of Incomplete Cure in Primary Aldosteronism.

PURPOSE: Laparoscopic adrenalectomy is standard treatment for patients with unilateral aldosterone-producing adenomas, but surgeons are increasingly tempted to perform partial adrenalectomy, disregarding potential multinodularity of the adrenal. We assess the diagnostic value of endoscopic ultrasound for differentiating solitary adenomas from multinodularity by examining in-depth adrenal pathology with ex vivo 11.7 T magnetic resonance imaging and immunohistochemistry. MATERIALS AND METHODS: In 15 primary aldosteronism patients, we performed intraoperative endoscopic ultrasound, ex vivo magnetic resonance imaging and histopathological examination. Every adrenal was intraoperatively and postoperatively assessed for solitary adenomas or multinodular hyperplasia. After unblinding for ex vivo magnetic resonance imaging results a second detailed histopathological examination, including immunohistochemistry analysis with CYP11B2 (aldosterone synthase) and chemokine receptor 4 (CXCR4), a new marker for aldosterone-producing adenomas, was performed. Finally, presence of somatic mutations linked to aldosterone-producing adenomas was assessed. RESULTS: The sensitivity and specificity of endoscopic ultrasound to identify multinodularity were 46% and 50%, respectively. We found multinodular hyperplasia in 87% of adrenals with ex vivo magnetic resonance imaging combined with detailed histopathology, and 6 adrenals contained multiple CYP11B2-producing nodules. Every CYP11B2 positive nodule and 61% of CYP11B2 negative nodules showed CXCR4 staining. Finally, in 4 adrenals (27%) we found somatic mutations. In multinodular glands, only 1 nodule harbored this mutation. CONCLUSIONS: Intraoperative endoscopic ultrasound in primary aldosteronism patients has low accuracy to identify multinodularity. Ex vivo magnetic resonance imaging can serve as a tool to direct detailed histopathological examination, which frequently shows CYP11B2 production in multiple nodules. Therefore, partial adrenalectomy is inappropriate in primary aldosteronism as multiple aldosterone-producing nodules easily stay behind.

Liquid biopsy reveals KLK3 mRNA as a prognostic marker for progression free survival in patients with metastatic castration-resistant prostate cancer undergoing first-line abiraterone acetate and prednisone treatment.

Circulating RNAs extracted from liquid biopsies represent a promising source of cancer- and therapy-related biomarkers. We screened whole blood from patients with metastatic castration-resistant prostate cancer (mCRPC) following their first-line treatment with abiraterone acetate and prednisone (AA-P) to identify circulating RNAs that may correlate with progression-free survival (PFS). In a prospective multicenter observational study, 53 patients with mCRPC were included after they started first-line AA-P treatment. Blood was drawn at baseline, 1, 3, and 6 months after treatment initiation. The levels of predefined circulating RNAs earlier identified as being upregulated in patients with mCRPC (e.g., microRNAs, long noncoding RNAs, and mRNAs), were analyzed. Uni- and multivariable Cox regression and Kaplan-Meier analyses were used to analyze the prognostic value of the various circulating RNAs for PFS along treatment. Detectable levels of kallikrein-related peptidase 3 (KLK3) mRNA at baseline were demonstrated to be an independent prognostic marker for PFS (201 vs 501 days, P = 0.00054). Three months after AA-P treatment initiation, KLK3 could not be detected in the blood of responding patients, but was still detectable in 56% of the patients with early progression. Our study confirmed that KLK3 mRNA detection in whole blood is an independent prognostic marker in mCRPC patients receiving AA-P treatment. Furthermore, the levels of circulating KLK3 mRNA in patients receiving AA-P treatment might reflect treatment response or early signs of progression.

The Identification of Small Molecule Inhibitors That Reduce Invasion and Metastasis of Aggressive Cancers.

Transformed epithelial cells can activate programs of epithelial plasticity and switch from a sessile, epithelial phenotype to a motile, mesenchymal phenotype. This process is linked to the acquisition of an invasive phenotype and the formation of distant metastases. The development of compounds that block the acquisition of an invasive phenotype or revert the invasive mesenchymal phenotype into a more differentiated epithelial phenotype represent a promising anticancer strategy. In a high-throughput assay based on E-cadherin (re)induction and the inhibition of tumor cell invasion, 44,475 low molecular weight (LMW) compounds were screened. The screening resulted in the identification of candidate compounds from the PROAM02 class. Selected LMW compounds activated E-cadherin promoter activity and inhibited cancer cell invasion in multiple metastatic human cancer cell lines. The intraperitoneal administration of selected LMW compounds reduced the tumor burden in human prostate and breast cancer in vivo mouse models. Moreover, selected LMW compounds decreased the intra-bone growth of xenografted human prostate cancer cells. This study describes the identification of the PROAM02 class of small molecules that can be exploited to reduce cancer cell invasion and metastases. Further clinical evaluation of selected candidate inhibitors is warranted to address their safety, bioavailability and antitumor efficacy in the management of patients with aggressive cancers.

Prognostic value of PSMA, c-MET and E-cadherin in salivary duct carcinoma.

OBJECTIVES: Salivary duct carcinoma (SDC) is a rare and aggressive subtype of salivary gland cancer. Androgen receptor (AR) (96%) and HER2 (29-46%) expression, and a high propensity for regional lymph node metastases are hallmarks of the disease. We hypothesized that c-MET, E-cadherin, PSMA tumor and PSMA neovascular expression may be prognostic factors in SDC. MATERIALS AND METHODS: Expression levels of these proteins were established on tissue microarrays containing 165 primary SDC tumor specimens. Association with survival was studied with Kaplan-Meier curves, and univariable and multivariable Cox regression models. Furthermore, association with lymph node status, AR and HER2 expression, and gender was studied. RESULTS: We found that patients with high PSMA tumor expression showed a significantly longer overall survival (OS) (median 83 vs. 43 months, P = 0.022), a trend towards a longer DFS (median 51 vs. 22 months, P = 0.094), and significantly reduced hazard ratio for death in the univariable Cox regression model (HR 0.46, P = 0.034). In the multivariable model only a high number of tumor-positive lymph nodes and high age (>80) at diagnosis were prognostic for poor OS. High PSMA tumor expression was also significantly associated with low N-stage (P = 0.001) and expression was higher in women versus men (P = 0.029). High PSMA tumor expression and E-cadherin loss were significantly associated with strong and weak AR-expression, respectively (P = 0.033 and P = 0.007). None of the factors were significantly associated with HER2 expression. CONCLUSION: c-MET, E-cadherin, and tumor and neovascular PSMA expression are no independent prognostic factors in SDC.

Prognostic Value of Novel Liquid Biomarkers in Patients with Metastatic Castration-Resistant Prostate Cancer Treated with Enzalutamide: A Prospective Observational Study.

BACKGROUND: Several treatment options were recently added for metastatic castration-resistant prostate cancer (mCRPC). However, response to therapy is variable, and biomarkers that can guide treatment selection and response evaluation are lacking. Circulating RNAs are a promising source of biomarkers. We explored messenger RNAs (mRNAs), microRNAs (miRNAs), and long noncoding RNAs (lncRNAs) as potential biomarkers in liquid biopsies of patients with mCRPC treated with enzalutamide. METHODS: Forty patients were included in this prospective multicenter observational study. Whole blood was drawn at baseline and 1, 3, and 6 months after start of therapy. Four mRNAs, 6 miRNAs, and 5 lncRNAs were analyzed by quantitative PCR. RNA levels in 30 healthy individuals were used as controls. RNA expression data were analyzed by Kaplan-Meier and Cox regression analyses, and the primary end point was progression-free survival. Clinical factors were included in the multivariable Cox regression analysis. RESULTS: Levels of 2 miRNAs, miR-375 and miR-3687, and 1 lncRNA, N-acetylated alpha-linked acidic dipeptidase like 2 antisense RNA 2 (NAALADL2-AS2), were more than 2-fold higher in patients with mCRPC compared with healthy volunteers. Patients with higher levels of miR-375 or miR-3687 showed a shorter time to progression. Patients with higher levels of NAALADL2-AS2 showed a longer time to progression. In the multivariable Cox regression analysis, higher miR-375, miR-3687 and serum prostate-specific antigen concentrations were shown to be independent predictors for shorter time to progression. CONCLUSIONS: We identified miR-3687 as a novel prognostic marker for response in patients with CRPC treated with enzalutamide, and we confirmed the prognostic value of miR-375.

68Ga-PSMA-HBED-CC PET/CT imaging for adenoid cystic carcinoma and salivary duct carcinoma: a phase 2 imaging study.

Rationale: Treatment options for recurrent and/or metastatic (R/M) adenoid cystic carcinoma (ACC) and salivary duct carcinoma (SDC), major subtypes of salivary gland cancer, are limited. Both tumors often show overexpression of prostate-specific membrane antigen (PSMA). In prostate cancer, PSMA-ligands labeled with 68Ga or 177Lu are used for imaging and therapy, respectively. Primary aim of this study in R/M ACC and SDC patients was to systematically investigate 68Ga-PSMA-uptake by PET/CT imaging to determine if PSMA radionuclide therapy could be a treatment option. Methods: In a prospective phase II study, PET/CT imaging was performed 1 h post injection of 68Ga-PSMA-HBED-CC in 15 ACC patients and 10 SDC patients. Maximum standardized uptake values (SUV) were determined in tumor lesions. Immunohistochemical PSMA expression was scored in primary tumors and metastatic tissue. Standard imaging (MRI or CT) was performed for comparison. Results: In ACC patients, SUVmax ranged from 1.1 to 30.2 with a tumor/liver-ratio >1 in 13 out of 14 evaluable patients (93%). In SDC patients, SUVmax ranged from 0.3 to 25.9 with a tumor/liver-ratio >1 in 4 out of 10 patients (40%). We found a large intra-patient inter-metastatic variation in uptake of 68Ga-PSMA, and immunohistochemistry did not predict ligand uptake in ACC and SDC. Finally, PSMA-PET detected additional bone metastases compared to CT in 2 ACC patients with unexplained pain. Conclusion: In 93% of ACC patients and 40% of SDC patients we detected relevant PSMA-ligand uptake, which warrants to study PSMA radionuclide therapy in these patients. Additionally, our data provide arguments for patient selection and treatment timing. Finally, PSMA-PET imaging has added diagnostic value compared to CT in selected patients.