A novel methylated cell-free DNA marker panel to monitor treatment response in metastatic prostate cancer.

Aim: This study examined circulating cell-free DNA (cfDNA) biomarkers associated with androgen treatment resistance in metastatic castration resistance prostate cancer (mCRPC). Materials & methods: We designed a panel of nine candidate cfDNA methylation markers using droplet digital PCR (Methyl-ddPCR) and assessed methylation levels in sequentially collected cfDNA samples from patients with mCRPC. Results: Increased cfDNA methylation in eight out of nine markers during androgen-targeted treatment correlated with a faster time to clinical progression. Cox proportional hazards modeling and logistic regression analysis further confirmed that higher cfDNA methylation during treatment was significantly associated with clinical progression. Conclusion: Overall, our findings have revealed a novel methylated cfDNA marker panel that could aid in the clinical management of metastatic prostate cancer.

Distinct DNA methylation patterns associated with treatment resistance in metastatic castration resistant prostate cancer.

Androgens are a major driver of prostate cancer (PCa) and continue to be a critical treatment target for advanced disease, which includes castration therapy and antiandrogens. However, resistance to these therapies leading to metastatic castration-resistant prostate cancer (mCRPC), and the emergence of treatment-induced neuroendocrine disease (tNEPC) remains an ongoing challenge. Instability of the DNA methylome is well established as a major hallmark of PCa development and progression. Therefore, investigating the dynamics of the methylation changes going from the castration sensitive to the tNEPC state would provide insights into novel mechanisms of resistance. Using an established xenograft model of CRPC, genome-wide methylation analysis was performed on cell lines representing various stages of PCa progression. We confirmed extensive methylation changes with the development of CRPC and tNEPC using this model. This included key genes and pathways associated with cellular differentiation and neurodevelopment. Combined analysis of methylation and gene expression changes further highlighted genes that could potentially serve as therapeutic targets. Furthermore, tNEPC-related methylation signals from this model were detectable in circulating cell free DNA (cfDNA) from mCRPC patients undergoing androgen-targeting therapies and were associated with a faster time to clinical progression. These potential biomarkers could help with identifying patients with aggressive disease.

Investigating Urinary Circular RNA Biomarkers for Improved Detection of Renal Cell Carcinoma.

Renal cell carcinomas (RCC) are usually asymptomatic until late stages, posing several challenges for early detection of malignant disease. Non-invasive liquid biopsy biomarkers are emerging as an important diagnostic tool which could aid with routine screening of RCCs. Circular RNAs (circRNAs) are novel non-coding RNAs that play diverse roles in carcinogenesis. They are promising biomarkers due to their stability and ease of detection in small quantities from non-invasive sources such as urine. In this study, we analyzed the expression of various circRNAs that were previously identified in RCC tumors (circEGLN3, circABCB10, circSOD2 and circACAD11) in urinary sediment samples from non-neoplastic controls, patients with benign renal tumors, and clear cell RCC (ccRCC) patients. We observed significantly reduced levels of circEGLN3 and circSOD2 in urine from ccRCC patients compared to healthy controls. We also assessed the linear variant of EGLN3 and found differential expression between patients with benign tumors compared to ccRCC patients. These findings highlight the potential of circRNA markers as non-invasive diagnostic tools to detect malignant RCC.

Dynamics of the cell-free DNA methylome of metastatic prostate cancer during androgen-targeting treatment.

Aim: We examined methylation changes in cell-free DNA (cfDNA) in metastatic castration-resistant prostate cancer (mCRPC) during treatment. Patients & methods: Genome-wide methylation analysis of sequentially collected cfDNA samples derived from mCRPC patients undergoing androgen-targeting therapy was performed. Results: Alterations in methylation states of genes previously implicated in prostate cancer progression were observed and patients that maintained methylation changes throughout therapy tended to have a longer time to clinical progression. Importantly, we also report that markers associated with a highly aggressive form of the disease, neuroendocrine-CRPC, were associated with a faster time to clinical progression. Conclusion: Our findings highlight the potential of monitoring the cfDNA methylome during therapy in mCRPC, which may serve as predictive markers of response to androgen-targeting agents.

Impaired resolution of inflammation in the Endoglin heterozygous mouse model of chronic colitis.

Endoglin is a coreceptor of the TGF-ß superfamily predominantly expressed on the vascular endothelium and selective subsets of immune cells. We previously demonstrated that Endoglin heterozygous (Eng (+/-)) mice subjected to dextran sulfate sodium (DSS) developed persistent gut inflammation and pathological angiogenesis. We now report that colitic Eng (+/-) mice have low colonic levels of active TGF-ß1, which was associated with reduced expression of thrombospondin-1, an angiostatic factor known to activate TGF-ß1. We also demonstrate dysregulated expression of BMPER and follistatin, which are extracellular regulators of the TGF-ß superfamily that modulate angiogenesis and inflammation. Heightened colonic levels of the neutrophil chemoattractant and proangiogenic factor, CXCL1, were also observed in DSS-treated Eng (+/-) mice. Interestingly, despite increased macrophage and neutrophil infiltration, a gut-specific reduction in expression of the key phagocytic respiratory burst enzymes, NADPH oxidase 2 (Nox-2) and myeloperoxidase, was seen in Eng (+/-) mice undergoing persistent inflammation. Taken together, these findings suggest that endoglin is required for TGF-ß superfamily mediated resolution of inflammation and fully functional myeloid cells.

Anti-VEGF therapy reduces intestinal inflammation in Endoglin heterozygous mice subjected to experimental colitis.

Chronic intestinal inflammation is associated with pathological angiogenesis that further amplifies the inflammatory response. Vascular endothelial growth factor (VEGF), is a major angiogenic cytokine that has been implicated in chronic colitis and inflammatory bowel diseases. Endoglin (CD105), a transforming growth factor-ß superfamily co-receptor expressed on endothelial and some myeloid cells, is a modulator of angiogenesis involved in wound healing and potentially in resolution of inflammation. We showed previously that Endoglin heterozygous (Eng (+/-)) mice subjected to dextran sodium sulfate developed severe colitis, abnormal colonic vessels and high tissue VEGF. We therefore tested in the current study if treatment with a monoclonal antibody to VEGF could ameliorate chronic colitis in Eng (+/-) mice. Tissue inflammation and microvessel density (MVD) were quantified on histological slides. Colonic wall thickness, microvascular hemodynamics and targeted MAdCAM-1(+) inflamed vessels were assessed in vivo by ultrasound. Mediators of angiogenesis and inflammation were measured by Milliplex and ELISA assays. Colitic Eng (+/-) mice showed an increase in intestinal inflammation, MVD, colonic wall thickness, microvascular hemodynamics and the number of MAdCAM-1(+) microvessels relative to colitic Eng (+/+) mice; these parameters were all attenuated by anti-VEGF treatment. Of all factors up-regulated in the inflamed gut, granulocyte colony-stimulating factor (G-CSF) and amphiregulin were further increased in colitic Eng (+/-) versus Eng (+/+) mice. Anti-VEGF therapy decreased tissue VEGF and inflammation-induced endoglin, IL-1ß and G-CSF in colitic Eng (+/-) mice. Our results suggest that endoglin modulates intestinal angiogenic and inflammatory responses in colitis. Furthermore, contrast-enhanced ultrasound provides an excellent non-invasive imaging modality to monitor gut angiogenesis, inflammation and responses to anti-angiogenic treatment.

Endoglin and activin receptor-like kinase 1 heterozygous mice have a distinct pulmonary and hepatic angiogenic profile and response to anti-VEGF treatment.

Hereditary hemorrhagic telangiectasia (HHT) is a vascular dysplasia associated with dysregulated angiogenesis and arteriovascular malformations. The disease is caused by mutations in endoglin (ENG; HHT1) or activin receptor-like kinase 1 (ALK1; HHT2) genes, coding for transforming growth factor ß (TGF-ß) superfamily receptors. Vascular endothelial growth factor (VEGF) has been implicated in HHT and beneficial effects of anti-VEGF treatment were recently reported in HHT patients. To investigate the systemic angiogenic phenotype of Endoglin and Alk1 mutant mice and their response to anti-VEGF therapy, we assessed microvessel density (MVD) in multiple organs after treatment with an antibody to mouse VEGF or vehicle. Lungs were the only organ showing an angiogenic defect, with reduced peripheral MVD and secondary right ventricular hypertrophy (RVH), yet distinctly associated with a fourfold increase in thrombospondin-1 (TSP-1) in Eng (+/-) versus a rise in angiopoietin-2 (Ang-2) in Alk1 (+/-) mice. Anti-VEGF treatment did reduce lung VEGF levels but interestingly, led to an increase in peripheral pulmonary MVD and attenuation of RVH; it also normalized TSP-1 and Ang-2 expression. Hepatic MVD, unaffected in mutant mice, was reduced by anti-VEGF therapy in heterozygous and wild type mice, indicating a liver-specific effect of treatment. Contrast-enhanced micro-ultrasound demonstrated a reduction in hepatic microvascular perfusion after anti-VEGF treatment only in Eng (+/-) mice. Our findings indicate that the mechanisms responsible for the angiogenic imbalance and the response to anti-VEGF therapy differ between Eng and Alk1 heterozygous mice and raise the need for systemic monitoring of anti-angiogenic therapy effects in HHT patients.

Dextran sulfate sodium leads to chronic colitis and pathological angiogenesis in Endoglin heterozygous mice.

BACKGROUND: Pathological angiogenesis is an intrinsic component of chronic intestinal inflammation, which results in remodeling and expansion of the gut microvascular bed. Endoglin is essential for endothelial cell function and physiological angiogenesis. In this study we investigated its potential role in the regulation of inflammation by testing the response of Endoglin heterozygous (Eng(+/-)) mice to experimental colitis. METHODS: C57BL/6 Eng(+/-) and littermate control mice drank water supplemented with 3% dextran sulfate sodium (DSS) for 5 days and were monitored for up to 26 days for clinical signs of colitis. Inflammation, crypt damage, and angiogenic index were scored on histological sections of distal colon. Levels of the vascular endothelial growth factor (VEGF) and angiopoietins were measured by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and/or Western blots. Vascular permeability was assessed using Evans Blue. RESULTS: Eng(+/-) and control mice developed acute colitis, which peaked at day 9. While control mice recovered by days 19-26, Eng(+/-) mice progressed to chronic colitis and showed numerous vascular protrusions penetrating into the serosa of the inflamed distal colon. Prior to DSS induction, VEGF levels and vascular permeability were higher in the distal colon of Eng(+/-) mice, while angiopoietin 1 and 2 levels were unchanged. In the chronic phase of colitis, VEGF levels were increased in both groups of mice and remained significantly higher in the Eng(+/-) mice. CONCLUSIONS: Higher VEGF levels and increased vascular permeability in the distal colon may predispose Eng(+/-) mice to progress to chronic and persistent bowel inflammation, associated with pathological angiogenesis.