Unveiling the epigenomic mechanisms of acquired platinum-resistance in high-grade serous ovarian cancer.

Resistance to platinum-based chemotherapy is the major cause of death from high-grade serous ovarian cancer (HGSOC). We hypothesise that detection of specific DNA methylation changes may predict platinum resistance in HGSOC. Using a publicly available "discovery" dataset we examined epigenomic and transcriptomic alterations between primary platinum-sensitive (n = 32) and recurrent acquired drug resistant HGSOC (n = 28) and identified several genes involved in immune and chemoresistance-related pathways. Validation via high-resolution melt analysis of these findings, in cell lines and HGSOC tumours, demonstrated the most consistent changes were observed in three of the genes: APOBEC3A, NKAPL and PDCD1. Plasma samples from an independent HGSOC cohort (n = 17) were analysed using droplet digital PCR. Hypermethylation of NKAPL was detected in 46% and hypomethylation of APOBEC3A in 69% of plasma samples taken from women with relapsed HGSOC (n = 13), with no alterations identified in disease-free patients (n = 4). Following these results, and using a CRISPR-Cas9 approach, we were also able to demonstrate that in vitro NKAPL promoter demethylation increased platinum sensitivity by 15%. Overall, this study demonstrates the importance of aberrant methylation, especially of the NKAPL gene, in acquired platinum resistance in HGSOC.

Cannabidiol Inhibits the Proliferation and Invasiveness of Prostate Cancer Cells.

Prostate cancer is the fifth leading cause of cancer death in men, responsible for over 375,000 deaths in 2020. Novel therapeutic strategies are needed to improve outcomes. Cannabinoids, chemical components of the cannabis plant, are a possible solution. Preclinical evidence demonstrates that cannabinoids can modulate several cancer hallmarks of many tumor types. However, the therapeutic potential of cannabinoids in prostate cancer has not yet been fully explored. The aim of this study was to investigate the antiproliferative and anti-invasive properties of cannabidiol (CBD) in prostate cancer cells in vitro. CBD inhibited cell viability and proliferation, accompanied by reduced expression of key cell cycle proteins, specifically cyclin D3 and cyclin-dependent kinases CDK2, CDK4, and CDK1, and inhibition of AKT phosphorylation. The effects of CBD on cell viability were not blocked by cannabinoid receptor antagonists, a transient receptor potential vanilloid 1 (TRPV1) channel blocker, or an agonist of the G-protein-coupled receptor GPR55, suggesting that CBD acts independently of these targets in prostate cancer cells. Furthermore, CBD reduced the invasiveness of highly metastatic PC-3 cells and increased protein expression of E-cadherin. The ability of CBD to inhibit prostate cancer cell proliferation and invasiveness suggests that CBD may have potential as a future chemotherapeutic agent.

Development of a multivariable risk model integrating urinary cell DNA methylation and cell-free RNA data for the detection of significant prostate cancer.

BACKGROUND: Prostate cancer exhibits severe clinical heterogeneity and there is a critical need for clinically implementable tools able to precisely and noninvasively identify patients that can either be safely removed from treatment pathways or those requiring further follow up. Our objectives were to develop a multivariable risk prediction model through the integration of clinical, urine-derived cell-free messenger RNA (cf-RNA) and urine cell DNA methylation data capable of noninvasively detecting significant prostate cancer in biopsy naïve patients. METHODS: Post-digital rectal examination urine samples previously analyzed separately for both cellular methylation and cf-RNA expression within the Movember GAP1 urine biomarker cohort were selected for a fully integrated analysis (n = 207). A robust feature selection framework, based on bootstrap resampling and permutation, was utilized to find the optimal combination of clinical and urinary markers in a random forest model, deemed ExoMeth. Out-of-bag predictions from ExoMeth were used for diagnostic evaluation in men with a clinical suspicion of prostate cancer (PSA ≥ 4 ng/mL, adverse digital rectal examination, age, or lower urinary tract symptoms). RESULTS: As ExoMeth risk score (range, 0-1) increased, the likelihood of high-grade disease being detected on biopsy was significantly greater (odds ratio = 2.04 per 0.1 ExoMeth increase, 95% confidence interval [CI]: 1.78-2.35). On an initial TRUS biopsy, ExoMeth accurately predicted the presence of Gleason score ≥3 + 4, area under the receiver-operator characteristic curve (AUC) = 0.89 (95% CI: 0.84-0.93) and was additionally capable of detecting any cancer on biopsy, AUC = 0.91 (95% CI: 0.87-0.95). Application of ExoMeth provided a net benefit over current standards of care and has the potential to reduce unnecessary biopsies by 66% when a risk threshold of 0.25 is accepted. CONCLUSION: Integration of urinary biomarkers across multiple assay methods has greater diagnostic ability than either method in isolation, providing superior predictive ability of biopsy outcomes. ExoMeth represents a more holistic view of urinary biomarkers and has the potential to result in substantial changes to how patients suspected of harboring prostate cancer are diagnosed.

Epigenetics of malignant melanoma.

Patients with malignant melanoma generally have a good prognosis if the disease presents prior to metastasis. Due to progress with targeted and immunotherapies, the median survival of metastatic melanoma patients is now over 2 years. The disease is characterised by one of the highest somatic mutation rates observed amongst cancer types, with a specific mutational signature based on UV radiation damage evident. Highly prevalent mutations, such as the BRAFV600E, in the MAPK cascade indicate truncal involvement of this pathway in the earliest stage of melanoma. The molecular sub-classification of melanoma based on genetic alterations is now well established. This has paved the way for researchers in epigenetics to investigate specific pathways of known importance, and the involvement of the diverse range of epigenetic mechanisms. Herein, we review the literature to highlight that epigenetic alterations are integrally involved in this malignancy. We focus on the most current evidence around the epigenetic mechanisms: DNA methylation and demethylation including 5-hydroxy-methylcytosine; histone post-translational modifications including variant histones; chromatin remodelling complexes and in particular the polycomb-repressive complex PRC2 and its histone methyltransferase subunit EZH2; and non-coding RNAs. Each mechanism is described generally, studies involving melanoma are assessed and clinical relevance is highlighted where possible.

A four-group urine risk classifier for predicting outcomes in patients with prostate cancer.

OBJECTIVES: To develop a risk classifier using urine-derived extracellular vesicle (EV)-RNA capable of providing diagnostic information on disease status prior to biopsy, and prognostic information for men on active surveillance (AS). PATIENTS AND METHODS: Post-digital rectal examination urine-derived EV-RNA expression profiles (n = 535, multiple centres) were interrogated with a curated NanoString panel. A LASSO-based continuation ratio model was built to generate four prostate urine risk (PUR) signatures for predicting the probability of normal tissue (PUR-1), D'Amico low-risk (PUR-2), intermediate-risk (PUR-3), and high-risk (PUR-4) prostate cancer. This model was applied to a test cohort (n = 177) for diagnostic evaluation, and to an AS sub-cohort (n = 87) for prognostic evaluation. RESULTS: Each PUR signature was significantly associated with its corresponding clinical category (P < 0.001). PUR-4 status predicted the presence of clinically significant intermediate- or high-risk disease (area under the curve = 0.77, 95% confidence interval [CI] 0.70-0.84). Application of PUR provided a net benefit over current clinical practice. In an AS sub-cohort (n = 87), groups defined by PUR status and proportion of PUR-4 had a significant association with time to progression (interquartile range hazard ratio [HR] 2.86, 95% CI 1.83-4.47; P < 0.001). PUR-4, when used continuously, dichotomized patient groups with differential progression rates of 10% and 60% 5 years after urine collection (HR 8.23, 95% CI 3.26-20.81; P < 0.001). CONCLUSION: Urine-derived EV-RNA can provide diagnostic information on aggressive prostate cancer prior to biopsy, and prognostic information for men on AS. PUR represents a new and versatile biomarker that could result in substantial alterations to current treatment of patients with prostate cancer.

Improving multivariable prostate cancer risk assessment using the Prostate Health Index.

OBJECTIVES: To analyse the clinical utility of a prediction model incorporating both clinical information and a novel biomarker, p2PSA, in order to inform the decision for prostate biopsy in an Irish cohort of men referred for prostate cancer assessment. PATIENTS AND METHODS: Serum isolated from 250 men from three tertiary referral centres with pre-biopsy blood draws was analysed for total prostate-specific antigen (PSA), free PSA (fPSA) and p2PSA. From this, the Prostate Health Index (PHI) score was calculated (PHI = (p2PSA/fPSA)*√tPSA). The men's clinical information was used to derive their risk according to the Prostate Cancer Prevention Trial (PCPT) risk model. Two clinical prediction models were created via multivariable regression consisting of age, family history, abnormality on digital rectal examination, previous negative biopsy and either PSA or PHI score, respectively. Calibration plots, receiver-operating characteristic (ROC) curves and decision curves were generated to assess the performance of the three models. RESULTS: The PSA model and PHI model were both well calibrated in this cohort, with the PHI model showing the best correlation between predicted probabilities and actual outcome. The areas under the ROC curve for the PHI model, PSA model and PCPT model were 0.77, 0.71 and 0.69, respectively, for the prediction of prostate cancer (PCa) and 0.79, 0.72 and 0.72, respectively, for the prediction of high grade PCa. Decision-curve analysis showed a superior net benefit of the PHI model over both the PSA model and the PCPT risk model in the diagnosis of PCa and high grade PCa over the entire range of risk probabilities. CONCLUSION: A logical and standardized approach to the use of clinical risk factors can allow more accurate risk stratification of men under investigation for PCa. The measurement of p2PSA and the integration of this biomarker into a clinical prediction model can further increase the accuracy of risk stratification, helping to better inform the decision for prostate biopsy in a referral population.

IMPROVER (Involving Men with Prostate Cancer in Engaged Research): Assessing Patient Experience With Testing, Diagnosis, And Surveillance.

BACKGROUND: Our understanding of patient experiences with prostate cancer testing for diagnosis and surveillance is limited. The aim of this study was to collaborate with patients and clinicians to understand their lived experience and unmet needs around the early detection, diagnosis and monitoring (active surveillance) of prostate cancer. METHODS: Two focus groups were held with patients (n = 20) and healthcare professionals (n = 16), to identify the main challenges in prostate cancer detection, diagnosis, and monitoring. This information formed the basis of an online questionnaire for broader dissemination. RESULTS: A total of 1138 analyzable responses were obtained from people tested for prostate cancer (69% tested positive) in Europe and the US. Only 29 healthcare professionals completed the survey. Almost one-third of people reported knowing very little/nothing about prostate cancer prior to testing. Prior disease awareness was significantly higher in those who tested negative (P < .0001). Most respondents (n = 857; 75%) felt informed about the steps involved in testing. Receiving written information was a key factor; 91% of those who felt uninformed were not given any written information. Overall, most people felt "satisfied" with the typical prostate cancer tests: PSA, DRE, mpMRI, and biopsy. However, dissatisfaction for prostate biopsy (12%) was almost double that of other tests (P < .0001). Most patients understood why each test was done, and felt that their results and next steps were clearly explained to them; though PSA scored lowest in all of these fields. Apart from PSA, test satisfaction was lower when used repeatedly for surveillance, compared to once-off detection/diagnosis. CONCLUSIONS: Greater public awareness and education around prostate cancer, as well as clear and accessible written information for patients at the beginning of their cancer journey is needed. Further research is needed into alternative, less invasive tests, particularly when used repeatedly in the surveillance population.

Gene expression and epigenetic discovery screen reveal methylation of SFRP2 in prostate cancer.

Aberrant activation of Wnts is common in human cancers, including prostate. Hypermethylation associated transcriptional silencing of Wnt antagonist genes SFRPs (Secreted Frizzled-Related Proteins) is a frequent oncogenic event. The significance of this is not known in prostate cancer. The objectives of our study were to (i) profile Wnt signaling related gene expression and (ii) investigate methylation of Wnt antagonist genes in prostate cancer. Using TaqMan Low Density Arrays, we identified 15 Wnt signaling related genes with significantly altered expression in prostate cancer; the majority of which were upregulated in tumors. Notably, histologically benign tissue from men with prostate cancer appeared more similar to tumor (r = 0.76) than to benign prostatic hyperplasia (BPH; r = 0.57, p < 0.001). Overall, the expression profile was highly similar between tumors of high (≥ 7) and low (≤ 6) Gleason scores. Pharmacological demethylation of PC-3 cells with 5-Aza-CdR reactivated 39 genes (≥ 2-fold); 40% of which inhibit Wnt signaling. Methylation frequencies in prostate cancer were 10% (2/20) (SFRP1), 64.86% (48/74) (SFRP2), 0% (0/20) (SFRP4) and 60% (12/20) (SFRP5). SFRP2 methylation was detected at significantly lower frequencies in high-grade prostatic intraepithelial neoplasia (HGPIN; 30%, (6/20), p = 0.0096), tumor adjacent benign areas (8.82%, (7/69), p < 0.0001) and BPH (11.43% (4/35), p < 0.0001). The quantitative level of SFRP2 methylation (normalized index of methylation) was also significantly higher in tumors (116) than in the other samples (HGPIN = 7.45, HB = 0.47, and BPH = 0.12). We show that SFRP2 hypermethylation is a common event in prostate cancer. SFRP2 methylation in combination with other epigenetic markers may be a useful biomarker of prostate cancer.

Gene expression analysis in prostate cancer: the importance of the endogenous control.

BACKGROUND: Aberrant gene expression is a hallmark of cancer. Quantitative reverse-transcription PCR (qRT-PCR) is the gold-standard for quantifying gene expression, and commonly employs a house-keeping gene (HKG) as an endogenous control to normalize results; the choice of which is critical for accurate data interpretation. Many factors, including sample type, pathological state, and oxygen levels influence gene expression including putative HKGs. The aim of this study was to determine the suitability of commonly used HKGs for qRT-PCR in prostate cancer. METHODS: Prostate cancer (LNCaP, 22Rv1, PC3, and DU145) and normal (PWR1E and RWPE1) cell lines were cultured in air and hypoxia. The performance of 16 HKGs was assessed using Normfinder and coefficient of variation. In silico promoter analysis was performed to identify putative hypoxia response elements (HREs). The impact of the endogenous control on expression levels of HIF1A and GSTP1 was investigated by qRT-PCR in cell lines and tissue specimens respectively. RESULTS: Hypoxia altered expression of several HKGs: IPO8, B2M, and PGK1. The most stably expressed HKGs were ACTB, PPIA, and UBC. Both UBC and ACTB showed constitutive expression of HIF1A in air and hypoxia, while PGK1 falsely implied a sixfold hypoxia-induced down-regulation. In prostate tumors, UBC and PGK1 both revealed down-regulation of GSTP1 relative to matched benign, whereas ACTB showed variability. CONCLUSIONS: This study demonstrates that no universal endogenous control exists for gene expression studies, even within one disease type. It highlights the importance of validating expression of intended HKGs between different sample types and environmental exposures.

Localized hypermutation and associated gene losses in legume chloroplast genomes.

Point mutations result from errors made during DNA replication or repair, so they are usually expected to be homogeneous across all regions of a genome. However, we have found a region of chloroplast DNA in plants related to sweetpea (Lathyrus) whose local point mutation rate is at least 20 times higher than elsewhere in the same molecule. There are very few precedents for such heterogeneity in any genome, and we suspect that the hypermutable region may be subject to an unusual process such as repeated DNA breakage and repair. The region is 1.5 kb long and coincides with a gene, ycf4, whose rate of evolution has increased dramatically. The product of ycf4, a photosystem I assembly protein, is more divergent within the single genus Lathyrus than between cyanobacteria and other angiosperms. Moreover, ycf4 has been lost from the chloroplast genome in Lathyrus odoratus and separately in three other groups of legumes. Each of the four consecutive genes ycf4-psaI-accD-rps16 has been lost in at least one member of the legume "inverted repeat loss" clade, despite the rarity of chloroplast gene losses in angiosperms. We established that accD has relocated to the nucleus in Trifolium species, but were unable to find nuclear copies of ycf4 or psaI in Lathyrus. Our results suggest that, as well as accelerating sequence evolution, localized hypermutation has contributed to the phenomenon of gene loss or relocation to the nucleus.

Involving patients in healthcare research is well documented but can it work in lab-based research?

Public and Patient Involvement in research is becoming a requirement on most research funding applications; this includes both healthcare and lab-based research. Whilst case studies and practical guides have been developed and are well documented for PPI in healthcare research, there is very little guidance available for PPI in lab-based research. In this piece we discuss our experience of how we have successfully involved patients in our translational cancer research, which is focused on developing personalised treatment for high-grade serous ovarian cancer. We discuss the benefits it has made to both our research and to us as researchers. The patients involved write about their experience, what they enjoyed, and the benefits they felt. Although PPI is quite topical and is being widely discussed, there is hesitancy among researchers, especially those in lab-based research about getting started because of a lack of practical guidance about how to implement it. Here, we have shared our experience, hopefully providing a practical example of how PPI can be incorporated into a lab-based research project.

This piece is co-authored by researchers and ovarian cancer patients and presents their experience of patient involvement in a laboratory-based cancer research project focused on the personalised treatment of high-grade serous ovarian cancer. Discussions with five ovarian cancer patients about their treatment experience highlighted the fact that drugs showing equivalent clinical efficacy are not necessarily tolerated equally by individual patients. This led researchers to alter their original experimental design, by including a number of the same drug type instead of focusing on only one. The researchers also discuss the benefits it has made to both the research and to them as researchers. The patients involved write about their experience, what they enjoyed, and the benefits they felt.

IGFBP7 promoter methylation and gene expression analysis in prostate cancer.

PURPOSE: IGFBP7 belongs to a family of insulin-like growth factor-1 regulatory binding proteins. IGFBP7 hypermethylation is associated with its down-regulation in various carcinomas. In prostate cancer IGFBP7 down-regulation has been widely reported but to our knowledge the mechanisms behind this event are unknown. We performed a denaturing high performance liquid chromatography screening and validation strategy to profile the methylation status of IGFBP7 in prostate cancer. MATERIALS AND METHODS: We combined denaturing high performance liquid chromatography and bisulfite sequencing to examine IGFBP7 methylation in a panel of prostate cancer cell lines. Quantitative methylation specific polymerase chain reaction was used to determine methylation levels in prostate tissue specimens of primary prostate cancer, histologically benign prostate adjacent to tumor, high grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia. IGFBP7 gene expression was measured by quantitative methylation specific polymerase chain reaction in cell lines and tissue specimens. RESULTS: IGFBP7 was methylated in the 4 prostate cancer cell lines DU145, LNCaP, PC-3 and 22RV1. Quantitative methylation specific polymerase chain reaction analysis revealed that promoter methylation was associated with decreased IGFBP7 expression. Quantitative methylation specific polymerase chain reaction showed that IGFBP7 methylation was more frequently detected in prostate cancer (60% (31/52)) and high grade prostatic intraepithelial neoplasia (40% (6/15)) samples compared to histologically benign prostate adjacent to tumor (10%) and benign prostatic hyperplasia (0%) samples. CONCLUSIONS: To our knowledge this is the first report of aberrant IGFBP7 promoter hypermethylation and concurrent IGFBP7 gene silencing in prostate cancer cell lines. Results demonstrate that CpG methylation of IGFBP7 may represent a novel biomarker of prostate cancer and pre-invasive neoplasms. Thus, future examination of IGFBP7 methylation and expression in a larger patient cohort, including bodily fluids, is justified to further evaluate its role in a diagnostic and prognostic setting.

Plant-derived cannabinoids as anticancer agents.

Substantial preclinical evidence demonstrates the antiproliferative, cytotoxic, and antimetastatic properties of plant-derived cannabinoids (phytocannabinoids) such as cannabidiol and tetrahydrocannabinol. The cumulative body of research into the intracellular mechanisms and phenotypic effects of these compounds supports a logical, judicious progression to large-scale phase II/III clinical trials in certain cancer types to truly assess the efficacy of phytocannabinoids as anticancer agents.

In silico analysis and DHPLC screening strategy identifies novel apoptotic gene targets of aberrant promoter hypermethylation in prostate cancer.

BACKGROUND: Aberrant DNA methylation has been implicated as a key survival mechanism in cancer, whereby promoter hypermethylation silences genes essential for many cellular processes including apoptosis. Limited data is available on the methylation profile of apoptotic genes in prostate cancer (CaP). The aim of this study was to profile methylation of apoptotic-related genes in CaP using denaturing high performance liquid chromatography (DHPLC). METHODS: Based on an in silico selection process, 13 genes were screened for methylation in CaP cell lines using DHPLC. Quantitative methylation specific PCR was employed to determine methylation levels in prostate tissue specimens (n = 135), representing tumor, histologically benign prostate, high-grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia. Gene expression was measured by QRT-PCR in cell lines and tissue specimens. RESULTS: The promoters of BIK, BNIP3, cFLIP, TMS1, DCR1, DCR2, and CDKN2A appeared fully or partially methylated in a number of malignant cell lines. This is the first report of aberrant methylation of BIK, BNIP3, and cFLIP in CaP. Quantitative methylation analysis in prostate tissues identified 5 genes (BNIP3, CDKN2A, DCR1, DCR2 and TMS1) which were frequently methylated in tumors but were unmethylated in 100% of benign tissues. Furthermore, 69% of tumors were methylated in at least one of the five-gene panel. In the case of all genes, except BNIP3, promoter hypermethylation was associated with concurrent downregulation of gene expression. CONCLUSION: Future examination of this "CaP apoptotic methylation signature" in a larger cohort of patients is justified to further evaluate its value as a diagnostic and prognostic marker.

The role of secreted frizzled-related protein 2 expression in prostate cancer.

AIMS: Improved prostate cancer (PCa)-specific biomarkers are urgently required to distinguish between indolent and aggressive disease, in order to avoid overtreatment. In this study, we investigated the prostatic tissue expression of secreted frizzled-related protein (SFRP)-2. METHODS AND RESULTS: Following immunohistochemical analysis on PCa tissue microarrays with samples from 216 patients, strong/moderate SFRP-2 expression was observed in epithelial cells of benign prostatic hyperplasia, and negative/weak SFRP-2 expression was observed in the majority of tumour epithelia. However, among Gleason grade 5 carcinomas, 40% showed strong/moderate SFRP-2 expression and 60% showed negative SFRP-2 expression in epithelial cells. Further microscopic evaluation of Gleason grade 5 tumours revealed different morphological patterns, corresponding with differential SFRP-2 expression. The first subgroup (referred to as Type A) appeared to have a morphologically solid growth pattern, whereas the second subgroup (referred to as Type B) appeared to have a more diffuse pattern. Furthermore, 100% (4/4) of Type A patients experienced biochemical recurrence, as compared with 0% (0/6) of Type B patients. CONCLUSIONS: These results imply: (i) that there is a loss of SFRP-2 expression from benign to malignant prostate glands; and (ii) differential SFRP-2 expression among two possible subgroups of Gleason grade 5 tumours.

5-AZA-dC induces epigenetic changes associated with modified glycosylation of secreted glycoproteins and increased EMT and migration in chemo-sensitive cancer cells.

BACKGROUND: Glycosylation, one of the most fundamental post-translational modifications, is altered in cancer and is subject in part, to epigenetic regulation. As there are many epigenetic-targeted therapies currently in clinical trials for the treatment of a variety of cancers, it is important to understand the impact epi-therapeutics have on glycosylation. RESULTS: Ovarian and triple negative breast cancer cells were treated with the DNA methyltransferase inhibitor, 5-AZA-2-deoxycytidine (5-AZA-dC). Branching and sialylation were increased on secreted N-glycans from chemo-sensitive/non-metastatic cell lines following treatment with 5-AZA-dC. These changes correlated with increased mRNA expression levels in MGAT5 and ST3GAL4 transcripts in ovarian cancer cell lines. Using siRNA transient knock down of GATA2 and GATA3 transcription factors, we show that these regulate the glycosyltransferases ST3GAL4 and MGAT5, respectively. Moreover, 5-AZA-dC-treated cells displayed an increase in migration, with a greater effect seen in chemo-sensitive cell lines. Western blots showed an increase in apoptotic and senescence (p21) markers in all 5-AZA-dC-treated cells. The alterations seen in N-glycans from secreted glycoproteins in 5-AZA-dC-treated breast and ovarian cancer cells were similar to the N-glycans previously known to potentiate tumour cell survival. CONCLUSIONS: While the FDA has approved epi-therapeutics for some cancer treatments, their global effect is still not fully understood. This study gives insight into the effects that epigenetic alterations have on cancer cell glycosylation, and how this potentially impacts on the overall fate of those cells.

Longitudinal analysis of individual cfDNA methylome patterns in metastatic prostate cancer.

BACKGROUND: Disease progression and therapeutic resistance are hallmarks of advanced stage prostate cancer (PCa), which remains a major cause of cancer-related mortality around the world. Longitudinal studies, coupled with the use of liquid biopsies, offer a potentially new and minimally invasive platform to study the dynamics of tumour progression. Our aim was to investigate the dynamics of personal DNA methylomic profiles of metastatic PCa (mPCa) patients, during disease progression and therapy administration. RESULTS: Forty-eight plasma samples from 9 mPCa patients were collected, longitudinally, over 13-21 months. After circulating cell-free DNA (cfDNA) isolation, DNA methylation was profiled using the Infinium MethylationEPIC BeadChip. The top 5% most variable probes across time, within each individual, were utilised to study dynamic methylation patterns during disease progression and therapeutic response. Statistical testing was carried out to identify differentially methylated genes (DMGs) in cfDNA, which were subsequently validated in two independent mPCa (cfDNA and FFPE tissue) cohorts. Individual cfDNA global methylation patterns were temporally stable throughout the disease course. However, a proportion of CpG sites presented a dynamic temporal pattern that was consistent with clinical events, including different therapies, and were prominently associated with genes linked to immune response pathways. Additionally, study of the tumour fraction of cfDNA identified > 2000 DMGs with dynamic methylation patterns. CONCLUSIONS: Longitudinal assessment of cfDNA methylation in mPCa patients unveiled dynamic patterns associated with disease progression and therapy administration, thus highlighting the potential of using liquid biopsies to study PCa evolution at a methylomic level.

Evaluating liquid biopsies for methylomic profiling of prostate cancer.

BACKGROUND: Liquid biopsies offer significant potential for informing on cancer progression and therapeutic resistance via minimally invasive serial monitoring of genetic alterations. Although the cancer epigenome is a central driving force in most neoplasia, the accuracy of monitoring the tumor methylome using liquid biopsies remains relatively unknown. OBJECTIVES: to investigate how well two types of liquid biopsy (urine and blood) capture the prostate cancer methylome, and may thus serve as a non-invasive surrogate for studying the tumor epigenome. METHODS: A cohort of four metastatic treatment naïve prostate cancer (PCa) patients was selected. Matched biopsy cores (tumor and histologically matched-normal), post-DRE, pre-biopsy urine, and peripheral blood plasma were available for each subject. DNA methylation was profiled utilizing the Infinium® MethylationEPIC BeadChip (Illumina) and analysed using the RnBeads software. Significantly (FDR adjusted P < 0.05) differentially methylated probes (DMPs) between tumor and MN were identified and examined in the liquids (done at a grouped and individual subject level). RESULTS: DNA methylation analysis of urine and blood in men with metastatic PCa showed highly correlated patterns between the different liquid types (ρ = 0.93, P < 0.0001), with large contributions from non-tumor sources. DNA methylation profiles of liquids were more similar between subjects, than intra-individual liquid-tumor correlations. Overall, both urine and plasma are viable surrogates for tumor tissue biopsies, capturing up to 39.40% and 64.14% of tumor-specific methylation alterations, respectively. CONCLUSION: We conclude that both urine and blood plasma are easily accessible and sensitive biofluids for the study of PCa epigenomic alterations.

Novel Strategies for Cancer Treatment: Highlights from the 55th IACR Annual Conference.

While conventional cancer treatments, such as surgery, radiotherapy and chemotherapy, have been combined for decades in an effort to treat cancer patients, the emergence of novel fields of cancer research have led to a renewed interest in combining conventional treatments with more innovative approaches. The realisation that cancer progression is not exclusively due to changes in the cancer epithelial cells, but also involves changes in the tumour microenvironment, has opened new avenues for combination treatments. Here we discuss the use of combination therapies presented at the 55th Irish Association for Cancer Research (IACR) Annual Conference, highlighting examples of novel therapeutic strategies which, combined with conventional therapies, may greatly enhance not only the overall outcome for patients, but also the quality of life for cancer survivors. Among the novel treatment strategies, immune metabolism, epigenetic therapies and physical exercise are presented. In addition, novel technologies in the field of precision medicine, which will be useful to discover new therapeutics and to stratify patients for combination treatments, are also discussed.