Cell-free DNA Methylation as a Predictive Biomarker of Response to Neoadjuvant Chemotherapy for Patients with Muscle-invasive Bladder Cancer in SWOG S1314.

BACKGROUND: Neoadjuvant chemotherapy (NAC) is the standard of care in muscle-invasive bladder cancer (MIBC). However, treatment is intense, and the overall benefit is small, necessitating effective biomarkers to identify patients who will benefit most. OBJECTIVE: To characterize cell-free DNA (cfDNA) methylation in patients receiving NAC in SWOG S1314, a prospective cooperative group trial, and to correlate the methylation signatures with pathologic response at radical cystectomy. DESIGN, SETTING, AND PARTICIPANTS: SWOG S1314 is a prospective cooperative group trial for patients with MIBC (cT2-T4aN0M0, ≥5 mm of viable tumor), with a primary objective of evaluating the coexpression extrapolation (COXEN) gene expression signature as a predictor of NAC response, defined as achieving pT0N0 or ≤pT1N0 at radical cystectomy. For the current exploratory analysis, blood samples were collected prospectively from 72 patients in S1314 before and during NAC, and plasma cfDNA methylation was measured using the Infinium MethylationEPIC BeadChip array. INTERVENTION: No additional interventions besides plasma collection. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Differential methylation between pathologic responders (≤pT1N0) and nonresponders was analyzed, and a classifier predictive of treatment response was generated using the Random Forest machine learning algorithm. RESULTS AND LIMITATIONS: Using prechemotherapy plasma cfDNA, we developed a methylation-based response score (mR-score) predictive of pathologic response. Plasma samples collected after the first cycle of NAC yielded mR-scores with similar predictive ability. Furthermore, we used cfDNA methylation data to calculate the circulating bladder DNA fraction, which had a modest but independent predictive ability for treatment response. In a model combining mR-score and circulating bladder DNA fraction, we correctly predicted pathologic response in 79% of patients based on their plasma collected at baseline and after one cycle of chemotherapy. Limitations of this study included a limited sample size and relatively low circulating bladder DNA levels. CONCLUSIONS: Our study provides the proof of concept that cfDNA methylation can be used to generate classifiers of NAC response in bladder cancer patients. PATIENT SUMMARY: In this exploratory analysis of S1314, we demonstrated that cell-free DNA methylation can be profiled to generate biomarker signatures associated with neoadjuvant chemotherapy response. With validation in additional cohorts, this minimally invasive approach may be used to predict chemotherapy response in locally advanced bladder cancer and perhaps also in metastatic disease.

Integration of Liquid Biopsies in Clinical Management of Metastatic Prostate Cancer.

PURPOSE OF REVIEW: The field of liquid biopsies is constantly evolving through novel technologies. This review outlines current data on liquid biopsies and application to clinical management of metastatic prostate cancer. RECENT FINDINGS: To date, there are three platforms with FDA approval for use in the setting of metastatic prostate cancer and others which have been clinically validated. There is substantial evidence supporting the use of circulating tumor cell (CTC) enumeration to guide prognosis in metastatic castration-resistant prostate cancer (mCRPC). Additional evidence supports targeted sequencing of CTC and cell-free DNA (cfDNA) to guide androgren-directed therapy, identify candidates for treatment with PARP inhibitors, and monitor development of resistance. As a real-time and minimally invasive approach, utilization of liquid biopsies has the potential to drastically impact the treatment of metastatic prostate cancer and improve overall survival. With further clinical validation, additional liquid biopsy is likely to enter standard clinical practice.

Non-Invasive Profiling of Advanced Prostate Cancer via Multi-Parametric Liquid Biopsy and Radiomic Analysis.

Integrating liquid biopsies of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) with other minimally invasive measures may yield more comprehensive disease profiles. We evaluated the feasibility of concurrent cellular and molecular analysis of CTCs and cfDNA combined with radiomic analysis of CT scans from patients with metastatic castration-resistant PC (mCRPC). CTCs from 22 patients were enumerated, stained for PC-relevant markers, and clustered based on morphometric and immunofluorescent features using machine learning. DNA from single CTCs, matched cfDNA, and buffy coats was sequenced using a targeted amplicon cancer hotspot panel. Radiomic analysis was performed on bone metastases identified on CT scans from the same patients. CTCs were detected in 77% of patients and clustered reproducibly. cfDNA sequencing had high sensitivity (98.8%) for germline variants compared to WBC. Shared and unique somatic variants in PC-related genes were detected in cfDNA in 45% of patients (MAF > 0.1%) and in CTCs in 92% of patients (MAF > 10%). Radiomic analysis identified a signature that strongly correlated with CTC count and plasma cfDNA level. Integration of cellular, molecular, and radiomic data in a multi-parametric approach is feasible, yielding complementary profiles that may enable more comprehensive non-invasive disease modeling and prediction.

Baseline Circulating Tumor Cell Count as a Prognostic Marker of PSA Response and Disease Progression in Metastatic Castrate-Sensitive Prostate Cancer (SWOG S1216).

PURPOSE: In metastatic castrate-sensitive prostate cancer (mCSPC), combined androgen axis inhibition is a standard of care. Noninvasive biomarkers that guide initial therapy decisions are needed. We hypothesized that CellSearch circulating tumor cell (CTC) count, an FDA-cleared assay in metastatic castrate-resistant prostate cancer (mCRPC), is a relevant biomarker in mCSPC. EXPERIMENTAL DESIGN: SWOG S1216 is a phase III prospective randomized trial of androgen deprivation therapy (ADT) combined with orteronel or bicalutamide for mCSPC. CellSearch CTC count was measured at registration (baseline). Prespecified CTC cut-off points of 0, 1-4, and ≥5 were correlated with baseline patient characteristics and, in a stratified subsample, were also correlated with two prespecified trial secondary endpoints: 7-month PSA ≤0.2 ng/mL versus 0.2-4.0 versus >4.0 (intermediate endpoint for overall survival); and progression-free survival (PFS) ≤ versus >2 years. RESULTS: A total of 523 patients submitted baseline samples, and CTCs were detected (median 3) in 33%. Adjusting for two trial stratification factors (disease burden and timing of ADT initiation), men with undetectable CTCs had nearly nine times the odds of attaining 7-month PSA ≤ 0.2 versus > 4.0 [OR 8.8, 95% confidence interval (CI), 2.7-28.6, P < 0.001, N = 264] and four times the odds of achieving > 2 years PFS (OR 4.0, 95% CI, 1.9-8.5, P < 0.001, N = 336) compared with men with baseline CTCs ≥5. CONCLUSIONS: Baseline CTC count in mCSPC is highly prognostic of 7-month PSA and 2-year PFS after adjusting for disease burden and discriminates men who are likely to experience poor survival outcomes.

Design and Implementation of a Smart Home in a Box to Monitor the Wellbeing of Residents With Dementia in Care Homes.

There is a global challenge related to the increasing number of People with Dementia (PwD) and the diminishing capacity of governments, health systems, and caregivers to provide the best care for them. Cost-effective technology solutions that enable and ensure a good quality of life for PwD via monitoring and interventions have been investigated comprehensively in the literature. The objective of this study was to investigate the challenges with the design and deployment of a Smart Home In a Box (SHIB) approach to monitoring PwD wellbeing within a care home. This could then support future SHIB implementations to have an adequate and prompt deployment allowing research to focus on the data collection and analysis aspects. An important consideration was that most care homes do not have the appropriate infrastructure for installing and using ambient sensors. The SHIB was evaluated via installation in the rooms of PwD with varying degrees of dementia at Kirk House Care Home in Belfast. Sensors from the SHIB were installed to test their capabilities for detecting Activities of Daily Living (ADLs). The sensors used were: (i) thermal sensors, (ii) contact sensors, (iii) Passive Infrared (PIR) sensors, and (iv) audio level sensors. Data from the sensors were collected, stored, and handled using a 'SensorCentral' data platform. The results of this study highlight challenges and opportunities that should be considered when designing and implementing a SHIB approach in a dementia care home. Lessons learned from this investigation are presented in addition to recommendations that could support monitoring the wellbeing of PwD. The main findings of this study are: (i) most care home buildings were not originally designed to appropriately install ambient sensors, and (ii) installation of SHIB sensors should be adapted depending on the specific case of the care home where they will be installed. It was acknowledged that in addition to care homes, the homes of PwD were also not designed for an appropriate integration with ambient sensors. This study provided the community with useful lessons, that will continue to be applied to improve future implementations of the SHIB approach.

Epigenetic plasticity potentiates a rapid cyclical shift to and from an aggressive cancer phenotype.

Highly tumorigenic, drug-resistant cancer stem-like cells drive cancer progression. These aggressive cells can arise repeatedly from bulk tumor cells independently of mutational events, suggesting an epigenetic mechanism. To test this possibility, we studied bladder cancer cells as they cyclically shifted to and from a cancer stem-like phenotype, and we discovered that these two states exhibit distinct DNA methylation and chromatin accessibility. Most differential chromatin accessibility was independent of methylation and affected the expression of driver genes such as E2F3, a cell cycle regulator associated with aggressive bladder cancer. Cancer stem-like cells exhibited increased E2F3 promoter accessibility and increased E2F3 expression that drove cell migration, invasiveness and drug resistance. Epigenetic interference using a DNA methylation inhibitor blocked the transition to a cancer stem-like state and reduced E2F3 expression. Our findings indicate that epigenetic plasticity plays a key role in the transition to and from an aggressive, drug-resistant phenotype.

Cancer transcriptomic profiling from rapidly enriched circulating tumor cells.

Transcriptomic profiling of metastatic cancer can illuminate mechanisms of progression and lead to new therapies, but standard biopsy is invasive and reflects only a single metastatic site. In contrast, circulating tumor cell (CTC) profiling is noninvasive and repeatable, reflecting the dynamic and systemic nature of advanced disease. To date, transcriptomic profiling of CTCs has not delivered on its full potential, because white blood cells (WBCs) vastly outnumber CTCs. Current profiling strategies either lack cancer sensitivity and specificity or require specialized CTC capture protocols that are not readily scalable to large patient cohorts. Here, we describe a new strategy for rapid CTC enrichment and transcriptomic profiling using commercially available WBC depletion, microfluidic enrichment and RNA sequencing. When applied to blood samples from patients with advanced prostate cancer (PC), transcriptomes from enriched samples cluster with cancer positive controls and previously undetectable prostate-specific transcripts become readily measurable. Gene set enrichment analysis reveals multiple significantly enriched signaling pathways associated with PC, as well as novel pathways that merit further study. This accessible and scalable approach yields cancer-specific transcriptomic data and can be applied repeatedly and noninvasively in large cancer patient cohorts to discover new therapeutic targets in advanced disease.

Multiparametric liquid biopsy analysis in metastatic prostate cancer.

Molecular profiling of prostate cancer with liquid biopsies, such as circulating tumor cells (CTCs) and cell-free nucleic acid analysis, yields informative yet distinct data sets. Additional insights may be gained by simultaneously interrogating multiple liquid biopsy components to construct a more comprehensive molecular disease profile. We conducted an initial proof-of-principle study aimed at piloting this multiparametric approach. Peripheral blood samples from men with metastatic castrate-resistant prostate cancer were analyzed simultaneously for CTC enumeration, single-cell copy number variations, CTC DNA and matched cell-free DNA mutations, and plasma cell-free RNA levels of androgen receptor (AR) and AR splice variant (ARV7). In addition, liquid biopsies were compared with matched tumor profiles when available, and a second liquid biopsy was drawn and analyzed at disease progression in a subset of patients. In this manner, multiparametric liquid biopsy profiles were successfully generated for each patient and time point, demonstrating the feasibility of this approach and highlighting shared as well as unique cancer-relevant alterations. With further refinement and validation in large cohorts, multiparametric liquid biopsies can optimally integrate disparate but clinically informative data sets and maximize their utility for molecularly directed, real-time patient management.

A Thermal Imaging Solution for Early Detection of Pre-ulcerative Diabetic Hotspots.

Foot ulcers are a common complication of diabetes and are the leading cause of amputation amongst those with diabetes. Research has shown that, an increase of two degrees Celsius in the skin temperature on the plantar surface of the foot can be an early indication of injury or inflammation. Early detection and treatment of a hotspot region may reduce the risk of an ulcer developing. This paper presents a thermography-based approach for detecting temperature hotspots on the foot. The system comprises a bespoke application and a thermal camera attachment which captures RGB images and a temperature matrix. Web-based services process the captured data and detect whether any regions of higher temperature are present on the foot, in comparison to the other foot. The accuracy of this system has been verified through a pilot study. Hotspots were simulated on the feet of 10 healthy participants. The results indicated that hotspots were correctly detected for 60% of the participants. We discuss some reasons why the results were inaccurate for the remaining four participants. Furthermore, we also suggest some potential enhancements to the system with the aim of increasing the precision of the results.

Development and Application of Liquid Biopsies in Metastatic Prostate Cancer.

PURPOSE OF REVIEW: Metastatic prostate cancer is a lethal and highly heterogeneous malignancy, associated with a broad spectrum of potentially actionable molecular alterations. In the past decade, disease profiling has expanded to include not only traditional tumor tissue, but also liquid biopsies of cells and genetic material circulating in the blood. These liquid biopsies offer a minimally invasive, repeatable source of tumor material for longitudinal disease profiling but also raise new technical and biological challenges. Here we will summarize recent advances in liquid biopsy strategies and the role they have played in biomarker development and disease management. RECENT FINDINGS: Technologies for analysis of circulating tumor cells (CTCs) continue to evolve rapidly, and the latest high content scanning platforms have underscored the phenotypic heterogeneity of CTC populations. Among liquid biopsies, CTC enumeration remains the most extensively validated prognostic marker to date, but other clinically relevant phenotypes like androgen receptor (AR) localization or presence of AR-V7 splice variant are important new predictors of therapy response. Serial genomic profiling of CTCs or circulating tumor DNA (ctDNA) is helping to define primary and acquired resistance mechanisms and helping to guide patient selection for targeted therapies such as poly(adenosine diphosphate [ADP] ribose) polymerase (PARP) inhibition. The era of liquid biopsy-based biomarkers has arrived, driven by powerful new enrichment and analysis techniques. As new blood-based markers are identified, their biological significance as disease drivers must be elucidated to advance new therapeutic strategies, and their clinical impact must be translated through assay standardization, followed by analytical and clinical validation. These efforts, already ongoing on multiple fronts, constitute the critical steps toward more effective precision management of advanced prostate cancer.

Doorstep: A doorbell security system for the prevention of doorstep crime.

Safety and security rank highly in the priorities of older people on both an individual and policy level. Older people are commonly targeted as victims of doorstep crime, as they can be perceived as being vulnerable. As a result, this can have a major effect on the victim's health and wellbeing. There have been numerous prevention strategies implemented in an attempt to combat and reduce the number of doorstep crimes. There is, however, little information available detailing the effectiveness of these strategies and how they impact on the fear of crime, particularly with repeat victims. There is therefore clear merit in the creation and piloting of a technology based solution to combat doorstep crime. This paper presents a developed solution to provide increased security for older people within their home.

Considerations in the identification of endogenous substrates for protein L-isoaspartyl methyltransferase: the case of synuclein.

Protein L-isoaspartyl methyltransferase (PIMT) repairs abnormal isoaspartyl peptide bonds in age-damaged proteins. It has been reported that synuclein, a protein implicated in neurodegenerative diseases, is a major target of PIMT in mouse brain. To extend this finding and explore its possible relevance to neurodegenerative diseases, we attempted to determine the stoichiometry of isoaspartate accumulation in synuclein in vivo and in vitro. Brain proteins from PIMT knockout mice were separated by 2D electrophoresis followed by on-blot [(3)H]-methylation to label isoaspartyl proteins, and by immunoblotting to confirm the coincident presence of synuclein. On-blot (3)H-methylation revealed numerous isoaspartyl proteins, but no signal in the position of synuclein. This finding was corroborated by immunoprecipitation of synuclein followed by on-blot (3)H-methylation. To assess the propensity of synuclein to form isoaspartyl sites in vitro, samples of recombinant mouse and human α-synucleins were aged for two weeks by incubation at pH 7.5 and 37 °C. The stoichiometries of isoaspartate accumulation were extremely low at 0.02 and 0.07 mol of isoaspartate per mol of protein respectively. Using a simple mathematical model based on the first order kinetics of isoaspartyl protein methyl ester hydrolysis, we ascribe the discrepancy between our results and the previous report to methodological limitations of the latter stemming from an inherent, and somewhat counterintuitive, relationship between the propensity of proteins to form isoaspartyl sites and the instability of the (3)H-methyl esters used to tag them. The results presented here indicate that synuclein is not a major target of PIMT in vivo, and emphasize the need to minimize methyl ester hydrolysis when using methylation to assess the abundance of isoaspartyl sites in proteins.

Efficient production of human beta-defensin 2 (HBD2) in Escherichia coli.

Human beta-defensin 2 (HBD2) has been shown to interact with pathogenic bacteria and components of the mammalian innate and adaptive immune response. We describe a quick and reliable method for the production of HBD2 in Escherichia coli. HBD2 was expressed as an insoluble fusion, chemically cleaved and oxidised to give a single, folded HBD2 beta-isoform. The purified peptide was analysed by high resolution mass spectrometry, displayed a well-dispersed (1)H NMR spectrum, was a chemoattractant to HEK293 cells expressing CCR6 and acted as an antimicrobial agent against E. coli, P. aeruginosa, C. albicans and S. aureus.

Has the incidence of deep vein thrombosis in patients undergoing total hip/knee arthroplasty changed over time? A systematic review of randomized controlled trials.

BACKGROUND: There is a perception in the orthopaedic and thromboembolism community that the incidence of deep vein thrombosis (DVT) has decreased in patients undergoing total knee arthroplasty (TKA) or total hip arthroplasty (THA). OBJECTIVES: To assess the incidence of DVT with warfarin thromboprophylaxis over time in patients undergoing elective TKA or THA. METHODS: The MEDLINE, EMBASE, and Cochrane Library databases were searched to October 2006, supplemented by a manual search of reference lists. Two reviewers independently extracted data on study characteristics, quality and the frequency of total, symptomatic and proximal DVT. RESULTS: Fourteen studies (4,423 patients) were included. Total and proximal DVT after TKA declined over time (r=-0.75, p=0.031; r=-0.86, p=0.007 respectively). Total and proximal DVT after THA did not change. The risk of developing DVT after TKA was significantly higher than after THA (OR 1.85, 95% CI 1.6-2.14; p<0.0001). The risk of developing symptomatic DVT after THA was significantly higher than after TKA (OR 2.18, 95% CI 1.11-4.27; p=0.012). CONCLUSIONS: The incidence of DVT in patients undergoing elective TKA appears to have declined in patients receiving warfarin thromboprophylaxis.