Phosphorylated RB Promotes Cancer Immunity by Inhibiting NF-κB Activation and PD-L1 Expression.

Aberrant expression of programmed death ligand-1 (PD-L1) in tumor cells promotes cancer progression by suppressing cancer immunity. The retinoblastoma protein RB is a tumor suppressor known to regulate the cell cycle, DNA damage response, and differentiation. Here, we demonstrate that RB interacts with nuclear factor κB (NF-κB) protein p65 and that their interaction is primarily dependent on CDK4/6-mediated serine-249/threonine-252 (S249/T252) phosphorylation of RB. RNA-seq analysis shows a subset of NF-κB pathway genes including PD-L1 are selectively upregulated by RB knockdown or CDK4/6 inhibitor. S249/T252-phosphorylated RB inversely correlates with PD-L1 expression in patient samples. Expression of a RB-derived S249/T252 phosphorylation-mimetic peptide suppresses radiotherapy-induced upregulation of PD-L1 and augments therapeutic efficacy of radiation in vivo. Our findings reveal a previously unrecognized tumor suppressor function of hyperphosphorylated RB in suppressing NF-κB activity and PD-L1 expression and suggest that the RB-NF-κB axis can be exploited to overcome cancer immune evasion triggered by conventional or targeted therapies.

Single-cell analysis of treatment-resistant prostate cancer: Implications of cell state changes for cell surface antigen-targeted therapies.

Targeting cell surface molecules using radioligand and antibody-based therapies has yielded considerable success across cancers. However, it remains unclear how the expression of putative lineage markers, particularly cell surface molecules, varies in the process of lineage plasticity, wherein tumor cells alter their identity and acquire new oncogenic properties. A notable example of lineage plasticity is the transformation of prostate adenocarcinoma (PRAD) to neuroendocrine prostate cancer (NEPC)-a growing resistance mechanism that results in the loss of responsiveness to androgen blockade and portends dismal patient survival. To understand how lineage markers vary across the evolution of lineage plasticity in prostate cancer, we applied single-cell analyses to 21 human prostate tumor biopsies and two genetically engineered mouse models, together with tissue microarray analysis on 131 tumor samples. Not only did we observe a higher degree of phenotypic heterogeneity in castrate-resistant PRAD and NEPC than previously anticipated but also found that the expression of molecules targeted therapeutically, namely PSMA, STEAP1, STEAP2, TROP2, CEACAM5, and DLL3, varied within a subset of gene-regulatory networks (GRNs). We also noted that NEPC and small cell lung cancer subtypes shared a set of GRNs, indicative of conserved biologic pathways that may be exploited therapeutically across tumor types. While this extreme level of transcriptional heterogeneity, particularly in cell surface marker expression, may mitigate the durability of clinical responses to current and future antigen-directed therapies, its delineation may yield signatures for patient selection in clinical trials, potentially across distinct cancer types.

Characterization of HOXB13 expression patterns in localized and metastatic castration-resistant prostate cancer.

HOXB13 is a key lineage homeobox transcription factor that plays a critical role in the differentiation of the prostate gland. Several studies have suggested that HOXB13 alterations may be involved in prostate cancer development and progression. Despite its potential biological relevance, little is known about the expression of HOXB13 across the disease spectrum of prostate cancer. To this end, we validated a HOXB13 antibody using genetic controls and investigated HOXB13 protein expression in murine and human developing prostates, localized prostate cancers, and metastatic castration-resistant prostate cancers. We observed that HOXB13 expression increases during later stages of murine prostate development. All localized prostate cancers showed HOXB13 protein expression. Interestingly, lower HOXB13 expression levels were observed in higher-grade tumors, although no significant association between HOXB13 expression and recurrence or disease-specific survival was found. In advanced metastatic prostate cancers, HOXB13 expression was retained in the majority of tumors. While we observed lower levels of HOXB13 protein and mRNA levels in tumors with evidence of lineage plasticity, 84% of androgen receptor-negative castration-resistant prostate cancers and neuroendocrine prostate cancers (NEPCs) retained detectable levels of HOXB13. Notably, the reduced expression observed in NEPCs was associated with a gain of HOXB13 gene body CpG methylation. In comparison to the commonly used prostate lineage marker NKX3.1, HOXB13 showed greater sensitivity in detecting advanced metastatic prostate cancers. Additionally, in a cohort of 837 patients, 383 with prostatic and 454 with non-prostatic tumors, we found that HOXB13 immunohistochemistry had a 97% sensitivity and 99% specificity for prostatic origin. Taken together, our studies provide valuable insight into the expression pattern of HOXB13 during prostate development and cancer progression. Furthermore, our findings support the utility of HOXB13 as a diagnostic biomarker for prostate cancer, particularly to confirm the prostatic origin of advanced metastatic castration-resistant tumors. © 2023 The Pathological Society of Great Britain and Ireland.

MDM2 E3 ligase activity is essential for p53 regulation and cell cycle integrity.

MDM2 and MDM4 are key regulators of p53 and function as oncogenes when aberrantly expressed. MDM2 and MDM4 partner to suppress p53 transcriptional transactivation and polyubiquitinate p53 for degradation. The importance of MDM2 E3-ligase-mediated p53 regulation remains controversial. To resolve this, we generated mice with an Mdm2 L466A mutation that specifically compromises E2 interaction, abolishing MDM2 E3 ligase activity while preserving its ability to bind MDM4 and suppress p53 transactivation. Mdm2L466A/L466A mice exhibit p53-dependent embryonic lethality, demonstrating MDM2 E3 ligase activity is essential for p53 regulation in vivo. Unexpectedly, cells expressing Mdm2L466A manifest cell cycle G2-M transition defects and increased aneuploidy even in the absence of p53, suggesting MDM2 E3 ligase plays a p53-independent role in cell cycle regulation and genome integrity. Furthermore, cells bearing the E3-dead MDM2 mutant show aberrant cell cycle regulation in response to DNA damage. This study uncovers an uncharacterized role for MDM2's E3 ligase activity in cell cycle beyond its essential role in regulating p53's stability in vivo.

Correction: MDM2 E3 ligase activity is essential for p53 regulation and cell cycle integrity.

[This corrects the article DOI: 10.1371/journal.pgen.1010171.].

Extracellular Vesicles from Pancreatic Cancer Stem Cells Lead an Intratumor Communication Network (EVNet) to fuel tumour progression.

OBJECTIVE: Intratumor heterogeneity drives cancer progression and therapy resistance. However, it has yet to be determined whether and how subpopulations of cancer cells interact and how this interaction affects the tumour. DESIGN: We have studied the spontaneous flow of extracellular vesicles (EVs) between subpopulations of cancer cells: cancer stem cells (CSC) and non-stem cancer cells (NSCC). To determine the biological significance of the most frequent communication route, we used pancreatic ductal adenocarcinoma (PDAC) orthotopic models, patient-derived xenografts (PDXs) and genetically engineered mouse models (GEMMs). RESULTS: We demonstrate that PDAC tumours establish an organised communication network between subpopulations of cancer cells using EVs called the EVNet). The EVNet is plastic and reshapes in response to its environment. Communication within the EVNet occurs preferentially from CSC to NSCC. Inhibition of this communication route by impairing Rab27a function in orthotopic xenographs, GEMMs and PDXs is sufficient to hamper tumour growth and phenocopies the inhibition of communication in the whole tumour. Mechanistically, we provide evidence that CSC EVs use agrin protein to promote Yes1 associated transcriptional regulator (YAP) activation via LDL receptor related protein 4 (LRP-4). Ex vivo treatment of PDXs with antiagrin significantly impairs proliferation and decreases the levels of activated YAP.Patients with high levels of agrin and low inactive YAP show worse disease-free survival. In addition, patients with a higher number of circulating agrin+ EVs show a significant increased risk of disease progression. CONCLUSION: PDAC tumours establish a cooperation network mediated by EVs that is led by CSC and agrin, which allows tumours to adapt and thrive. Targeting agrin could make targeted therapy possible for patients with PDAC and has a significant impact on CSC that feeds the tumour and is at the centre of therapy resistance.

The Successful Measurement of Clitoral Pulse Amplitude Using a New Clitoral Photoplethysmograph: A Pilot Study.

BACKGROUND: Although the clitoris is more sensitive to stimulation and its innervation more conducive to sensory feedback than the vagina, the field of sexual psychophysiology, which uses psychophysiological methods including genital response measures to study sexual arousal, relies heavily on the measurement of vaginal, rather than clitoral, pulse amplitude. AIM: To develop and test a new clitoral photoplethysmograph for the measurement of clitoral pulse amplitude (CPA). METHODS: 2 versions of the new device, which differed in the orientation of the sensor and light source (parallel vs angled), were tested in 15 premenopausal, sexually functional women. Vaginal pulse amplitude (VPA) was assessed simultaneously. The women viewed a 3-minute erotic and an anxiety-inducing film segment with each clitoral sensor, interspersed with neutral video excerpts. In addition, they were asked to indicate their subjective level of sexual arousal during and after erotic video presentations. OUTCOMES: The main outcome measures are CPA, VPA, and subjective sexual arousal. RESULTS: The clitoral photoplethysmograph successfully detected CPA. The quality of the signals was best for the angled sensor. Main effects of the film and the interaction between the film and epoch were stronger for the clitoral than for the vaginal device. In addition, CPA followed more closely changes in intensity of sexual films than VPA. Within- and between-subject correlations between genital response and subjective sexual arousal were higher for the clitoral than for the vaginal device. CLINICAL TRANSLATION: Comparison of CPA with other genital blood flow measures in clinical samples is indicated and may contribute to improved physiological assessments of sexual response in women. STRENGTHS AND LIMITATIONS: Our sample was small and consisted of healthy volunteers. Future research could examine test-retest reliability, by including multiple recording sessions, and further explore the specificity of CPA by comparing sexual and non-sexual stimuli with positive valence. CONCLUSION: This study presents the first instrument to successfully measure CPA. Mechelmans DJ, Sachtler WL, von Wiegand TE, et al. The Successful Measurement of Clitoral Pulse Amplitude Using a New Clitoral Photoplethysmograph: A Pilot Study. J Sex Med 2020;17:1118-1125.

Riluzole induces AR degradation via endoplasmic reticulum stress pathway in androgen-dependent and castration-resistant prostate cancer cells.

BACKGROUND: Prostate cancer (PCa) is diagnosed at the highest rate of all non-cutaneous male cancers in the United States. The androgen-dependent (AD) transcription factor, androgen receptor (AR), drives PCa-but inhibiting AR or androgen biosynthesis induces remission for only a short time. At which point, patients acquire more aggressive castration-resistant (CR) disease with re-activated AR-dependent signaling. To combat treatment resistance, down-regulating AR protein expression has been considered as a potential treatment strategy for CR-PCa. METHODS: AD- and CR-PCa cell lines were treated with the well-tolerated FDA-approved oral medicine, riluzole. Expression of full-length or wild-type AR (AR-FL) and constitutively active AR-splice variant 7 (AR-V7) was assessed by immunoblotting. AR-FL/AR-V7 activity was measured using qRT-PCR of AR-target genes. Cytoplasmic [Ca2+ ] levels were measured using a fluorescent Ca2+ indicator microplate assay. Markers of the endoplasmic reticulum stress (ERS) pathway and autophagy were assessed by immunoblotting. Direct interaction between AR and selective autophagy receptor p62 was demonstrated by co-immunoprecipitation. RESULTS: We demonstrate that riluzole downregulates AR-FL, mutant ARs, and AR-V7 proteins expression by protein degradation through ERS pathway and selective autophagy. Riluzole also significantly inhibited AR transcription activity by decreasing its target genes expression (PSA, TMPRSS2, and KLK2). CONCLUSIONS: We provide key mechanistic insights by which riluzole exerts its anti-tumorigenic effects and induces AR protein degradation via ERS pathways. Our findings support the potential utility of riluzole for treatment of PCa.

Quality Enhancement Research Initiative Implementation Roadmap: Toward Sustainability of Evidence-based Practices in a Learning Health System.

BACKGROUND: Learning Health Systems strive to continuously integrate innovations and evidence-based practices (EBPs) into routine care settings. Few models provide a specified pathway to accelerate adoption and spread of EBPs across diverse settings. OBJECTIVE: The US Department of Veterans Affairs Quality Enhancement Research Initiative (QUERI) Implementation Roadmap facilitates uptake of EBPs in routine practice by aligning research and health system priorities. METHODS: The Roadmap is based on earlier iterations of the QUERI translational research pipeline, incorporating recent advancements in quality improvement and implementation science. Progressive, dynamic phases were operationalized to form an implementation process that promoted a participatory approach which enables stakeholders (health care consumers, clinicians, administrators, and leaders) to systematically plan, deploy, evaluate, and sustain EBPs using implementation strategies within a Learning Health System framework. RESULTS: The Roadmap consists of Preimplementation, Implementation, and Sustainment phases. Preimplementation identifies a high-priority need, selects EBPs to address the need, engages stakeholders to build implementation capacity, specifies needed EBP adaptions and evaluation goals, and activates leadership support. During Implementation, clinical and research leaders use implementation strategies to promote EBP technical competency and adaptive skills to motivate providers to own and sustain EBPs. Sustainment includes evaluation analyses that establish the EBP business case, and hand-off to system leadership to own EBP implementation maintenance over time. CONCLUSIONS: The QUERI Implementation Roadmap systematically guides identification, implementation, and sustainment of EBPs, demystifying implementation science for stakeholders in a Learning Health System to ensure that EBPs are more rapidly implemented into practice to improve overall consumer health.

Research Lifecycle to Increase the Substantial Real-world Impact of Research: Accelerating Innovations to Application.

BACKGROUND: US health care systems face a growing demand to incorporate innovations that improve patient outcomes at a lower cost. Funding agencies increasingly must demonstrate the impact of research investments on public health. The Learning Health System promotes continuous institutional innovation, yet specific processes to develop innovations for further research and implementation into real-world health care settings to maximize health impacts have not been specified. OBJECTIVE: We describe the Research Lifecycle and how it leverages institutional priorities to support the translation of research discoveries to clinical application, serving as a broader operational approach to enhance the Learning Health System. METHODS: Developed by the US Department of Veterans Affairs Office of Research and Development Research-to-Real-World Workgroup, the Research Lifecycle incorporates frameworks from product development, translational science, and implementation science methods. The Lifecycle is based on Workgroup recommendations to overcome barriers to more direct translation of innovations to clinical application and support practice implementation and sustainability. RESULTS: The Research Lifecycle posits 5 phases which support a seamless pathway from discovery to implementation: prioritization (leadership priority alignment), discovery (innovation development), validation (clinical, operational feasibility), scale-up and spread (implementation strategies, performance monitoring), and sustainability (business case, workforce training). An example of how the Research Lifecycle has been applied within a health system is provided. CONCLUSIONS: The Research Lifecycle aligns research and health system investments to maximize real-world practice impact via a feasible pathway, where priority-driven innovations are adapted for effective clinical use and supported through implementation strategies, leading to continuous improvement in real-world health care.

An approach for controlling the timing and order of engineered mutations in mice.

Significant advances in our understanding of normal development and disease have been facilitated by engineered mice in which genes can be altered in a spatially, temporally, or cell type restricted manner using site specific recombinase systems like Cre-loxP or Flp-frt. In many circumstances it is important to understand how interactions between multiple genes influence a given phenotype. Robust approaches for precisely controlling multiple genetic alterations independently are limited, however, thus the impact of mutation order and timing on phenotype is generally unknown. Here we describe and validate a novel Gt(ROSA)26Sor targeted transgene allowing precise control over the order and timing of multiple genetic mutations in the mouse. The transgene expresses an optimized, Flp-estrogen receptor fusion protein (Flpo-ERT2) under the control of a loxP-stop-loxP cassette. In this system, genes modified by loxP sites are altered first upon expression of Cre. Cre also eliminates the loxP-stop-loxP cassette, permitting widespread expression of Flpo-ERT2. Because of the estrogen receptor fusion, Flp activity remains inert until administration of tamoxifen, allowing genes modified by frt sites to be modified subsequently with controllable timing. This mouse transgene will be useful in a wide variety of applications where independent control of different mutations in the mouse is desirable.

Child Sexual Abuse and Negative Affect as Shared Risk Factors for Sexual Aggression and Sexual HIV Risk Behavior in Heterosexual Men.

Previous research has suggested that sexually aggressive behavior and sexual HIV risk behavior are associated. Childhood sexual abuse (CSA) is a well-established risk factor for both types of problematic sexual behavior. Negative affect (i.e., anxiety, depression, and anger) is a less well-studied risk factor, but it has been theorized to relate to both sexual aggression and HIV risk behavior. Thus, this study sought to (1) confirm the relationship between sexual aggression and HIV risk behavior, (2) establish CSA and negative affect as shared risk factors for sexual aggression and HIV risk behavior, and (3) evaluate whether negative affect mediates the relationship between CSA and sexual aggression and between CSA and HIV sexual risk in a sample of heterosexual men. We recruited 18- to 30-year-old heterosexual men (N = 377) from urban sexually transmitted infection clinics. Men completed measures of sexual HIV risk history (number of partners and condom use), sexual aggression history, CSA history, and trait negative affect (anger, anxiety, and depression). Structural equation modeling was used to examine hypothesized direct and indirect relationships. In the final SEM model, sexual aggression history and sexual HIV risk behavior were correlated. CSA was associated with both types of problematic sexual behavior. Anxiety significantly mediated the relationship between CSA and sexual aggression and between CSA and sexual HIV risk behavior (χ 2[1300] = 2121.79, p < .001; CFI = 0.905; RMSEA [90% CI] = .044 [.041-.047]). Sexual aggression appears to be part of a constellation of sexual risk behaviors; thus, it may be possible to develop prevention programs that target both sexual HIV risk and sexual aggression. CSA is a shared risk factor for sexual aggression and HIV risk behavior through the pathway of anxiety. Thus, anxiety might be one promising target for intervention.

Behavioral Treatment for Veterans with Obesity: 24-Month Weight Outcomes from the ASPIRE-VA Small Changes Randomized Trial.

BACKGROUND: Small Changes (SC) is a weight management approach that demonstrated superior 12-month outcomes compared to the existing MOVE!® Weight Management Program at two Veterans Affairs (VA) sites. However, approaches are needed to help graduates of treatment continue to lose or maintain their weight over the longer term. OBJECTIVE: The purpose of the present study was to examine the effectiveness of a second year of low-intensity SC support compared to support offered by the usual care MOVE! programs. DESIGN: Following participation in the year-long Aspiring to Lifelong Health in VA (ASPIRE-VA) randomized controlled trial, participants were invited to extend their participation in their assigned program for another year. Three programs were extended to include six SC sessions delivered via telephone (ASPIRE-Phone) or an in-person group (ASPIRE-Group), or 12 sessions offered by the MOVE! programs. PARTICIPANTS: Three hundred thirty-two overweight/obese veterans who consented to extend their participation in the ASPIRE-VA trial by an additional year. MAIN MEASURES: Twenty-four-month weight change (kg). KEY RESULTS: Twenty-four months after baseline, participants in all three groups had modest weight loss (-1.40 kg [-2.61 to -0.18] in the ASPIRE-Group, -2.13 kg [-3.43 to -0.83] in ASPIRE-Phone, and -1.78 kg [-3.07 to -0.49] in MOVE!), with no significant differences among the three groups. Exploratory post hoc analyses revealed that participants diagnosed with diabetes initially benefited from the ASPIRE-Group program (-2.6 kg [-4.37 to 0.83]), but experienced significant weight regain during the second year (+2.8 kg [0.92-4.69]) compared to those without diabetes. CONCLUSIONS: Participants in all three programs lost weight and maintained a statistically significant, though clinically modest, amount of weight loss over a 24-month period. Although participants in the ASPIRE-Group initially had greater weight loss, treatment was not sufficient to sustain weight loss through the second year, particularly in veterans with diabetes. Consistent, continuous-care treatment is needed to address obesity in the VA.

Implementing Effective Policy in a National Mental Health Reengagement Program for Veterans.

Policy is a powerful motivator of clinical change, but implementation success can depend on organizational characteristics. This article used validated measures of organizational resources, culture, and climate to predict uptake of a nationwide Veteran's Health Administration (VA) policy aimed at implementing Re-Engage, a brief care management program that reestablishes contact with veterans with serious mental illness lost to care. Patient care databases were used to identify 2738 veterans lost to care. Local recovery coordinators (LRCs) were to update disposition for 2738 veterans at 158 VA facilities and, as appropriate, facilitate a return to care. Multivariable regression was used to assess organizational culture and climate as predictors of early policy compliance (via LRC presence) and uptake at 6 months. Higher composite climate and culture scores were associated with higher odds of having a designated LRC but were not predictive of higher uptake. Sites with LRCs had significantly higher rates of updated documentation than sites without LRCs.

Military and veteran health behavior research and practice: challenges and opportunities.

There are 2.1 million current military servicemembers and 21 million living veterans in the United States. Although they were healthier upon entering military service compared to the general U.S. population, in the longer term veterans tend to be of equivalent or worse health than civilians. One primary explanation for the veterans' health disparity is poorer health behaviors during or after military service, especially areas of physical activity, nutrition, tobacco, and alcohol. In response, the Department of Defense and Department of Veterans Affairs continue to develop, evaluate, and improve health promotion programs and healthcare services for military and veteran health behavior in an integrated approach. Future research and practice is needed to better understand and promote positive health behavior during key transition periods in the military and veteran life course. Also paramount is implementation and evaluation of existing interventions, programs, and policies across the population using an integrated and person centered approach.

Surface Soil Moisture Retrieval Using the L-Band Synthetic Aperture Radar Onboard the Soil Moisture Active-Passive Satellite and Evaluation at Core Validation Sites.

This paper evaluates the retrieval of soil moisture in the top 5-cm layer at 3-km spatial resolution using L-band dual-copolarized Soil Moisture Active-Passive (SMAP) synthetic aperture radar (SAR) data that mapped the globe every three days from mid-April to early July, 2015. Surface soil moisture retrievals using radar observations have been challenging in the past due to complicating factors of surface roughness and vegetation scattering. Here, physically based forward models of radar scattering for individual vegetation types are inverted using a time-series approach to retrieve soil moisture while correcting for the effects of static roughness and dynamic vegetation. Compared with the past studies in homogeneous field scales, this paper performs a stringent test with the satellite data in the presence of terrain slope, subpixel heterogeneity, and vegetation growth. The retrieval process also addresses any deficiencies in the forward model by removing any time-averaged bias between model and observations and by adjusting the strength of vegetation contributions. The retrievals are assessed at 14 core validation sites representing a wide range of global soil and vegetation conditions over grass, pasture, shrub, woody savanna, corn, wheat, and soybean fields. The predictions of the forward models used agree with SMAP measurements to within 0.5 dB unbiased-root-mean-square error (ubRMSE) and -0.05 dB (bias) for both copolarizations. Soil moisture retrievals have an accuracy of 0.052 m3/m3 ubRMSE, -0.015 m3/m3 bias, and a correlation of 0.50, compared to in situ measurements, thus meeting the accuracy target of 0.06 m3/m3 ubRMSE. The successful retrieval demonstrates the feasibility of a physically based time series retrieval with L-band SAR data for characterizing soil moisture over diverse conditions of soil moisture, surface roughness, and vegetation.

Evaluation of comprehensive chromosome screening platforms for the detection of mosaic segmental aneuploidy.

PURPOSE: A subset of preimplantation embryos identified as euploid may in fact possess both whole and sub-chromosomal mosaicism, raising concerns regarding the predictive value of current comprehensive chromosome screening (CCS) methods utilizing a single biopsy. Current CCS methods may be capable of detecting sub-chromosomal mosaicism in a trophectoderm biopsy by examining intermediate levels of segmental aneuploidy within a biopsy. This study evaluates the sensitivity and specificity of segmental aneuploidy detection by three commercially available CCS platforms utilizing a cell line mixture model of segmental mosaicism in a six-cell trophectoderm biopsy. METHODS: Two cell lines with known karyotypes were obtained and mixed together at specific ratios of six total cells (0:6, 1:5, 2:4, 3:3, 4:2, 5:1, and 6:0). A female cell line containing a 16.2 Mb deletion on chromosome 5 and a male cell line containing a 25.5 Mb deletion on chromosome 4 were used to create mixtures at each level. Six replicates of each mixture were prepared, randomized, and blinded for analysis by one of the three CCS platforms (SNP-array, VeriSeq NGS, or NexCCS). Sensitivity and specificity of segmental aneuploidy at each level of mosaicism was determined and compared between each platform. Additionally, an alternative VeriSeq NGS analysis method utilizing previously published criteria was evaluated. RESULTS: Examination of the default settings of each platform revealed that the sensitivity was significantly different between NexCCS and SNP up to 50% mosaicism, custom VeriSeq, and SNP-array up to 66% mosaicism, and between NexCCS and custom VeriSeq up to 50% mosaicism. However, no statistical difference was observed in mixtures with >50% mosaicism with any platform. No comparison was made between default VeriSeq, as it does not report segmental imbalances. Furthermore, while the use of previously published criteria for VeriSeq NGS significantly increased sensitivity at low levels of mosaicism, a significant decrease in specificity was observed (66% false positive prediction of segmental aneuploidy). CONCLUSION: These results demonstrate the potential of NGS-based detection methods to detect segmental mosaicism within a biopsy. However, these data also demonstrate that a balance between sensitivity and specificity should be more carefully considered. These results emphasize the importance of vigorous preclinical evaluation of new testing criteria prior to clinical implementation providing a point of departure for further algorithm development and improved detection of mosaicism within preimplantation embryos.

Generation of a C57BL/6 MYC-Driven Mouse Model and Cell Line of Prostate Cancer.

INTRODUCTION: Transgenic mouse modeling is a favorable tool to reflect human prostate tumorigenesis and interactions between prostate cancer and the microenvironment. The use of GEMMs and derived cell lines represent powerful tools to study prostate cancer initiation and progression with an associated tumor microenvironment. Notably, such models provide the capacity for rapid preclinical therapy studies including immune therapies for prostate cancer treatment. METHODS: Backcrossing FVB Hi-MYC mice with C57BL/6N mice, we established a Hi-MYC transgenic mouse model on a C57BL/6 background (B6MYC). In addition, using a conditional reprogramming method, a novel C57BL/6 MYC driven prostate adenocarcinoma cell line was generated. RESULTS: Our results demonstrate that disease progression is significantly delayed in B6MYC when compared to their FVB counterparts. Current data also indicates infiltrating immune cells are present in pre-cancer lesions, prostate intraepithelial neoplasia (PIN). Further, immunophenotyping of this immune infiltrate demonstrates the predominant population as myeloid-derived suppressor cells (MDSC). Also, we successfully generated a B6MYC-CaP cell line, and determined that this new PCa cell line express markers of luminal epithelial lineage. DISCUSSION: This novel model of PCa provides a new platform to understand the cross talk between MYC driven prostate cancer and the microenvironment. Importantly, these models will be an ideal tool to support the clinical development of immunotherapy as well as other novel therapeutic strategies for prostate cancer treatment. Prostate 76:1192-1202, 2016. © 2016 Wiley Periodicals, Inc.

Long-Term Effects of an Internet-Mediated Pedometer-Based Walking Program for Chronic Obstructive Pulmonary Disease: Randomized Controlled Trial.

BACKGROUND: Regular physical activity (PA) is recommended for persons with chronic obstructive pulmonary disease (COPD). Interventions that promote PA and sustain long-term adherence to PA are needed. OBJECTIVE: We examined the effects of an Internet-mediated, pedometer-based walking intervention, called Taking Healthy Steps, at 12 months. METHODS: Veterans with COPD (N=239) were randomized in a 2:1 ratio to the intervention or wait-list control. During the first 4 months, participants in the intervention group were instructed to wear the pedometer every day, upload daily step counts at least once a week, and were provided access to a website with four key components: individualized goal setting, iterative feedback, educational and motivational content, and an online community forum. The subsequent 8-month maintenance phase was the same except that participants no longer received new educational content. Participants randomized to the wait-list control group were instructed to wear the pedometer, but they did not receive step-count goals or instructions to increase PA. The primary outcome was health-related quality of life (HRQL) assessed by the St George's Respiratory Questionnaire Total Score (SGRQ-TS); the secondary outcome was daily step count. Linear mixed-effect models assessed the effect of intervention over time. One participant was excluded from the analysis because he was an outlier. Within the intervention group, we assessed pedometer adherence and website engagement by examining percent of days with valid step-count data, number of log-ins to the website each month, use of the online community forum, and responses to a structured survey. RESULTS: Participants were 93.7% male (223/238) with a mean age of 67 (SD 9) years. At 12 months, there were no significant between-group differences in SGRQ-TS or daily step count. Between-group difference in daily step count was maximal and statistically significant at month 4 (P<.001), but approached zero in months 8-12. Within the intervention group, mean 76.7% (SD 29.5) of 366 days had valid step-count data, which decreased over the months of study (P<.001). Mean number of log-ins to the website each month also significantly decreased over the months of study (P<.001). The online community forum was used at least once during the study by 83.8% (129/154) of participants. Responses to questions assessing participants' goal commitment and intervention engagement were not significantly different at 12 months compared to 4 months. CONCLUSIONS: An Internet-mediated, pedometer-based PA intervention, although efficacious at 4 months, does not maintain improvements in HRQL and daily step counts at 12 months. Waning pedometer adherence and website engagement by the intervention group were observed. Future efforts should focus on improving features of PA interventions to promote long-term behavior change and sustain engagement in PA. CLINICALTRIAL: Clinicaltrials.gov NCT01102777; https://clinicaltrials.gov/ct2/show/NCT01102777 (Archived by WebCite at http://www.webcitation.org/6iyNP9KUC).

A randomized and blinded comparison of qPCR and NGS-based detection of aneuploidy in a cell line mixture model of blastocyst biopsy mosaicism.

PURPOSE: A subset of preimplantation stage embryos may possess mosaicism of chromosomal constitution, representing a possible limitation to the clinical predictive value of comprehensive chromosome screening (CCS) from a single biopsy. However, contemporary methods of CCS may be capable of predicting mosaicism in the blastocyst by detecting intermediate levels of aneuploidy within a trophectoderm biopsy. This study evaluates the sensitivity and specificity of aneuploidy detection by two CCS platforms using a cell line mixture model of a mosaic trophectoderm biopsy. METHODS: Four cell lines with known karyotypes were obtained and mixed together at specific ratios of six total cells (0:6, 1:5, 2:4, 3:3, 4:2, 5:1, and 6:0). A female euploid and a male trisomy 18 cell line were used for one set, and a male trisomy 13 and a male trisomy 15 cell line were used for another. Replicates of each mixture were prepared, randomized, and blinded for analysis by one of two CCS platforms (quantitative polymerase chain reaction (qPCR) or VeriSeq next-generation sequencing (NGS)). Sensitivity and specificity of aneuploidy detection at each level of mosaicism was determined and compared between platforms. RESULTS: With the default settings for each platform, the sensitivity of qPCR and NGS were not statistically different, and 100 % specificity was observed (no false positives) at all levels of mosaicism. However, the use of previously published custom criteria for NGS increased sensitivity but also significantly decreased specificity (33 % false-positive prediction of aneuploidy). CONCLUSIONS: By demonstrating increased false-positive diagnoses when reducing the stringency of predicting an abnormality, these data illustrate the importance of preclinical evaluation of new testing paradigms before clinical implementation.