Protocol for Delivery of CRISPR/dCas9 Systems for Epigenetic Editing into Solid Tumors Using Lipid Nanoparticles Encapsulating RNA.

Genome editing tools, particularly the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems (e.g., CRISPR/Cas9), and their repurposing into epigenetic editing platforms, offer enormous potential as safe and customizable therapies for cancer. Specifically, various transcriptional abnormalities in human malignancies, such as silencing of tumor suppressors and ectopic re-expression of oncogenes, have been successfully targeted with virtually no off-target effects using CRISPR activation and repression systems. In these systems, the nuclease-deactivated Cas9 protein (dCas9) is fused to one or more domains inducing selective activation or repression of the targeted genes. Despite these advances, the efficient in vivo delivery of these molecules into the target cancer cells represents a critical barrier to accomplishing translation into a clinical therapy setting for cancer. Major obstacles include the large size of dCas9 fusion proteins, the necessity of multimodal delivery of protein and gRNAs, and the potential of these formulations to elicit detrimental immune responses.In this context, viral methods for delivering CRISPR face several limitations, such as the packaging capacity of the viral genome, the potential for integration of the nucleic acids into the host cells genome, and immunogenicity of viral proteins, posing serious safety concerns. The rapid development of mRNA vaccines in response to the COVID-19 pandemic has rekindled interest in mRNA-based approaches for CRISPR/dCas9 delivery. Simultaneously, due to their high loading capacity, scalability, customizable surface modification for cell targeting, and low immunogenicity, lipid nanoparticles (LNPs) have been widely explored as nonviral vectors. In this chapter, we first describe the design of optimized dCas9-effector mRNAs and gRNAs for epigenetic editing. We outline formulations of LNPs suitable for dCas9 mRNA delivery. Additionally, we provide a protocol for the co-encapsulation of the dCas9-effector mRNAs and gRNA into these LNPs, along with detailed methods for delivering these formulations to both cell lines (in vitro) and mouse models of breast cancer (in vivo).

Financial Distress in Genitourinary Cancer: Insights From CDC National Health Interview Survey.

PURPOSE: This study leverages CDC National Health Interview Survey data to examine Financial Distress (FD) among genitourinary (GU) cancer survivors, specifically prostate cancer (PC), kidney cancer (KC), and bladder cancer (BC). It investigates the economic impacts faced by these patients, especially in relation to disparities in insurance coverage and its effects on material, psychological, and behavioral aspects of FD. METHODS: We retrospectively analyzed responses from GU cancer survivors, stratifying by cancer status and age (18-64 years, ≥65 years). Medical financial hardship was divided into three domains: material, psychological, and behavioral. Associations between cancer history, hardship, and clinical factors were assessed using generalized ordinal logistic regressions. RESULTS: Significant health care access disparities were found, particularly for mental health services, with 25% of younger BC survivors and 4.7% of younger KC survivors reporting affordability issues, in contrast to 2.7% of noncancer individuals. Dental care was also problematic, with higher avoidance rates among younger BC (27%) and KC (15%) survivors compared with the general population. Surprisingly, noncancer individuals reported more difficulty in affording prescriptions than BC survivors across both age groups. PC survivors, however, showed lower FD across all domains versus noncancer controls, indicating fewer concerns about medical bills and a lesser tendency to forgo care. CONCLUSION: The study underscores significant gaps in the financial support system for GU cancer survivors, with urgent needs in mental and dental health care access. Policy interventions, including comprehensive insurance reforms, are imperative to alleviate the financial burdens on these individuals.

Superstition in Surgery: A Population-Based Cohort Study to Assess the Association Between Surgery on Friday the 13th and Postoperative Outcomes.

Objective: We sought to examine whether the outcomes of patients who receive a surgical procedure on Friday the 13th differ from patients who receive surgery on flanking Fridays. Background: Numerous studies have demonstrated that increased anxiety from the provider or patient around the time of surgery can lead to worse outcomes. Superstitious patients often express significant concern and anxiety when undergoing a surgical procedure on Friday the 13th. Methods: A retrospective, population-based cohort study of 19,747 adults undergoing 1 of 25 common surgical procedures on Friday the 13th or flanking control Fridays (Friday the 6th and Friday the 20th) between January 1, 2007, and December 31, 2019, with 1 year of follow-up. The main outcomes included death, readmission, and complications at 30 days (short-term), 90 days (intermediate-term), and 1 year (long-term). Results: A total of 7,349 (37.2%) underwent surgery on Friday the 13th, and 12,398 (62.8%) underwent surgery on a flanking Friday during the study period. Patient characteristics were similar between the 2 groups. We found no evidence that patients receiving surgery on Friday the 13th group were more likely to experience the composite primary outcome at 30 days [adjusted odds ratio (aOR) = 1.02 (95% CI = 0.94-1.09)], 90 days [aOR = 0.97 (95% CI = 0.90-1.04)], and 1 year [aOR = 0.99 (95% CI = 0.94-1.04)] after surgery. Conclusion: Patients receiving surgery on Friday the 13th do not appear to fare worse than those treated on ordinary Fridays with respect to the composite outcome.

Development of an effective predictive screening tool for prostate cancer using the ClarityDX machine learning platform.

The current prostate cancer (PCa) screen test, prostate-specific antigen (PSA), has a high sensitivity for PCa but low specificity for high-risk, clinically significant PCa (csPCa), resulting in overdiagnosis and overtreatment of non-csPCa. Early identification of csPCa while avoiding unnecessary biopsies in men with non-csPCa is challenging. We built an optimized machine learning platform (ClarityDX) and showed its utility in generating models predicting csPCa. Integrating the ClarityDX platform with blood-based biomarkers for clinically significant PCa and clinical biomarker data from a 3448-patient cohort, we developed a test to stratify patients' risk of csPCa; called ClarityDX Prostate. When predicting high risk cancer in the validation cohort, ClarityDX Prostate showed 95% sensitivity, 35% specificity, 54% positive predictive value, and 91% negative predictive value, at a ≥ 25% threshold. Using ClarityDX Prostate at this threshold could avoid up to 35% of unnecessary prostate biopsies. ClarityDX Prostate showed higher accuracy for predicting the risk of csPCa than PSA alone and the tested model-based risk calculators. Using this test as a reflex test in men with elevated PSA levels may help patients and their healthcare providers decide if a prostate biopsy is necessary.

Chemotherapy, immunotherapy, or combination first-line treatment for metastatic urothelial carcinoma of the bladder: A large real-world experience.

INTRODUCTION: First-line systemic therapy for metastatic urothelial carcinoma of the bladder (mUC) consists of platinum-based chemotherapy in most patients and PD1/L1 inhibitors in selected patients. Multiple combination chemoimmunotherapy trials failed to show a clear benefit over chemotherapy alone. We used real-world data to evaluate clinical and sociodemographic factors associated with receipt of first-line chemotherapy, immunotherapy, or combination chemoimmunotherapy treatment for metastatic bladder cancer and examined differences in overall survival (OS). MATERIALS AND METHODS: We used the National Cancer Database to identify patients with stage IV mUC diagnosed between 2014 and 2018, who were treated with first-line immunotherapy, chemotherapy, or combination treatment. We performed multivariable logistic regression modeling to determine factors associated with treatment receipt Adjusted Kaplan-Meier survival analysis and multivariable Cox proportional hazards regression were used to evaluate the association between treatment and OS. RESULTS: In our cohort of 4,169 patients, multivariable analysis identified increasing age (RRR: 1.07, 95%CI, 1.06-1.08) and comorbidity burden (, as independent predictors of receiving immunotherapy. Treatment at an academic facility was associated with increased likelihood of combination treatment (RRR: 1.29, 95%CI, 1.01-1.65). After IPTW, we found that combination therapy (hazard ratio [HR]: 0.72; 95%CI, 0.62-0.83) was associated with improved survival compared to chemotherapy. CONCLUSIONS: Patients with older age and more comorbidities were more likely to receive immunotherapy than chemotherapy for first-line treatment of metastatic urothelial carcinoma of the bladder. Utilization of chemoimmunotherapy was observed to be higher in academic centers and was associated with improved survival compared to chemotherapy.

Comparison of Postoperative outcomes Among Patients Treated by Male Versus Female Surgeons: A Systematic Review and Meta-analysis.

OBJECTIVE: To compare clinical outcomes of patients treated by female surgeons versus those treated by male surgeons. SUMMARY BACKGROUND DATA: It remains unclear as to whether surgical performance and outcomes differ between female and male surgeons. METHODS: We conducted a meta-analysis to compare patients' clinical outcomes-including patients' postoperative mortality, readmission, and complication rates-between female versus male surgeons. MEDLINE, Embase, CENTRAL, ICTRP, and ClinicalTrials.gov were searched from inception to September 8, 2022. The update search was conducted on July 19, 2023. We used random-effects models to synthesize data and GRADE to evaluate the certainty. RESULTS: A total of 15 retrospective cohort studies provided data on 5,448,121 participants. We found that patients treated by female surgeons experienced a lower post-operative mortality compared with patients treated by male surgeons (8 studies; adjusted odds ratio [aOR], 0.93; 95%CI, 0.88 - 0.97; I2=27%; moderate certainty of the evidence). We found a similar pattern for both elective and non-elective (emergent or urgent) surgeries, although the difference was larger for elective surgeries (test for subgroup difference P=0.003). We found no evidence that female and male surgeons differed for patient readmission (3 studies; aOR, 1.20; 95%CI, 0.83 - 1.74; I2=92%; very low certainty of the evidence) or complication rates (8 studies; aOR, 0.94; 95%CI, 0.88 - 1.01: I2=38%; very low certainty of the evidence). CONCLUSIONS: This systematic review and meta-analysis suggests that patients treated by female surgeons have a lower mortality compared with those treated by male surgeons.

Contemporary morbidity and mortality of open versus robotic cystectomy for bladder cancer: An analysis of the National Surgical Quality Improvement Program (NSQIP) procedure targeted cystectomy database.

OBJECTIVES: To evaluate the association between surgical modality (RARC vs. ORC) and the risk of 30-day complications. MATERIALS AND METHODS: We utilized the American College of Surgeons-National Surgical Quality Improvement Program (ACS-NSQIP) Cystectomy-Targeted database from 2019 to 2021. The primary outcome was a composite of major complications including 30-day mortality, reoperation, cardiac events, and stroke. Secondary outcomes included individual major and cystectomy-specific complications. Propensity score matching (PSM) was employed to minimize inherent differences within our cohort. We performed logistic regression to assess the association between outcomes of interest and operative modality. RESULTS: We found no difference between operative modality and the primary outcome, however, RARC was associated with a 70% lower risk of 30-day mortality (OR 0.30, 95% CI 0.13-0.70) and had favorable outcomes with respect to respiratory, deep venous thrombosis, wound complications, and length of stay. Limitations are related to residual confounding given the observational methodology. CONCLUSIONS: RARC was associated with reduced risk of multiple 30-day complications, including mortality, as well as organ system and cystectomy-specific outcomes. These data support the clinical benefit of increased adoption of RARC.

Prostate Radiotherapy in Low-volume Metastatic Hormone-sensitive Prostate Cancer: A Network Meta-analysis.

BACKGROUND AND OBJECTIVE: The utility of prostate radiotherapy (RT) is unclear in men with metastatic hormone-sensitive prostate cancer (mHSPC) receiving intensified systemic therapy with androgen deprivation therapy (ADT) and androgen receptor pathway inhibitors (ARPIs). We performed a network meta-analysis of randomized controlled trials (RCTs) to investigate the role of prostate RT in low-volume mHSPC. METHODS: Bibliographic databases and conference proceedings were searched through July 2023 for RCTs evaluating the addition of ARPIs or prostate RT to standard of care (SOC) systemic therapy, defined as ADT or ADT plus docetaxel, for the initial treatment of mHSPC. We focused exclusively on aggregate data from the low-volume mHSPC subpopulation in these trials. We pooled the treatment arms into four groups: SOC, SOC plus ARPI, SOC plus RT, and SOC plus ARPI plus RT. The primary outcome was overall survival (OS). To compare treatment strategies, a fixed-effects Bayesian network meta-analysis was undertaken, while a Bayesian network meta-regression was performed to account for across-trial differences in docetaxel use as part of SOC and in proportions of patients with de novo presentation. KEY FINDINGS AND LIMITATIONS: Ten RCTs comprising 4423 patients were eligible. The Surface Under the Cumulative Ranking Curve scores were 0.0006, 0.45, 0.62, and 0.94 for SOC, SOC plus RT, SOC plus ARPI, and SOC plus ARPI plus RT, respectively. On a meta-regression, in a population with de novo mHSPC and no docetaxel use, we did not find sufficient evidence of a difference in OS between SOC plus ARPI plus RT versus SOC plus ARPI (hazard ratio [HR]: 0.76; 95% credible interval: 0.51-1.16) and SOC plus RT versus SOC plus ARPI (HR: 1.10; 95% credible interval: 0.92-1.42). CONCLUSIONS AND CLINICAL IMPLICATIONS: There was some evidence that SOC plus ARPI plus RT reduced mortality compared with the next best strategy of SOC plus ARPI in patients with low-volume de novo mHSPC. A meta-analysis with individual patient data or an RCT is needed to confirm these findings.

Pre-inoculation water deficit effects on grapevine physiology, Xylella fastidiosa titers, and Pierce's disease progression.

Drought and Pierce's disease are common throughout many grapevine-growing regions such as Mexico and the United States. Yet, how ongoing water deficits affect infections of Xylella fastidiosa, the causal agent of Pierce's disease, is poorly understood. Symptoms were observed to be significantly more severe in water-stressed plants one month after X. fastidiosa inoculation, and, in one experiment, titers were significantly lower in water-stressed than well-watered grapevines. Host chemistry examinations revealed overall amino acid and phenolic levels did not statistically differ due to water deficits, but sugar levels were significantly greater in water stressed than well-watered plants. Results highlight the need to especially manage Pierce's disease spread in grapevines experiencing drought.

Predicting Peak Productivity in Urologic Medicare Practice via Work-Relative Value Units.

OBJECTIVE: To explore factors associated with productivity in urologic practice. Work-relative value units (wRVUs), the basis for Center for Medicare & Medicaid Services (CMS) and private payer reimbursements, commonly serve to estimate physician productivity. Limited data describes which practice factors predict increased wRVU productivity. METHODS: The 2017 and 2018 CMS databases were retrospectively queried for urologic Medicare provider demographics and procedural/service details. Medical school graduation year was used to estimate years in practice and generation (Millennial, Gen X, Baby Boomer, or Post-War). Treated patients' demographics were obtained. Adjusted and unadjusted linear mixed models were performed to predict wRVU production. RESULTS: Included were 6773 Medicare-participating urologists across the United States. Millennials produced 1115 wRVUs per year, while Gen X and Baby Boomers produced significantly more (1997 and 2104, respectively, P <.01). Post-War urologists produced numerically more (1287, P = .88). In adjusted analyses, predictors of Medicare wRVU productivity included female and pelvic medicine and reconstructive surgery (exponentiated beta estimate (ß) 1.46, 95% CI 1.32-1.60), men's health (ß 1.22, 95% CI 1.13-1.32), and oncologic subspecialization (ß 1.08, 95% CI 1.02-1.14), female gender (ß 0.87, 95% CI 0.82-0.92), wRVUs generated from inpatient procedures (ß 1.08, 95% CI 1.06-1.09) and office visits (ß 0.88, 95% CI 0.87-0.89), and the level of education (ß 1.10, 95% CI 1.07-1.14) and percent impoverished patients (ß 0.85, 95% CI 0.83-0.88) in provider's practice zip code. CONCLUSION: Urologic experience, specialization, demographics, practice patterns, and patient demographics are significantly associated with wRVU productivity in Medicare settings. Further work should incorporate quality metrics into wRVUs and ensure patient demographics do not affect reimbursement.

Days alive and out of hospital for adult female and male cardiac surgery patients: a population-based cohort study.

BACKGROUND: Research shows women experience higher mortality than men after cardiac surgery but information on sex-differences during postoperative recovery is limited. Days alive and out of hospital (DAH) combines death, readmission and length of stay, and may better quantify sex-differences during recovery. This main objective is to evaluate (i) how DAH at 30-days varies between sex and surgical procedure, (ii) DAH responsiveness to patient and surgical complexity, and (iii) longer-term prognostic value of DAH. METHODS: We evaluated 111,430 patients (26% female) who underwent one of three types of cardiac surgery (isolated coronary artery bypass [CABG], isolated non-CABG, combination procedures) between 2009 - 2019. Primary outcome was DAH at 30 days (DAH30), secondary outcomes were DAH at 90 days (DAH90) and 180 days (DAH180). Data were stratified by sex and surgical group. Unadjusted and risk-adjusted analyses were conducted to determine the association of DAH with patient-, surgery-, and hospital-level characteristics. Patients were divided into two groups (below and above the 10th percentile) based on the number of days at DAH30. Proportion of patients below the 10th percentile at DAH30 that remained in this group at DAH90 and DAH180 were determined. RESULTS: DAH30 were lower for women compared to men (22 vs. 23 days), and seen across all surgical groups (isolated CABG 23 vs. 24, isolated non-CABG 22 vs. 23, combined surgeries 19 vs. 21 days). Clinical risk factors including multimorbidity, socioeconomic status and surgical complexity were associated with lower DAH30 values, but women showed lower values of DAH30 compared to men for many factors. Among patients in the lowest 10th percentile at DAH30, 80% of both females and males remained in the lowest 10th percentile at 90 days, while 72% of females and 76% males remained in that percentile at 180 days. CONCLUSION: DAH is a responsive outcome to differences in patient and surgical risk factors. Further research is needed to identify new care pathways to reduce disparities in outcomes between male and female patients.

Characterization of data observing Meloidogyne incognita, Neofusicoccum parvum, and Xylella fastidiosa infection effects on development of grapevine phenolic compound levels and resistance to subsequent Neofusicoccum parvum infections.

Grapevines encounter many different pathogens throughout their lifespans, including the bacterial pathogen Xylella fastidiosa, which causes Pierce's disease that results in vascular occlusion and eventual plant host death, the fungal pathogen Neofusicoccum parvum, which causes stem cankers that kill individual vines and reduce fruit yields, and the root knot nematode Meloidogyne incognita, which destroys root tissues that impacts host vigour. To date, little research has been conducted to examine how one infection could impact subsequent infections by the same or different pathogens despite this is important to ensure healthy vineyards. Therefore, grapevines initially infected with either X. fastidiosa, N. parvum, or M. incognita were subsequently infected with N. parvum eight weeks later to observe developing lesion lengths, which were assessed to determine grapevine resistance to infections. Collected data shows that when prior infections were present, the N. parvum lesions lengths were smaller. This suggests grapevines had induced resistance to combat infections. Further, defence-associated phenolics were measured by high-performance liquid chromatography to determine roles in observed resistance to the secondary N. parvum infections. Data shows that of the different phenolics examined, only stilbenoids were different due to infections, with lowered levels observed in plants that were infected compared with non-infected controls. These data provide insight into how infections by different pathogens could impact grapevine host resistance to new, subsequent pathogen infections.

Functional Outcomes After Localized Prostate Cancer Treatment.

Importance: Adverse outcomes associated with treatments for localized prostate cancer remain unclear. Objective: To compare rates of adverse functional outcomes between specific treatments for localized prostate cancer. Design, Setting, and Participants: An observational cohort study using data from 5 US Surveillance, Epidemiology, and End Results Program registries. Participants were treated for localized prostate cancer between 2011 and 2012. At baseline, 1877 had favorable-prognosis prostate cancer (defined as cT1-cT2bN0M0, prostate-specific antigen level <20 ng/mL, and grade group 1-2) and 568 had unfavorable-prognosis prostate cancer (defined as cT2cN0M0, prostate-specific antigen level of 20-50 ng/mL, or grade group 3-5). Follow-up data were collected by questionnaire through February 1, 2022. Exposures: Radical prostatectomy (n = 1043), external beam radiotherapy (n = 359), brachytherapy (n = 96), or active surveillance (n = 379) for favorable-prognosis disease and radical prostatectomy (n = 362) or external beam radiotherapy with androgen deprivation therapy (n = 206) for unfavorable-prognosis disease. Main Outcomes and Measures: Outcomes were patient-reported sexual, urinary, bowel, and hormone function measured using the 26-item Expanded Prostate Cancer Index Composite (range, 0-100; 100 = best). Associations of specific therapies with each outcome were estimated and compared at 10 years after treatment, adjusting for corresponding baseline scores, and patient and tumor characteristics. Minimum clinically important differences were 10 to 12 for sexual function, 6 to 9 for urinary incontinence, 5 to 7 for urinary irritation, and 4 to 6 for bowel and hormone function. Results: A total of 2445 patients with localized prostate cancer (median age, 64 years; 14% Black, 8% Hispanic) were included and followed up for a median of 9.5 years. Among 1877 patients with favorable prognosis, radical prostatectomy was associated with worse urinary incontinence (adjusted mean difference, -12.1 [95% CI, -16.2 to -8.0]), but not worse sexual function (adjusted mean difference, -7.2 [95% CI, -12.3 to -2.0]), compared with active surveillance. Among 568 patients with unfavorable prognosis, radical prostatectomy was associated with worse urinary incontinence (adjusted mean difference, -26.6 [95% CI, -35.0 to -18.2]), but not worse sexual function (adjusted mean difference, -1.4 [95% CI, -11.1 to 8.3), compared with external beam radiotherapy with androgen deprivation therapy. Among patients with unfavorable prognosis, external beam radiotherapy with androgen deprivation therapy was associated with worse bowel (adjusted mean difference, -4.9 [95% CI, -9.2 to -0.7]) and hormone (adjusted mean difference, -4.9 [95% CI, -9.5 to -0.3]) function compared with radical prostatectomy. Conclusions and Relevance: Among patients treated for localized prostate cancer, radical prostatectomy was associated with worse urinary incontinence but not worse sexual function at 10-year follow-up compared with radiotherapy or surveillance among people with more favorable prognosis and compared with radiotherapy for those with unfavorable prognosis. Among men with unfavorable-prognosis disease, external beam radiotherapy with androgen deprivation therapy was associated with worse bowel and hormone function at 10-year follow-up compared with radical prostatectomy.