Preformed Vesicle Approach to LNP Manufacturing Enhances Retinal mRNA Delivery.

Complete encapsulation of nucleic acids by lipid-based nanoparticles (LNPs) is often thought to be one of the main prerequisites for successful nucleic acid delivery, as the lipid environment protects mRNA from degradation by external nucleases and assists in initiating delivery processes. However, delivery of mRNA via a preformed vesicle approach (PFV-LNPs) defies this precondition. Unlike traditional LNPs, PFV-LNPs are formed via a solvent-free mixing process, leading to a superficial mRNA localization. While demonstrating low encapsulation efficiency in the RiboGreen assay, PFV-LNPs improved delivery of mRNA to the retina by up to 50% compared to the LNP analogs across several benchmark formulations, suggesting the utility of this approach regardless of the lipid composition. Successful mRNA and gene editors' delivery is observed in the retinal pigment epithelium and photoreceptors and validated in mice, non-human primates, and human retinal organoids. Deploying PFV-LNPs in gene editing experiments result in a similar extent of gene editing compared to analogous LNP (up to 3% on genomic level) in the Ai9 reporter mouse model; but, remarkably, retinal tolerability is significantly improved for PFV-LNP treatment. The study findings indicate that the LNP formulation process can greatly influence mRNA transfection and gene editing outcomes, improving LNP treatment safety without sacrificing efficacy.

Lipid nanoparticles with PEG-variant surface modifications mediate genome editing in the mouse retina.

Ocular delivery of lipid nanoparticle (LNPs) packaged mRNA can enable efficient gene delivery and editing. We generated LNP variants through the inclusion of positively charged-amine-modified polyethylene glycol (PEG)-lipids (LNPa), negatively charged-carboxyl-(LNPz) and carboxy-ester (LNPx) modified PEG-lipids, and neutral unmodified PEG-lipids (LNP). Subretinal injections of LNPa containing Cre mRNA in the mouse show tdTomato signal in the retinal pigmented epithelium (RPE) like conventional LNPs. Unexpectedly, LNPx and LNPz show 27% and 16% photoreceptor transfection, respectively, with striking localization extending from the photoreceptor synaptic pedicle to the outer segments, displaying pan-retinal distribution in the photoreceptors and RPE. LNPx containing Cas9 mRNA and sgAi9 leads to the formation of an oval elongated structure with a neutral charge resulting in 16.4% editing restricted to RPE. Surface modifications of LNPs with PEG variants can alter cellular tropism of mRNA. LNPs enable genome editing in the retina and in the future can be used to correct genetic mutations that lead to blindness.

Environmental contamination and evaluation of healthcare-associated SARS-CoV-2 transmission risk in temporary isolation wards during the COVID-19 pandemic.

BACKGROUND: Temporary isolation wards have been introduced to meet demands for airborne-infection-isolation-rooms (AIIRs) during the COVID-19 pandemic. Environmental sampling and outbreak investigation was conducted in temporary isolation wards converted from general wards and/or prefabricated containers, in order to evaluate the ability of such temporary isolation wards to safely manage COVID-19 cases over a period of sustained use. METHODS: Environmental sampling for SARS-CoV-2 RNA was conducted in temporary isolation ward rooms constructed from pre-fabricated containers (N = 20) or converted from normal-pressure general wards (N = 47). Whole genome sequencing (WGS) was utilized to ascertain health care-associated transmission when clusters were reported amongst HCWs working in isolation areas from July 2020 to December 2021. RESULTS: A total of 355 environmental swabs were collected; 22.4% (15/67) of patients had at least one positive environmental sample. Patients housed in temporary isolation ward rooms constructed from pre-fabricated containers (adjusted-odds-ratio, aOR = 10.46, 95% CI = 3.89-58.91, P = .008) had greater odds of detectable environmental contamination, with positive environmental samples obtained from the toilet area (60.0%, 12/20) and patient equipment, including electronic devices used for patient communication (8/20, 40.0%). A single HCW cluster was reported amongst staff working in the temporary isolation ward constructed from pre-fabricated containers; however, health care-associated transmission was deemed unlikely based on WGS and/or epidemiological investigations. CONCLUSION: Environmental contamination with SARS-CoV-2 RNA was observed in temporary isolation wards, particularly from the toilet area and smartphones used for patient communication. However, despite intensive surveillance, no healthcare-associated transmission was detected in temporary isolation wards over 18 months of prolonged usage, demonstrating their capacity for sustained use during succeeding pandemic waves.

Differential Spatial Gene and Protein Expression Associated with Recurrence Following Chemoradiation for Localized Anal Squamous Cell Cancer.

The identification of transcriptomic and protein biomarkers prognosticating recurrence risk after chemoradiation of localized squamous cell carcinoma of the anus (SCCA) has been limited by a lack of available fresh tissue at initial presentation. We analyzed archival FFPE SCCA specimens from pretreatment biopsies prior to chemoradiation for protein and RNA biomarkers from patients with localized SCCA who recurred (N = 23) and who did not recur (N = 25). Tumor cells and the tumor microenvironment (TME) were analyzed separately to identify biomarkers with significantly different expression between the recurrent and non-recurrent groups. Recurrent patients had higher mean protein expression of FoxP3, MAPK-activation markers (BRAF, p38-MAPK) and PI3K/Akt activation (phospho-Akt) within the tumor regions. The TME was characterized by the higher protein expression of immune checkpoint biomarkers such as PD-1, OX40L and LAG3. For patients with recurrent SCCA, the higher mean protein expression of fibronectin was observed in the tumor and TME compartments. No significant differences in RNA expression were observed. The higher baseline expression of immune checkpoint biomarkers, together with markers of MAPK and PI3K/Akt signaling, are associated with recurrence following chemoradiation for patients with localized SCCA. These data provide a rationale towards the application of immune-based therapeutic strategies to improve curative-intent outcomes beyond conventional therapies for patients with SCCA.

Outcomes and Toxicities of Modern Combined Modality Therapy with Atezolizumab Plus Bevacizumab and Radiation Therapy for Hepatocellular Carcinoma.

Atezolizumab plus bevacizumab has become frontline therapy for unresectable HCC. The compatibility of atezolizumab/bevacizumab with liver-directed RT has not been reported. Methods: HCC patients treated with liver-directed RT and atezolizumab/bevacizumab between 1/2020−11/2021 were included. Toxicity and outcomes were retrospectively recorded. For ALCs, we matched the analysis to a previously cohort of RT-treated HCC patients who did not receive atezolizumab/bevacizumab. Survival and time-to-liver-failure were analyzed using Kaplan−Meier. Results: Of 21 patients, with a median follow-up of 9.5 months, the median OS was 16.1 months. Post-RT, all patients had reduced tumors or treatment response. There were no ≥Grade 3 RT-related toxicities. Autoimmune complications occurred in two patients (9.5%), and GI bleeding in three patients (14.3%). Liver function remained stable post-RT. There was a marked decrease in ALCs immediately post-RT (post-RT/pre-RT ratio 47.3%, p < 0.0001), restored by 1 month to pre-treatment baseline (1-month post-RT/pre-RT ratio 95.1%, n.s.). Compared to HCC patients treated with RT alone, post-RT ALC recovery was faster with atezolizumab/bevacizumab (p = 0.009). Conclusion: In this first reported experience of RT with modern systemic therapy for HCC, combination therapy is safe and well-tolerated. As a favorable prognosticator, there appears to be faster recovery of ALC among patients who received RT with atezolizumab/bevacizumab.

Global Impact of the COVID-19 Pandemic on Cerebral Venous Thrombosis and Mortality / 대한뇌졸중학회지

Background@#and Purpose Recent studies suggested an increased incidence of cerebral venous thrombosis (CVT) during the coronavirus disease 2019 (COVID-19) pandemic. We evaluated the volume of CVT hospitalization and in-hospital mortality during the 1st year of the COVID-19 pandemic compared to the preceding year. @*Methods@#We conducted a cross-sectional retrospective study of 171 stroke centers from 49 countries. We recorded COVID-19 admission volumes, CVT hospitalization, and CVT in-hospital mortality from January 1, 2019, to May 31, 2021. CVT diagnoses were identified by International Classification of Disease-10 (ICD-10) codes or stroke databases. We additionally sought to compare the same metrics in the first 5 months of 2021 compared to the corresponding months in 2019 and 2020 (ClinicalTrials.gov Identifier: NCT04934020). @*Results@#There were 2,313 CVT admissions across the 1-year pre-pandemic (2019) and pandemic year (2020); no differences in CVT volume or CVT mortality were observed. During the first 5 months of 2021, there was an increase in CVT volumes compared to 2019 (27.5%; 95% confidence interval [CI], 24.2 to 32.0; P<0.0001) and 2020 (41.4%; 95% CI, 37.0 to 46.0; P<0.0001). A COVID-19 diagnosis was present in 7.6% (132/1,738) of CVT hospitalizations. CVT was present in 0.04% (103/292,080) of COVID-19 hospitalizations. During the first pandemic year, CVT mortality was higher in patients who were COVID positive compared to COVID negative patients (8/53 [15.0%] vs. 41/910 [4.5%], P=0.004). There was an increase in CVT mortality during the first 5 months of pandemic years 2020 and 2021 compared to the first 5 months of the pre-pandemic year 2019 (2019 vs. 2020: 2.26% vs. 4.74%, P=0.05; 2019 vs. 2021: 2.26% vs. 4.99%, P=0.03). In the first 5 months of 2021, there were 26 cases of vaccine-induced immune thrombotic thrombocytopenia (VITT), resulting in six deaths. @*Conclusions@#During the 1st year of the COVID-19 pandemic, CVT hospitalization volume and CVT in-hospital mortality did not change compared to the prior year. COVID-19 diagnosis was associated with higher CVT in-hospital mortality. During the first 5 months of 2021, there was an increase in CVT hospitalization volume and increase in CVT-related mortality, partially attributable to VITT.

First-in-Human Study of Bamlanivimab in a Randomized Trial of Hospitalized Patients With COVID-19.

Therapeutics for patients hospitalized with coronavirus disease 2019 (COVID-19) are urgently needed during the pandemic. Bamlanivimab is a potent neutralizing monoclonal antibody that blocks severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) attachment and entry into human cells, which could potentially lead to therapeutic benefit. J2W-MC-PYAA was a randomized, double-blind, sponsor unblinded, placebo-controlled, single ascending dose first-in-human trial (NCT04411628) in hospitalized patients with COVID-19. A total of 24 patients received either placebo or a single dose of bamlanivimab (700 mg, 2,800 mg, or 7,000 mg). The primary objective was assessment of safety and tolerability, including adverse events and serious adverse events, with secondary objectives of pharmacokinetic (PK) and pharmacodynamic analyses. Treatment-emergent adverse event (TEAE) rates were identical in the placebo and pooled bamlanivimab groups (66.7%). There were no apparent dose-related increases in the number or severity of TEAEs. There were no serious adverse events or deaths during the study, and no discontinuations due to adverse events. PKs of bamlanivimab is linear and exposure increased proportionally with dose following single i.v. administration. The half-life was ~ 17 days. These results demonstrate the favorable safety profile of bamlanivimab, and provided the initial critical evaluation of safety, tolerability, and PKs in support of the development of bamlanivimab in several ongoing clinical trials.

Resource utilization across the continuum of HIV care: An emergency department-based cohort study.

BACKGROUND: The objective of this study was to determine the healthcare resource utilization for people living with HIV (PLWH) presenting to the emergency department (ED) across the HIV Care Continuum. METHODS: This prospective study enrolled PLWH presenting to an urban ED between June 2016 and March 2017. Subjects were categorized as being linked to care, retained in care, on antiretroviral therapy (ART), and virally suppressed (<200 copies/ml). Data on ED visit rates, duration of stay, and hospital admission rates were compared to local metrics. RESULTS: Overall, 94.3% of 159 enrollees had been linked to care, 75.5% retained in care, 81.1% on ART, and 62.8% virally suppressed. Compared to the general population of the city and of the ED, participants had a higher ED visit rate (3.0 v. 1.2 visits per person-per year) in the past two years, a higher median duration of ED stay (12.6 v. 7.6 h), and a higher hospital admission rate (36.5% v. 24.9%) during their index ED visit. Viral suppression was negatively associated with admission (OR = 0.35, 95% CI: 0.17, 0.72). Forty-eight (30.2%) participants who had at least eight ED visits in the past two years were more likely to have a diagnosed mental health disorder (79.2% v. 62.2%, p=0.036). CONCLUSIONS: Our results showed that PLWH use more ED resources than the general population and a better engagement in HIV care is linked to lesser ED resource utilization for PLWH, indicating the importance of improved HIV care engagement in healthcare utilization management.

Contraception and Pre-Conception Counselling in Cardiac Patients: We Can Do Better. Experience From a Tertiary Centre in New Zealand.

BACKGROUND: Physiological changes in pregnancy can precipitate decompensation in women with pre-existing cardiac disease leading to suboptimal fetal outcome in addition to maternal risk. Many women born with congenital heart disease are living into childbearing years, and rheumatic heart disease (RHD) remains a significant problem within Maori and Pasifika communities in New Zealand. AIMS: To assess documentation of contraception advice and pre-conception counselling in women with pre-existing cardiac disease of childbearing potential and to explore potential barriers to these conversations. METHODS: We conducted a retrospective review of electronic clinic letters of 194 women with modified World Health Organization (mWHO) class 2 or above heart disease. This was followed by a survey of our cardiology team. RESULTS: Fifty-one (51) women with RHD and 143 women with non-RHD were identified. Thirty-eight per cent (38%) of women had documented discussions about contraception and pre-conception counselling. Women with RHD were less likely to receive discussions about contraception than women with non-RHD. All surveyed members of our cardiology team agreed that women with cardiac disease should have planned pregnancies and the majority reported always or usually discussing contraception. Factors such as lack of time, cultural barriers and presence of family members were identified. Many felt that the subject was outside of their expertise or admitted that they simply did not think about it. CONCLUSIONS: Advice regarding contraception in addition to pre-pregnancy counselling should be given to all patients with pre-existing cardiovascular disease of potential child-bearing potential. Our study shows much room for improvement.

Clinical implementation of plasma EGFR T790M testing using droplet digital PCR in TKI-resistant NSCLC patients.

BACKGROUND: Majority of non-small cell lung cancer (NSCLC) patients progressed on epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) due to acquired T790M mutation. Blood sample is increasingly used in clinical setting for EGFR T790M detection and our laboratory employed the droplet digital PCR (ddPCR) methodology for testing. This study investigated the positive rate, specimen type for rebiopsy and clinical impact of blood-based EGFR T790M testing. METHODS: We retrospectively evaluated clinical samples that underwent plasma EGFR T790M testing in TTSH Molecular Diagnostic Laboratory from August 2017 to September 2019. Data on diagnosis, EGFR activating and T790M mutations, and treatment strategies were recorded. RESULTS: A total of 104 progressive NSCLC cases were included in this study. Overall, 46 patients (44.2%) were tested T790M positive, and 47.8% of these tested positive had low levels (defined as ≤3% fractional abundance and <50 copies/mL plasma), which may be missed by the conventional methods with lower sensitivity. Of these tested with low T790M abundance, 77.3% subsequently received osimertinib. Activating mutations were not detected in 42 (40.4%) cases, indicating that the tumors were not actively shedding ctDNA. Among these, 24 patients underwent repeat testing with tissue or blood specimens. Thirteen patients were subsequently tested T790M positive and 12 of them switched treatment to osimertinib. The recommendation to repeat testing with a different biopsy or after a suitable interval increased the overall positive rate to 56.7% (59/104). CONCLUSION: The use of a highly sensitive platform such as ddPCR for the detection of low abundance T790M, and the approach of repeat testing in cases with insufficient ctDNA increased the positive rate. This in turn identified more patients who are eligible for targeted therapy.

Validation of a Drug-Related Problem Classification System for the Intermediate and Long-Term Care Setting in Singapore.

Background: This study aims to evaluate the inter-rater reliability and perceived usability of a newly developed drug-related problem (DRP) classification system for use by pharmacists in the intermediate and long-term care (ILTC) setting in Singapore. METHODS: This was a cross-sectional survey study involving the use of a self-administered questionnaire. All 55 pharmacists affiliated to the Pharmaceutical Society of Singapore (PSS) ILTC Pharmacists Workgroup who were above 21 years old and not authors of the classification system were invited to participate. The inter-rater reliability of participants' classification of 46 mock DRP cases using the new DRP classification system was determined using Fleiss's kappa (κ). Participants' perceived usability of the classification system was evaluated using six items with five-point Likert scales (1-"strongly disagree", 5-"strongly agree"). Results: Thirty-three pharmacists responded to the survey. Overall inter-rater reliability was found to be substantial (κ = 0.614; 95% CI: 0.611⁻0.617). All usability items received positive ratings ("strongly agree" or "agree") from at least 69% of participants. Conclusion: The new DRP classification system has substantial external validity and appears to be suitable for use by pharmacists to document and report DRPs in the ILTC setting in Singapore and facilitate evaluation of the impact of pharmaceutical care in the ILTC setting.

Non-invasive detection of actionable mutations in advanced non-small-cell lung cancer using targeted sequencing of circulating tumor DNA.

OBJECTIVE: To evaluate the feasibility of detecting actionable gene mutations in circulating tumor DNA (ctDNA) in patients with advanced non-small-cell lung cancer (NSCLC) using targeted next-generation sequencing (NGS). MATERIALS AND METHODS: In total 50 plasma samples from patients newly diagnosed with advanced NSCLC or resistant to first-line tyrosine kinase inhibitors (TKIs) were subjected to deep sequencing on a seven-gene panel (BRAF, EGFR, ERBB2, KRAS, NRAS, PIK3CA, PTEN) incorporated with molecular barcodes to improve accuracy in variant detection. When possible, results were compared with those from matched tissue samples. RESULTS: At least one alteration in the ctDNA was detected in 44 out of 50 patients (88%); EGFR was the most frequently mutated gene. Half the total number of patients (50%, 25 of 50) had at least one actionable genetic alteration with targeted therapies available for treatment. Our results showed a high concordance rate of 81% in detection of EGFR mutation between 26 matched tissue and plasma samples. For progressive patients, from whom tissue is mostly unavailable, the resistant EGFR T790 M mutation was validated using the droplet digital polymerase chain reaction (ddPCR), yielding a concordance of 92% between alternative platforms. CONCLUSION: Our study demonstrated that therapeutically actionable mutations can be detected with high accuracy in ctDNA using NGS. This promising approach offers alternative and non-invasive diagnostic methods for treatment guidance and clinical monitoring.

The Future Is Now-Prospective Study of Radiosurgery for More Than 4 Brain Metastases to Start in 2018!

Stereotactic radiosurgery (SRS) has replaced whole brain radiotherapy (WBRT) as standard therapy for most patients with four or fewer brain metastases due to improved cognitive outcomes and more favorable health related quality of life (QoL). Whether SRS or WBRT is the optimal radiation modality for patients with five to fifteen brain metastases remains an open question. Efforts are underway to develop prospective evidence to answer this question. One of the planned trials is a Canadian Cancer Trials Group (CCTG)-lead North American intergroup trial. In general cancer treatments must have two basic aims: prolonging and improving QoL. In this vein, the selection of overall survival and QoL metrics as outcomes appear obvious. Potential secondary outcomes are numerous: patient/disease related, treatment related, economic, translational, imaging, and dosimetric. In designing a trial, one must also ponder what is standard WBRT-specifically, whether it should be associated with memantine. With the rapid accrual of an intergroup trial of hippocampal-sparing WBRT, we may find that the standard WBRT regimen changes in the course of planned trials. As up-front radiosurgery is increasingly used for more than 4 brain metastases without high level evidence, we have a window of opportunity to develop high quality evidence which will help guide our future clinical and policy decisions.

Metabolic Bone Disease in Primary Biliary Cirrhosis.

Primary biliary cirrhosis (PBC) is a liver-specific autoimmune disease that primarily affects women (female-to-male ratio, 10:1) between 40 and 60 years of age. Metabolic bone disease is a common complication of PBC, affecting 14% to 52% of patients, depending on the duration and severity of liver disease. The osteoporosis seen in PBC seems mainly due to low bone formation, although increased bone resorption may contribute. Treatment of osteoporosis consists primarily of antiresorptive agents. Additional large prospective, long-term studies in patients with PBC are needed to determine efficacy in improving bone density as well as reducing fracture risk.

Incidence and predictors of Lhermitte's sign among patients receiving mediastinal radiation for lymphoma.

PURPOSE: To prospectively examine the risk of developing Lhermitte's sign (LS) in patients with lymphoma treated with modern-era chemotherapy followed by consolidation intensity-modulated radiation therapy. METHODS: We prospectively interviewed all patients with lymphoma who received irradiation to the mediastinum from July 2011 through April 2014. We extracted patient, disease, and treatment-related variables from the medical records of those patients and dosimetric variables from treatment-planning systems and analyzed these factors to identify potential predictors of LS with Pearson chi-square tests. RESULTS: During the study period 106 patients received mediastinal radiation for lymphoma, and 31 (29 %) developed LS. No correlations were found between LS and any of the variables examined, including total radiation dose, maximum point dose to the spinal cord, volume receiving 105 % of the dose, and volumes receiving 5 or 15 Gy. CONCLUSION: In this group of patients, treatment with chemotherapy followed by intensity-modulated radiation therapy led to 29 % developing LS; this symptom was independent of radiation dose and seemed to be an idiosyncratic reaction. This relatively high incidence could have resulted from prospective use of a structured interview.

Relationship between atypical T- and B-cell size predicts survival in peripheral T-cell lymphomas with large B-cells.

BACKGROUND: Pathological prognostication for peripheral T-cell lymphomas (PTCLs) complicated by large B-cell (LBC) proliferations has not been previously devised. METHODS: Forty-six cases of PTCL with LBCs were reviewed immunohistologically, by in situ hybridisation for Epstein-Barr virus encoded RNA and polymerase chain reaction analyses for T-cell receptor (TCR) clonality. Follow-up intervals ranged from 1 to 149 months (mean 40 months). RESULTS: Cases with atypical T-cell size equal to or exceeding that of interspersed LBCs (ATEB+, n = 12, including an ALK negative anaplastic large cell lymphoma) had significantly inferior disease-specific survival compared to ATEB negative (ATEB-, n = 34) cases (p = 0.002 for all cases and p = 0.014 when restricted to TCR clonal cases, n = 30) [hazard ratio 11.2; 95% confidence interval (CI) 0.94-85.0, p = 0.019]. All recorded deaths amongst ATEB+ cases occurred in 26 months, while TCR polyclonal ATEB- cases (n = 9) had none; TCR clonal ATEB- cases (n = 22) had 62% 5-year survival (95% CI 33-91%, p = 0.001). There was no survival separation between angioimmunoblastic (n = 25) and unspecified (n = 20) subsets of PTCL (p = 0.957). CONCLUSION: In PTCLs, cytological grading using harboured LBCs as internal yardsticks, as well as molecular genotypic measures of lymphoma cell burden, have prognostic value.