Risk, prevention, screening and management of carotid artery stenosis in head & neck cancer patients-An evidence based review.

Our review aims to clarify the incidence of carotid artery stenosis, risks of development, screening, management, and primary prevention strategies documented in the literature after radiation therapy for head and neck cancers. The high prevalence of carotid stenosis after radiation therapy for head and neck cancers has made surveillance and risk stratification critical. In addition to general cardiovascular risk factors such as smoking, diabetes, and dyslipidemia, risk factors for carotid artery stenosis after head and neck radiation included total plaque score, radiotherapy use and dosage, length of time after radiotherapy, and age greater than 50. Cancer subtype, namely nasopharyngeal cancer, may be correlated with increased risk as well, though contrasting results have been found. Interestingly, however, no significant relationship has been found between radiotherapy dose and stroke risk. Surgical management of post-radiation carotid stenosis is similar to that of stenosis unrelated to radiation, with carotid endarterectomy considered to be the gold standard treatment and carotid artery stenting being an acceptable, less-invasive alternative. Medical management of these patients has not been well-studied, but antiplatelet therapy, statins, and blood pressure control may be beneficial. The mainstay of screening for radiation-induced stenosis has been Doppler ultrasound, with measurement of changes in the intima-media thickness being a primary marker of disease development. A literature review was carried out using the MeSH terms "Carotid Artery Stenosis," "Head and Neck Neoplasms," and "Radiotherapy."

Multilevel thrombotic or embolic burden and its role in sex-related outcomes in acute limb ischemia.

OBJECTIVE: The impact of sex upon outcomes in acute limb ischemia (ALI) remains disputed. We aim to quantify the effect of sex upon amputation-free survival (AFS) after a percutaneous-first approach for ALI. METHODS: This was a two-center retrospective review of ALI managed via a percutaneous-first approach. Demographics, comorbidities, and clinical characteristics were analyzed. The Kaplan-Meier and Cox regression were used to estimate AFS, limb salvage, and overall survival. RESULTS: Over 9 years, 170 patients (n = 87, 51% males; median age, 67 [interquartile range (IQR), 59-77 years) presented with ALI. Rutherford classification was I in 56 (33%); IIa in 85 (50%); IIb in 20 (12%), and III in 9 (5%). Thirty-day mortality, major amputation rate, and fasciotomy rates were 8% (n = 13); 6.5% (n = 11), and 4.7% (n = 8), respectively. Among revascularized limbs, 92% were patent at 30 days. Length of stay was 7 days (IQR, 3-11 days). Complications included 13 bleeding episodes (8%), four cases of atrial fibrillation (2%), and three re-thrombosis/clot extension events (1.7%). No differences were noted in complication rates when stratified by sex. Females were older than males (median age, 70 [IQR, 62-79] vs 65 [IQR, 56-76 years]; P = .02) and more likely to present with atrial fibrillation (20.5% vs 8%; P = .02) and hyperlipidemia (72% vs 57%; P = .04). Females also more frequently presented with multi-level thrombotic/embolic burden compared with males (56% vs 43%; P = .03) and required both aspiration thrombectomy and thrombolysis (27% vs 14%; P = .02). Kaplan-Meier estimated median AFS, limb salvage, and overall survival were 425 days (IQR, 140-824 days); 314 days (IQR, 72-727 days); and 342 days (IQR, 112-762 days). When stratified by sex, females had worse survival (median, 270 days [IQR, 92-636 days] vs 406 days [IQR, 140-937 days]; P = .005) and limb salvage (median, 241 days [IQR, 88-636 days] vs 363 days [IQR, 49-822 days]; P = .04) compared with males. Univariate Cox regression showed female sex (hazard ratio [HR], 1.46; 95% confidence interval [CI], 1.04-2.05; P = .03), multi-level thrombotic/embolic burden (HR, 1.64; 95% CI, 1.17-2.31; P = .004), and Rutherford class (HR, 1.37; 95% CI, 1.08-1.73; P = .009) predicted major amputation/death. By multivariable Cox regression, multi-level thrombotic/embolic burden (HR, 1.54; 95% CI, 1.09-2.17; P = .01), Rutherford class (HR, 1.34; 95% CI, 1.07-1.69; P = .01), and female sex (HR, 1.45; 95% CI, 1.03-2.05; P = .03) were each independently predictive of major amputation/death. CONCLUSIONS: A percutaneous-first strategy is safe and efficacious in the overall ALI population. Similar to prior works, female vs male patients with ALI in our cohort have higher rates of mortality and major amputation. In our multivariable model, multi-level thrombotic/embolic burden was independently associated with a greater than 45% increased hazard of major amputation/death at last follow-up. Further prospective analysis is warranted to elucidate the underlying factors contributing to the higher prevalence of multi-level thrombotic/embolic burden in female patients with ALI, and to further define the optimal percutaneous-first approach for ALI in consideration of patient sex and extent of clot burden.

Cutdown is Associated with Higher 30-day Unplanned Readmissions and Wound Complications than Percutaneous Access for EVAR.

BACKGROUND: A 2023 Cochrane review showed no difference in bleeding/wound infection complications, short-term mortality and aneurysm exclusion between the percutaneous and cut-down approach for femoral access in endovascular aortic aneurysm repair (EVAR). In contrast, single-center studies have shown bilateral cutdown resulting in higher readmission rates due to higher rates of groin wound infections. Whether 30-day readmission rates vary by type of access during EVAR procedures is unknown. The goal of this study was to ascertain which femoral access approach for EVAR is associated with the lowest risk of 30-day readmission. METHODS: The Targeted Vascular Module from the American College of Surgeons National Surgical Quality Improvement Program was queried to identify patients undergoing EVAR for aortic disease from 2012-2021. All ruptures and other emergency cases were excluded. Cohorts were divided into bilateral cutdown, unilateral cutdown, failed percutaneous attempt converted to open and successful percutaneous access. The primary 30-day outcomes were unplanned readmission and wound complications. Univariate analyses were performed using the Fisher's exact test, Chi-Square test and the Student's t-test. Multivariable analysis was performed using logistic regression. RESULTS: From 2012 to 2021, 14,002 patients met study criteria. Most (7,395 [53%]) underwent completely percutaneous access, 5,616 (40%) underwent bilateral cutdown, 849 (6%) underwent unilateral cutdown, and 146 (1%) had a failed percutaneous access which was converted to open. Unplanned readmissions by access strategy included 7.6% for bilateral cutdown, 7.3% for unilateral cutdown, 7.8% for attempted percutaneous converted to cutdown, and 5.7% for completely percutaneous access (P < 0.001, Figure 1). After multivariable analysis, unplanned readmissions compared to percutaneous access yielded: percutaneous converted to cutdown adjusted odds ratio (AOR): 1.38, 95% CI [0.76-2.53], P = 0.29; unilateral cutdown AOR: 1.18, 95% CI [0.92-1.51], P = 0.20; bilateral cutdown AOR: 1.26, 95% CI [1.09-1.43], P = 0.001. Bilateral cutdown was also associated with higher wound complications compared to percutaneous access (AOR: 4.41, CI [2.86-6.79], P < 0.001), as was unilateral cutdown (AOR: 3.04, CI [1.46-6.32], P = 0.003). CONCLUSIONS: Patients undergoing cutdown for EVAR are at higher risk for 30-day readmission compared to completely percutaneous access. If patient anatomy allows for percutaneous EVAR, this access option should be prioritized.

In vivo affinity maturation of mouse B cells reprogrammed to express human antibodies.

Mice adoptively transferred with mouse B cells edited via CRISPR to express human antibody variable chains could help evaluate candidate vaccines and develop better antibody therapies. However, current editing strategies disrupt the heavy-chain locus, resulting in inefficient somatic hypermutation without functional affinity maturation. Here we show that these key B-cell functions can be preserved by directly and simultaneously replacing recombined mouse heavy and kappa chains with those of human antibodies, using a single Cas12a-mediated cut at each locus and 5' homology arms complementary to distal V segments. Cells edited in this way to express the human immunodeficiency virus type 1 (HIV-1) broadly neutralizing antibody 10-1074 or VRC26.25-y robustly hypermutated and generated potent neutralizing plasma in vaccinated mice. The 10-1074 variants isolated from the mice neutralized a global panel of HIV-1 isolates more efficiently than wild-type 10-1074 while maintaining its low polyreactivity and long half-life. We also used the approach to improve the potency of anti-SARS-CoV-2 antibodies against recent Omicron strains. In vivo affinity maturation of B cells edited at their native loci may facilitate the development of broad, potent and bioavailable antibodies.

Detailed DNA methylation characterisation of phyllodes tumours identifies a signature of malignancy and distinguishes phyllodes from metaplastic breast carcinoma.

Phyllodes tumours (PTs) are rare fibroepithelial lesions of the breast that are classified as benign, borderline, or malignant. As little is known about the molecular underpinnings of PTs, current diagnosis relies on histological examination. However, accurate classification is often difficult, particularly for distinguishing borderline from malignant PTs. Furthermore, PTs can be misdiagnosed as other tumour types with shared histological features, such as fibroadenoma and metaplastic breast cancers. As DNA methylation is a recognised hallmark of many cancers, we hypothesised that DNA methylation could provide novel biomarkers for diagnosis and tumour stratification in PTs, whilst also allowing insight into the molecular aetiology of this otherwise understudied tumour. We generated whole-genome methylation data using the Illumina EPIC microarray in a novel PT cohort (n = 33) and curated methylation microarray data from published datasets including PTs and other potentially histopathologically similar tumours (total n = 817 samples). Analyses revealed that PTs have a unique methylome compared to normal breast tissue and to potentially histopathologically similar tumours (metaplastic breast cancer, fibroadenoma and sarcomas), with PT-specific methylation changes enriched in gene sets involved in KRAS signalling and epithelial-mesenchymal transition. Next, we identified 53 differentially methylated regions (DMRs) (false discovery rate < 0.05) that specifically delineated malignant from non-malignant PTs. The top DMR in both discovery and validation cohorts was hypermethylation at the HSD17B8 CpG island promoter. Matched PT single-cell expression data showed that HSD17B8 had minimal expression in fibroblast (putative tumour) cells. Finally, we created a methylation classifier to distinguish PTs from metaplastic breast cancer samples, where we revealed a likely misdiagnosis for two TCGA metaplastic breast cancer samples. In conclusion, DNA methylation alterations are associated with PT histopathology and hold the potential to improve our understanding of PT molecular aetiology, diagnostics, and risk stratification. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

In vivo affinity maturation of the HIV-1 Env-binding domain of CD4.

Many human proteins have been repurposed as biologics for clinical use. These proteins have been engineered with in vitro techniques that improve affinity for their ligands. However, these approaches do not select against properties that impair efficacy such as protease sensitivity or self-reactivity. Here we engineer the B-cell receptor of primary murine B cells to express a human protein biologic without disrupting their ability to affinity mature. Specifically, CD4 domains 1 and 2 (D1D2) of a half-life enhanced-HIV-1 entry inhibitor CD4-Ig (CD4-Ig-v0) were introduced into the heavy-chain loci of murine B cells, which were then adoptively transferred to wild-type mice. After immunization, transferred B cells proliferated, class switched, affinity matured, and efficiently produced D1D2-presenting antibodies. Somatic hypermutations found in the D1D2-encoding region of engrafted B cells improved binding affinity of CD4-Ig-v0 for the HIV-1 envelope glycoprotein (Env) and the neutralization potency of CD4-Ig-v0 by more than ten-fold across a global panel of HIV-1 isolates, without impairing its pharmacokinetic properties. Thus, affinity maturation of non-antibody protein biologics in vivo can guide development of more effective therapeutics.

Mesothelin antigen density influences anti-mesothelin chimeric antigen receptor T cell cytotoxicity.

BACKGROUND AIMS: Several anti-mesothelin (MSLN) chimeric antigen receptor (CAR) T cells are in phase 1/2 clinical trials to treat solid-organ malignancies. The effect of MSLN antigen density on MSLN CAR cytotoxicity against tumor cells has not been examined previously, nor are there data regarding the effect of agents that increase MSLN antigen density on anti-MSLN CAR T cell efficacy. METHODS: MSLN antigen density was measured on a panel of pancreatic cancer and mesothelioma cell lines by flow cytometry. In parallel, the cytotoxicity and specificity of two anti-MSLN CAR T cells (m912 and SS1) were compared against these cell lines using a real-time impedance-based assay. The effect of two MSLN 'sheddase' inhibitors (lanabecestat and TMI-1) that increase MSLN surface expression was also tested in combination with CAR T cells. RESULTS: SS1 CAR T cells were more cytotoxic compared with m912 CAR T cells against cell lines that expressed fewer than ∼170 000 MSLN molecules/cell. A comparison of the m912 and amatuximab (humanized SS1) antibodies identified that amatuximab could detect and bind to lower levels of MSLN on pancreatic cancer and mesothelioma cell lines, suggesting that superior antibody/scFv affinity was the reason for the SS1 CAR's superior cytotoxicity. The cytotoxicity of m912 CAR T cells was improved in the presence of sheddase inhibitors, which increased MSLN antigen density. CONCLUSIONS: These data highlight the value of assessing CAR constructs against a panel of cells expressing varying degrees of target tumor antigen as occurs in human tumors. Furthermore, the problem of low antigen density may be overcome by concomitant administration of drugs that inhibit enzymatic shedding of MSLN.

In vivo affinity maturation of the HIV-1 Env-binding domain of CD4.

Many human proteins have been repurposed as biologics for clinical use. These proteins have been engineered with in vitro techniques that improve affinity for their ligands. However, these approaches do not select against properties that impair efficacy such as protease sensitivity or self-reactivity. Here we engineer the B-cell receptor of primary murine B cells to express a human protein biologic without disrupting their ability to affinity mature. Specifically, CD4 domains 1 and 2 (D1D2) of a half-life enhanced-HIV-1 entry inhibitor CD4-Ig (CD4-Ig-v0) were introduced into the heavy-chain loci of murine B cells, which were then adoptively transferred to wild-type mice. After immunization, transferred B cells proliferated, class switched, affinity matured, and efficiently produced D1D2-presenting antibodies. Somatic hypermutations found in the D1D2-encoding region of engrafted B cells improved binding affinity of CD4-Ig-v0 for the HIV-1 envelope glycoprotein (Env) and the neutralization potency of CD4-Ig-v0 by more than ten-fold across a global panel of HIV-1 isolates, without impairing its pharmacokinetic properties. Thus, affinity maturation of non-antibody protein biologics in vivo can guide development of more effective therapeutics.

Exogenous Klotho Extends Survival in COVID-19 Model Mice.

A striking feature of COVID-19 disease is the broad spectrum of risk factors associated with case severity, as well as the diversity of clinical manifestations. While no central agent has been able to explain the pathogenesis of SARS-CoV-2 infection, the factors that most robustly correlate with severity are risk factors linked to aging. Low serum levels of Klotho, an anti-aging protein, strongly correlate with the pathogenesis of the same risk factors and manifestations of conditions similar to those expressed in severe COVID-19 cases. The current manuscript presents original research on the effects of the exogenous application of Klotho, an anti-aging protein, in COVID-19 model mice. Klotho supplementation resulted in a statistically significant survival benefit in parametric and non-parametric models. Further research is required to elucidate the mechanistic role Klotho plays in COVID-19 pathogenesis as well as the possible modulation SARS-CoV-2 may have on the biological aging process.

The 4-1BBζ costimulatory domain in chimeric antigen receptors enhances CD8+ T-cell functionality following T-cell receptor stimulation.

BACKGROUND: Chimeric antigen receptor (CAR) T-cells have revolutionized the treatment of CD19- and B-cell maturation antigen-positive haematological malignancies. However, the effect of a CAR construct on the function of T-cells stimulated via their endogenous T-cell receptors (TCRs) has yet to be comprehensively investigated. METHODS: Experiments were performed to systematically assess TCR signalling and function in CAR T-cells using anti-mesothelin human CAR T-cells as a model system. CAR T-cells expressing the CD28 or 4-1BB costimulatory endodomains were manufactured and compared to both untransduced T-cells and CAR T-cells with a non-functional endodomain. These cell products were treated with staphylococcal enterotoxin B to stimulate the TCR, and in vitro functional assays were performed by flow cytometry. RESULTS: Increased proliferation, CD69 expression and IFNγ production were identified in CD8+ 4-1BBζ CAR T-cells compared to control untransduced CD8+ T-cells. These functional differences were associated with higher levels of phosphorylated ZAP70 after stimulation. In addition, these functional differences were associated with a differing immunophenotype, with a more than two-fold increase in central memory cells in CD8+ 4-1BBζ CAR T-cell products. CONCLUSION: Our data indicate that the 4-1BBζ CAR enhances CD8+ TCR-mediated function. This could be beneficial if the TCR targets epitopes on malignant tissues or infectious agents, but detrimental if the TCR targets autoantigens.

Testing an Automated Approach to Identify Variation in Outcomes among Children with Type 1 Diabetes across Multiple Sites.

Introduction: Efficient methods to obtain and benchmark national data are needed to improve comparative quality assessment for children with type 1 diabetes (T1D). PCORnet is a network of clinical data research networks whose infrastructure includes standardization to a Common Data Model (CDM) incorporating electronic health record (EHR)-derived data across multiple clinical institutions. The study aimed to determine the feasibility of the automated use of EHR data to assess comparative quality for T1D. Methods: In two PCORnet networks, PEDSnet and OneFlorida, the study assessed measures of glycemic control, diabetic ketoacidosis admissions, and clinic visits in 2016-2018 among youth 0-20 years of age. The study team developed measure EHR-based specifications, identified institution-specific rates using data stored in the CDM, and assessed agreement with manual chart review. Results: Among 9,740 youth with T1D across 12 institutions, one quarter (26%) had two or more measures of A1c greater than 9% annually (min 5%, max 47%). The median A1c was 8.5% (min site 7.9, max site 10.2). Overall, 4% were hospitalized for diabetic ketoacidosis (min 2%, max 8%). The predictive value of the PCORnet CDM was >75% for all measures and >90% for three measures. Conclusions: Using EHR-derived data to assess comparative quality for T1D is a valid, efficient, and reliable data collection tool for measuring T1D care and outcomes. Wide variations across institutions were observed, and even the best-performing institutions often failed to achieve the American Diabetes Association HbA1C goals (<7.5%).